Food Analysis Laboratory Manual

Food Science Text Series
S. Suzanne Nielsen
Food Analysis
Laboratory Manual
Third Edition
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Food Science
Text Series
Third Edition
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Series editor:
Dennis R. Heldman
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Mason, Ohio, USA
The Food Science Text Series provides faculty with the leading teaching tools. The Editorial Board has
outlined the most appropriate and complete content for each food science course in a typical food science
program and has identified textbooks of the highest quality, written by the leading food science educators.
Series Editor Dennis R. Heldman, Professor, Department of Food, Agricultural, and Biological Engineering,
The Ohio State University. Editorial Board; John Coupland, Professor of Food Science, Department of Food
Science, Penn State University, David A. Golden, Ph.D., Professor of Food Microbiology, Department of Food
Science and Technology, University of Tennessee, Mario Ferruzzi, Professor, Food, Bioprocessing and
Nutrition Sciences, North Carolina State University, Richard W. Hartel, Professor of Food Engineering,
Department of Food Science, University of Wisconsin, Joseph H. Hotchkiss, Professor and Director of the
School of Packaging and Center for Packaging Innovation and Sustainability, Michigan State University,
S. Suzanne Nielsen, Professor, Department of Food Science, Purdue University, Juan L. Silva, Professor,
Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Martin
Wiedmann, Professor, Department of Food Science, Cornell University, Kit Keith L. Yam, Professor of Food
Science, Department of Food Science, Rutgers University
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Food Analysis
Laboratory Manual
Third Edition
edited by
S. Suzanne Nielsen
Purdue University
West Lafayette, IN, USA
S. Suzanne Nielsen
Department of Food Science
Purdue University
West Lafayette
Indiana
USA
ISSN 1572-0330 ISSN 2214-7799 (electronic)

Food Science Text Series
ISBN 978-3-319-44125-2 ISBN 978-3-319-44127-6 (eBook)
DOI 10.1007/978-3-319-44127-6
Library of Congress Control Number: 2017942968
© Springer International Publishing 2017, corrected publication 2019

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Preface and Acknowledgments
This laboratory manual was written to accompany able for teaching food analysis laboratory
the textbook, Food Analysis, fifth edition. The labora- sessions vary considerably between schools,
tory exercises are tied closely to the text and cover 21 as do student numbers and their level in
of the 35 chapters in the textbook. Compared to the school. Therefore, instructors may need to
second edition of this laboratory manual, this third modify the laboratory procedures (e.g., num-
edition contains four introductory chapters with ber of samples analyzed, replicates) to fit their
basic information that compliments both the text- needs and situation. Some experiments
book chapters and the laboratory exercises (as include numerous parts/methods, and it is
described below). Three of the introductory chapters not assumed that an instructor uses all parts
include example problems and their solutions, plus of the experiment as written. It may be logical
additional practice problems at the end of the chap- to have students work in pairs to make things
ter (with answers at the end of the laboratory man- go faster. Also, it may be logical to have some
ual). This third edition also contains three new students do one part of the experiment/one
laboratory exercises, and previous experiments have type of sample and other students to another
been updated and corrected as appropriate. Most of part of the experiment/type of sample.
the laboratory exercises include the following: back- 4. Use of Chemicals: The information on hazards
ground, reading assignment, objective, principle of and precautions in the use of the chemicals for
method, chemicals (with CAS number and hazards), each experiment is not comprehensive but
reagents, precautions and waste disposal, supplies, should make students and a laboratory assis-
equipment, procedure, data and calculations, ques- tant aware of major concerns in handling and
tions, and resource materials. disposing of the chemicals.
Instructors using these laboratory exercises 5. Reagent Preparation: It is recommended in the
should note the following: text of the experiments that a laboratory assis-
tant prepare many of the reagents, because of
1. Use of Introductory Chapters: the time limitations for students in a laboratory
• Chap. 1, “Laboratory Standard Operating session. The lists of supplies and equipment for
Procedures” – recommended for students experiments do not necessarily include those
prior to starting any food analysis labora- needed by the laboratory assistant in preparing
tory exercises reagents for the laboratory session.
• Chap. 2, “Preparation of Reagents and 6. Data and Calculations: The laboratory exer-
Buffers” – includes definition of units of cises provide details on recording data and
concentrations, to assist in making chemi- doing calculations. In requesting laboratory
cal solutions reports from students, instructors will need to
• Chap. 3, “Dilution and Concentration specify if they require just sample calculations
Calculations” – relevant for calculations in or all calculations.
many laboratory exercises
• Chap. 4, “Use of Statistics in Food Even though this is the third edition of this labo-
Analysis” – relevant to data analysis ratory manual, there are sure to be inadvertent omis-
2. Order of Laboratory Exercises: The order of sions and mistakes. I will very much appreciate
laboratory exercises has been changed to be receiving suggestions for revisions from instructors,
fairly consistent with the reordering of chap- including input from lab assistants and students.
ters in the textbook, Food Analysis, fifth edition I maintain a website with additional teaching
(i.e., chromatography and spectroscopy near materials related to both the Food Analysis textbook
the front of the book). However, each labora- and laboratory manual. Instructors are welcome to
tory exercise stands alone, so they can be cov- contact me for access to this website. To compliment
ered in any order. the laboratory manual, the website contains more
3. Customizing Laboratory Procedures: It is rec- detailed versions of select introductory chapters and
ognized that the time and equipment avail- Excel sheets related to numerous laboratory exercises.
v
vi Preface and Acknowledgments
I am grateful to the food analysis instructors much appreciated. Special thanks go to Baraem
identified in the text who provided complete labora- (Pam) Ismail and Andrew Neilson for their input
tory experiments or the materials to develop the and major contributions toward this edition of the
experiments. For this edition, I especially want to laboratory manual. My last acknowledgment goes to
thank the authors of the new introductory chapters my former graduate students, with thanks for their
who used their experience from teaching food analy- help in working out and testing all experimental pro-
sis to develop what I hope will be very valuable cedures written for the initial edition of the labora-
chapters for students and instructors alike. The input tory manual.
I received from other food analysis instructors, their
students, and mine who reviewed these new intro- West Lafayette, IN, USA S. Suzanne Nielsen
ductory chapters was extremely valuable and very
The original version of this book was revised.

The correction to this book can be found at DOI 10.1007/978-3-319-44127-6_32
www.dbooks.org
Contents
Preface and Acknowledgments v 4.6 t-Scores 58
4.7 t-Tests 59
4.8 Practical Considerations 61
Part 1 Introductory Chapters 4.9 Practice Problems 62
4.10 Terms and Symbols 62
1 Laboratory Standard Operating Procedures 3
1.1 Introduction 5
1.2 Precision and Accuracy 5 Part 2 Laboratory Exercises
1.3 Balances 6
1.4 Mechanical Pipettes 7 5 Nutrition Labeling Using a Computer
1.5 Glassware 9 Program 65
1.6 Reagents 16 5.1 Introduction 67
1.7 Data Handling and Reporting 18 5.2 Preparing Nutrition Labels for Sample
1.8 Basic Laboratory Safety 19 Yogurt Formulas 67
5.3 Adding New Ingredients to a Formula
2 Preparation of Reagents and Buffers 21 and Determining How They Influence
2.1 Preparation of Reagents of Specified the Nutrition Label 68
Concentrations 22 5.4 An Example of Reverse Engineering
2.2 Use of Titration to Determine in Product Development 69
Concentration of Analytes 24 5.5 Questions 70
2.3 Preparation of Buffers 25
2.4 Notes on Buffers 30 6 Accuracy and Precision Assessment 71
2.5 Practice Problems 31 6.1 Introduction 72
6.2 Procedure 73
3 Dilutions and Concentrations 33 6.3 Data and Calculations 74
3.1 Introduction 34 6.4 Questions 74
3.2 Reasons for Dilutions
and Concentrations 34 7 High-Performance Liquid
3.3 Using Volumetric Glassware Chromatography 77
to Perform Dilutions 7.1 Introduction 79
and Concentrations 34 7.2 Determination of Caffeine in Beverages
3.4 Calculations for Dilutions By HPLC 79
and Concentrations 34 7.3 Solid-Phase Extraction and HPLC Analysis
3.5 Special Cases 40 of Anthocyanidins from Fruits
3.6 Standard Curves 41 and Vegetables 81
3.7 Unit Conversions 44
3.8 Avoiding Common Errors 45 8 Gas Chromatography 87
3.9 Practice Problems 46 8.1 Introduction 89
8.2 Determination of Methanol and Higher
4 Statistics for Food Analysis 49 Alcohols in Wine by Gas
4.1 Introduction 50 Chromatography 89
4.2 Population Distributions 50 8.3 Preparation of Fatty Acid Methyl
4.3 Z-Scores 51 Esters (FAMEs) and Determination
4.4 Sample Distributions 54 of Fatty Acid Profile of Oils by Gas
4.5 Confidence Intervals 55 Chromatography 91
vii
viii Contents
9 Mass Spectrometry with High-Performance 16 Water Hardness Testing by Complexometric

Liquid Chromatography 97 Determination of Calcium 147
9.1 Introduction 98 16.1 Introduction 149
9.2 Procedure 100 16.2 EDTA Titrimetric Method for Testing
9.3 Data and Calculations 101 Hardness of Water 149
9.4 Questions 102 16.3 Test Strips for Water Hardness 151
9.5 Case Study 102
17 Phosphorus Determination by Murphy-Riley
10 Moisture Content Determination 105 Method 153
10.2 Forced Draft Oven 107 17.2 Procedure 155
10.3 Vacuum Oven 109 17.3 Data and Calculations 155
10.4 Microwave Drying Oven 110 17.4 Questions 155
10.5 Rapid Moisture Analyzer 111
10.6 Toluene Distillation 111
10.7 Karl Fischer Method 112 18 Iron Determination by Ferrozine Method 157
10.8 Near-Infrared Analyzer 114 18.1 Introduction 158
10.9 Questions 114 18.2 Procedure 158
18.3 Data and Calculations 159
18.4 Question 159
11 Ash Content Determination 117
11.1 Introduction 118
11.2 Procedure 118 19 Sodium Determination Using Ion-Selective
11.3 Data and Calculations 118 Electrodes, Mohr Titration, and Test Strips 161
11.4 Questions 119 19.1 Introduction 163
19.2 Ion-Selective Electrodes 163
19.3 Mohr Titration 165
12 Fat Content Determination 121 19.4 Quantab® Test Strips 167
12.1 Introduction 123 19.5 Summary of Results 169
12.2 Soxhlet Method 123 19.6 Questions 170
12.3 Goldfish Method 125
12.4 Mojonnier Method 125
12.5 Babcock Method 127 20 Sodium and Potassium Determinations
by Atomic Absorption Spectroscopy
and Inductively Coupled Plasma-Optical
13 Protein Nitrogen Determination 131 Emission Spectroscopy 171
13.2 Kjeldahl Nitrogen Method 132 20.2 Procedure 174
13.3 Nitrogen Combustion Method 135 20.3 Data and Calculations 176
20.4 Questions 177
14 Total Carbohydrate by Phenol-Sulfuric
Acid Method 137 21 Standard Solutions and Titratable Acidity 179
14.2 Procedure 139 21.2 Preparation and Standardization of Base
14.3 Data and Calculations 140 and Acid Solutions 180
14.4 Questions 141 21.3 Titratable Acidity and pH 182
15 Vitamin C Determination by Indophenol 22 Fat Characterization 185

Method 143 22.1 Introduction 187
15.1 Introduction 144 22.2 Saponification Value 187
15.2 Procedure 145 22.3 Iodine Value 188
15.3 Data and Calculations 145 22.4 Free Fatty Acid Value 190
15.4 Questions 146 22.5 Peroxide Value 191
22.6 Thin-Layer Chromatography Separation
of Simple Lipids 193
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Contents ix
23 Proteins: Extraction, Quantitation, 27 CIE Color Specifications Calculated

and Electrophoresis 195 from Reflectance or Transmittance Spectra 219
23.2 Reagents 197 27.2 Procedure 222
23.3 Supplies 198 27.3 Questions 224
23.4 Procedure 198
23.6 Questions 200 28 Extraneous Matter Examination 225
28.2 Extraneous Matter in Soft Cheese 227
24 Glucose Determination by Enzyme 28.3 Extraneous Matter in Jam 228
Analysis 203 28.4 Extraneous Matter in Infant Food 229
24.1 Introduction 204 28.5 Extraneous Matter in Potato Chips 229
24.2 Procedure 205 28.6 Extraneous Matter in Citrus Juice 230
24.3 Data and Calculations 205 28.7 Questions 230
24.4 Questions 205
Part 3 Answers to Practice Problems
25 Gliadin Detection by Immunoassay 207
29 Answers to Practice Problems in Chap. 2,
Preparation of Reagents and Buffers 233
25.2 Procedure 209
25.4 Questions 211 30 Answers to Practice Problems in Chap. 3,
Dilutions and Concentrations 239
26 Viscosity Measurements of Fluid Food
Products 213 31 Answers to Practice Problems in Chap. 4,
26.1 Introduction 214 Use of Statistics in Food Analysis 247
26.2 Procedure 214
26.3 Data 216
26.4 Calculations 216 Correction to: Food Analysis Laboratory Manual C1
26.5 Questions 217
Contributors
Charles E. Carpenter Department of Nutrition, Oscar A. Pike Department of Nutrition, Dietetics,
Dietetics and Food Sciences, Utah State University, and Food Science, Brigham Young University, Provo,
Logan, UT, USA UT, USA
Young-Hee Cho Department of Food Science, Michael C. Qian Department of Food Science and
Purdue University, West Lafayette, IN, USA Technology, Oregon State University, Corvallis, OR,
USA
M. Monica Giusti Department of Food Science and
Technology, The Ohio State University, Columbus, Qinchun Rao Department of Nutrition, Food and
OH, USA Exercise Sciences, Florida State University,
Tallahassee, FL, USA
Y.H. Peggy Hsieh Department of Nutrition, Food
and Exercise Sciences, Florida State University, Ann M. Roland Owl Software, Columbia, MO, USA
Daniel E. Smith Department of Food Science and
Baraem P. Ismail Department of Food Science and Technology, Oregon State University, Corvallis, OR,
Nutrition, University of Minnesota, St. Paul, MN, USA
USA
Denise M. Smith School of Food Science,
Helen S. Joyner School of Food Science, University Washington State University, Pullman, WA, USA
of Idaho, Moscow, ID, USA
Stephen T. Talcott Department of Nutrition and
Dennis A. Lonergan The Vista Institute, Eden Food Science, Texas A&M University, College
Prairie, MN, USA Station, TX, USA
Lloyd E. Metzger Department of Dairy Science, Catrin Tyl Department of Food Science and
University of South Dakota, Brookings, SD, USA Nutrition, University of Minnesota, St. Paul, MN,
USA
Andrew P. Neilson Department of Food Science
and Technology, Virginia Polytechnic Institute and Robert E. Ward Department of Nutrition, Dietetics
State University, Blacksburg, VA, USA and Food Sciences, Utah State University, Logan, UT,
USA
S. Suzanne Nielsen Department of Food Science,
Purdue University, West Lafayette, IN, USA Ronald E. Wrolstad Department of Food Science
and Technology, Oregon State University, Corvallis,
Sean F. O’Keefe Department of Food Science and
OR, USA
Technology, Virginia Tech, Blacksburg, VA, USA
xi
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1
part
Introductory Chapters
1
chapter
Laboratory Standard
Operating Procedures
Andrew P. Neilson (*)
Department of Food Science and Technology,
Virginia Polytechnic Institute and State University,
Blacksburg, VA, USA
e-mail: [email protected]
Dennis A. Lonergan
The Vista Institute,
Eden Prairie, MN, USA
S. Suzanne Nielsen
Department of Food Science, Purdue University,
S.S. Nielsen, Food Analysis Laboratory Manual, Food Science Text Series, 3
DOI 10.1007/978-3-319-44127-6_1, © Springer International Publishing 2017
www.dbooks.org
1.1 Introduction 1.6 Reagents
1.2 Precision and Accuracy 1.6.1 Acids
1.3 Balances 1.6.2 Distilled Water
1.3.1 Types of Balances 1.6.3 Water Purity
1.3.2 Choice of Balance 1.6.4 Carbon Dioxide-Free Water
1.3.3 Use of Top Loading Balances 1.6.5 Preparing Solutions and Reagents
1.3.4 Use of Analytical Balances 1.7 Data Handling and Reporting
1.3.5 Additional Information 1.7.1 Significant Figures
1.4 Mechanical Pipettes 1.7.2 Rounding Off Numbers
1.4.1 Operation 1.7.3 Rounding Off Single Arithmetic
1.4.2 Pre-rinsing Operations
1.4.3 Pipetting Solutions of Varying Density or 1.7.4 Rounding Off the Results of a Series
Viscosity of Arithmetic Operations
1.4.4 Performance Specifications 1.8 Basic Laboratory Safety
1.4.5 Selecting the Correct Pipette 1.8.1 Safety Data Sheets
1.5 Glassware 1.8.2 Hazardous Chemicals
1.5.1 Types of Glassware/Plasticware 1.8.3 Personal Protective
1.5.2 Choosing Glassware/Plasticware Equipment and Safety Equipment
1.5.3 Volumetric Glassware 1.8.4 Eating, Drinking, Etc.
1.5.4 Using Volumetric Glassware to 1.8.5 Miscellaneous Information
Perform Dilutions and Concentrations
1.5.5 Conventions and Terminology
1.5.6 Burets
1.5.7 Cleaning of Glass and Porcelain
Chapter 1 • Laboratory Standard Operating Procedures 5
1.1 INTRODUCTION cal relationship between the analyte concentration and

the analytical response). There are a number of differ-
This chapter is designed to cover “standard operating ent methods available for the determination of preci-
procedures” (SOPs), or best practices, for a general sion. One method follows:
food analysis laboratory. The topics covered in this
chapter include balances, mechanical pipettes, glass- 1. Three separate concentration levels should be
ware, reagents, precision and accuracy, data handling, studied, including a low concentration near the
data reporting, and safety. These procedures apply to sensitivity level of the method, an intermediate
all the laboratory experiments in this manual, and concentration, and a concentration near the
therefore a thorough review of general procedures will upper limit of application of the method.
be invaluable for successful completion of these labo- 2. Seven replicate determinations should be made
ratory exercises. at each of the concentrations tested.
This manual covers many of the basic skills and 3. To allow for changes in instrument conditions,
information that are necessary for one to be a good the precision study should cover at least 2 h of
analytical food chemist. Much of this material is the normal laboratory operation.
type that one “picks up” from experience. Nothing can 4. To permit the maximum interferences in sequen-
replace actual lab experience as a learning tool, but tial operation, it is suggested that the samples be
hopefully this manual will help students learn proper run in the following order: high, low, and inter-
lab techniques early rather than having to correct mediate. This series is then repeated seven times
improper habits later. When one reads this manual, to obtain the desired replication.
your reaction may be “is all of this attention to detail 5. The precision statement should include a range
necessary?” Admittedly, the answer is “not always.” of standard deviations over the tested range of
This brings to mind an old Irish proverb that “the best concentration. Thus, three standard deviations
person for a job is the one that knows what to ignore.” will be obtained over a range of three
There is much truth to this proverb, but a necessary concentrations.
corollary is that one must know what they are ignor-
ing. The decision to use something other than the Accuracy refers to the degree (absolute or relative)
“best” technique must be conscious decision and not of difference between observed and “actual” values.
one made from ignorance. This decision must be based The “actual” value is often difficult to ascertain. It may
not only upon knowledge of the analytical method be the value obtained by a standard reference method
being used but also on how the resulting data will be (the accepted manner of performing a measurement).
used. Much of the information in this manual has been Another means of evaluating accuracy is by the addi-
obtained from an excellent publication by the US tion of a known amount of the material being analyzed
Environmental Protection Agency entitled Handbook for the food sample and then calculation of % recov-
for Analytical Quality Control in Water and Wastewater ery. This latter approach entails the following steps:
Laboratories. 1. Known amounts of the particular constituent
are added to actual samples at concentrations
for which the precision of the method is satis-
1.2 PRECISION AND ACCURACY factory. It is suggested that amounts be added
to the low-concentration sample, sufficient to
To understand many of the concepts in this chapter, a double that concentration, and that an amount
rigorous definition of the terms “precision” and “accu- be added to the intermediate concentration, suf-
racy” is required here. Precision refers to the ficient to bring the final concentration in the
reproducibility of replicate observations, typically sample to approximately 75 % of the upper limit
measured as standard deviation (SD), standard error of application of the method.
(SE), or coefficient of variation (CV). Refer to Chap. 4 2. Seven replicate determinations at each concen-
in this laboratory manual and Smith, 2017, for a more tration are made.
complete discussion of precision and accuracy. The 3. Accuracy is reported as the percent recovery at
smaller these values are, the more reproducible or pre- the final concentration of the spiked sample.
cise the measurement is. Precision is determined not on Percent recovery at each concentration is the
reference standards, but by the use of actual food sam- mean of the seven replicate results.
ples, which cover a range of concentrations and a vari-
ety of interfering materials usually encountered by the A fast, less rigorous means to evaluate precision
analyst. Obviously, such data should not be collected and accuracy is to analyze a food sample and replicate
until the analyst is familiar with the method and has a spiked food sample, and then calculate the recovery
obtained a reproducible standard curve (a mathemati- of the amount spiked. An example is shown in Table 1.1.
www.dbooks.org
6 A.P. Neilson et al.
balances are usually sensitive to 0.001–0.00001 g,

1. 1 Measured calcium content (g/L) of milk and
depending on the specific model. It should be remem-
table bered, however, that sensitivity (ability to detect small
spiked milk
differences in mass) is not necessarily equal to accuracy
Replicate Milk Milk + 0.75 g Ca/L (the degree to which the balances correctly report the
actual mass). The fact that a balance can be read to
1 1.29 2.15
0.01 mg does not necessarily mean it is accurate to
2 1.40 2.12
0.01 mg. What this means is that the balance can distin-
3 1.33 2.20
4 1.24 2.27 guish between masses that differ by 0.01 mg, but may
5 1.23 2.07 not accurately measure those masses to within 0.01 mg
6 1.40 2.10 of the actual masses (because the last digit is often
7 1.24 2.20 rounded). The accuracy of a balance is independent of
8 1.27 2.07 its sensitivity.
9 1.24 1.74
10 1.28 2.01 1.3.2 Choice of Balance
11 1.33 2.12
Mean 1.2955 2.0955 Which type of balance to use depends on “how much
SD 0.062 0.138 accuracy” is needed in a given measurement. One way
%CV 4.8 6.6 to determine this is by calculating how much relative
(%) error would be introduced by a given type of bal-
ance. For instance, if 0.1 g of a reagent was needed,
The accuracy can then be measured by calculating weighing it on a top loading balance accurate to within
the % of the spike (0.75 g/L) detected by comparing only ± 0.02 g of the actual mass would introduce
the measured values from the unspiked and spiked approximately 20 % error:
samples:
% error in measured mass
accuracy ≈ % recovery = (1.1)
absolute error in measured mass
measured spiked sample 100% (1.2)
× 100% measured mass
measured sample + amount of spike
0.02 g
2.0955 g / L % error in measured mass 100% 20%
accuracy % recovery 0.1 g
1.2955 g / L 0.75 g / L
100% 102.44% This would clearly be unacceptable in most situations.
The method measured the spike to within 2.44 %. By Therefore, a more accurate balance would be needed.
adding 0.75 g/L Ca to a sample that was measured to However, the same balance (with accuracy to
have 1.2955 g/L Ca, a perfectly accurate method within ± 0.02 g) would probably be acceptable for
would result in a spiked sample concentration of 1.295 weighing out 100 g of reagent, as the error would be
5 g/L + 0.75 g/L = 2.0455 g/L. The method actually approximately 0.02 %:
measured the spiked sample at 2.0955 g/L, which is 0.02 g
2.44 % greater than it should be. Therefore, the accu- % error in measured mass 100% 0.02%
100 g
racy is estimated at ~2.44 % relative error.
The decision on “how much accuracy” is needed can
only be answered when one knows the function of the
1.3 BALANCES reagent in the analytical method. This is one reason
why it is necessary to understand the chemistry
1.3.1 Types of Balances
involved in an analytical method, and not to simply
Two general types of balances are used in most laborato- approach an analytical method in a cookbook fashion.
ries. These are top loading balances and analytical Therefore, a general guideline regarding which bal-
balances. Top loading balances usually are sensitive to ance to use is hard to define.
0.1–0.001 g, depending on the specific model in use (this Another situation in which care must be exercised
means that they can measure differences in the mass of a in determining what type of balance to use is when a
sample to within 0.1–0.001 g). In, general, as the capacity difference in masses is to be calculated. For instance, a
(largest mass that can be measured) increases, the sensi- dried crucible to be used in a total ash determination
tivity decreases. In other words, balances that can mea- may weigh 20.05 g on a top loading balance, crucible
sure larger masses generally measure differences in plus sample = 25.05 g, and the ashed crucible 20.25 g. It
those masses to fewer decimal places. Analytical may appear that the use of the top loading balance
with its accuracy of ± 0.02 g would introduce approxi- with the vessel. The mass of the vessel must be
mately 0.1 % error, which would often be acceptable. known so that it can be subtracted from the
Actually, since a difference in weight (0.20 g) is being final mass to get the mass of the dried sample or
determined, the error would be approximately 10 % ash. Therefore, make sure to obtain the mass of
and thus unacceptable. In this case, an analytical bal- the vessel before the analysis. This can be done
ance is definitely required because sensitivity is by either weighing the vessel before taring the
required in addition to accuracy. balance and then adding the sample or obtain-
ing the mass of the vessel and then the mass of
1.3.3 Use of Top Loading Balances the vessel plus the sample.
2. The accumulation of moisture from the air or
These instructions are generalized but apply to the use
fingerprints on the surface of a vessel will add a
of most models of top loading balances:
small mass to the sample. This can introduce
1. Level the balance using the bubble level and the errors in mass that affect analytical results, par-
adjustable feet (leveling is required so that the ticularly when using analytical balances.
balance performs correctly). Therefore, beakers, weigh boats, and other
2. Either zero the balance (so the balance reads 0 weighing vessels should be handled with tongs
with nothing on the pan) or tare the balance so or with gloved hands. For precise measure-
that the balance reads 0 with a container that ments (moisture, ash, and other measurements),
will hold the sample (empty beaker, weighing weighing vessels should be pre-dried and
boat, etc.) on the weighing pan. The tare func- stored in a desiccator before use, and then
tion is conveniently used for “subtracting” the stored in a desiccator after drying, ashing, etc.
weight of the beaker or weighing boat into prior to weighing the cooled sample.
which the sample is added. 3. Air currents or leaning on the bench can cause
3. Weigh the sample. appreciable error in analytical balances. It is
best to take the reading after closing the side
1.3.4 Use of Analytical Balances doors of an analytical balance.
4. Most balances in modern laboratories are elec-
It is always wise to consult the specific instruction
tric balances. Older lever-type balances are no
manual for an analytical balance before using it.
longer in wide use, but they are extremely
Speed and accuracy are both dependent on one being
reliable.
familiar with the operation of an analytical balance. If
it has been a while since you have used a specific type
of analytical balance, it may be helpful to “practice”
1.4 MECHANICAL PIPETTES
before actually weighing a sample by weighing a
spatula or other convenient article. The following Mechanical pipettes (i.e., automatic pipettors) are
general rules apply to most analytical balances and standard equipment in many analytical laboratories.
should be followed to ensure that accurate results are This is due to their convenience, precision, and accept-
obtained and that the balance is not damaged by able accuracy when used properly and when calibrated.
improper use: Although these pipettes may be viewed by many as
1. Analytical balances are expensive precision being easier to use than conventional glass volumetric
instruments; treat them as such. pipettes, this does not mean that the necessary accuracy
2. Make sure that the balance is level and is on a and precision can be obtained without attention to
sturdy table or bench free of vibrations. proper pipetting technique. Just the opposite is the
3. Once these conditions are met, the same proce- case; if mechanical pipettes are used incorrectly, this
dure specified above for top loading balances is will usually cause greater error than the misuse of glass
used to weigh the sample on an analytical volumetric pipettes. The proper use of glass volumetric
balance. pipettes is discussed in the section on glassware. The
4. Always leave the balance clean. PIPETMAN mechanical pipette (Rainin Instrument
Co., Inc.) is an example of a continuously adjustable
1.3.5 Additional Information design. The proper use of this type of pipette, as recom-
mended by the manufacturer, will be described here.
Other points to be aware of regarding the use of bal- Other brands of mechanical pipettes are available, and
ances are the following: although their specific instructions should be followed,
1. Many analyses (moisture, ash, etc.) require their proper operation is usually very similar to that
weighing of the final dried or ashed sample described here.
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1.4.1 Operation 10. With plunger fully depressed, withdraw

mechanical pipette from vessel carefully with
1. Set the desired volume on the digital microme-
tip sliding along wall of vessel.
ter/volumeter. For improved precision, always
11. Allow plunger to return to top position.
approach the desired volume by dialing down-
12. Discard tip by depressing tip-ejector button
ward from a larger volume setting. Make sure
smartly.
not to wind it up beyond its maximum capacity;
13. A fresh tip should be used for the next measure-
this will break it beyond repair.
ment if:
2. Attach a disposable tip to the shaft of the pipette
and press on firmly with a slight twisting (a) A different solution or volume is to be
motion to ensure a positive, airtight seal. pipetted.
3. Depress the plunger to the first positive stop. (b) A significant residue exists in the tip (not to
This part of the stroke is the calibrated volume be confused with the visible “film” left by
displayed. Going past the first positive stop will some viscous or organic solutions).
cause inaccurate measurement.
4. Holding the mechanical pipette vertically,
1.4.2 Pre-rinsing
immerse the disposable tip into sample liquid
to a depth indicated (Table 1.2), specific to the Pipetting very viscous solutions or organic solvents
maximum volume of the pipette (P-20, 100, 200, will result in a significant film being retained on the
500, 1000, 5000, correspond to maximum vol- inside wall of the tip. This will result in an error that
umes of 20, 100, 200, 500, 1000, and 5000 μL, will be larger than the tolerance specified if the tip is
respectively). only filled once. Since this film remains relatively con-
5. Allow plunger to slowly return to the “up” posi- stant in successive pipettings with the same tip, accu-
tion. Never permit it to snap up (this will suck liquid racy may be improved by filling the tip, dispensing
up into the pipette mechanism, causing inaccu- the volume into a waste container, refilling the tip a
rate measurement and damaging the pipette). second time, and using this quantity as the sample.
6. Wait 1–2 s to ensure that full volume of sample This procedure is recommended in all pipetting oper-
is drawn into the tip. If the solution is viscous ations when critical reproducibility is required,
such as glycerol, you need to allow more time. whether or not tips are reused (same solution) or
7. Withdraw tip from sample liquid. Should any changed (different solutions/different volumes).
liquid remain on outside of the tip, wipe care- Note that the “non-wettability” of the polypropylene
fully with a lint-free cloth, taking care not to tip is not absolute and that pre-rinsing will improve
touch the tip opening. the precision and accuracy when pipetting any
8. To dispense sample, place tip end against side solution.
wall of vessel and depress plunger slowly past
the first stop until the second stop (fully 1.4.3 Pipetting Solutions of Varying
depressed position) is reached. Density or Viscosity
9. Wait (Table 1.3).
Compensation for solutions of varying viscosity or
density is possible with any adjustable pipette by
setting the digital micrometer slightly higher or
1. 2 Appropriate pipette depth for automatic lower than the required volume. The amount of
table pipettors compensation is determined empirically. Also, when
dispensing viscous liquids, it will help to wait 1 s
Pipette Depth (mm) longer at the first stop before depressing to the sec-
P-20D, P-100D, P-200D 1–2
ond stop.
P-500D, P-1000D 2–4
P-5000D 3–6 1.4.4 Performance Specifications
The manufacturer of PIPETMAN mechanical pipettes
provides the information in Table 1.4, on the precision
1. 3 Appropriate dispense wait time for auto-
and accuracy of their mechanical pipettes.
table matic pipettors
1.4.5 Selecting the Correct Pipette
Pipette Time (s)
Although automatic pipettes can dispense a wide
P-20D, P-100D, P-200D 1 range of volumes, you may often have to choose the
P-500D, P-1000D 1–2 “best” pipette with the most accuracy/precision from
P-5000D 2–3 among several choices. For example, a P5000
1. 4 Accuracy and precision of PIPETMAN 1. 5 Recommended volume ranges for mechani-

table mechanical pipettes table cal pipettors
Reproducibilitya Maximum volume Lowest recommended volume

Model Accuracy a
(standard deviation)
5 mL (5000 μL) 1 mL (1000 μL)
P-2OD <0.l μL @ 1–10 μL <0.04 μL @ 2 μL 1 mL (1000 μL) 0.1–0.2 mL (100–200 μL)
<1 % @ 10–20 μL <0.05 μL @ 10 μL 0.2 mL (200 μL) 0.02–0.04 mL (20–40 μL)
P-200D <0.5 μL @ 20–60 μL <0.15 μL @ 25 μL 0.1 mL (100 μL) 0.01–0.02 mL (10–20 μL)
<0.8 % @ 60–200 μL <0.25 μL @ 100 μL 0.05 mL (50 μL) 0.005–0.01 mL (5–10 μL)
<0.3 μL @ 200 μL 0.02 mL (20 μL) 0.002–0.004 mL (2–4 μL)
P-1000D <3 μL @ 100–375 μL <0.6 μL @ 250 μL 0.01 mL (10 μL) 0.001–0.002 mL (1–2 μL)
<0.8 % @ 375–l000 μL <1.0 μL @ 500 μL
<1.3 μL @ 1000 μL
P-5000D <12 μL @ 0.5–2 mL <3 μL @ 1.0 mL student grade to others possessing specific properties
<0.6 % @ 2.0–5.0 mL <5 μL @ 2.5 mL such as resistance to thermal shock or alkali, low boron
<8 μL @ 5.0 mL content, and super strength. The most common type is
a
Aqueous solutions, tips prerinsed once a highly resistant borosilicate glass, such as that
manufactured by Corning Glass Works under the
name “Pyrex” or by Kimble Glass Co. as “Kimax.”
(i.e., 5 mL) automatic pipettor could theoretically
Brown/amber actinic glassware is available, which
pipette anywhere between 0 and 5 mL. However, there
blocks UV and IR light to protect light-sensitive solu-
are several limitations that dictate which pipettes to
tions and samples. The use of vessels, containers, and
use. The first is a practical limitation: mechanical
other apparatus made of Teflon, polyethylene, poly-
pipettes are limited by the graduations (the incre-
styrene, and polypropylene is common. Teflon stop-
ments) of the pipette. The P5000 and P1000 are typi-
cock plugs have practically replaced glass plugs in
cally adjustable in increments of 0.01 mL (10 μL).
burets, separatory funnels, etc., because lubrication to
Therefore, these pipettes cannot dispense volumes of
avoid sticking (called “freezing”) is not required.
<10 μL, nor can they dispense volumes with more pre-
Polypropylene, a methylpentene polymer, is available
cision that of 10 μL. However, just because these
as laboratory bottles, graduated cylinders, beakers,
pipettes can technically be adjusted to 10 μL does not
and even volumetric flasks. It is crystal clear, shatter-
mean that they should be used to measure volumes
proof, autoclavable, chemically resistant, but relatively
anywhere near this small. Most pipettes are labeled
expensive as compared to glass. Teflon (polytetrafluo-
with a working range that lists the minimum and max-
roethylene, PTFE) vessels are available, although they
imum volume, but this is not the range for ideal per-
are very expensive. Finally, most glassware has a polar
formance. Mechanical pipettes should be operated
surface. Glassware can be treated to derivatize the sur-
from 100 % down to 10–20 % of their maximum capac-
face (typically, tetramethylsilane, or TMS) to make it
ity (Table 1.5). Below 10–20 % of their maximum capac-
nonpolar, which is required for some assays. However,
ity, performance (accuracy and precision) suffers. A
acid washing will remove this nonpolar layer.
good way of thinking of this is to use the largest pipette
capable of dispensing the volume in a single aliquot.
Mechanical pipettes are invaluable pieces of labo- 1.5.2 Choosing Glassware/Plasticware
ratory equipment. If properly treated and maintained, Some points to consider in choosing glassware and/or
they can last for decades. However, improper use can plasticware are the following:
destroy them in seconds. Mechanical pipettes should
1. Generally, special types of glass are not required
be calibrated, lubricated, and maintained at least
to perform most analyses.
yearly by a knowledgeable pipette technician.
2. Reagents and standard solutions should be
Weighing dispensed water is often a good check to see
stored in borosilicate or polyethylene bottles.
if the pipette needs calibration.
3. Certain dilute metal solutions may plate out on
glass container walls over long periods of stor-
1.5 GLASSWARE age. Thus, dilute metal standard solutions
should be prepared fresh at the time of
1.5.1 Types of Glassware/Plasticware analysis.
4. Strong mineral acids (such as sulfuric acid) and
Glass is the most widely used material for construc- organic solvents will readily attack polyethyl-
tion of laboratory vessels. There are many grades and ene; these are best stored in glass or a resistant
types of glassware to choose from, ranging from plastic.
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5. Borosilicate glassware is not completely inert, meniscus should be tangent to the calibration mark.
particularly to alkalis; therefore, standard solu- There are other sources of error, however, such as
tions of silica, boron, and the alkali metals (such changes in temperature, which result in changes in the
as NaOH) are usually stored in polyethylene actual capacity of glass apparatus and in the volume of
bottles. the solutions. The volume capacity of an ordinary
6. Certain solvents dissolve some plastics, includ- 100 mL glass flask increases by 0.025 mL for each 1°
ing plastics used for pipette tips, serological rise in temperature, but if made of borosilicate glass,
pipettes, etc. This is especially true for acetone the increase is much less. One thousand mL of water
and chloroform. When using solvents, check (and of most solutions that are ≤ 0.1 N) increases in
the compatibility with the plastics you are volume by approximately 0.20 mL per 1 °C increase at
using. Plastics dissolved in solvents can cause room temperature. Thus, solutions must be measured
various problems, including binding/precipi- at the temperature at which the apparatus was cali-
tating the analyte of interest, interfering with brated. This temperature (usually 20 °C) will be indi-
the assay, clogging instruments, etc. cated on all volumetric ware. There may also be errors
7. Ground-glass stoppers require care. Avoid of calibration of the adjustable measurement appara-
using bases with any ground glass because the tus (e.g., measuring pipettes), that is, the volume
base can cause them to “freeze” (i.e., get stuck). marked on the apparatus may not be the true volume.
Glassware with ground-glass connections Such errors can be eliminated only by recalibrating the
(burets, volumetric flasks, separatory funnels, apparatus (if possible) or by replacing it.
etc.) are very expensive and should be handled A volumetric apparatus is calibrated “to contain”
with extreme care. or “to deliver” a definite volume of liquid. This will be
indicated on the apparatus with the letters “TC” (to
For additional information, the reader is referred contain) or “TD” (to deliver). Volumetric flasks are cali-
to the catalogs of the various glass and plastic manu- brated to contain a given volume, which means that the
facturers. These catalogs contain a wealth of informa- flask contains the specified volume ± a defined toler-
tion as to specific properties, uses, sizes, etc. ance (error). The certified TC volume only applies to
the volume contained by the flask and it does not take
1.5.3 Volumetric Glassware into account the volume of solution that will stick to
the walls of the flask if the liquid is poured out.
Accurately calibrated glassware for accurate and pre- Therefore, for example, a TC 250 mL volumetric flask
cise measurements of volume has become known as will hold 250 mL ± a defined tolerance; if the liquid is
volumetric glassware. This group includes volumet- poured out, slightly less than 250 mL will be dispensed
ric flasks, volumetric pipettes, and accurately cali- due to solution retained on the walls of the flask (this is
brated burets. Less accurate types of glassware, the opposite of “to deliver” or TD, glassware discussed
including graduated cylinders, serological pipettes, below). They are available in various shapes and sizes
and measuring pipettes, also have specific uses in the ranging from 1 to 2000 mL capacity. Graduated cylin-
analytical laboratory when exact volumes are unnec- ders, on the other hand, can be either TC or TD. For
essary. Volumetric flasks are to be used in preparing accurate work the difference may be important.
standard solutions, but not for storing reagents. The Volumetric pipettes are typically calibrated to
precision of an analytical method depends in part deliver a fixed volume. The usual capacities are
upon the accuracy with which volumes of solutions 1–100 mL, although micro-volumetric pipettes are also
can be measured, due to the inherent parameters of the available. The proper technique for using volumetric
measurement instrument. For example, a 10 mL volu- pipettes is as follows (this technique is for TD pipettes,
metric flask will typically be more precise (i.e., have which are much more common than TC pipettes):
smaller variations between repeated measurements)
than a 1000 mL volumetric flask, because the neck on 1. Draw the liquid to be delivered into the pipette
which the “fill to” line is located is narrower, and above the line on the pipette. Always use a
therefore smaller errors in liquid height above or pipette bulb or pipette aid to draw the liquid
below the neck result in smaller volume differences into the pipette. Never pipette by mouth.
compared to the same errors in liquid height for the 2. Remove the bulb (when using the pipette aid,
larger flask. However, accuracy and precision are often or bulbs with pressure release valves, you can
independent of each other for measurements on simi- deliver without having to remove it) and replace
lar orders of magnitude. In other words, it is possible it with your index finger.
to have precise results that are relatively inaccurate 3. Withdraw the pipette from the liquid and wipe
and vice versa. There are certain sources of error, off the tip with tissue paper. Touch the tip of the
which must be carefully considered. The volumetric pipette against the wall of the container from
apparatus must be read correctly; the bottom of the which the liquid was withdrawn (or a spare
beaker). Slowly release the pressure of your fin- standard for laboratory glassware. Class A glassware
ger (or turn the scroll wheel to dispense) on the has the tightest tolerances and therefore the best
top of the pipette and allow the liquid level in accuracy and precision. These flasks are rated
the pipette to drop so that the bottom of the TC. Therefore, volumetric flasks are used to bring
meniscus is even with the line on the pipette. samples and solutions up to a defined volume. They
4. Move the pipette to the beaker or flask into are not used to quantitatively deliver or transfer sam-
which you wish to deliver the liquid. Do not ples because the delivery volume is not known. Other
wipe off the tip of the pipette at this time. Allow types of glassware (non-Class A flasks, graduated
the pipette tip to touch the side of the beaker or cylinders, Erlenmeyer flasks, round-bottomed flasks,
flask. Holding the pipette in a vertical position, beakers, bottles, etc., Fig. 1.1b) are less accurate and
allow the liquid to drain from the pipette. less precise. They should not be used for quantitative
5. Allow the tip of the pipette to remain in contact volume dilutions or concentrations if Class A volu-
with the side of the beaker or flask for several metric flasks are available.
seconds. Remove the pipette. There will be a For transferring a known volume of a liquid sam-
small amount of liquid remaining in the tip of ple for a dilution or concentration, the “gold standard”
the pipette. Do not blow out this liquid with the providing maximal accuracy and precision is a Class A
bulb, as TD pipettes are calibrated to account glass volumetric pipette (Fig. 1.2a). These pipettes are
for this liquid that remains. rated “to deliver” (TD), which means that the pipette
will deliver the specified volume ± a defined tolerance
Note that some volumetric pipettes have calibra- (error). The certified TD volume takes into account the
tion markings for both TC and TD measurements. volume of solution that will stick to the walls of the
Make sure to be aware which marking refers to which pipette as well as the volume of the drop of solution
measurement (for transfers, use the TD marking). The that typically remains in the tip of the pipette after
TC marking will be closer to the dispensing end of the delivery (again, you should not attempt to get this
pipette (TC does not need to account for the volume drop out, as it is already accounted for). Therefore, for
retained on the glass surface, whereas TD does account example, a TD 5 mL pipette will hold slightly more
for this). than 5 mL but will deliver (dispense) 5 mL ± a defined
Measuring and serological pipettes should also be tolerance (the opposite of TC glassware). It is impor-
held in a vertical position for dispensing liquids; how- tant to note that volumetric pipettes are used only to
ever, the tip of the pipette is only touched to the wet deliver a known amount of solution. Typically they
surface of the receiving vessel after the outflow has should not be used to determine the final volume of
ceased. Some pipettes are designed to have the small the solution unless the liquids dispensed are the only
amount of liquid remaining in the tip blown out and components of the final solution. For example, if a
added to the receiving container; such pipettes have a sample is dried down and then liquid from a volumet-
frosted band near the top. If there is no frosted band ric pipette is used to resolubilize the solutes, it is
near the top of the pipette, do not blow out any remain- unknown if the solutes significantly affect the volume
ing liquid. of the resulting solution, unless the final volume is
measured, which may be difficult to do. Although the
effect is usually negligible, it is best to use volumetric
1.5.4 Using Volumetric Glassware
glassware to assure that the final volume of the result-
to Perform Dilutions
ing solution is known (the dried solutes could be dis-
and Concentrations
solved in a few mL of solvent and then transferred to a
Typically, dilutions are performed by adding a liq- volumetric flask for final dilution). However, it is
uid (water or a solvent) to a sample or solution. acceptable to add several solutions together using vol-
Concentrations may be performed by a variety of umetric pipettes and then add the individual volumes
methods, including rotary evaporation, shaking together to calculate the final volume. However, using
vacuum evaporation, vacuum centrifugation, boil- a single volumetric flask to dilute to a final volume is
ing, oven drying, drying under N2 gas, or freeze still the favored approach, as using one measurement
drying. for the final volume reduces the uncertainty. (The
For bringing samples or solutions up to a known errors, or tolerances, of the amounts added are also
volume, the “gold standard” providing maximal added together; therefore, using fewer pieces of glass-
accuracy and precision is a Class A glass volumetric ware lowers the uncertainty of the measurement even
flask (Fig. 1.1a). During manufacture, glassware to be if the tolerances of the glassware are the same.) For
certified as Class A is calibrated and tested to comply example, suppose you need to measure out 50 mL of
with tolerance specifications established by the solution. You have access to a 50 mL volumetric flask
American Society for Testing and Materials (ASTM, and a 25 mL volumetric pipette, both of which have
West Conshohocken, PA). These specifications are the tolerances of ± 0.06 mL. If you obtain 50 mL by filling
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a b c d e
1. 1 Class A volumetric flask (a) and other types of non-Class A volume measuring glassware: graduated cylinder
figure
(b), Erlenmeyer flask (c), beaker (d), and bottle (e)
the volumetric flask, the measured volume is Information typically printed on the side of the
50 mL ± 0.06 mL (or somewhere between 49.94 and pipette or flask includes the class of the pipette or
50.06 mL). If you pipette 25 mL twice into a beaker, the flask, whether the glassware is TD or TC, the TC or TD
tolerance of each measurement is 25 mL ± 0.06 mL, and volume, and the defined tolerance (error) (Fig. 1.3).
the tolerance of the combined volume is the sum of the Note that the specifications are typically valid at a
means and the errors: specified temperature, typically 20 °C. Although it is
rare that scientists equilibrate solutions to exactly
( 25 mL ± 0.06 mL ) + ( 25 mL ± 0.06 mL ) = 20 °C before volume measurement, this temperature is
50 mL ± 0.12 mL = 49.88 − 50.12 mL assumed to be approximate room temperature. Be
aware that the greater the deviation from room tem-
This additive property of tolerances, or errors, com- perature, the greater the error in volume measure-
pounds further as more measurements are combined; ment. The specific gravity (density) of water at 4, 20,
conversely, when the solution is brought to volume 60, and 80 °C relative to 4 °C is 1.000, 0.998, 0.983, and
using a volumetric flask, only a single tolerance factors 0.972. This means that a given mass of water has lower
into the error of the measurement. density (greater volume for given mass) at tempera-
Other types of pipettes (non-Class A volumetric tures above 20 °C. This is sometimes seen when a volu-
glass pipettes, adjustable pipettors, automatic pipet- metric flask is brought exactly to volume at room
tors, reed pipettors, serological pipettes, etc., Fig. 1.2b) temperature and then is placed in an ultrasonic bath to
and other glassware (graduated cylinders, etc.) are less help dissolve the chemicals, warming the solution. A
accurate and less precise. They should not be used for solution that was exactly at the volume marker at
quantitative volume transfers. Pipettes are available room temperature will be above the volume when the
(but rare) that are marked with lines for both TC and solution is warmer. To minimize this error, volumes
TD. For these pipettes, the TD line would represent the should be measured at room temperature.
volume delivered when the drop at the tip is dispensed Volumetric glassware (flasks and pipettes) should
and TC when the drop remains in the pipette. be used for quantitative volume measurements during
a b c d e
1. 2 Class A volumetric pipette (a) and non-volumetric pipettes: adjustable pipettors (b), reed pipettor (c), serological
figure
pipettes (d)
a b
1. 3 Image of the label on a Class A volumetric flask pipette (a) and Class A volumetric pipette (b)
figure
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volumetric measurements can result in significant

error being introduced into the measurement.
Typical tolerances for lab glassware are presented
in Tables 1.6 and 1.7. References for ASTM specifica-
tions are found at http://www.astm.org/.
A comparison of Tables 1.6 and 1.7 reveals some
important points. First, even for Class A glassware, the
tolerances for volumetric transfer pipettes (pipettes
with a single TD measurement) are much tighter than
for graduated measuring pipettes (pipettes with grad-
uations that can be used to measure a wide range of
volumes) of the same volume. Second, even for Class
A glassware, the tolerances for volumetric transfer
pipettes and volumetric flasks are much tighter than
1. 4 Image of a liquid meniscus at the line for a
figure
Class A volumetric flask
dilutions and concentrations whenever possible to 1. 6 Volume tolerances of Class A glassware

maximize the accuracy and precision of the procedure. table required by ASTM specifications
For both volumetric flasks and pipettes, the level of the Tolerance (± mL)
liquid providing the defined volume is indicated by a
Volumetric Measuring Volumetric
line (usually white or red) etched or printed on the Volume (transfer) (graduated) flask Graduated
neck of the glassware. To achieve the TD or TC vol- (mL) Buret pipette pipettes cylinder
ume, the bottom of the meniscus of the liquid should
0.5 0.006
be at the line as shown in Fig. 1.4.
1 0.006 0.010
For a volumetric flask, the proper technique for 2 0.006 0.01 0.015
achieving the correct volume is to pour the liquid into 3 0.01 0.02 0.015
the flask until the meniscus is close to the marking line, 4 0.01 0.03 0.020
and then add additional liquid dropwise (with a man- 5 0.01 0.05 0.020 0.05
ual pipette or Pasteur pipette) until the bottom (NOT 10 0.02 0.02 0.08 0.020 0.10
the top or middle) of the meniscus is at the line with 25 0.03 0.03 0.10 0.030 0.17
your eye level to the line. (If you do not look straight at 50 0.05 0.05 0.050 0.25
the line, occur, making it appear that so that your eye 100 0.10 0.08 0.080 0.50
and the line are at the same level, a phenomenon 250 0.012 1.00
500 0.013 2.00
known as “parallax” can occur, making it appear that
1000 0.015 3.00
the bottom of the meniscus is at the line when in fact it
is not, resulting in errors in volume measurement.) If
the level of the liquid is too high, liquid can be removed
using a clean pipette (or the liquid poured out and start
again). However, be aware that this cannot be done 1. 7 Volume tolerances of non-Class A glass-
table ware required by ASTM specifications
when preparing a reagent for which the solutes were
accurately measured into the flask and you are adding Tolerance (± mL)
liquid to make up to volume. In this case, you must Volume Volumetric Volumetric Graduated
start over. For this reason, the best practice is to add (mL) Buret (transfer) pipette flask cylinder
liquid slowly, and then use a pipette to add liquid
0.5 0.012
dropwise when approaching the desired volume. 1 0.012
For a volumetric pipette, the proper technique for 2 0.012
achieving the correct volume is to draw liquid into the 3 0.02
pipette until the meniscus is above the line, and then 4 0.02
withdraw the pipette from the liquid and dispense the 5 0.02 0.10
excess liquid from the pipette until the bottom of the 10 0.04 0.04 0.04 0.20
meniscus is at the line. It is critical that the pipette be 25 0.06 0.06 0.06 0.34
withdrawn from the solution for this step. If the level 50 0.10 0.10 0.24 0.50
of the liquid goes below the line, additional liquid is 100 0.20 0.16 0.40 1.00
250 0.60 2.00
drawn up, and the process is repeated. Proper volu-
500 4.00
metric measurements require practice and should be
1000 6.00
repeated until they are performed correctly. Improper
for graduated cylinders of the same volume. Therefore, salt solution is concentrated tenfold (10X), the volume is
volumetric transfer pipettes and volumetric flasks are decreased to 9 mL (either by reducing to 9 mL or drying
preferred for dilutions and concentrations. For exam- completely and reconstituting to 9 mL, tenfold or 10X
ple, a 1000 mL Class A volumetric flask has a tolerance lower than 90 mL), and the final concentration is 3.1 ppm
of ±0.015 mL (the actual TC volume is somewhere salt (tenfold or 10X more than 0.31 ppm). Although ten-
between 999.985 and 1000.015 mL), while a 1000 mL fold or 10X was used for these examples, any value can
graduated cylinder has a tolerance of ± 3.00 mL (the be used. In microbiology, values of 10X, 100X, 1000X, etc.
actual TC volume is somewhere between 997 and are commonly used due to the log scale used in that
1003 mL). This is a 200-fold larger potential error in the field. However, less standard dilutions of any value are
measurement of 1000 mL! Finally, tolerances for non- routinely used in analytical chemistry.
Class A glassware are much broader than for Class A, The last terminology system for dilutions and
and thus Class A should be used if available. concentrations involves ratios. This system is some-
what ambiguous and is not used in the Food Analysis
1.5.5 Conventions and Terminology text or lab manual. This system refers to dilutions as
“X:Y,” where X and Y are the masses or volumes of the
To follow the analytical procedures described in this
initial and final solutions/samples. For example, it
manual and perform calculations correctly, common
may be stated that “the solution was diluted 1:8.” This
terminology and conventions (a convention is a stan-
system is ambiguous for the following reasons:
dard or generally accepted way of doing or naming
something) must be understood. A common phrase in 1. The first and last numbers typically refer to the
dilutions and concentrations is “diluted to” or “diluted initial and final samples, respectively (there-
to a final volume of.” This means that the sample or fore, a 1:8 dilution would mean 1 part initial
solution is placed in a volumetric flask, and the final sample and 8 parts final sample). However,
volume is adjusted to the specified value. In contrast, there is no standard convention. Therefore, an
the phrase “diluted with” means that the specified “X:Y” dilution could be interpreted either way.
amount is added to the sample or solution. In this latter 2. There is no standard convention as to whether
case, the final mass/volume must be calculated by add- this system describes the “diluted to” or
ing the sample mass/volume and the amount of liquid “diluted with” (as described above) approach.
added. For example, suppose you take a 1.7 mL volume Therefore, diluting a sample 1:5 could be inter-
and either (1) dilute to 5 mL with methanol or (2) dilute preted as either (1) diluting 1 mL sample with
with 5 mL methanol. In the first case, this means that the 4 mL for a final volume of 5 mL (“diluted to”) or
sample (1.7 mL) is placed in a volumetric flask and (2) diluting 1 mL sample with 5 mL for a final
methanol (~3.3 mL) is added so that the final volume is volume of 6 mL (“diluted with”).
5 mL total. In the second case, the sample (1.7 mL) is
combined with 5 mL methanol, and the final volume is Because of these ambiguities, the ratio system is
6.7 mL. As you can see, these are very different values. discouraged in favor of the “X-fold” terminology.
This will always be the case except when one of the vol- However, ratio dilutions still appear in some litera-
umes is much larger than the other. For example, if you ture. If possible, it is recommended that you investi-
were working with a 10 μL sample, diluting it “to 1 L” gate to clarify what is meant by this terminology.
or “with 1 L” would result in final volumes of 1 L and Another factor to consider is that liquid volumes
1.00001 L, respectively. It is important to understand the are often not strictly additive. For example, exactly
differences between these two conventions to perform 500 ml 95 % v/v ethanol aq. added to 500 ml distilled
procedures correctly and interpret data accurately. water will not equal 1000 ml; in fact, the new volume
Another common term in dilutions/concentrations will be closer to 970 ml. Where did the missing 30 ml
is the term “fold” or “X.” This refers to the ratio of the go? Polar molecules such as water undergo different
final and initial concentrations (or volumes and masses) three-dimensional intermolecular bonding in a pure
of the sample or solution during each step. An “X-fold solution versus in a mixture with other solute or chemi-
dilution” means that the concentration of a sample cals such as ethanol. The difference in bonding causes
decreases (and typically the volume increases) by a an apparent contraction in this case. As well, addition of
given factor. For example, if 5 mL of an 18.9 % NaCl solu- solute to an exact volume of water will change the vol-
tion is diluted tenfold (or 10X) with water, 45 mL water ume after dissolved. To account for this effect, volumet-
is added so that the final volume is 50 mL (tenfold or 10X ric glassware is used to bring mixed solutions up to a
greater than 5 mL) and the final concentration is 1.89 % final volume after initial mixing. When two liquids are
NaCl (tenfold or 10X less than 18.9 %). Conversely, an mixed, the first liquid is volumetrically transferred into
“X-fold concentration” means that the concentration of a a volumetric flask, and then the second liquid is added
sample increases (and typically the volume decreases) to volume, with intermittent swirling or vortexing to
by the stated factor. For example, if 90 mL of a 0.31 ppm mix the liquids as they are being combined. For mixing
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solids into solvents, the chemicals are first placed in a “ultrapure” grades. The purity of these materials
volumetric flask, dissolved in a partial volume, and required in analytical chemistry varies with the type
then brought to exact volume with additional solvent. of analysis. The parameter being measured and the
sensitivity and specificity of the detection system are
1.5.6 Burets important factors in determining the purity of the
reagents required. Technical grade is useful for mak-
Burets are used to deliver definite volumes. The more
ing cleaning solutions, such as the nitric acid and
common types are usually of 25 or 50 ml capacity,
alcoholic potassium hydroxide solutions mentioned
graduated to tenths of a milliliter, and are provided
previously. For many analyses, analytical reagent
with stopcocks. For precise analytical methods in
grade is satisfactory. Other analyses, e.g., trace
microchemistry, microburets are also used. Microburets
organic and HPLC, frequently require special “ultra-
generally are of 5 or 10 ml capacity, graduated in hun-
pure” reagents and solvents. In methods for which
dredths of a milliliter division. General rules in regard
the purity of reagents is not specified, it is intended
to the manipulation of a buret are as follows:
that analytical reagent grade be used. Reagents of
1. Do not attempt to dry a buret that has been lesser purity than that specified by the method should
cleaned for use, but rather rinse it two or three not be used.
times with a small volume of the solution with There is some confusion as to the definition of the
which it is to be filled. terms analytical reagent grade, reagent grade, and
2. Do not allow alkaline solutions to stand in a buret, ACS analytical reagent grade. A review of the litera-
because the glass will be attacked, and the stop- ture and chemical supply catalogs indicates that the
cock, unless made of Teflon, will tend to freeze. three terms are synonymous. National Formulary
3. A 50 ml buret should not be emptied faster than (NF), US Pharmaceutical (USP), and Food Chemicals
0.7 ml per second; otherwise, too much liquid Codex (FCC) are grades of chemicals certified for use
will adhere to the walls; as the solution drains as food ingredients. It is important that only NF, USP,
down, the meniscus will gradually rise, giving a or FCC grades be used as food additives if the product
high false reading. is intended for consumption by humans, rather than
for chemical analysis.
It should be emphasized that improper use of
and/or reading of burets can result in serious calcula- 1.6.1 Acids
tion errors.
The concentration of common commercially available
acids is given in Table 1.8.
1.5.7 Cleaning of Glass and Porcelain
In the case of all apparatus for delivering liquids, 1.6.2 Distilled Water
the glass must be absolutely clean so that the film of
Distilled or demineralized water is used in the lab-
liquid never breaks at any point. Careful attention
oratory for dilution, preparation of reagent solu-
must be paid to this fact or the required amount of
tions, and final rinsing of washed glassware.
solution will not be delivered. The method of clean-
ing should be adapted to both the substances that
are to be removed and the determination to be per-
formed. Water-soluble substances are simply 1. 8 Concentration of common commercial
washed out with hot or cold water, and the vessel is table strength acids
finally rinsed with successive small amounts of dis-
tilled water. Other substances more difficult to Molecular
remove, such as lipid residues or burned material, weight Concentration Specific
may require the use of a detergent, organic solvent, Acid (g/mol) (M) gravity
nitric acid, or aqua regia (25 % v/v conc. HNO3 in Acetic acid, glacial 60.05 17.4 1.05
conc. HCl). In all cases it is good practice to rinse a Formic acid 46.02 23.4 1.20
vessel with tap water as soon as possible after use. Hydriodic acid 127.9 7.57 1.70
Material allowed to dry on glassware is much more Hydrochloric acid 36.5 11.6 1.18
difficult to remove. Hydrofluoric acid 20.01 32.1 1.167
Hypophosphorous acid 66.0 9.47 1.25
Lactic acid 90.1 11.3 1.2
1.6 REAGENTS Nitric acid 63.02 15.99 1.42
Perchloric acid 100.5 11.65 1.67
Chemical reagents, solvents, and gases are available Phosphoric acid 98.0 14.7 1.70
Sulfuric acid 98.0 18.0 1.84
in a variety of grades of purity, including technical
Sulfurous acid 82.1 0.74 1.02
grade, analytical reagent grade, and various
Ordinary distilled water is usually not pure. It may 1.6.4 Carbon Dioxide-Free Water
be contaminated by dissolved gases and by materi-
Carbon dioxide (CO2) dissolved in water can interfere
als leached from the container in which it has been
with many chemical measurements. Thus, CO2-free
stored. Volatile organics distilled over from the orig-
water may need to be produced. CO2-free water may
inal source feed water may be present, and nonvola-
be prepared by boiling distilled water for 15 min and
tile impurities may occasionally be carried over by
cooling to room temperature. As an alternative, dis-
the steam, in the form of a spray. The concentration
tilled water may be vigorously aerated with a stream
of these contaminants is usually quite small, and
of inert gas (e.g., N2 or He2) for a period sufficient to
distilled water is used for many analyses without
achieve CO2 removal. The final pH of the water should
further purification. There are a variety of methods
lie between 6.2 and 7.2. It is not advisable to store CO2-
for purifying water, such as distillation, filtration,
free water for extended periods. To ensure that CO2-
and ion exchange. Distillation employs boiling of
free water remains that way, an ascarite trap should be
water and condensation of the resulting steam, to
fitted to the container such that air entering the con-
eliminate nonvolatile impurities (such as minerals).
tainer (as boiled water cools) is CO2-free. Ascarite is
Ion exchange employs cartridges packed with ionic
silica coated with NaOH, and it removes CO2 by the
residues (typically negatively charged) to remove
following reaction:
charged contaminants (typically positively charged
minerals) when water is passed through the car- 2NaOH CO 2 Na 2 CO 3 H 2 O
tridge. Finally, filtration and reverse osmosis remove
Ascarite should be sealed from air except when water
insoluble particulate matter above a specific size.
is being removed from the container.
1.6.3 Water Purity
1.6.5 Preparing Solutions and Reagents
Water purity has been defined in many different ways,
The accurate and reproducible preparation of labora-
but one generally accepted definition states that high
tory reagents is essential to good laboratory practice.
purity water is water that has been distilled and/or
Liquid reagents are prepared using volumetric glass-
deionized so that it will have a specific resistance of
ware (pipettes and flasks) as appropriate.
500,000 Ω (2.0 μΩ/cm conductivity) or greater. This defi-
To prepare solutions from solid reagents (such as
nition is satisfactory as a base to work from, but for more
sodium hydroxide):
critical requirements, the breakdown shown in Table 1.9
has been suggested to express degrees of purity. 1. Determine the amount of solid reagent needed.
Distilled water is usually produced in a steam- 2. Fill the TC volumetric flask ~ ¼–½ full with the
heated metal still. The feed water is (or should be) soft- solvent.
ened to remove calcium and magnesium to prevent 3. Add the solid reagent (it is best to pre-dissolve
scale (Ca or Mg carbonate) formation. Several compa- solids in a beaker with a small amount of liquid,
nies produce ion-exchange systems that use resin- and then add this to the flask; rinse the smaller
packed cartridges for producing “distilled water.” The beaker thoroughly and also put the rinses into
lifespan of an ion-exchange cartridge is very much a flask).
function of the mineral content of the feed water. Thus, 4. Swirl to mix until essentially dissolved.
the lifespan of the cartridge is greatly extended by using 5. Fill the flask to volume with the solvent.
distilled or reverse osmosis-treated water as the incom- 6. Cap and invert the flask ~10–20 times to com-
ing stream. This procedure can also be used for prepar- pletely mix the solution.
ing ultrapure water, especially if a low flow rate is used
and the ion-exchange cartridge is of “research” grade. Note that it is not appropriate to simply combine
the solid reagent with the final volume and assume
that the final volume does not change. This is particu-
larly true for high % concentrations. For example, 1 L
1. 9 of a 10 % aqueous NaOH solution is correctly made by
table Classification of water purity filling a 1 L flask with ~25–500 mL water, adding 100 g
NaOH, mixing until dissolved, and diluting to 1 L. It
Maximum Approximate
conductivity concentration of
would be incorrect to simply combine 100 g NaOH
Degree of purity (μΩ /cm) electrolytes (mg/L) with 1 L water, as the dissolved solid will take up some
volume in solution. (Note that solid NaOH is difficult
Pure 10 2–5 to dissolve, requires a stir bar, and is exothermic,
Very pure 1 0.2–0.5 releasing heat upon dissolution; therefore, do not han-
Ultrapure 0.1 0.01–0.02
dle the glass with bare hands.) Additionally, if a stir
Theoretically pure 0.055 0.00
bar is used, make sure to remove this after the solution
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is dissolved but BEFORE diluting to volume. Note that 2. Zeros before a decimal point with other preced-
sonication is preferred to using a stir bar in a volumet- ing digits are significant. With no preceding
ric flask. digit, a zero before the decimal point is not
The following similar procedures are used to pre- significant.
pare reagents from two or more liquids: 3. If there are no digits preceding a decimal point,
the zeros after the decimal point but preceding
1. Determine the total volume of the final reagent.
other digits are not significant. These zeros only
2. Obtain a TC volumetric flask (if possible) equal
indicate the position of the decimal point.
to the final volume.
4. Final zeros in a whole number may or may not
3. Use TD volumetric glassware to add the correct
be significant. In a conductivity measurement
amount of the liquids with the smallest
of 1000 μΩ/cm, there is no implication that the
volumes.
conductivity is 1000 ± 1 μΩ/cm. Rather, the
4. Dilute to volume with the liquid with the larg-
zeros only indicate the magnitude of the
est volume, gently swirling during addition.
number.
5. Cap and invert the flask ~10–20 times to com-
pletely mix the solution.
A good measure of the significance of one or more
zeros before or after another digit is to determine
Note that a TC volumetric flask should be used
whether the zeros can be dropped by expressing the
whenever possible to bring the solution to final vol-
number in exponential form. If they can, the zeros are
ume. For example, the correct way to prepare 1 L of a
not significant. For example, no zeros can be dropped
5 % ethanol in water solution is to use a 50 mL TD
when expressing a weight of 100.08 g is exponential
pipette to dispense 50 mL ethanol into a 1L TC flask
form; therefore the zeros are significant. However, a
and then fill the flask to volume with water. It would
weight of 0.0008 g can be expressed in exponential
be incorrect to simply combine 50 mL ethanol and
form as 8 × 10−4 g, and the zeros are not significant.
950 mL water, since complex physical properties gov-
Significant figures reflect the limits of the particular
ern the volume of a mixture of liquids, and it cannot be
method of analysis. If more significant figures are
assumed that two liquids of different densities and
needed, selection of another method will be required
polarities will combine to form a volume equal to the
to produce an increase in significant figures.
sum of their individual volumes. If the final volume is
Once the number of significant figures is estab-
not a commonly available TC flask size, then use TD
lished for a type of analysis, data resulting from such
glassware to deliver all reagents.
analyses are reduced according to the set rules for
The use of graduated cylinders and beakers
rounding off.
should be avoided for measuring volumes for reagent
preparation.
1.7.2 Rounding Off Numbers
Rounding off numbers is a necessary operation in all
1.7 DATA HANDLING AND REPORTING analytical areas. However, it is often applied in chemi-
cal calculations incorrectly by blind rule or prema-
1.7.1 Significant Figures turely and, in these instances, can seriously affect the
The term significant figure is used rather loosely to final results. Rounding off should normally be applied
describe some judgment of the number of reportable only as follows:
digits in a result. Often the judgment is not soundly 1. If the figure following those to be retained is
based and meaningful digits are lost or meaningless less than 5, the figure is dropped, and the
digits are accepted. Proper use of significant figures retained figures are kept unchanged. As an
gives an indication of the reliability of the analytical example, 11.443 is rounded off to 11.44.
method used. Thus, reported values should contain 2. If the figure following those to be retained is
only significant figures. A value is made up of signifi- greater than 5, the figure is dropped, and the
cant figures when it contains all digits known to be last retained figure is raised by 1. As an exam-
true and one last digit in doubt. For example, if a value ple, 11.446 is rounded off to 11.45.
is reported at 18.8 mg/l, the “18” must be a firm value, 3. When the figure following those to be retained
while the “0.8” is somewhat uncertain and may be is 5 and there are no figures other than zeros
between “0.7” or “0.9.” The number zero may or may beyond the 5, the figure is dropped, and the last
not be a significant figure: place figure retained is increased by 1 if it is an
1. Final zeros after a decimal point are always sig- odd number, or it is kept unchanged if an even
nificant figures. For example, 9.8 g to the near- number. As an example, 11.435 is rounded off to
est mg is reported as 9.800 g. 11.44, while 11.425 is rounded off to 11.42.
1.7.3 Rounding Off Single Arithmetic reactivity, storage, disposal, protective equipment, and
Operations spill-handling procedures” (http://en.wikipedia.org/
wiki/Material_safety_data_sheet#United_States).
Addition: When adding a series of numbers, the sum
SDSs are available for all reagents, chemicals, sol-
should be rounded off to the same numbers of decimal
vents, gases, etc. used in your laboratory. You can con-
places as the addend with the smallest number of
sult these documents if you have questions regarding
places. However, the operation is completed with all
how to safely handle a material, the potential risks of
decimal places intact and rounding off is done after-
the material, how to properly clean up a spill, etc. They
ward. As an example:
should be available to you in a centralized location
11.1 11.12 11.13 33.35 (typically, a binder) in the lab. If not available, you
Thesum is rounded off to 33.4 may request these from your instructor or find them
online. Generally, the following information is avail-
able on a MSDS or SDS in a 16-section format:
Multiplication: When two numbers of unequal
digits are to be multiplied, all digits are carried 1. Identification of the substance/mixture
through the operation, and then the product is 2. Hazard identification
rounded off to the number of significant digits 3. Composition/information on ingredients
of the less accurate number. 4. First aid measures
5. Firefighting measures
Division: When two numbers of unequal digits 6. Accidental release measures
are to be divided, the division is carried out on 7. Handling and storage
the two numbers using all digits. Then the quo- 8. Exposure controls/personal protection
tient is rounded off to the lower number of sig- 9. Physical and chemical properties
nificant digits between the two values. 10. Stability and reactivity
Powers and roots: When a number contains n 11. Toxicological information
significant digits, its root can be relied on for n 12. Ecological information
digits, but its power can rarely be relied on for n 13. Disposal considerations
digits. 14. Transport information
15. Regulatory information
1.7.4 Rounding Off the Results of a Series 16. Other information
of Arithmetic Operations
1.8.2 Hazardous Chemicals
The rules for rounding off are reasonable for simple
calculations. However, when dealing with two nearly Food analysis laboratories, like any chemical labora-
equal numbers, there is a danger of loss of all signifi- tory, often contain hazardous compounds, including:
cance when applied to a series of computations that 1. Acids (hydrochloric acid, sulfuric acid, etc.)
rely on a relatively small difference in two values. 2. Bases (e.g., sodium hydroxide)
Examples are calculation of variance and standard 3. Corrosives and oxidizers (sulfuric acid, nitric
deviation. The recommended procedure is to carry acid, perchloric acid, etc.)
several extra figures through the calculation and then 4. Flammables (organic solvents such as hexane,
to round off the final answer to the proper number of ether, alcohols)
significant figures. This operation is simplified by
using the memory function on calculators, which for 1.8.3 Personal Protective Equipment
most calculators is a large number, often 10 or more, and Safety Equipment
digits.
It is important to understand the location and use of
lab safety equipment. The purpose of this is threefold:
1.8 BASIC LABORATORY SAFETY 1. To prevent accidents and/or injuries in the lab
2. To quickly and effectively respond to any acci-
1.8.1 Safety Data Sheets dent and/or injury in the lab
Safety Data Sheets (SDSs), formerly called Material 3. Be able to perform laboratory procedures with-
Safety Data Sheets (MSDSs), are informational packets out excessive worrying about lab hazards
that are “intended to provide workers and emergency
personnel with procedures for handling or working Your laboratory instructor should provide instruc-
with that substance in a safe manner and include infor- tion regarding basic laboratory safety equipment. You
mation such as physical data (melting point, boiling should be aware of these general rules and the exis-
point, flash point, etc.), toxicity, health effects, first aid, tence of this equipment.
www.dbooks.org
Proper clothing is required to work in any chemi- laboratory: food, water, beverages, tobacco, and cos-
cal laboratory. The following standards and rules metics. Some unconscious activities (e.g., touching
regarding dress are generally applicable, although your face and eyes) are difficult to avoid. However,
standards may vary between laboratories: wearing gloves in the laboratory may minimize these
actions.
1. Close-toed shoes (no flip-flops, sandals, or other
“open” footwear).
1.8.5 Miscellaneous Information
2. Long pants (dresses, skirts, and shorts may be
allowed in some laboratories). The following general rules and guidelines apply to
3. No excessively loose clothing or accessories. working in the laboratory:
4. Long hair should be pulled back from the face
1. When combining acid and water, always add
into a ponytail or otherwise restrained.
the acid to water (instead of adding water to the
acid). When acid dissolves in water, heat is
You should be able to obtain and wear the follow-
released. This can cause splattering of the solu-
ing personal protective equipment (PPE) and under-
tion. By adding the acid to water, the heat is dis-
stand their proper use:
sipated, and splattering is reduced or
1. Safety glasses, goggles, and face shields eliminated.
2. Lab coat or apron 2. Be aware that dissolving sodium hydroxide in
3. Shoe covers water generates heat. Making high concentra-
4. Latex or acetonitrile gloves tions of aqueous sodium hydroxide can lead to
5. Puncture-resistant gloves very hot solutions that can burn bare hands.
6. Heat-resistant gloves Allow these solutions to cool, or handle with
heat-resistant gloves.
You should be aware of the locations of the follow- 3. Broken glass and other sharps (razor blades,
ing safety equipment items and their proper use: scalpel blades, needles, etc.) should be disposed
of in puncture-resistant sharps containers.
1. First aid kit
4. Do not pour waste or chemicals down the drain.
2. Bodily fluids cleanup kit
This practice can damage the building’s plumb-
3. Acid, base, and solvent spill kits
ing and harm the environment. Dispose of liq-
4. Fire extinguisher and fire blanket
uid, solid, chlorinated, radioactive, and
5. Safety shower and eyewash station
biohazard wastes into the appropriate contain-
6. Solid, liquid, chlorinated, and biohazard waste
ers provided by the lab instructor. If you are
disposal containers, if applicable
unsure how to properly dispose of waste, ask
7. Sharps and broken glass disposal containers, if
your instructor or teaching assistant.
applicable
5. Handle volatile, noxious, or corrosive com-
pounds in the fume hood with appropriate PPE.
1.8.4 Eating, Drinking, Etc.
Your hands may become contaminated with sub-
stances used in the lab simply by touching lab benches, RESOURCE MATERIALS
glassware, etc. This may happen even without your
knowledge. Even if you are not handling hazardous Analytical Quality Control Laboratory. 2010. Handbook for
substances, previous lab occupants may not have analytical quality control in water and wastewater labora-
cleaned benches and glassware, leaving behind haz- tories. U.S. Environmental Protection Agency, Technology
ardous substances that you are unaware of. To avoid Transfer.
spreading potentially harmful substances from your Anonymous. 2010. Instructions for Gilson Pipetman. Rainin
Instrument Co., Inc., Washburn, MA.
hands to your face, eyes, nose, and mouth (where they
Applebaum, S.B. and Crits, G.J. 1964. “Producing High
may irritate sensitive or be introduced to circulation Purity Water”. Industrial Water Engineering.
by mucus membranes, ingestion, or inhalation), the Smith JS. 2017. Evaluation of analytical data, Ch. 4, In: Nielsen
following activities are prohibited in chemical labora- SS (ed.) Food analysis, 5th edn. Springer, New York.
tories: eating, drinking, smoking, chewing tobacco or Willare, H.H. and Furman, W.H. 1947. Elementary
snuff, and applying cosmetics ( e.g., lip balm). The fol- Quantitative Analysis – Theory and Practice. Van
lowing should not even be brought into a chemical Norstrand Co., Inc., New York.
2
chapter
Preparation
of Reagents and Buffers
Catrin Tyl (*) • Baraem P. Ismail
Department of Food Science and Nutrition, University of Minnesota,
St. Paul, MN, USA
e-mail: [email protected]; [email protected]
2.1 Preparation of Reagents of Specified 2.4 Notes on Buffers

Concentrations 2.5 Practice Problems
2.2 Use of Titration to Determine References
Concentration of Analytes
2.3 Preparation of Buffers
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22 C. Tyl and B.P. Ismail
2.1 PREPARATION OF REAGENTS

OF SPECIFIED CONCENTRATIONS Example A2 How much would you need to
weigh out if you were using the dihydrate (i.e.,
Virtually every analytical method involving wet chem- CaCl2.2H2O) to prepare 2 L of a 4 mM solution of
istry starts with preparing reagent solutions. This usu- calcium chloride?
ally involves dissolving solids in a liquid or diluting
from stock solutions. The concentration of analytes in Solution
solution can be expressed in weight (kg, g, or lower During crystallization of salts, water may be
submultiples) or in the amount of substance (mol), per incorporated into the crystal lattice. Examples
a unit volume (interchangeably L or dm3, mL or cm3, include phosphate salts, calcium chloride, and
and lower submultiples). Preparing reagents of correct certain sugars. The names of these compounds
concentrations is crucial for the validity and reproduc- are amended by the number of bound water mol-
ibility of any analytical method. Below is a sample cal- ecules. For example, Na2HPO4.7H2O is called
culation to prepare a calcium chloride reagent of a sodium monophosphate heptahydrate, and
particular concentration. CaCl2.2H2O is called calcium chloride dihydrate.
The water is tightly bound and is not visible (the
dry reagents do not look clumped). Many com-
mercially available salts are sold as hydrates that
can be used analogously to their dry counterparts
after adjusting for their increased molecular
Example A1 How much calcium chloride do you weight, which includes the bound water mole-
need to weigh out to get 2 L of a 4 mM solution? cules. The molecular weight of CaCl2 changes
from 110.98 g/mol to 147.01 g/mol to account for
Solution two molecules of water at about 18 g/mol. Hence,
The molarity (M) equals the number of Eq. 2.6 needs to be modified to:
moles (n) in the volume (v) of 1 L:
æ mol ö æ g ö
æ mol ö n ( mol ) m ( g ) = 0.004 ç ÷ ´ 2 ( L ) ´ 147.01ç ÷
Mç ÷= (2.1) è L ø è mol ø
è L ø v [L ] = 1.176 [ g ] (2.7)
The desired molarity is 4 mM = 0.004 M; the
desired volume is 2 L. Rearrange Eq. 2.1 so that: Thus, 1.176 g of CaCl2 would be weighed
out and the volume made up to 2 L in a volu-
æ mol ö metric flask.
n ( mol ) = M ç ÷´ v ( L ) (2.2)
è L ø
The mass (m) of 1 mole CaCl2 (110.98 g/mol)
is specified by the molecular weight (MW) as Other commonly used ways to express concentra-
defined through Eq. 2.3. The mass to be weighed tions are listed in Table 2.1. For instance, for very low
is calculated by rearranging into Eq. 2.4: concentrations as encountered in residue analysis,
æ g ö m(g ) parts per million (e.g., μg/mL or mg/L) and parts per
MW ç ÷= (2.3)
è mol ø n ( mol ) billion (e.g., μg/L) are preferred units. Concentrated
acids and bases are often labeled in percent mass by
æ g ö
m ( g ) = n ( mol ) ´ MW ç ÷ (2.4) mass or percent mass per volume. For instance, a 28 %
è mol ø wt/wt solution of ammonia in water contains 280 g
Now substitute Eq. 2.2 into Eq. 2.4. Some ammonia per 1000 g of solution. On the other hand,
of the units then cancel out, indicated as 32 % wt/vol NaOH solution contains 320 g of NaOH
strike-throughs: per L. For dilute solutions in water, the density is
approximately 1 kg/L at room temperature (the den-
æ mol ö æ g ö
m(g ) = M ç ÷ ´ v ( L ) ´ MW ç ÷ (2.5) sity of water is exactly 1 kg/L only at 5 °C), and thus
è L ø è mol ø wt/wt and wt/vol are almost equal. However, concen-
æ mol ö æ g ö trated solutions or solutions in organic solvents can
m ( g ) = 0.004 ç ÷ ´ 2 ( L ) ´ 110.98 ç ÷
è L ø è mol ø deviate substantially in their density. Therefore, for
= 0.888 [g ] (2.6) concentrated reagents, the correct amount of reagent
needed for dilute solutions is found by accounting for
the density, as illustrated in Example A3 below:
Chapter 2 • Preparation of Reagents and Buffers 23
2.1
table Concentration expression terms
Unit Symbol Definition Relationship

Molarity M Number of moles of solute per liter of mol
M=
solution liter
Normality N Number of equivalents of solute per liter equivalents
N=
of solution liter
Percent by weight (parts wt % Ratio of weight of solute to weight of wt solute ´ 100
wt % =
per hundred) solute plus weight of solvent × 100 total wt
wt/vol % Ratio of weight of solute to total wt solute ´ 100
volume × 100 wt / vol % =
total volume
Percent by volume vol % Ratio of volume of solute to total vol of solute ´ 100
vol % =
volume × 100 total volume
Parts per million ppm Ratio of solute (wt or vol) to total weight mg solute
ppm =
or volume × 1,000,000 kg solution
mg solute
=
g solution
mg solute
=
L solution
mg solute
=
mL solution
Parts per billion ppb Ratio of solute (wt or vol) to total weight mg solute
ppb =
or volume × 1,000,000,000 kg solution
ng solute
=
g solution
mg solute
=
L solution
ng solute
=
mL solution
Example A3 Prepare 500 mL of a 6 M sulfuric in terms of known quantities (MW and d).
acid solution from concentrated sulfuric acid. However, because molarity is specified in moles
The molecular weight is 98.08 g/mol, and the per L, the density needs to be multiplied by 1000
manufacturer states that it is 98 % wt/wt and has to obtain the g per L (the unit of density is g per
a density (d) of 1.84 g/mL. mL or kg per L).
m(g )
Solution Rearrange Eq. 3 : n ( mol ) = (2.9)
The density specifies the mass per volume of æ g ö
MW ç ÷
the concentrated H2SO4: è mol ø
mæ g ö
d= ç ÷ (2.8)
ægö
v è mL ø Rearrange Eq. 8 : m ( g ) = d ç ÷ ´ 1000 ´ v ( L ) (2.10)
The molarity of the concentrated sulfuric èLø
acid is determined by rearranging Eqs. 2.3 and
2.8 to express the unknown number of moles (n)
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2.2 USE OF TITRATION TO DETERMINE

Substitute Eq. 10 into Eq. 9 : CONCENTRATION OF ANALYTES
ægö
d ´ 1000 ç ÷ ´ v ( L ) A wide range of standard methods in food analysis,
n ( mol ) = èLø (2.11) such as the iodine value, peroxide value and titratable
æ mol ö acidity, involve the following concept:
MW ç ÷
è g ø
• A reagent of known concentration (i.e., the titrant)
Substitute Eq. 2.11 into Eq. 2.1. The 98 % wt/wt is titrated into a solution of analyte with unknown
(see Table 2.1) can be treated like a proportional- concentration. The used up volume of the reagent
ity factor: every g of solution contains 0.98 g solution is measured.
H2SO4. To account for this, multiply Eq. 2.1 with • The ensuing reaction converts the reagent and ana-
the wt %: lyte into products.
ægö • When all of the analyte is used up, there is a mea-
æ mol ö d ´ v ´1000 1
Mç ÷= ´ ´ %w ç ÷ = surable change in the system, e.g., in color or pH.
è L ø MW v ègø (2.12)
• The concentration of the titrant is known; thus the
amount of converted reactant can be calculated.
ægö
d ´ 1000 ç ÷ The stoichiometry of the reaction allows for the cal-
æ mol ö è L ø ´ %w æ g ö (2.13) culation of the concentration of the analyte, for
Mç ÷= ç ÷
è L ø MW æ g ö ègø example, in the case of iodine values, the absorbed
ç ÷
è mol ø grams of iodine per 100 g of sample; in the case of
peroxide value, milliequivalents of peroxide per kg
ægö ægö of sample; or in the case of titratable acidity, % wt/
1.84 ç ÷ ´1000 ´ 0.98 ç ÷ vol acidity.
æ mol ö è ø
L ègø
Mç ÷=
è L ø æ g ö
98.08 ç ÷ There are two principle types of reactions for
è mol ø which this concept is in widespread use: acid-base
æ mol ö (2.14) and redox reactions. For both reaction types, the con-
= 18.39 ç ÷
è L ø cept of normality (N) plays a role, which signifies the
The necessary volume to supply the desired number of equivalents of solute per L of solution. The
amount of moles for 500 mL of a 6 M solution is number of equivalents corresponds to the number of
found by using Eq. 2.15: transferred H+ for acid-base reactions and transferred
electrons for redox reactions. Normality equals the
æ mol ö æ mol ö product of the molarity with the number of equiva-
v1 ( L ) ´ M1 ç ÷ = v2 ( L ) ´ M 2 ç ÷ (2.15)
è L ø è L ø lents (typically 1–3 for common acids and bases with
low molecular weight), i.e., it is equal to or higher
v stock solution ( L ) = than the molarity. For instance, a 0.1 M sulfuric acid
v of diluted M of diluted solution would be 0.2 N, because two H+ are donated
× per molecule H2SO4. On the other hand, a 0.1 M NaOH
solution ( L ) solution ( mol /−L) solution would still be 0.1 N, as indicated by Eq. 2.18:
(2.16)
 mol 
M of stock solution   Normality = Molarity ´ number of equivalents (2.18)
 −L 
Substituting N for M, reaction equivalence can be
0.5 ( L ) ´ 6 expressed through a modified version of Eq. 2.15
v(L) = (which is the same as Refr. [4], Sect. 22.2.2, Eq. 2.1):
18.39
= 0.163 L or 163 mL (2.17) v of titrant ´ N of titrant = v of analyte solution
´ N of analyte solution (2.19)
Hence, to obtain 500 mL, 163 mL concentrated
sulfuric acid would be combined with 337 mL of Some analyses (such as in titratable acidity) require the
water (500–163 mL). The dissolution of concen- use of equivalent weights instead of molecular
trated sulfuric acid in water is an exothermic pro- weights. These can be obtained by dividing the molec-
cess, which may cause splattering, and glassware ular weight by the number of equivalents transferred
can get very hot (most plastic containers are not over the course of the reaction.
suited for this purpose!). The recommended pro- Equivalent weight ( g ) =
cedure would be to add some water to a 500 mL
volumetric glass flask, e.g., ca. 250 mL, then add æ g ö
molecular weight ç ÷
the concentrated acid, allow the mixture to cool è mol ø
down, mix, and bring up to volume with water. number of equivalents ( mol ) (2.20)
Using equivalent weights can facilitate calculations,

because it accounts for the number of reactive groups Example B2 Assume that 100 mL of apple juice
of an analyte. For H2SO4, the equivalent weight would are titrated with 1 M NaOH, and the volume
98.08 used is 36 mL. What is the molarity of malic acid
be = 49.04 g/mol, whereas for NaOH it would in the apple juice?
2
be equal to the molecular weight since there is only
one OH group. Solution Malic acid contains two carboxylic
To illustrate the concept, the reaction of acetic acid groups, and thus for every mole of malic acid,
with sodium hydroxide in aqueous solution is stated two moles of NaOH are needed to fully ionize it.
below: Therefore, the normality of malic acid is two
times the molarity. Again, use Eq. 2.19 to solve
CH 3 COOH + NaOH ® CH 3 COO - + H 2 O + Na + Example B2:
This reaction can form the basis for quantifying acetic æ mol ö
36 ( mL ) ´ 1ç ÷ = 100 ( mL ) ´ 2
acid contents in vinegar (for which it is the major è L ø
acid): æ mol ö
´ M of malic acidsolution ç ÷ (2.23)
è L ø
Example B1 100 mL of vinegar is titrated with a

æ mol ö
solution of NaOH that is exactly 1 M. (Chapter 21, M of malic acid in apple juice ç ÷=
Sect. 21.2, in this laboratory manual describes è L ø
how to standardize titrants.) If 18 mL of NaOH 36 ( mL ) æ mol ö 1 æ mol ö
´ 1ç ÷ ´ = 0.18 ç ÷ (2.24)
are used up, what is the corresponding acetic 100 ( mL ) è L ø 2 è L ø
acid concentration in vinegar?
This is the same value as obtained above for ace-
Solution The reaction equation shows that both
tic acid. However, as a direct consequence of
NaOH and CH3COOH have an equivalence
malic acid having two carboxyl groups instead
number of 1, because they each have only one
of one, twice the amount of NaOH was needed.
reactive group. Thus, their normality and molar-
ity are equal. Equation 2.19 can be used to solve
Example B1, and the resulting N will, in this
case, equal the M: Tables, calculators, and other tools for calculat-
æ mol ö ing molarities, normalities, and % acidity are avail-
18 ( mL ) ´ 1ç ÷ = 100 ( mL ) ´ Normality able in print and online literature. However, it is
è L ø
important for a scientist to know the stoichiometry of
æ mol ö (2.21) the reactions involved, and the reactivity of reaction
of acetic acid solution ç ÷
è L ø partners to correctly interpret these tables.
The concept of normality also applies to redox
æ mol ö
M and N of acetic acid in vinegar ç ÷= reactions; only electrons instead of protons are trans-
è L ø ferred. For instance, potassium dichromate, K2Cr2O7,
18 ( mL ) æ mol ö æ mol ö can supply six electrons, and therefore the normality
´ 1ç ÷ = 0.18 ç ÷
100 ( mL ) è L ø è L ø (2.22) of a solution would be six times its molarity. While
the use of the term normality is not encouraged by
the IUPAC, the concept is ubiquitous in food analysis
because it can simplify and speed up calculations.
For a practical application of how normality is
used to calculate results of titration experiments, see
Chap. 21 in this laboratory manual.
Sometimes the stoichiometry of a reaction is different,
such as when NaOH reacts with malic acid, the main
acid found in apples and other fruits. 2.3 PREPARATION OF BUFFERS
A buffer is an aqueous solution containing compara-

ble molar amounts of either a weak acid and its corre-
sponding base or a weak base and its corresponding
acid. A buffer is used to keep a pH constant. In food
analysis, buffers are commonly used in methods that
utilize enzymes, but they also arise whenever weak
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acids or bases are titrated. To explain how a weak acid respectively. Most acids found in foods are weak acids;
or base and its charged counterpart manage to main- hence, once the equilibrium has been reached, only
tain a certain pH, the “comparable molar amounts” trace amounts have dissociated, and the vast majority
part of the definition is key: For a buffer to be effective, of the acid is in its initial, undissociated state. For the
its components must be present in a certain molar ratio. purpose of this discussion, we will refer to the buffer
This section is intended to provide guidance on how components as acid AH, undissociated state, and cor-
to solve calculation and preparation problems relat- responding base A−, concentrations can be measured
ing to buffers. While it is important for food scientists and are published in the form of dissociation constants,
to master calculations of buffers, the initial focus will Ka, or their negative logarithms to base 10, pKa values.
be on developing an understanding of the chemistry. These values can be found on websites of reagent man-
Figures 2.1 and 2.2 show an exemplary buffer system, ufacturers as well as numerous other websites and text-
and how the introduction of strong acid would affect it. books. Table 2.2 lists the pKa values of some common
Only weak acids and bases can form buffers. The acids either present in foods or often used to prepare
distinction of strong versus weak acids/bases is made buffers, together with the acid’s molecular and equiva-
based on how much of either H3O+ or OH− is generated, lent weights. However, reported literature values for
Ka/pKa can be different for the same compound. For
instance, they range between 6.71 and 7.21 for H2PO4−.
Dissociation constants depend on the ionic strength of
the system, which is influenced by the concentrations of
all ions in the system, even if they do not buffer. In addi-
O O
Na+
tion, the pH in a buffer system is, strictly speaking,
O- OH
determined by activities, not by concentrations (see
O Na+ O Na+ Refr. [4], Sect. 22.3.2.1). However, for concentrations
OH O-
O <0.1 M, activities are approximately equal to concentra-
Na+ O OH tions, especially for monovalent ions. For H2PO4−, the
O
OH O value 7.21 is better suited for very dilute systems. For
O- O- buffers intended for media or cell culture, the system
typically contains several salt components, and 6.8
would be a commonly used value. If literature specifies
2.1 A buffer solution composed of the weak acetic several pKa values, try finding information on the ionic
figure
acid, CH3COOH, and a salt of its correspond- strength where these values were obtained and calcu-
ing base, sodium acetate, CH3COO−Na+. In late/estimate the ionic strength of the solution where
aqueous solution, the sodium acetate the buffer is to be used. However, even for the same
dissociates into CH3COO− (acetate ions) and ionic strength, there may be different published values,
Na+ (sodium ions), and for this reason, these
depending on the analytical method. When preparing a
ions are drawn spatially separated. The Na+
ions do not participate in buffering actions
buffer (see notes below), always test and, if necessary,
and can be ignored for future considerations. adjust the pH, even for commercially available dry buffer
Note that the ratio of acetic acid and acetate is mixes that only need to be dissolved. The values listed
equal in our example. Typical buffer systems in Table 2.2 are for temperatures of 25 °C and ionic
have concentrations between 1 and 100 mM strengths of 0 M, as well as 0.1 M, if available.
HCI
HCI
2.2 Changes induced by addition of the strong acid
figure
HCl, to the buffer from Fig. 2.1. HCl can be
considered as completely dissociated into
Cl− and H+. The H+ combines with CH3COO−
O O
instead of H2O, because CH3COO− is the Na+ O Na+ Na+ O CI-
O- OH
stronger base. Thus, instead of H3O+, additional OH OH
O
CH3COOH is formed. This alters the ratio Na+ Na+ Na+ Na+ Na+
O O
O-
between CH3COOH and CH3COO−, resulting in OH CI-
O O OH O
a different pH as illustrated in problem C2. The O OH O
O O- O OH
Cl− ions are merely counterions to balance O- O OH OH O OH
charges but do not participate in buffer reactions O- O-
and can thus be ignored
2.2
table Properties of common food acids
Molecular Equivalent
Formula pKa1 pKa2 pKa3 weight weight
Acetic acid CH3COOH 4.75b – – 60.06 60.06
Carbonic acid H2CO3 6.4b 10.3b – 62.03 31.02
6.1c 9.9c
Citric acid HOOCCH2C(COOH)OHCH2COOH 3.13b 4.76b 6.40b 192.12 64.04
2.90c 4.35c 5.70c
Formic acid HCOOH 3.75b – – 46.03 46.03
Lactic acid CH3CH(OH)COOH 3.86 – – 90.08 90.08
Malic acid HOOCCH2CHOHCOOH 3.5b 5.05b – 134.09 67.05
3.24c 4.68c
Oxalic acid HOOCCOOH 1.25b 4.27b – 90.94 45.02
1.2c 3.80c
Phosphoric acid H3PO4 2.15b 7.20b 12.38b 98.00 32.67
1.92c 6.71c 11.52c
Potassium acid HOOCC6H4COO−K+ 5.41b – – 204.22 204.22
phthalate
Tartaric acida HOOCCH(OH)CH(OH)COOH 3.03 4.45 – 150.09 75.05
Data from Refs. [1, 2]
a
Dissociation constants depend on the stereoisomer (D/L vs. meso-form). Values given for the naturally occurring R,R enantio-
mer (L-form)
b
At ionic strength of 0 M
c
At ionic strength of 0.1 M
Maximum buffering capacity always occurs of A− gives the same result, as the pKb is related to the
around the pKa value of the acid component. At the pKa through 14 − pKb = pKa.
pKa, the ratio of acid/corresponding base is 1:1 (not, as Below are several examples of buffer preparation
often erroneously assumed, 100:0). Therefore, a certain using the Henderson-Hasselbalch equation:
acid/corresponding base pair is suitable for buffering
a pH range of pKa ± 1. The molarity of a buffer refers to
the sum of concentration for acid and corresponding
base. The resulting pH of a buffer is governed by their
concentration ratio, as described through the
Henderson-Hasselbalch equation: Example C1 What is the pH of a buffer obtained by
- mixing 36 mL of a 0.2 M Na2HPO4 solution and
éA ù
pH = pK a + log ë û (2.25) 14 mL of a 0.2 M NaH2PO4 solution, after adding
[ AH] water to bringing the volume to 100 mL to obtain a
Weak bases such as ammonia, NH3, can also form buf- 0.1 M buffer?
fers with their corresponding base, in this case NH4+.
The Henderson-Hasselbalch equation would actually Solution The Henderson-Hasselbalch equation
not change, since NH4+ would serve as the acid, denoted requires knowledge of the acidic component’s
BH, and the base NH3 is denoted as B. The acid compo- pKa value and the concentrations of both buffer
nent is always the form with more H+ to donate. components. Rearrange Eq. 2.15 so that:
However, you may find the alternative equation:
æ mol ö
M in buffer ç ÷=
éë BH + ùû è L ø
pOH = pK b + log (2.26)
[ B] M of stock v of stock
æ mol ö ´ solutions æç mol ö÷
The pH would then be calculated as 14 − pOH. The solutions ç ÷
è L ø è L ø
term pOH refers to the concentration of OH−, which (2.27)
increases when bases are present in the system (see v of buffer ( L )
Ref. [4], Sect. 22.3, for details). However, using
Eq. 2.25 and BH+ instead of AH as well as B in place
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æ mol ö n of Na 2HPO 4 = 0.072 ´ 0.1 = 0.0072 ( mol ) (2.32)

0.2 ç ÷ ´ 0.036 ( L )
æ mol ö è L ø
M of Na 2HPO 4 ç ÷=
è L ø 0.1( L ) n of NaH 2PO 4 = 0.028 ´ 0.1 = 0.0028 ( mol ) (2.33)
æ mol ö (2.28)
= 0.072 ç ÷ n of HCl ( mol ) = M ´ v = 2 ´ 0.001
è L ø
= 0.002 ( mol ) (2.34)
æ mol ö n of Na 2HPO 4 after HCl addition ( mol ) :

0.2 ç ÷ ´ 0.014 ( L ) 0.0072 − 0.002 = 0.0052 (2.35)
æ mol ö è L ø
M of NaH 2PO 4 ç ÷=
è L ø 0.1( L )
n of NaH 2PO 4 after HCl addition ( mol ) :
æ mol ö (2.36)
= 0.028 ç ÷ (2.29) 0.0028 + 0.002 = 0.0048
è L ø
pH of buffer after HCl addition and conversion
of n into M through Eq. 2.1:
The other necessary step to solve Example C1 is
finding the correct pKa.
pH = 6.71 + log
[0.052] = 6.74 (2.37)
k1 [0.048]
H 3PO 4  H 2PO 4 – + H +
k –1 Note: The ratio of A− to AH stays the same
k2 k3 whether concentrations or amounts are inserted,
 HPO 4 2 – + H +  PO 4 3 – + H + since units are canceled, so technically n does not
k –2 k –3
need to be converted into M to obtain the correct
−
In this buffer, H2PO4 acts as the acid, as it result.
donates H+ more strongly than HPO42−. The acid
component in a buffer is always the one with
more acidic H+ attached. Hence the relevant pKa
value is pk2. As listed in Table 2.2, this value is
6.71. Example C3 Prepare 250 mL of 0.1 M acetate
The solution to Example C1 is now only a buffer with pH 5. The pKa of acetic acid is 4.76
matter of inserting values into Eq. 2.25: (see Table 2.2). The molecular weights of acetic
pH = 6.71 + log
[0.072] (2.30)
acid and sodium acetate are 60.06 and 82.03 g/
[0.028] mol, respectively.
pH = 6.71 + 0.41 = 7.12 (2.31) Solution This example matches the tasks at
hand in a lab better than Example C1. One needs
a buffer to work at a certain pH, looks up the
pKa value, and decides on the molarity and vol-
ume needed. The molarity of a buffer equals
Example C2 How would the pH of the buffer in [A−] + [AH]. To solve Example C3, one of those
Example C1 change upon addition of 1 mL of concentrations needs to be expressed in terms of
2 M HCl? Note: You may ignore the slight change the other, so that the equation only contains one
in volume caused by the HCl addition. unknown quantity. For this example, it will be
[A−], but the results would be the same if [AH]
Solution Calculate the moles of HCl supplied had been chosen. Together with the target pH
using Eq. 2.1. HCl converts HPO42− into H2PO4−, (5) and the pKa (4.76), the values are inserted
because HPO42− is a stronger base than H2PO4−, into Eq. 2.25:
as apparent by its higher pKa value. This changes æ mol ö
÷ = [ AH ] + [A ]
-
the ratio of [AH]:[A−]. The new ratio needs to be Molarity of buffer ç (2.38)
è L ø
inserted into Eq. 2.24. To calculate the new ratio,
account for the volume of the buffer, 0.1 L, and
0.1 = [A - ] + [ AH ] (2.39)
calculate the amount of HPO42− and
H2PO4− present:
n of buffer components ( mol ) = M ´ v (2.2) [A – ] = 0.1 – [ AH ] (2.40)
v of aceticacid ( mL ) =
0.1 - [ AH ]
5 = 4.76 + log (2.41) æ g ö æ mol ö
[ AH] 60.06 ç ÷ ´ 0.1ç ÷ ´ 1( L )
è mol ø è L ø = 5.72 ( mL ) (2.53)
æ g ö
0.1 - [ AH ] 1.05 ç ÷
0.24 = log (2.42) è mL ø
[ AH]
Dissolving each of these amounts of sodium
0.1 - [ AH ] acetate and acetic acid in 1 L of water gives two
1.7378 = (2.43) 1 L stock solutions with a concentration of
[ AH] 0.1 M. To calculate how to mix the stock solu-
tions, use their concentrations in the buffer, i.e.,
1.7378 ´[ AH ] = 0.1 - [ AH ] (2.44)
0.0365 as obtained from Eqs. 2.48 and 2.49 –
æ mol ö æ mol ö
1.7378 ´[ AH ] + [ AH ] = 0.1 (2.45) 0.0365 ç ÷ for acetic acid and 0.0635 ç ÷
è L ø è L ø
for sodium acetate and Eq. 2.15:
[ AH]´ (1.7378 + 1) = 0.1 (2.46)
v of acetic acid stock solution ( L )
0.1
[ AH] = (2.47) æ mol ö
1.7378 + 1 M of aceticacidin buffer ç ÷ ´ v of buffer ( L )
= è L ø (2.54)
æ mol ö
[ AH] = 0.0365 æç
mol ö M of stock solution ç ÷
÷ (2.48) è L ø
è L ø
v of acetic acid stock solution ( L ) =
- æ mol ö 0.0365 ´ 0.25
[A ] = 0.1 - 0.0365 = 0.0635 ç ÷ (2.49) = 0.091( L ) (2.55)
è L ø 0.1
v of sodium acetate stock solution ( L ) =
There are three ways to prepare such a buffer: 0.0635 ´ 0.25

= 0.159 ( L ) (2.56)
1. Prepare 0.1 M acetic acid and 0.1 M sodium ace- 0.1
tate solutions. For our example, 1 L will be pre-
Combining the 0.091 L of acetic acid stock and
pared. Sodium acetate is a solid and can be
0.159 L of sodium acetate stock solution gives
weighed out directly using Eq. 2.5. For acetic
0.25 L of buffer with the correct pH and molarity.
acid, it is easier to pipet the necessary amount.
2. Directly dissolve appropriate amounts of both
Rearrange Eq. 2.8 to calculate the volume of
components in the same container. Equations 2.48
concentrated acetic acid (density = 1.05) needed
and 2.49 yield the molarities of acetic acid and
to prepare a volume of 1 L. Then express the
sodium acetate in a buffer, i.e., the moles per 1
mass through Eq. 2.5.
liter. Just like for approach 1 above, use Eqs. 2.5
m of sodium acetate ( g ) = M ´ v ´ MW (2.5) and 2.52 to calculate the m for sodium acetate
and v for acetic acid, but this time use the buffer
mof sodium acetate ( g ) = volume of 0.25 [L] to insert for v:
æ mol ö æ g ö For sodium acetate :
0.1ç ÷ ´1( L ) ´ 82 ç ÷ = 8.2 ( g ) (2.50)
è L ø è mol ø æ mol ö æ g ö
m(g ) = M ç ÷ ´ v ( L ) ´ MW ç ÷
è L ø è mol ø
m (g )
v of acetic acid ( mL ) = (2.51)
æ g ö = 0.0635 ´ 0.25 ´ 82.03 = 1.3 ( g ) (2.57)
dç ÷
è mL ø
For acetic acid :
v of aceticacid ( mL ) =
æ mol ö æ g ö
æ mol ö æ g ö Mç ÷ ´ v ( L ) ´ MW ç ÷
Mç ÷ ´ v ( L ) ´ MW ç ÷
v ( mL ) = è ø è mol ø
L
è L ø è mol ø (2.52) æ g ö
æ g ö dç ÷
dç ÷ è mL ø
è mL ø
0.0365 ´ 0.25 ´ 60.02
= = 0.52 ( mL ) (2.58)
1.05
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Dissolve both reagents in the same glassware in tional factors may need to be considered, as detailed
200 mL water, adjust the pH if necessary, and below (listed in no particular order of importance):
bring up to 250 mL after transferring into a vol-
1. The buffer components need to be well soluble
umetric flask.
in water. Some compounds require addition of
Note: It does not matter in how much water you
acids or bases to fully dissolve.
initially dissolve these compounds, but it
2. Buffer recipes may include salts that do not par-
should be > 50 % of the total volume. Up to a
ticipate in the buffering process, such as sodium
degree, buffers are independent of dilution;
chloride for phosphate-buffered saline.
however, you want to ensure complete solubili-
However, the addition of such salts changes the
zation and leave some room to potentially
ionic strength and affects the acid’s pKa.
adjust the pH.
Therefore, combine all buffer components
3. Pipet the amount of acetic acid necessary for
before adjusting the pH.
obtaining 250 mL of a 0.1 M acetic acid solution,
3. If a buffer is to be used at a temperature other
but dissolve in < 250 mL, e.g., 200 mL. Then add
than room temperature, heat or cool it to this
concentrated NaOH solution drop-wise until
intended temperature before adjusting the
pH 5 is reached, and make up the volume to
pH. Some buffer systems are more affected than
250 mL. The amount of acetic acid is found
others, but it is always advisable to check. For
analogously to Eq. 2.52:
instance, 2-[4-(2-hydroxyethyl)piperazin-1-yl]
M ´ v ´ MW ethanesulfonic acid [HEPES] is a widely used
v acetic acid ( mL ) = =
d buffer component for cell culture experiments.
0.1´ 0.25 ´ 60.02 At 20 °C, its pKa is 7.55, but its change in pH
= 1.43 ( mL ) (2.59) from 20 to 37 °C is −0.014 ΔpH/°C [3]. Thus, the
1.05
pKa at 37 °C would be:
You will find all three approaches described above if
you search for buffer recipes online and in published pK a at 37 °C = pHat 20 °C – DpH ´ ( T1 – T 2 ) (2.60)
methods. For instance, AOAC Method 991.43 for total
dietary fiber involves approach 3. It requires dissolving
19.52 g of 2-(N-morpholino)ethanesulfonic acid (MES) 7.55 - 0.014 ´ ( 37 - 20 ) = 7.31 (2.61)
and 12.2 g of 2-amino-2-hydroxymethyl-propane- 4. Do not bring the buffer up to volume before
1,3-diol (Tris) in 1.7 L water, adjusting the pH to 8.2 with adjusting the pH. Use relatively concentrated
6 M NaOH, and then making up the volume to 2 L. acids and bases for this purpose, so that
the volumes needed for pH adjustment are
However, the most common approach for prepar- small.
ing buffers is approach 1. It has the advantage that 5. Ensure that buffer components do not interact
once stock solutions are prepared, they can be mixed with the test system. This is especially impor-
in different ratios to obtain a range of pH values, tant when performing experiments on living
depending on the experiment. One disadvantage is systems, such as cell cultures, but even
that if the pH needs to be adjusted, then either some in vitro systems are affected, particularly
acid or some base needs to be added, which slightly when enzymes are used. For instance, phos-
alters the volume and thus the concentrations. This phate buffers tend to precipitate with calcium
problem can be solved by preparing stock solutions of salts or affect enzyme functionality. For this
higher concentrations and adding water to the correct reason, a range of zwitterionic buffers with
volume, like in Example C1, for which 36 mL and sulfonic acid and amine groups has been
14 mL of 0.2 M stock solutions were combined and developed for use at physiologically relevant
brought to a volume of 100 mL to give a 0.1 M buffer. pH values.
This way, one also corrects for potential volume con- 6. Appropriate ranges for pH and molarities of
traction effects that may occur when mixing solutions. buffer systems that can be described through
Another potential disadvantage of stock solutions is the Henderson-Hasselbalch equation are
that they are often not stable for a long time (see Notes roughly 3–11 and 0.001–0.1 M, respectively.
below). 7. The calculations and theoretical background
described in this chapter apply to aqueous sys-
tems. Consult appropriate literature if you wish
2.4 NOTES ON BUFFERS to prepare a buffer in an organic solvent or
water/organic solvent mixtures.
When choosing an appropriate buffer system, the most 8. If you store non-autoclaved buffer or salt solu-
important selection criterion is the pKa of the acid tions, be aware that over time microbial growth
component. However, depending on the system, addi- or precipitation may occur. Visually inspect the
solutions before use, and discard if cloudy or dis- 11. You want to prepare a standard for atomic
colored. If autoclaving is an option, check if the absorption spectroscopy measurements con-
buffer components are suitable for this process. taining 1000 ppm Ca. How much CaCl2 do you
9. When developing a standard operating proce- need to weigh out for 1000 mL stock solution?
dure for a method that includes a buffer, include The atomic mass unit of Ca is 40.078, and the
all relevant details (e.g., reagent purity) and cal- molecular weight of CaCl2 is 110.98 g/mol.
culations. This allows tracing mishaps back to 12. Outline how you would prepare 250 mL of
an inappropriate buffer. 0.1 M acetate buffer at pH 5.5 for enzymatic glu-
10. There are numerous online tools that can help cose analysis.
with preparing buffer recipes. They can save time 13. Complexometric determination of calcium
and potentially allow you to verify calculations. requires an ammonium buffer with 16.9 g
However, they are no substitute for knowing the ammonium chloride (molecular weight, 53.49)
theory and details about the studied system. in 143 mL concentrated ammonium hydroxide
solution (28 % wt/wt, density = 0.88; molecular
weight = 17; pKb = 4.74). It is also to contain
2.5 PRACTICE PROBLEMS 1.179 g of Na2EDTA.2H2O (molecular
weight = 372.24 g/mol) and 780 mg
(Note: Answers to problems are in the last section of the MgSO4.7H2O (molecular weight = 246.47 g/mol).
laboratory manual.) After combining all reagents, the volume is
1. (a) How would you prepare 500 mL of 0.1 M brought up to 250 mL. What are the molarities
NaH2PO4 starting with the solid salt? for EDTA and MgSO4, and what pH does this
buffer have?
(b) When you look for NaH2PO4 in your lab, 14. Your lab uses 0.2 M stock solutions of NaH2PO4
you find a jar with NaH2PO4. 2H2O. Can you and Na2HPO4 for buffer preparation.
use this chemical instead, and if so, how
(a) Calculate the amounts weighed out for
much do you need to weigh out?
preparing 0.5 L of fresh 0.2 M stock solu-
2. How many g of dry NaOH pellets (molecular
tions of NaH2PO4.H2O (molecular weight,
weight: 40 g/mol) would you weigh out for
138) and Na2HPO4.7H2O (molecular
150 mL of 10 % wt/vol sodium hydroxide?
weight, 268).
3. What is the normality of a 40 % wt/vol sodium
(b) You want to make 200 mL of a 0.1 M buffer
hydroxide solution?
with pH 6.2 from these stock solutions.
4. How many mL of 10 N NaOH would be
How many mL of each stock solution do
required to neutralize 200 mL of 2 M H2SO4?
5. How would you prepare 250 mL of 2 N HCl you have to take?
starting with concentrated HCl? The supplier (c) How would the pH change if 1 mL of a 6 M
states that its concentration is 37 % wt/wt, and NaOH solution was added (note: you may
the density is 1.2. ignore the volume change)?
6. How would you prepare 1 L of 0.04 M acetic
acid starting with concentrated acetic acid (den- 15. Tris (2-amino-2-hydroxymethyl-propane-1,3-diol)
sity = 1.05)? The manufacturer states that the is an amino compound suitable for preparation of
concentrated acetic acid is >99.8 %, so you may buffers in physiological pH ranges such as for the
assume that it is 100 % pure. dietary fiber assay; however, its pKa is highly
7. Is a 1 % wt/vol acetic acid solution the same as affected by temperature. The reported pKa at
a 0.1 M solution? Show calculations. 25 °C is 8.06 [3]. Assuming a decline in pKa of
8. Is a 10 % wt/vol NaOH solution the same as a approximately 0.023 ΔpH/°C, what pH would a
1 N solution? Show calculations. Tris buffer with a molar acid/base ratio of 4:1
9. What would the molarity and normality of a have at 60 °C versus at 25 °C?
solution of 0.2 g potassium dichromate (molec- 16. Ammonium formate buffers are useful for
ular weight = 294.185) in 100 mL water be? It LC-MS experiments. How would you prepare
acts as an oxidizer that can transfer six electrons 1 L of a 0.01 M buffer of pH 3.5 with formic acid
per reaction. (pKa = 3.75, 98 % wt/wt, density = 1.2) and
10. How would you make 100 mL of a 0.1 N KHP ammonium formate (molecular weight 63.06 g/
solution? Note: For this practice problem, you mol)? Note: You may ignore the contribution of
only need to calculate the amount to weigh out, ammonium ions to the pH and focus on the
not explain how to standardize it. Chapter 21 of ratio of formic acid/formate anion. You may
this laboratory manual provides further infor- treat the formic acid as pure for the calculation,
mation about standardization of acids and bases. i.e., ignore the % wt/wt.
www.dbooks.org
REFERENCES 3. Harakany AA, Abdel Halim FM, Barakat AO (1984)

Dissociation constants and related thermodynamic quan-
1. Albert A, Serjeant EP (1984) The determination of ioniza- tities of the protonated acid form of tris-(hydroxymethyl)-
tion constants. A laboratory manual, 3rd edn. Chapman aminomethane in mixtures of 2-methoxyethanol and
and Hall, New York water at different temperatures. J. Electroanalytical
2. Harris, D (2015) Quantitative Chemical Analysis, 9th edn Chemistry and Interfacial Electrochemistry 162:285–305
W. H. Freeman and Company, New York 4. Tyl C, Sadler GD (2017) pH and titratable acidity. Ch. 22.
In: Nielsen SS(ed) Food analysis 5th edn. Springer, New
York
3
chapter
Dilutions
and Concentrations
Andrew P. Neilson (*) • Sean F. O’Keefe
Blacksburg, VA, USA
3.1 Introduction 3.5 Special Cases

3.2 Reasons for Dilutions and Concentrations 3.5.1 Extraction
3.3 Using Volumetric Glassware 3.5.2 Homogenization/Blending/Mixing
to Perform Dilutions 3.6 Standard Curves
3.6.1 Sequential Versus Parallel Dilutions
and Concentrations 3.6.2 Designing Dilution Schemes
3.4 Calculations for Dilutions 3.7 Unit Conversions
and Concentrations 3.8 Avoiding Common Errors
3.4.1 Introduction 3.8.1 Draw a Picture
3.4.2 Expressing Concentration 3.8.2 Unit Analysis
3.4.3 Forward Calculations 3.8.3 “Sniff Test”
3.4.4 Back Calculations
3.4.5 Multiple-Step Dilutions 3.9 Practice Problems
and Concentrations
www.dbooks.org
34 A.P. Neilson and S.F. O’Keefe
3.1 INTRODUCTION (c) Post-extraction evaporation of solvent (see

Practice Problem 1)
This chapter builds upon the information presented in
the previous Chap. 1 (Laboratory Standard Operating
Procedures). This chapter covers the following topics: 3.3 USING VOLUMETRIC GLASSWARE
TO PERFORM DILUTIONS
1. Reasons for performing dilutions and concen- AND CONCENTRATIONS
trations in food analysis
2. Basic calculations and strategies for calculating The use of laboratory glassware was covered in
the final concentration, given the initial concen- Chap. 1. However, a few key points are sufficiently
tration and a known dilution/concentration important for calculations regarding dilutions and
scheme (and vice versa) concentrations to mention again:
3. Strategies for designing and performing dilu-
tions to obtain standard curves 1. Class A glass volumetric flasks are used for
4. Example and practice problems bringing samples or solutions up to a known
“to contain” (TC) volume.
This information will be used repeatedly in a food 2. Class A glass volumetric pipettes are used for
analysis course (e.g., homework, laboratories, and transferring (delivering) a known “to deliver”
exams). More importantly, the principles described in (TD) volume of sample.
this chapter are essential for virtually all laboratory or 3. Other types of pipettes (non-class A volumetric
bench work in food science, including quality assur- glass pipettes, adjustable pipettors, automatic
ance/quality control, analysis for food labeling, and pipettors, reed pipettors, graduated measuring
product formulation. pipettes) and other glassware (graduated cylin-
ders, beakers, etc.) are less accurate and less
precise and should not be used for quantitative
3.2 REASONS FOR DILUTIONS volume measurements.
AND CONCENTRATIONS 4. Dilutions can be performed as “diluted to” or
“diluted to a final volume of” or as “diluted
There are various reasons why dilutions and concen- with.” These are very different terms (see Chap.
trations are performed, including: 1 for definitions). It is critical to understand the
differences between these types of dilutions to
1. Dilution: perform lab procedures and calculations
(a) To reduce high analyte concentrations in the correctly.
sample down to levels within the operating
range or optimal range of a method/
instrument 3.4 CALCULATIONS FOR DILUTIONS
(b) To reduce high analyte concentrations in AND CONCENTRATIONS
the sample down to levels within the linear
region of a method/instrument or to levels 3.4.1 Introduction
within a defined standard curve (see Practice Calculations involving dilutions or concentrations are
Problems 5 and 7) critical for quantitative measurements. Many analytical
(c) To dilute the background matrix to levels methods (such as spectrophotometric assays, chroma-
that do not interfere with the analysis tography, titrations, protein determinations, etc.) are
(d) Reagent addition, which dilutes the sample performed on diluted or concentrated samples, and the
by increasing the volume analytical result must be converted to the concentration
(e) Solvent extraction, which dilutes the sam- in the undiluted or unconcentrated sample for labeling
ple by using large volumes of solvent to or research purposes. Instances for which these calcula-
favor transfer of the analyte from sample to tions are used include, but are not limited to:
solvent (see Practice Problem 1)
2. Concentration: 1. Preparation of standard curves (see Practice
(a) To increase low analyte concentrations in Problems 3 and 4).
the sample up to levels within the operating 2. Determining the necessary dilution required to
range or optimal range of a method/ obtain a sample concentration within a speci-
instrument fied range (see Practice Problems 5 and 7)
(b) To increase low analyte concentrations in 3. Determining the necessary range of dilutions
the sample up to levels in the linear region for preparing a range of concentrations for a
of a method or to within a standard curve standard curve from a stock standard solution
(see Practice Problem 1) (see Practice Problems 5 and 7)
Chapter 3 • Dilutions and Concentrations 35
4. For converting an analytical result obtained

from a diluted or concentrated sample to the X 168 mg thiamine 1.12 mg thiamine
undiluted or concentrated food (see Practice =
C = =
V 150 mL solution mL solution
Problems 1 and 6)
The amount of thiamine in the 0.25 mL that is
3.4.2 Expressing Concentration diluted is:
Recall that the concentration of an analyte in a sample 1.12 mg thiamine

X CV 0.25 mL solution
or solution is defined as follows:
mL solution
X X
= C = or C (3.1) 0.28 mg thiamine
m V
C  concentration Note that we are only concerned with the

X  amount of analyte g , mol , etc. amount of analyte in the portion of the sample
that is diluted/concentrated (0.25 mL in this
V  sample volume
example) and not the amount of analyte in the
m  mass whole initial sample (150 mL in this case).
Note that concentrations also can be expressed in When the 0.25 mL of the stock solution (con-
terms of percentages (%), parts per million (ppm), etc. taining a total of 0.28 mg thiamine) is diluted to
(see Chap. 2, Table 2.1). Percentage is a particular a final volume of 200 mL, the total amount of
problem as it can refer to ratios expressed as weight thiamine (0.28 mg) does not change because
per weight, weight per volume, or volume per vol- the added water does not contain any thia-
ume. Simply saying 5 % ethanol is unclear as it could mine. Therefore:
represent 5 % w/w, w/v, or v/v. Therefore, % are typi- X= X= 0.28 mg thiamine
1 2
cally expressed with the accompanying notation
(w/w, w/v, etc.) to clarify the meaning of the % value.
Rearranging Eq. 3.1, the amount of analyte can be However, the concentration of thiamine changes,
expressed as: because now the 0.28 mg thiamine is present in
200 mL instead of 0.25 mL:
= =
X Cm or X CV (3.2)
For each step in a dilution or concentration, a portion
X 0.28 mg thiamine 0.0014 mg thiamine
(mass or volume) of a sample or solution is either =
C = =
diluted with additional liquid or reduced in volume. V 200 mL solution mL solution
n
The mass, volume, and concentration of the sample
change, but the amount (mol, g, etc.) of the analyte Therefore, the thiamine solution has been diluted,
present in the amount of the sample that is diluted or as the final concentration (0.0014 mg/mL) is less
concentrated does not change. Therefore, the follow- than the initial concentration (1.68 mg/mL).
ing is true:
X1 = X 2 (3.3)
X1 = the amount of analyte in the sample before “Concentration” and “amount” are very distinct
the dilution / concentration step concepts. Concentration is a ratio of the amount of ana-
lyte to the amount of sample. The concentration (mg/
X 2 = the amount of analyte in the sample after the mL, M, N, %, ppm, etc.) of an analyte present in a sample
dilution / concentration step is not dependent on the amount (g, mL, etc.) of sample.
Example D2 If a pesticide is present at 2.8 parts

per billion (ppb) (note that 1 ppb = 1 μg/kg) in a
Example D1 Suppose that a stock solution of sample of applesauce, the pesticide is uniformly
thiamine is made by dissolving 168 mg thiamine distributed throughout the applesauce, and
to a volume of 150 mL, and then 0.25 mL of that therefore the concentration is 2.8 ppb regardless
stock solution is added to a volumetric flask and of whether 1 μg, 1 mg, 1 g, 1 kg, 1 mL, or 1 L of
water is added so that the final volume is applesauce is analyzed. However, the total
200 mL. The concentration of thiamine in the amount of the analyte present does depend on
stock solution is: the size of the sample. We can easily see that
www.dbooks.org
CiVi V Ci mi m
0.063 and 2.2 kg of applesauce contain the same Cf Ci i or Cf Ci i (3.8)
Vf Vf mf mf
concentration, but vastly different total amounts,
of the pesticide: This is intuitive, as the ratio of the final to the initial
X = Cm (3.4) volumes, masses, or concentrations is referred to as
the “fold” of the dilution. The ratio of the starting and
2.8 g pesticide final masses or volumes for each step is referred to as
0.063 kg applesauce
kg applesauce the dilution factor (DF) for that step (see Practice
0.176 g pesticide Problem 2):
Vi
DF = (3.9)
2.8 g pesticide Vf
2.2 kg applesauce
kg applesauce The final concentration is the product of the initial con-
6.16 g pesticide centration and the DF (the DF is a “multiplier” that can
be used to convert the initial concentration to the final
concentration):
Once we know the concentration of a sample or CiVi V

Cf Ci i Ci DF (3.10)
solution, we can take any part, or all, of the sample. Vf Vf

This changes the amount of the analyte but not the
concentration. Then, diluting or concentration the ali- Another way to think of the DF is the ratio of the final
quot chosen changes the concentration, but not the and initial concentrations for each step:
amount, of the analyte. These are two critical concepts Cf
that must be implicitly understood to master the con- DF = (3.11)
Ci
cepts of dilution and concentrations. Since the total
amount of analyte does not change during each step, Some conventions that apply to the term “dilution fac-
the following equations can be derived: tor” are as follows:
1. DF always refers to the forward direction (ini-
Xi = X f (3.5)
tial mass or volume divided by final mass or
volume for each step).
= =
and X Cm or X CV , therefore :
2. The term DF is usually used even if the step is a
concentration; less commonly, the term “con-
=CiVi C=
f Vf or Ci mi C f m f (3.6)
centration factor” may be used.
3. Since DF is a multiplier of the initial
i initial before dilution or concentration concentration:
(a) If the step is a dilution, DF < 1.
f final after dilution or concentration
(b) If the step is a concentration, DF > 1.
3.4.3 Forward Calculations The “fold” or “X” of a dilution is defined as
follows:
The relationship between the initial and final concen-
tration can be used to determine the starting and/or 1
final concentration if one concentration and the masses dilution " fold " or " X " = (3.12)
DF
or volumes used in the dilution are known. If the start-
ing concentration is known, the final concentration can
be calculated (a “forward” calculation):
CiVi Ci mi
=Cf = or C f (3.7) Example D3 For the thiamine solution described
Vf mf earlier, if 1 mL of the solution is added to a 10 mL
Similarly, if the final concentration is known, the starting volumetric flask and diluted to volume, the ratio
concentration can be calculated (a “back” calculation). of the final to initial volumes is 10:1 or a “ten-
From this relationship, it becomes evident that the final fold” or “10X” dilution, and therefore the final
concentration can be expressed as the initial concentra- concentration is tenfold (10X) lower than, or
tion multiplied by the ratio of the initial and final con- 1/10th of, the initial concentration:
centrations (or vice versa for the initial concentration):
V 1.12 mg thiamine 1 mL Example D4 Suppose that soybean oil contains

CiVi
Cf Ci i 175 mg oleic acid per g, and 2.5 g of the oil is
Vf Vf mL solution 10 mL
diluted with hexane to a final volume of
0.112 mg thiamine 75 mL. The concentration of oleic acid in the final
solution can be determined as follows:
mL solution
Ci mi = C f Vf
For a tenfold dilution:
1 m 175 mg oleic acid 2.5 g oil
dilution " fold " or " X " = C f Ci i
DF Vf g oil 75 mL

1
Therefore, DF = 5.83 mg oleic acid
dilution " fold " or " X "
mL
1
=
DF = 0.1
10
3.4.4 Back Calculations
1.12 mg thiamine
C f Ci DF 0.1 The examples worked up to this point involve “for-
mL solution
ward” calculations (calculating the final concentration
0.112 mg thiamine from a known initial concentration, see Practice

mL solution Problems 3 and 4). However, many food analysis calcu-
lations will involve “back” calculations (calculating
As discussed above, note that this is different
the initial sample concentration from a final concentra-
than adding 10 mL water to 1 mL of the thiamine
tion obtained by analysis of the diluted or concen-
solution, which makes the final volume 11 mL
trated sample, see Practice Problems 1 and 6). This
for an 11-fold or 11X dilution:
calculation is simply the reverse of the “forward” cal-
V 1.12 mg thiamine 1 mL culation, for which the initial concentration is solved
C f Ci i for as opposed to the final concentration:
Vf mL solution 1 mL 10 mL

=CiVi C=
f Vf or Ci mi C f m f , therefore :
1.12 mg thiamine 1 mL

mL solution 11 mL C f Vf Vf
0.102 mg thiamine Ci C f or
Vi Vi
mL solution
Cf mf mf
For concentration processes, the calculation is the Ci Cf (3.16)
mi mi
same except the analyte concentration increases
from initial to final. For example, suppose that Note that for back calculations, we flip the DF:
12.8 mL of the thiamine solution is reduced (by C f Vf Vf 1
boiling, freeze drying, rotary evaporation, etc.) to Ci Cf Cf or
3.9 mL. The final concentration is: Vi V
i DF
Cf mf 1
V 1.12 mg thiamine 12.8 mL Ci Cf (3.17)
C f Ci i mi DF
Vf mL solution 3.9 mL

3.68 mg thiamine
Example D5 Suppose that 50 mL of water is
mL solution
added to a 35 mL sample of apple juice (there-
fore, initial volume = 35 mL and final vol-
ume = 85 mL), and titration indicates that the
Note that masses can be used instead of volumes: concentration of malic acid in the diluted sample
is 0.24 % w/v. What is the concentration of malic
Ci mi = C f m f (3.13) acid in the undiluted juice? This can be deter-
Furthermore, both masses and volumes can be used: mined using Eq. 3.17 above:
=Ci mi C=
f Vf or CiVi C f m f (3.14 and 3.15) Vf 85 mL
Ci C f 0.24% w / v 0.583% w / v
Vi 35 mL
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A special case of concentration is when a solution 3.4.5 Multiple-Step Dilutions

is completely evaporated or dried (so that there is no and Concentrations
liquid left, only nonvolatile solutes) and then reconsti-
Up to this point, we have dealt exclusively with single-
tuted with a volume of liquid smaller than the initial
step dilutions and concentrations. However, real-
volume. This is technically composed of two distinct
world analytical practice often requires that several
steps: a concentration step (drying the sample or solu-
dilutions and/or concentrations be performed (see
tion until only solutes, usually a very small mass, are
Practice Problems 1, 2, and 6). There are two ways to
left) followed by a dilution step (diluting the residual
approach this problem. Suppose that three dilutions
dry mass to a define volume).
are used as follows:
Step 1 : C1 → C2 by performing V1 → V2
Example D6 Suppose that 10 mL apple juice,
containing 0.025 g malic acid/mL, is freeze Step 2 : C2 → C3 by performing V3 → V4
dried, leaving a residue of 0.09 g. This residue is
then reconstituted to a final volume of 3 mL with Step 3 : C3 → C4 by performing V5 → V6
water. What is the concentration of malic acid in The obvious approach is to solve for the final (or ini-
the final solution? For the first step, the problem tial) concentration of the first step, use the final (or ini-
is set up as: tial) concentration of the first step as the initial (or
final) concentration of the second step, and so forth:
V 0.025 g malicacid 10 mL juice
C f Ci i
Vf mL juice due V V V
0.09 g resid C2 C1 1 then C3 C2 3 then C4 C3 5
2.78 g malicacid V
2 V
4 V6

g residue This can be time-consuming. Furthermore, perform-
ing multiple calculations can introduce rounding
For the second step, the problem is set up as:
errors and increases the probability of making other
V 2.78 g malicacid 0.09 g residue errors. Notice that we can substitute the formula used
C f Ci i to calculate a concentration in one step for that value
Vf g residue 3 mL
in the next step:
0.0833 g malic acid V
C2 C1 1
mL
V2
However, in actual laboratory practice, this is typi-
cally treated as a single step (starting with the ini-
V V V
tial volume and ending with the final diluted C3 C2 3 C1 1 3
volume) for two reasons. First, the whole dried V4 V2 V4
sample is typically reconstituted, so the final mass
for the first step and the initial mass of the second V V V V
step are the same and thus cancel each other out in C4 C3 5 C1 1 3 5
V6 V2 V4 V6
the calculation. Second, for most concentrations
involving solutions, the mass of the solutes Therefore, as long as we have the volumes (or masses)
remaining after complete drying is much too small from each step, we can reduce this multistep calcula-
to be measured accurately with balances com- tion to a single-step calculation that goes directly from
monly found in most food analysis laboratories, the initial to the final concentration (or vice versa),
and it is difficult to get all of the residue out accu- keeping in mind that when going forward (from initial
rately for weighing. Therefore, these problems are sample to final solution), the initial concentration is
typically solved in a single step as follows: multiplied by a series of dilution factors for each step,
with the starting mass/volume divided by the final
V 0.025 g malic acid 10 mL juice mass/volume for each step. If we have a dilution or
C f Ci i
Vf mL juice 3 mL concentration scheme with n steps, we can set up the
calculations so that:
0.0833 g malic acid
Step 1 : Ci → C2 by performing V1 → V2
mL
Step 2 : C2 → C3 by performing V3 → V4
and so forth until the last step, where:
As long as the entire residue is reconstituted, the
calculation can be treated as a single step. Step n : C3 C f by performing Vk 1 Vk
Then, a general formula for multistep forward calcula-

tions is: Step 3 : 75 mL V5 concentrated to 50 mL V6
m or V1 m or V3 m or Vk 1
C f Ci Step 4 : 50 mL V7 diluted to 100 mL
m or V2 m or V4 m or Vk (3.18)
Ci DF1 DF2 DFn
V8 , 50 mL 50 mL ,
12.9 g
We can simplify this by calculating the “overall DF” Cf
mL
(DFΣ), the product of DFs from each step:
The calculation is a back calculation of the initial
DF DF1 DF2 DFn (3.19) concentration from the final calculation:
C f Ci DF (3.19) m or V2 m or V4 m or Vk
Ci C f
When going from final solution to initial sample, the m or V1 m or V3 m or Vk 1
calculation is reversed: the initial concentration is mul- 1 1 1
Cf
tiplied by dilution factors for each step, with the final DF1 DF2 DFn
mass/volume divided by the initial mass/volume for
each step. A general formula for multistep back calcu- There are four steps, so there must be four indi-
lations is: vidual DFs (this is a good way to make sure that
you are doing the problem correctly; does the
m or V2 m or V4 m or Vk
Ci C f number of DFs you used equal the number of
m or V1 m or V3 m or Vk 1 steps?):
1 1 1
Cf (3.20)
DF1 DF2 DFn m or V2 m or V4 m or V6 m or V8
Ci C f
m or V1 m or V3 m or V5 m or V7
1
C f Ci (3.21)
DF 12.9 g caffeine 500 mL
Ci
Cf mL 0. 56 g green teaextract
DF (3.22)
Ci 125 mL 50 mL 100 mL

This approach of solving dilution or concentration 25 mL L 75 mL 50 mL
problems in a single step can be used for dilutions, 76 , 800 g caffeine

concentrations, and mixed procedures involving both g green teaextract
dilutions and concentrations.
Another way to make sure that this calculation
has been done correctly is to assign each solution
Example D7 Suppose that 0.56 g of green tea a letter or number, and those should “cancel each
extract is diluted in boiling water to a volume of other out” along with the units. If the units and/
500 mL, 25 mL of the resulting solution is mixed or solution identities don’t cancel out properly,
with 100 mL phosphate buffer, 75 mL of this solu- one or more concentrations, volumes, or masses
tion is freeze dried, and the residue is dissolved have been used incorrectly. For this problem, the
to a final volume of 50 mL in methanol, and all of solutions could be identified as follows:
this solution is then combined with 50 mL ether.
The final concentration of caffeine is analyzed by Step 1 : Ci ? m 0.56 g m1 diluted to 500 mL
HPLC and is 12.9 μg/mL. What is the concentra- V2 , solution A
tion of caffeine in the green tea extract (in μg/g)?
We can set the problem up like this: Step 2 : 25 mL V3 , solution A diluted to 125 mL
V4 , solution B, 25 mL 100 mL
Step 1 : Ci ? 0.56 g m1 diluted to 500 mL V2
Step 2 : 25 mL V3 diluted to 125 mL Step 3 : 75 mL V5 , solution B concentratedto 50 mL

V4 , 25 mL 100 mL V6 ,solution C
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Step 4 : 50 mL V7 , solution C diluted to 100 mL Example E1 Suppose that peanut butter (PB)
contains 0.37 g fat/g, and 1.4 g peanut butter is
V8 , solution D, 50 mL 50 mL extracted with 100 mL hexane. What is the con-
centration of fat in the hexane (the assumption
12.9 g here is that the volume of the hexane doesn’t
Ci
mL change)?
The problem would then be solved the same This problem can be solved using the stan-
way with the solutions identified: dard procedure:
12.9 g caffeine 500 mL solution A m 0.37 g fat 1.4 g PB 0.00518 g fat
Ci C f Ci i
mL solution D 0.56 g green tea extrract Vf
g PB 100 mL mL
125 mL solution B 50 mL solution C

25 mL solution A 75 mL solution B Note that this is the same as if the entire sample
100 mL solution D 76 , 785.7 g caffeine was diluted into the solvent, but for an extrac-
tion only a part of the sample (but presumably
50 mL solution C g green tea extract
all of the analyte) is actually transferred.
In reality, only a fraction (generally unknown) of

3.5 SPECIAL CASES the analyte is transferred from the sample to the sol-
vent. In order to overcome this, repeated extractions
3.5.1 Extraction are often performed. These are the same as single
extractions, except the extraction is repeated with
There are several special cases regarding dilution that
fresh solvent each time and the solvent volumes from
merit specific mention: extraction and homogeniza-
each extraction are combined into a single extract. In
tion/mixing. Regarding extraction, often, we use a sol-
this case, we can treat this as a single extraction where
vent for which an analyte has a high affinity (e.g., lipids
the combined solvent volume is the final volume, and
are more soluble in nonpolar solvents such as hexane or
again we assume that 100 % of the analyte is trans-
chloroform than the foods they are in, so these solvents
ferred to the solvent.
“pull” the lipid out of the food) to extract that analyte
from a sample. The solvent is typically immiscible with
the sample as a whole. This means that the sample and
the solvent do not mix and can be easily separated by Example E2 Suppose that the peanut butter
centrifugation or by using a separatory funnel. Most of example above is adjusted so that the peanut
the analyte is transferred to the solvent, while most of butter is extracted three times with 75 mL of
the sample remains behind. For dilution and concentra- fresh hexane, and the three extracts are com-
tion purposes, we assume that 100 % of the desired ana- bined. What is the concentration of fat in the
lyte is quantitatively transferred from the sample into hexane?
the solvent (see Practice Problem 1). While this assump- In this case, the final volume would be
tion is never actually true, it is necessary for our pur- 3 × 75 mL = 225 mL:
poses. Further dilutions and/or concentrations may
then be performed on the extraction solvent. m 0.37 g fat 1.4 g PB 0.00230 g fat
C f Ci i
In some cases, we use a single extraction step. In Vf g PB 225 mL mL

this situation, the sample is mixed with the solvent
to achieve transfer (extraction) of the analyte from
the solvent. The sample and solvent are then sepa-
rated. This is different than the simple dilution Extraction procedures often specify that the
problems we have done previously, in that the sam- extraction solvent be brought up to a known final vol-
ple remains behind while only the analyte is trans- ume after extraction. This accounts for potential loss of
ferred to the solvent. However, the principle is still solvent, or significant changes in volume due to extrac-
the same: 100 % of the analyte that was in the sample tion of sample components, during extraction and also
is assumed to now reside in the solvent, and we can increases the precision and accuracy of the extraction.
use the equations previously discussed (Eqs. 3.16 This is handled by simply making this final volume
and 3.17). the final volume for the dilution calculation.
Example E3 Suppose that the peanut butter Example E5 Suppose that 22.7 g swordfish fillet
example above is adjusted so that the peanut is homogenized in a lab blender with 150 mL
butter is extracted 2X with 100 mL hexane, and denaturing buffer. Following homogenization,
the pooled hexane extract is diluted to a final the liquid is decanted into a 500 mL flask. Fresh
volume of 250 mL. What is the concentration of denaturing buffer (~25 mL) is used to rinse the
fat in the hexane? blender, and the rinse liquid is also decanted into
the flask. The flask is brought to volume with
In this case, the final volume would simply
denaturing buffer. The concentration of methyl-
be 250 mL, as the pooled extracts (~200 mL) are
amines in the diluted homogenate is 0.92 μmol/
further diluted with ~50 mL to an exact total vol-
mL. What is the concentration of methylamines
ume of 250 mL. The problem would then be
in the fish?
solved as shown above, using 250 mL as the final
volume. In this case, the sum of the homogenization
process is that 22.7 g fish is diluted into 500 mL
final volume. Therefore, the calculation is per-
In summary, extraction can be thought of as a formed as follows:
dilution (or concentration) in which the final volume is
V f 0.92 mol methylamines 500 mL
the total volume of solvent. Regardless of the number Ci C f
of extractions, it can be viewed as a single step as long V mL 22.7 g fish
i
as all the solvent extracted from the sample is kept and 20.3 mol methylamines
the final volume into which the sample is extracted is
g fish
known. After extraction, dilutions or concentrations of
the extract may be performed; these are handled as
any other dilution or concentration.
In summary, mixing processes can be thought of
3.5.2 Homogenization/Blending/Mixing as a dilution (or concentration) step for which the final
The other scenario that warrants consideration is volume is the total volume of solvent. Regardless of
homogenization, blending, and mixing. Often, sam- the number of steps involved, it can be viewed as a
ples need to be dispersed or blended into a solvent for single step as long as all the homogenate from whole
sample preparation. This is typically done using a lab sample is kept and the final volume into which the
blender, food processor, Polytron homogenizer, stom- sample is diluted is known. Keep in mind that, after
acher, sonicator, etc. These processes cannot typically mixing and quantitative dilution, subsequent dilu-
be done in volumetric glassware. Furthermore, the tions or concentrations of the homogenate may be per-
problem with these processes, from a calculation stand- formed, and these are handled as any other dilution or
point, is that homogenization, blending, and mixing of concentration.
solids with liquids typically change the volume of a
sample.
3.6 STANDARD CURVES
It is very common for analytical procedures to require

a standard curve, which is a set of solutions containing
Example E4 Suppose you homogenize 2.5 g
different concentrations of the analyte. Typically, a
cheese (of an unknown volume) in 100 mL phos-
stock solution containing a high concentration of the
phate buffer. What is the final volume?
analyte is prepared, and various dilutions are per-
The final volume of the liquid will be formed to get the desired standard curve solutions (see
>100 mL and is not easily measured by volumet- Practice Problems 3, 4, 5, and 7). These dilutions can be
ric glassware. Therefore, in this example, the done with either sequential dilutions (Fig. 3.1a) or par-
final volume is unknown. This poses a challenge allel dilutions (Fig. 3.1b).
for accurate dilution calculations. This process is
usually overcome similarly to the extractions 3.6.1 Sequential Versus Parallel Dilutions
described above: the sample is homogenized,
In the case of sequential dilutions, each dilution is
quantitatively transferred to a volumetric flask,
used to prepare the next dilution in the series:
and then diluted to a known final volume with
the same solvent used for homogenization. stock solution → solution 1 → solution 2
→ solution 3, etc.
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a
V 1 mL
Solution B : C f Ci i 0.333 M
Vf 3 mL

0.111 M
V 1 mL
Solution C : C f Ci i 0.111 M
stock solution solution solution Vf 3 mL
solution A B C
b 0.0370 M
V 1 mL
Solution D : C f Ci i 0.0370 M
Vf 3 mL

0.0123 M
solution
stock A For parallel dilutions, different DFs are applied to the
solution
stock to obtain the desired solutions:
DF 1
stock solution → solution 1
DF 2
solution
B stock solution → solution 2
DF 3
stock solution → solution 3
solution
C
Example F2 Suppose the 1 M stock solution of

Example standard citric acid is diluted as follows: 1 mL of stock
3.1 solution is diluted to volume in a 5 mL, 10 mL,
figure
curve dilution schemes for sequential
dilutions (a) and parallel dilutions (b) 25 mL, and 50 mL volumetric flask. What are the
citric acid concentrations in each solution? We
can solve this problem by assigning each solution
a name (Solutions A–D) and then calculating the
Example F1 Suppose a 1 M solution of citric acid concentration in each. For each dilution, the start-
is serially diluted as follows: 1 mL is diluted with ing concentration is the stock concentration (since
2 mL water, and the resulting solution is diluted they were made in parallel), and the final volume
the same way (1 mL + 2 mL water) until four is the volume of each volumetric flask:
diluted solutions are obtained. What are the con-
centrations in each solution? V 1 mL
We can solve this problem by assigning each Solution A : C f Ci i 1M 0.200 M
Vf 5 mL
solution a name (Solutions A–D) and then calcu-
lating the concentration in each. For each step,
the initial volume is 1 mL and the final volume is V 1 mL
3 mL, since 2 mL water is combined with each Solution B : C f Ci i 1M 0.100 M
Vf 10 mL
solution, and the initial concentration is the con-
centration of the previous solution (stock when
making Solution A, Solution A when making V 1 mL
Solution C : C f Ci i 1M 0.040 M
Solution B, etc.): Vf 25 mL

V 1 mL V 1 mL
Solution A : C f Ci i 1M 0.333 M Solution D : C f Ci i 1M 0.020 M
Vf 3 mL Vf
50 mL
Preparing standards in parallel is typically more 3.6.2 Designing Dilution Schemes

accurate than in series, as each dilution is performed
Occasionally, you may be given the stock solution con-
in a single step from the stock. This reduces error asso-
centration and the needed concentrations of standard
ciated with each solution.
curve solutions and asked to design a dilution scheme.
The way to do this is to calculate the needed DFs for
each solution and then design a scheme to achieve the
Example F3 If preparing a dilution involves DFs.
an error of ~1 % (a factor of 0.01), what is the
error in the third solution if the solutions are
prepared in parallel?
Example F5 Suppose you have a 0.2 % w/v
Performing three dilutions in parallel will result
NaCl solution and you need standard curve
in each solution being off by ~1 %:
solutions of 0.1, 0.04, 0.02, 0.01, and 0.002 % w/v.
DF 1 How would you make these?
stock solution solution 1 X 1% or 1.01X
First, start by calculating each DF from the stock
DF 2 solution: Cf
stock solution solution 2 X 1% or 1.01X DF =
Ci
DF 3
stock solution solution 3 X 1% or 1.01X
Cf 0.1% 1
DFA 0.5 i.e.,
Ci 0.2% 2
Cf 0.04% 1
If dilutions are performed in series, the error com- DFB 0.2 i.e.,
pounds because each solution is used to prepare the Ci 0.2% 5
next solution.
Cf 0.02% 1
DFC 0.1 i.e.,
Ci 0.2% 10
Example F4 For the same three dilutions, what
is the error in the third solution if the dilutions Cf 0.01% 1
are prepared in series? DFD 0.05 i.e.,
Ci 0.2% 20
When prepared in series, the error increases with
each subsequent dilution: Cf 0.002% 1
DFE 0.01 i.e.,
Ci 0.2% 100
stock solution solution 1 1% or 1.01X
From this point, there are several options. You
solution 2 1% or 1.01X
can dilute in series or in parallel. And, you can
solution 3 1% or 1.01X “dilute to” or “dilute with” to achieve the desired
concentrations. For simplicity, the parallel
error in solution 1 1.01X 1% “dilute to” example is shown in Table 3.1.
error in solution 2 1.01 1.01 X 3.1

1.0201X 2.01% table Dilution example for a standard curve
error in solution 3 1.01 1.01 1.01 X Ci Dilute To final Cf (%

Solution (% w/v) (mL) volume (mL) DF w/v)
1.030301X 3.0301%
A 0.2 1 2 0.5 1
B 0.2 1 5 0.2 0.04
C 0.2 1 10 0.1 0.02
D 0.2 1 20 0.05 0.01
Parallel dilutions are often preferred over series E 0.2 1 100 0.002 0.002
dilutions for accuracy and improved quantification for
standard curves. Regardless of how they are per-
formed, the principles of dilution calculations apply.
As with any dilution, these dilutions can be performed You should be able to design a scheme to dilute a
as “dilute to” or “dilute with” procedures, and the cal- stock solution to a wide array of diluted solutions
culations should be performed accordingly. using both parallel and series dilutions and perform
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either with “dilute to” or “dilute with” procedures.

One consideration to keep in mind is that class A volu-
metric glassware comes in discreet volumes, and only
3.2
table Dilution example for a standard curve
those volumes can be used for dilutions where maxi-
mum accuracy and precision are desired. The most Fe(II) Stock 0.1 N HCL Total volume
common volumes available are: (ppm) (mL) (mL) (mL)
1. Class A volumetric flasks: 5, 10, 25, 50, 100, 200, 1000 0.5 0.5 1.0
250, 500, and 1000 mL 750 0.375 0.625 1.0
2. Class A volumetric transfer pipettes: 1, 2, 3, 4, 5, 500 0.25 0.75 1.0
10, 20, 25, 50, and 100 mL 250 0.125 0.875 1.0
100 0.05 0.95 1.0
Therefore, your designed dilution scheme should use
only those volumes for transferring volumes (pipettes)
and bringing to volume (flasks), if possible, to optimize
In cases when an adjustable pipettor is used, accu-
accuracy and precision. Some schemes may require the
racy and precisions should be optimized by frequently
use of an adjustable pipettor (to transfer or add vol-
calibrating and maintaining the pipettors. Furthermore,
umes such as 0.1 mL, 950 μL, etc.), particularly when
using proper (and consistent) manual pipetting tech-
small volumes are used. This is often the case when pre-
nique is critical in these scenarios.
paring standard curves (see Practice Problems 3 and 4).
3.7 UNIT CONVERSIONS

Example F6 Suppose that you have a stock solu-
tion of 2000 ppm Fe(II) in 0.1 N HCl. You need to Up to this point, we have examined calculations for
create 1 mL of 1000, 750, 500, 250, and 100 ppm which the units of the dilutions and the units of the con-
Fe(II) by combining different volumes of the centrations that you have been given “match up” so that
stock solution and 0.1 N HCl. What volumes no conversions are required to get the correct answer.
should be used? However, this will not always be the case in actual lab
We know the starting (2000 ppm) and final con- practice. This will require you to perform unit conver-
centration (1000–100 ppm) and final volume sions to get an answer that is correct and makes sense.
(1 mL). Therefore, we must calculate the initial
volume of stock solution for each solution:
C f Vf Example G1 Suppose that you dilute 14.4 g of
= CiVi C= f V f andVi yogurt to a final volume of 250 mL with water.
Ci HPLC analysis of the diluted sample indicates a
riboflavin concentration of 0.0725 ng/μL. What
for 1000 ppm , Vi
C f Vf

1000 ppm 1 mL is the riboflavin content in the yogurt?
Ci 2000 ppm
This is a straightforward dilution problem that
0.5 mL can be solved as follows:
C f Vf 750 ppm 1 mL Vf

for 750 ppm , Vi Ci C f
Ci 2000 ppm Vi
0.375 mL However, if the calculation were performed
and so forth. Once the volume of stock solution without unit conversions, the answer obtained
is known for each dilution, we then calculate the would be as follows:
amount of 0.1 N HCl needed to dilute to 1 mL: V f 0.0725 ng riboflavin
Ci C f
for 1000 ppm , 0.5 mL 1 mL 0.5 mL V L diluted sample
i
0.5 mL 0.1 N HCl
250 mL diluted sample 1.26 ng riboflavin mL

for 750 ppm , 0.375 mL 1 mL 0.375 mL 14.4 mL yogurt g yogurt L
0.625 mL 0.1 N HCl
Obviously, (ng × mL)/(g × μL) is not an accept-
and so forth. The dilution scheme would thus be able unit for expressing the riboflavin concentra-
as shown in Table 3.2. tion in a product. Therefore, we must change
a
either μL to mL or mL to μL for the units to be
expressed correctly in the final answer. The cal-
culation is performed correctly as follows:
Vf 0.0725 ng riboflavin 1000 L

Ci C f
100 mL final volume
Vi Ldilutedsample mL 1.2 mL milk +
250 mLdilutedsample 1, 260 ng riboflavin 88.8 mL water

14.4 mL yogurt g yogurt
milk diluted solution
If the units are not converted, the magnitude
(number only) of the final answer is off by a fac-
tor of 1000 in this case, in addition to having the b
wrong units.
101.2 mL final volume
1.2 mL milk +
100 mL water
By keeping track of the units, you easily catch an

incorrect value and adjust the calculation.
milk diluted solution
3.8 AVOIDING COMMON ERRORS Dilution schemes for a “dilute to” (a) and
3.2
figure
“dilute with” (b) scenario
The most common errors associated with dilutions
and corrections are:
total volume 10 mL
1. Setting up the calculation incorrectly (incor-
100 mL
rectly using the information provided) 27.9% (w/w) glucose total volume 62.8 mL total volume 35 mL
2. Lack of needed unit conversion or incorrect unit dilute to

17 mL 5 mL+
conversion 12.8 mL honey + volume
30 mL
50 mL water freeze dried
methanol
Three strategies can be employed to avoid these mis- honey solution A solution B solution C
takes or catch them if they have been made:

Step 1 Step 2 Step 3
Ci=27.9% Vi=17 mL Vi=5 mL
1. Draw a picture of the dilution or concentration Vi=12.8 mL Vf=10 mL Vf=35 mL
Vf=62.8 mL
scheme. Cf=?
2. Perform unit analysis (also referred to as

3.3 Diagram of a complex multistep dilution and
dimensional analysis or the factor-label
figure
concentration scheme
method) and assign “names” to each solution in
a scheme.
3. Perform the “sniff test.” Drawing a picture can be particularly useful for visu-
alizing complex multistep processes.
3.8.1 Draw a Picture
Drawing a picture can be very useful for making sure
the problem has been set up correctly. This allows you Example H2 You have 100 mL honey (27.9 %
to visualize the dilution or concentration scheme, glucose by weight). 12.8 mL honey is dissolved
which is often helpful. Drawing a picture or diagram by adding 50 mL water, 17 mL of this solution is
can help clarify the meaning of the information pro- freeze dried and reconstituted to a total volume
vided and assist in setting up the calculation. of 10 mL with water, and 5 mL of this solution is
combined with 30 mL methanol. What is the %
glucose by weight in the final solution?
Example H1 Suppose you are told that 1.2 mL
skim milk is diluted to 100 mL with water. The This information can be diagramed as shown in
picture that would be drawn might look some- Fig. 3.3.
thing like Fig. 3.2a. By comparison, if you are
told that 1.2 mL skim milk is diluted with 100 mL
water, the picture that would be drawn might
look something like Fig. 3.2b.
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Figure 3.3 shows a fairly complex scheme, and orga- 2. If the procedure is an overall concentration,
nizing the information in your head can be challeng- then the final concentration should be higher
ing. Drawing this scheme allows you to assign names than the initial concentration.
to various solutions and organize the information so 3. With very few exceptions (such as % moisture
that it can easily be plugged into the calculation. expressed on a dry weight basis for some sam-
ples), the % of an analyte in a sample will never
3.8.2 Unit Analysis be ≥100 %.
4. Does the calculated value make sense? For
We have already discussed unit analysis, a key step in
example, one would not expect the following
verifying that the calculation has been set up correctly.
calculated values to be true:
Along with this, assigning names to each solution in a
scheme and incorporating those into the calculation (a) % moisture > a few % in very dry products
can assure that each step has been set up correctly in
(b) % solids > a few % in dilute solutions
the calculation. If the problem has been set up cor-
rectly, each solution name for the intermediate steps (c) % fat, protein, ash, or carbohydrates that
should be “canceled out,” leaving only the final solu- don’t make sense given what is known
tion or sample when the calculation is complete. about the product (such as vegetable oil
containing 35 % moisture or 12 % fat)
(d) Analyte levels that do not make sense given
Example H3 For the example in Fig. 3.3, the what is known about typical product com-
concentration of the final solution would be cal- position (particularly ≥1 order of magni-
culated as follows: tude greater than expected)
12.8 mL sample For the sniff test, it is important that you know a
C f 27.9% glucose little about the product you are analyzing, if possible.
62.8 mL solution A
If the “sniff test” suggests an error, carefully reexamine
on A 5 mL solution B
17 mL solutio how you set up the problem and how the calculation

10 mL solution B 35 mL solution C was performed.
1.38% glucose
In this example, we see that both the units and 3.9 PRACTICE PROBLEMS
the intermediate solution names cancel out.
However, it would be very easy to accidentally (Note: Answers to problems are in the last section of the
reverse one of the dilution factors. For example, laboratory manual.)
if we accidentally reversed the DF for step A → B:
The following example problems demonstrate
12.8 mL sample real-world calculations involving dilutions and
C f 27.9% glucose concentrations:
62.8 mL solution A
on B 5 mL solution B
10 mL solutio 1. You randomly select a cup of applesauce (con-
taining 113 g applesauce) from the processing
17 mL solution A 35 mL solution C line. You extract 10.3 g of the applesauce 3X
0.478% glucose with 50 mL ethyl acetate, pool the extracts, and
The units still cancel out, but you would get an dilute to 250 mL with ethyl acetate. You evapo-
incorrect answer. However, by keeping track of rate 25 mL of the extract to dryness under a
the assigned names of each solution, you would stream of N2 and redissolve the residue to 5 mL
quickly see that this step’s DF is reversed because with methanol. GC analysis of the methanol
the intermediate solution names do not cancel indicates a methoxyfenozide concentration of
out. Therefore, this is an easy way to quickly 0.00334 μg/mL. What is the concentration of
catch and correct errors in dilution calculations. methoxyfenozide in the applesauce (μg/g)?
What is the total amount of methoxyfenozide
in the applesauce cup (μg)?
2. You are given a stock solution of (-)-epicate-
3.8.3 “Sniff Test” chin (EC, MW = 290.26 g/mol) in water
Finally, use the “sniff test” on your calculations to (0.94 mg/mL). You prepare a series of solu-
detect any obvious errors. For example: tions by serial dilution as follows: (1) diluting
0.5 mL of the stock solution to 10 mL with
1. If the procedure is an overall dilution, then the water (solution A), (2) diluting 1.5 mL of solu-
final concentration should be lower than the tion A with 4 mL water (solution B), and (3)
initial concentration. diluting 3 mL solution B with 9 mL water
(solution C). What is the concentration of solu- standard curve described in Problem 4, the stan-
tion C (mg/mL and μM)? What is the overall dards cover the range of 0–0.5 mg/mL. Suppose
DF? How many “fold” has the stock solution you get a sample of a new type of juice into the
been diluted to solution C? QA lab and you have no idea how to dilute the
3. You are performing a spectrophotometric assay juice for analysis. Scientific literature suggests
for riboflavin using a commercial kit. The kit that this juice can have anthocyanin concentra-
comes with 2 mL of a solution of riboflavin tions of anywhere from 750 to 3000 μg/
(1.45 mg/mL). The instructions tell you to make a mL. Based on this information, design a dilu-
stock solution by diluting the riboflavin with tion scheme that will likely yield diluted juice
25 mL water. Then, the instructions tell you to samples within the range of the standard curve.
make a set of standard solutions by diluting 6. You are analyzing Ca content of milk using
100 μL of the stock solution with 0.5 mL (A), atomic absorption spectroscopy (AAS). This
0.75 mL (B), 1 mL (C), 2 mL (D), and 5 mL (E) analysis requires dry ashing to isolate the min-
water. What is the riboflavin concentration (mg/ erals from a sample (dry ashing is essentially a
mL) in the stock solution and the five standards? concentration and extraction step: the sample is
4. You are using a colorimetric method to measure incinerated to remove all organic mineral and
anthocyanin pigments in raspberry juice. The leave only the minerals). You dry ash a 2.8 mL
method requires a total sample volume of sample of milk, dissolve the ash in 12 mL 1 N
2 mL. You have a stock solution of 160 g/L HCl, and dilute the solution to 50 mL with 1 N
anthocyanins, and you need solutions of 0, 0.1, HCl. You further dilute 7 mL of this solution by
0.2, 0.3, and 0.5 mg/mL. Design a dilution adding 13 mL 1 N HCl. You then analyze 0.5 mL
scheme employing commonly available volu- sample and 0.5 mL of standards (10–100 ppm
metric flasks, volumetric pipettes, and/or an Ca) by AAS. You find that the diluted sample
adjustable pipettor (but do not use any volumes contains 28.2 ppm Ca. What is the Ca content of
less than 0.2 mL, and the final volume of each the undiluted milk in ppm?
solution must be at least 2 mL) to obtain the 7. You are measuring caffeine (194.2 g/mol) in
desired concentrations. drinks by high-performance liquid chromatog-
5. As discussed previously, samples often need to raphy (HPLC). You prepare standard solutions
be diluted or concentrated in order to obtain of 0–100 μM caffeine. Your method calls for
analyte concentrations in the range of the stan- dilution of the sample to 250 mL with water
dard curve. Ideally, the sample concentration prior to analysis. You know that a particular
would be near the middle of the standard curve drink contains 170 mg caffeine per 400 mL. How
values. This can be challenging, because you many mL should be diluted to 250 mL total to
often do not know the approximate sample obtain a concentration in the middle of your
concentration prior to the analysis. For the standard curve?
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4
Chapter
Statistics for Food

Analysis
Andrew P. Neilson ( ) • Sean F. O’Keefe *

Blacksburg, VA, USA
4.1 Introduction 4.8 Practical Considerations

4.2 Population Distributions 4.8.1 Sample Size
4.3 Z-Scores 4.8.2 Confidence
4.4 Sample Distributions 4.8.3 What Test to Use?
4.5 Confidence Intervals 4.9 Practice Problems
4.6 t-Scores 4.10 Terms and Symbols
4.7 t-Tests
4.1 INTRODUCTION 0.5
This chapter is a review of basic statistics and will 0.4

demonstrate how statistics is used in the context of
food analysis. It is meant to be a “survival guide” for 0.3
Proportion
reference. Students taking a food analysis course
should have already taken an undergraduate statistics
0.2
course or be taking one concurrently. A foundation for
this chapter is Chap. 4, Evaluation of Analytical Data,
in the Food Analysis textbook. The learning objectives 0.1
for this chapter are to be able to
0.0
1. Calculate a mean, standard deviation, Z-score,
Value
and t-score for a sample dataset.
2. Determine whether a population is significantly
4.1 Example normal distribution
different from a given value using a one-sample
t-test. figure
3. Calculate a confidence interval for a sample
mean using t-scores.
10
4. Determine if two populations are significantly
different using a two-sample t-test.
8
A significant component of a typical food analysis
Number of values
course and the use of food analysis in a professional set-

6
ting (industry, research, regulatory/government) is
evaluation of analytical data. In food analysis labora-
tory experiments, you first collect data (values), or you 4
are given data to work with (i.e., for laboratory exer-
cises, homework, exams, etc.). Then, you evaluate and 2
manipulate the data, answering questions such as these:
1. Are the values significantly different from a 0
1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
desired value or not?
sodium
2. Are two values significantly different from each (mg/500 mL)
other or not?
4.2 Example population histogram
In your food analysis course, you will use statis- figure
tics to solve problems typically encountered in the
food industry, research, and regulatory or government
scenarios. Data analysis concepts will be used in this that the y-axis of a histogram could be the absolute
chapter. number of population members with a given value, the
proportion (fraction or decimal) of population members
with a given value, or the percentage of population
members with a given value. Some populations are not
4.2 POPULATION DISTRIBUTIONS normally distributed, but that is a topic beyond the
scope of this chapter. For the purposes of this chapter,
The distribution of a given value in a population refers we will assume a normal distribution. Normal popula-
to how many members of a population have each value tion distributions are defined by two parameters
of the parameter measured. You can examine distribu- (Fig. 4.3):
tions by plotting a histogram, which is a graph with
parameter values on the x-axis and number of members 1. Population mean (μ): center of the plot
of the population having that value of the parameter 2. Population standard deviation (σ): a measure
(i.e., the “frequency” of each value) on the y-axis. of the spread of the plot
Suppose there were 100 bottles of water in a population
and you knew the sodium content of all of them. The For the example sodium data, you can plot the
histogram of this population might look like Fig. 4.1. histogram of the data with the population mean (μ)
Many populations are “normally distributed” (also and population standard deviation (σ). If you define
known as normal population). Normal populations sodium content as (x), you can calculate the mean and
have histograms that look roughly like Fig. 4.2. Note standard deviation of x:
www.dbooks.org
Chapter 4 • Statistics for Food Analysis 51
population mean µ – σ = 2.291 µ = 2.783 µ + σ = 3.275

(µ)
σ = 0.492 σ = 0.492
population standard
10
deviation (σ)
X=µ−σ
# of population members
X=µ+σ
8
Number of values
6
2
X values
Shape of a normal population 0

4.3 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0
figure sodium
(mg/500 mL)
4.4 Example population histogram with mean
Population mean of x x
xi figure
and standard deviation
n
x1 x2 xn 1 xn
(4.1)
n
1.0
2 σ = 0.5
x x
population members
Standard deviation of x x
i
(4.2) 0.8
n
Proportion of
0.6 σ=1
Population mean x 2.783 standard deviation
σ=2
2
0.4
x
xi 2.783 0.492
100
0.2
You can use these values to calculate the center and
spread of the data, as shown in Fig. 4.4, for example, 0.0
sodium data. Normal distributions can have differ- Value
ent shapes but are still always defined by N(μ, σ). The
notation “N(μ, σ)” indicates a normally distributed 4.5 Normal populations with the same mean but
(N) population with center μ and standard deviation figure
distinct standard deviations
σ. The shape of the curve is dictated by the standard
deviation. Figure 4.5 shows three populations, each
Assume you randomly sample four values from
with the same mean but different standard devia-
the 100 water bottle population, and 1.9, 2.7, 2.9, and
tions. For a normally distributed population, if you
2.9 mg/500 mL were selected. As seen in Fig. 4.7,
randomly pick (or sample) a member of the popu-
three of the randomly chosen values were close to
lation, the randomly selected member can have any
the mean, and one value was relatively far from the
value in the population. Since most of the population
mean. Remember, any of the 100 values could have
is clustered around the mean, the randomly selected
been selected at random. However, the values clos-
value is most likely to be close to the mean. The fur-
est to the mean have the highest probability of being
ther a value is from the mean, the less often it occurs
chosen.
in the population and the less likely it is to be ran-
domly selected from the population. If we know μ
and σ, we can predict the probability that any given
4.3 Z-SCORES
value will be selected at random from the popula-
tion, as shown in Fig. 4.6. Normal distributions have
Each normal population has a different μ, σ. However,
1. 50 % of values > μ and 50 % < μ this is inconvenient for statistical calculations. To make
2. 68, 95, and 99.7 % of values within ±1, 2, and 3 normal distributions easier to work with, you can
standard deviations (σ) of μ transform, or “standardize,” all normally distributed
68% within ±1σ οf µ

95% within ±2σ οf µ (34% on either side)
50% < µ 50% > µ (47.5% on either side)
99.7% within ±3σ οf µ
(49.85% on either side)
–3σ –2σ –1σ µ +1σ +2σ +3σ –3σ –2σ –1σ µ +1σ +2σ +3σ
X values X values
4.6 Densities of normal distributions

figure
µ=2.783 Z-score is the number of standard deviations that X

is away from μ. The more standard deviations (i.e.,
X=1.9 X=2.7 X=2.9 (twice)
multiples of σ or 1) that x (or Z) is from μ (or 0), the
10
less likely that value is to be randomly selected from
the population. You can calculate a Z-score for any x
8 value.
Number of values
6
Example C1 For the sodium data, calculate the
Z-scores for a hypothetical x value close to the
4 mean (2.45) and one that is far from the mean (3.8).
x 2.45 2.783 0.333
2 x 2.45, Z 0.677
0.492 0.492
0 x 3.8 2.783 1.017

1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 x 3.8 , Z 2.067
sodium 0.492 0.492
(mg/500 mL)
Therefore, for X ~ N(2.783, 0.492), x values of 2.45
4.7 Random values from the example distribution and 3.8 are 0.68 standard deviations below and 2.07
figure standard deviations above the mean, respectively.
populations by converting the population value (x) to The same distributions that apply to X ~ N(μ, σ)
a standardized variable, called Z: also apply to the Z ~ N(1, 0):
x 1. 0 % of Z values are > 0 and 50 % are < 0.
(4.3) z
2. 68, 95, and 99.7 % of Z values are within ±1, 2,
The variable x is normally distributed with mean and 3 of 0.
μ and standard deviation σ [x ~ N(μ, σ)]. The distribu-
tion of the Z-scores is Z ~ N(0, 1), i.e., “the standard Some other important properties of normal
normal distribution” (Fig. 4.8): populations:
1. The center of the Z-distribution is μ = 0. 1. The population curve = 100 % of population

2. The spread of the Z-distribution is defined by values
σ = 1. 2. The area under the population curve = 100 %
(percent) or 1 (fraction)
Each x value in a population has a correspond- 3. The % area under the distribution curve between
ing Z-score. For any x value, the corresponding any two points = probability of randomly selecting
www.dbooks.org
X~N(µ, σ) Z~N(0, 1)
1σ 1
standardize by converting x
values to Z-scores
–1σ µ +1σ –1 0 1
X values Z values
4.8 Transformation of x values to Z-scores

figure
Z-table value Example C2 Calculate the probability of observ-

=P(Z < Zobs) ing sodium values of less than 2.783 mg/500 mL
Zobs (the mean) and less than 3.5 mg/500 mL.
Calculate Z-scores for both:
x 2.783 2.783
When x 2.783 , z
0.492
0
0
0.492
x 3.5 2.783 0.717

When x 3.5, z 1.4
0.492 0.492
Z < Zobs
Find table values for those Z-scores:
4.9 Meaning of Z-table values
P(Z < 0) = table value for Z = 0 → 0.50 (50 %)
figure
P(Z < 1.46) = table value for Z = 1.46 → 0.9279
(92.79 %)
For the sodium population (normally distributed,
a value in that range from the population (see μ = 2.783, σ = 0.492), the probability of observing
below) sodium values < 2.783 and 3.5 is 50 % and 92.79 %,
respectively.
Based on these properties of normal populations,
statisticians have developed a “Z-table” (www.nor- Standardization to Z-scores eliminates the need
maltable.com), which contains the probability (P) of for a different Z-score table for each population.
getting a Z-score smaller than the observed Z-score if Although the Z-table contains P values of getting a
Z ~ N(1, 0) (Fig. 4.9). To use the Z-table: Z-score smaller than the observed Z-score, you can
1. Find your observed x value. calculate the P value of getting a Z-score larger than
2. Calculate a Z-score (Zobs) from the x value. the observed Z-score. The sum of the area = 1 (100 %).
3. Find the corresponding table value = P(Z < Zobs) Thus, the P value of getting a Z-score smaller than the
if Z ~ N(1, 0) = P(x < xobs) if x ~ N(μ, σ). observed Z-score plus the P value of getting a Z-score
larger than the observed Z-score equals 100 %:
For ease of use, the Z-table is organized with the
P Z Zobs P Z Zobs 1 100% (4.4)
first two digits of Z on the left and the third digit (sec-
ond decimal place: 0.00, 0.01, 0.02, etc.) across the top. Therefore:
Therefore, to find P(Z < 1.08), you would find the row
with 1.0 on the left, and then go across that row to the P Z Zobs 1 P Z Zobs (4.5)
0.08 column. The steps listed above can be applied to The full expression then becomes:
analysis of the sodium data.
P Z Zobs P Z Zobs P Z Zobs 1100% (4.6)
P(Zobs 1 < Z < Zobs 2)=(Z-table value for Zobs 1) - (Z-table value for Zobs 2)
P(Z > Zobs )=1-(Z-table value for Zobs)
P(Z < Zobs )=Z-table value for Zobs
P(Z < Zobs 2)=Z-table value for Zobs
P(Z > Zobs 1)=1- (Z-table value for Zobs 1)
Zobs 2
Zobs
Zobs 1
Z < Zobs Z > Zobs 4 . 11 The probability of observing Z between two

figure
Zobs values
4 . 10 The probability of observing Z smaller than

figure
Zobs
P Z1 Z Z2 P Z Z1 P Z Z2 (4.7)
This equation covers all possible Z-scores. Thus, the P

Example C4 Calculate the probability of observ-
value of getting a Z-score larger than the observed
ing sodium values between 1.9 and 3.1 mg/500 mL.
Z-score is simply 1− P value of getting a Z-score larger
than the observed Z-score. Therefore, to find the P Calculate the Z-scores:
value of getting a Z-score larger than the observed
Z-score (Fig. 4.10): x − m 1.9 − 2.783 −0.883
When x = 1.9, z = = = = −1.79
1. From the x value, calculate a Z-score (Zobs). s 0.492 0.492
2. From Zobs, find table value = P(Z < Zobs).
x 3.1 2.783 0.317
3. From P(Z < Zobs), calculate P(Z > Zobs) = 1 − table When x 3.1, z 0.64
value for Zobs = P(X > Xobs) if X ~ N(μ, σ). 0.492 0.492
Find the table values for the Z-scores:

Example C3 Calculate the probability of observ-
ing sodium values of greater than 2.9 mg/500 mL, P(Z < 0.64) = table value for Z = 0.64 → 0.7389
again following the steps listed previously. P(Z < 0.64) = table value for Z = −1.79 → 0.0376
Calculate the Z-score: Calculate the area between the two Z-scores:
x 2.9 2.783 0.117 P Z1 Z Z2 P Z Z2 P Z Z1

When x 2.9, z 0.24
0.492 0.492 0.7389 0.0376 0.7013 70.13%
Find the table value for the Z-score: So, for the sodium population which is normally
P(Z < 0.24) = table value for Z = 0.24 → 0.5948 (59.48 %). distributed with μ = 2.783 and σ = 0.492, the proba-
bility of observing a sodium values between 1.9
Convert to P(Z > Zobs) by subtracting from 1:
and 3.1 mg/500 mL is 70.13 %.
Z Zobs 1 P Z Zobs 1 0.5948
0.4052 40.52%
4.4 SAMPLE DISTRIBUTIONS
So, for the sodium population, normally distrib-
uted with μ = 2.783 and σ = 0.492, the probability of You almost never know the actual population values.
observing a sodium value > 2.9 is 40.52 %. This is due to several factors. First, the population is
often too large to sample all members. Second, sam-
How do you calculate the probability of getting an pling often means that the product is no longer avail-
x value (and corresponding Z-score) between two x able for use. You typically sample a few members (n)
values (with corresponding Z-scores)? The probability and estimate population parameters from sample
that a Z-score lies between two given Z-scores is sim- parameters. Population parameters are as follows:
ply the difference between the two table values for
P(Z < Zobs) (i.e., subtract the smaller table value from Population x mean x (4.8)
larger value) (Fig. 4.11):
www.dbooks.org
Population x standard deviation x (4.9) sx

SD of x distribution = s x− =
n
Sample parameters are as follows: Therefore as n ↑, s x− ↓
Sample size = n (4.10)
Everything you learned about Z-scores for pop-
Mean of x distribution x x (4.11) ulation values applies to sample mean values. You
can transform the sample mean distribution to the
x Z-distribution:
SD of x distribution x (4.12)
n
Sample parameters are related to population
x ~N , Z ~ N 0 , 1 (4.13)
n
parameters:
You can transform an observed sample mean into the
1. Sample mean ≈ population mean corresponding Z-score:
2. Sample standard deviation < population stan-
dard deviation x (4.14)
Z

This implies that sampling can be used to estimate n
population parameters. You can use the Z-table to calculate the probability of
getting a sample mean smaller or greater than an
Example D1 Determine the mean and standard observed mean or between two observed means (same
deviation of the sample mean distribution if you math as before).
sample five or ten members of the population.
Example D2 How likely would it be to have a
For both means: sample mean of greater than 3.0 mg/500 mL if you
x x 2.783 sampled four bottles?
Transform the x– into a Z-score:
The standard deviations of the sample means:
x 3 2.783 0.217
Z 0.88
0.492 0.492 0.246
x x for n 5, x x 0.220
n n 5 n 4
0.492 Find the table value for the Z-score (area to the left
for n 10 , x x 0.156
n 10 of Zobs):
P(Z < 0.88) = table value for Z = 0.88: 0.8106
You can see that: Find P(Z > Zobs):
1. The sample mean standard deviation < popula- P Z 0.88 1 P Z 0.88 1 0.8106 0.1894
tion standard deviation.
18.94 %
2. Larger n size decreases sample mean standard
deviation. So, for the sodium data, the probability of sam-
pling n = 4 bottles and getting a mean sodium con-
Sampling from a normal population results in a tent of >3.0 mg/500 mL is 18.9 %.
normally distributed population of all possible sample
means (x–). The population distribution refers to the
individual population values of x. The sample mean 4.5 CONFIDENCE INTERVALS
distribution refers to the values of sample means (x–)
generated by sampling x. Because the sample mean is How confident are you that the observed sample mean
an average of the population members: is close to the actual population mean? And how close?
1. The average sample mean is the same as the You can calculate the probability of getting a sample
average population member. mean as extreme as the one you observe, if you assume
2. The sample mean is less widely distributed the population is normally distributed and the sample
than the population (outliers in the population mean equals the population mean.
get diluted by taking a sample mean).
Procedure
The larger the sample size (n), the tighter (SD) the 1. Calculate Z-score for x–
sample mean distribution becomes: 2. Find area between + Z and − Z = P(Z < │Zobs│) = C
C
For the sodium population which is N(2.783,
α=1–C 0.492), there is an 82.3 % chance that you would
observe a sample mean within 1.35 standard devi-

ations (−1.35 < Z < +1.35) on either side of the mean
if you sampled three bottles from the population.
α/2=(1–C)/2 α/2=(1–C)/2
Now, find α:
1 C 1 0.823 0.177 17.7 %
0.492), there is a 17.7 % chance that you would
observe a sample mean > 1.35 standard deviations
Probability of getting a sample mean as (Z < −1.35 or Z > +1.35, i.e., Z > │1.35│) on either
4.12 side of the mean if you sampled three bottles from
figure
extreme as the observed mean
the population.
Now, find α/2:
The area (C) is the probability of getting a sample
mean as extreme as the one you observed (Fig. 4.12). 1 C 1 0.823
0.0885 8.85 %
You can calculate the probability of getting a sample 2 2 2
mean more extreme than the one you observed (α) on
either end, or getting a sample mean more extreme on
0.492), there is an 8.85 % chance that you would
a single end (α/2).
observe a sample mean > 1.35 standard deviations
C P Zobs Z Zobs (Z > +1.35) above the mean if you sampled three
bottles from the population.
P Z Zobs P Z Zobs (4.15)
1C (4.16) Usually you do not know the actual population

mean (μ) or standard deviation (σ). You sample and
1C calculate sample mean (observed x–) and sample
(4.17) standard deviation (sample SD = σx–) to estimate these
2 2
parameters (observed x– ≈ μx– = μx and SD ≈ σx– =
σx/√n). It is highly unlikely that the observed sample
Example E1 Suppose you sample n = 3 bottles and mean is exactly equal to the population mean. You can
get a mean of 2.4 mg/500 mL. Calculate the proba- calculate a range (i.e., an “interval”) around our
bilities of getting a sample mean this extreme, a observed sample that is likely to cover the true popu-
sample mean smaller than 1.8 mg/500 mL, and a lation mean with a given level of statistical confidence.
sample mean larger than 1.8 mg/500 mL. The confidence interval (CI) gives a margin of error
around our sample mean:
Calculate the Z-score for the observed population
mean: CI :x ± margin of error (4.18)
x 2.4 2.783 0.383 Suppose that you want to generate a CI that is within
Zobs 1.35 a specified number of standard deviations (i.e., num-
0.492 0.284
ber of Z-scores) from the mean. Using the Z-score
n 3
formula:
Find the + Z and − Z values:
x s
Z = −1.35, so you are interested in the
Z

Z x
n
range between Z = 1.35 and − 1.35 n
Find C = P(−Zobs < Z < +Zobs) from the table: Since it could be on either side of the mean, change to:
C P Zobs Z Zobs
Z x
P Z Zobs P Z Zobs n
Now, you have to choose how wide the sample margin of
C P Z Zobs P Z Zobs error should be. You do this by selecting the number of
table value Zobs table value Zobs standard deviations you desire to be within on each side,
which gives a maximum absolute value of Z. You could
C 0.9115 0.0885 0.823 82.3% decide that you want our CI to cover the true mean with
www.dbooks.org
1. Determine the desired C.

P(Z < Z1-α/2)=1–α/2=1–(1–C)/2=(1+C)/2 2. Calculate α/2.
3. Calculate 1−α/2.
4. Use this value to find Z1− α/2.
Z1-α/2 5. Calculate the CI.
P(Z > Z1-α/2)=α/2 6. Get from C → α/2 → 1 − α/2 → Z1 − α/2.
7. Plug these values into the formula and calculate
the CI.
Typically, you use 90, 95, or 99 % confidence in
food analysis. We can choose any desired level of con-
fidence that we want. However
4.13 Relationship between C, α/2, and Z-table Example E2 Suppose you sample n = 5 cans of soda,
figure
values and the mean caffeine content is 150 mg/can. You
know that the population has a standard deviation
(σ) of 15 mg caffeine/can. What is the 95 % confi-
a given percent confidence (C). This means that the prob- dence interval for the mean caffeine content per can?
ability that the CI does not cover the true mean would be
1 − C (which was defined previously as α): 1 C 1 0.95 0.05
Calculate α/2: 0.025
2 2 2 2
1 C C 1
Calculate 1 − α/2:
Then, you would find the “critical” Z-scores where:
P Z Z 1 1 0.025 0.975
1 2
1. P (Zcrit < Z < Zcrit) = C (the desired confidence 2
level)
2. P(Z > │Zcrit│) = α = 1 − C (the probability that the 1 − α/2 = P(Z < Z1−α/2) = table value for Z1−α/2…
CI does not cover the true mean) use this value to find Z1−α/2:
Table value 0.975 Z 1.96
1
The easiest way to do this is to find α/2 and then find 2
the corresponding Z-score:
Calculate the CI: x Z x
First, determine α/2 from the desired confidence (C): 1
2 n
1 C 1 0.823 15 mg / can
0.0885 8.85% 150 mg / can 1.96 x
2 2 2 5
150mg / can 13.1 mg / can
α/2 is an area to the right of the Zcrit needed to deter-
mine the area to the left of Zcrit (aka Z1−α/2), i.e.,
Lower and upper limits = 150 mg/can ± 13.1mg/can
P(Z < Z1−α/2) (Fig. 4.13):
= 136.9 and 163.1 mg / can
1C 1 C
P Z Zcrit P Z Z 1 1 Therefore, you estimate that the true population
1 2 2 2
2 mean is somewhere between 136.9 and 163.1 mg
caffeine/can with 95 % confidence.
P(Z < Z1−α/2) is the table value for Z1−α/2. Use this to find
Z1−α/2.
1. The more confidence you want, the broader the
Now you can calculate the “margin of error” for the CI: interval gets.
2. The less confidence you are willing to accept,

Margin of error : Z x (4.19) the narrower the range gets.
1
2 n
The confidence interval, with confidence level C, is: Example E3 For the soda data, calculate the 90 %
CI of the sample mean.

CI : x margin of error CI : x Z x (4.20) 1 C 1 0.90 0.10
1
2 n Calculate α/2: 0.05
2 2 2 2
Do not get flustered by the statistics details. You
Calculate 1 − α/2: 1 1 0.05 0.95
just need to be able to do the following: 2
in a table (www.normaltable.com), similar to Z. One

1 − α/2 = P(Z < Z1−α/2) = table value for Z1−α/2.
major difference between t and Z is that the t is based
Use this value to find Z1−α/2: on a value called degrees of freedom (df). This value
is based on the sample size:
Table value 0.95 Z 1.645
1
2 df n 1 (4.24)
SD Therefore, for each sample size value (n), there is a
Calculate the CI: x Z x
1
2 n unique t-score. Note that the t-table is divided into col-
umns for each α/2 values and, within those columns,
15 mg / can for each df value. Also, note that the t-table gives the
150 mg / can 1.645 x
5 area to the right of t (Fig. 4.14), whereas the Z-table
150 mg / can 11.0 mg / can gives the area to the left of Z. Although this is confusing
at first, it actually makes t easier to use for CIs than Z.
Lower and upper limits = 150 mg / can ± 11.0 mg / can First, determine α/2 from the desired confidence (C):
= 139.0 and 161.0 mg / can 1C

Therefore, you estimate that the true population 2 2
mean is between 139.0 and 161.0 mg/can with This is the table value for t. From the table value, find
90 % confidence. You see that our interval is tighter, tα/2, df = n – 1. Then, use this value to calculate the CI:
but you are therefore less confident that it contains
SD
the true population mean. CI : x t x (4.25)
2
, df n 1 n
In a food analysis course, t-scores (as opposed to
4.6 t-SCORES Z-scores) are used almost exclusively, and they have
practical use in the food industry. Because t is more
Usually you do not have large enough n and do not conservative for Z, the same confidence level will give
know σ, which are requirements for using the a wider interval if calculated using t.
Z-distribution. Therefore, statisticians developed the
t-score:
Example F1 Suppose that you sample n = 7 energy
x (4.21) bars and obtain a mean total carbohydrate content
t
SD of 47.2 % with a sample standard deviation of
n 3.22 %. Calculate the 99 % confidence interval.
Recall that the sample standard deviation (SD) is cal- Use the t-distribution because n is relatively small.
culated as follows:
1 C 1 0.99 0.01
Determine α/2: 0.005
2 2 2 2 2 2
SD n
x i x
or SD n 1
x i x
(4.22 and Therefore, you would look in the 0.005 column on
n n 1 4.23) the t-table.
You should use the “n − 1” formula for SD when

upper tail probability = P(t>tcritical)
n < 25–30. The t-score is similar to the Z-score. However,
there are some critical differences. The t-score:
1. Uses the SD of the sample (known) instead of
the population σ (usually unknown) tcritical
2. Is more conservative than Z-distribution (for a
given mean and SD, t is larger than Z)
3. Is typically only presented for a few selected
values of α/2 (typically 0.1, 0.05, 0.025, 0.01, and
0.005, which correspond to the commonly used
confidence values of 80, 90, 95, 98, and 99 %,
respectively)
4. Is only listed in positive values, not negative values t>tcritical
Typically, you should use the t-distribution (and 4.14 Interpretation of t-table values
sample SD) for n < 25–30, and the Z-distribution and figure
sample SD for n > 25–30. The t-distribution is presented
www.dbooks.org
The Example F2 demonstrates the utility of a CI. It is

Then, find tα/2, df = n – 1: df n 1 6 – 1 5 a range of values that have a specified likelihood of con-
taining the true population mean, whereas the point
Therefore, within the 0.005 column on the t-table,
estimate has a very small likelihood of being the true
you will look at the row for df = 5:
mean. You may wonder what use a CI is if it is a range of
values. However, you can often get very “tight” CIs (i.e.,
0.005 and df 5 t0.005 , df 5 4.032
2 a narrow range that is useful) by random sampling of a
Calculate the CI: population and choosing an appropriate level of confi-
SD 3.22% dence. As discussed in the textbook Chap 4, you can use
CI : x ± ta x → 47.2% ± 4.032 x
, df = n −1 n 7 the concept of a CI to determine how large the n size
2
should be to obtain a CI of a specific width. In addition
→ 47.2% ± 4.91
to CIs, another technique that is often used to express
SD 3.22% the observed sample mean as a range of values is to cal-
CI : x ± ta x → 47.2% ± 4.032 x
2
, df = n −1 n 7 culate the sample mean ± SD or the mean ± SEM. The
SEM, or standard error of the mean, is simply:
→ 47.2% ± 4.91
SD
Upper limit 47.2% 4.91% 52.1% SEM = (4.26)
n
Lower limit 47.2 % 4.91% 42.3%
You will notice that the SEM term appears in the
Therefore, you estimate that the true population mean t-test and CI formulae Eqs. 4.21 and 4.25. When you
is between 42.3 and 52.5 % with 99 % confidence. express the observed sample mean ± SD or SEM, you
are not giving a probability-based estimate (that would
The take-home message is that a sample mean is a require the t-term multiplier used for t-tests and CIs)
single point estimate that is probably not exactly cor- but rather simply presenting the data as an estimate of
rect. Therefore, a single point estimate is almost use- the mean based on the sample variability. The range
less in food analysis because it is almost always calculated based on SEM will always be narrower, and
incorrect. So, why even sample and test if it is almost hence less conservative, than the t-test and CI. This
always wrong? may not be true for the range calculated based on SD,
depending on the sample size. For the cheese powder
data (Example F2), you could express the estimate as:
Example F2 Suppose your company’s specifica-
tion says that the cheese powder to be used in your Mean SD 16.8 % 0.3% 16.5 17.1 %
products must contain 17.0 % protein by weight. 0.3%
You measure n = 10 bags of the cheese powder and Mean SEM 16.8 % 16.8 % 0.0949
10
calculate a mean of 16.8 % by weight and a SD of
0.3 %. The observed sample mean does not equal 16.705 16.895 %
the specification value (and it is unlikely to ever
fall exactly on 17.0 %). Do you reject the lot because Therefore, based on these ranges, we would accept the
it is not exactly 17.0 %? Do you keep sampling raw material based on mean ± SD but reject it based on
until the observed mean is 17.0 %? However, sup- mean ± SEM. Therefore, it is very important to know
pose your company specifies that the raw ingredi- how to use your analytical data once it is obtained.
ent must meet the specification, from n = 10 What are the specific rules you will use to evaluate
sampled units, using a 95 % CI. data and make decisions? Typically, these are specified
by company lab quality assurance/quality control
This CI is: manuals, industry standards, or regulatory agencies.
SD 0.3%
x ± ta x → 16.8% ± t0.025, df = 4 x
2
, df = n −1 n 10 4.7 t-TESTS
0.3%
→ 16.8% ± 2.776 x Sometimes you need to determine if the observed
10
sample mean indicates that the population is the same,
CI : 16.8% 0.263% or 16.537 % 17.063% or different from, a chosen value. You can use a proce-
dure called a t-test if you have the sample mean (x–),
Therefore, although the point estimate does not standard deviation (SD), sample sizes (n), and a
equal the specification value, you are 95 % confi- desired population mean (μ) that you want to compare
dent that the true mean lies somewhere between the same mean to. First, determine α/2 from the
16.537 % and 17.063 %, which contains the specifi- desired confidence (C) and the df value, as before:
cation value (17.05). Therefore, based on this, you
would accept the raw material. 1C
and df n 1
2 2
From these values, find the table value tα/2, df=n – 1. This
is the “critical value” for a t-test, labeled as “tcritical.” dence. If │tobs│ had been > tα/2, df=n − 1 (3.499), you
Then, calculate the observed t-score (“tobs”) for the would say that you have evidence the sample mean
sample mean, SD and n: was significantly different from 35 mg/capsule.
Note here that you are concerned with the absolute
x value of tobs (│tobs│) vs. tcritical. Given your chosen
t
SD confidence, you have a tcritical value of 3.499, so as
n long as your │tobs│ is <3.499 you can conclude that
the observed sample mean is not significantly dif-
Then, the tcritical and tobs values are compared.
ferent from the chosen μ value. Therefore, tobs could
be anywhere on the interval (−3.499, +3.499) and
If tobs t sample mean is significantly still be considered to be not significantly different
, df n 1
2 from the label value. So, the observed mean could
different from with confidence C
be above or below the chosen μ, as long as it is not
“too far” in either direction.
If tobs t sample mean is not significantly
, df n 1
2
different from with confidence C Confidence intervals and t-tests provide the same
information for a chosen confidence level. Either proce-
A few notes are worth mentioning: dure may be used to compare the sample mean and
chosen μ value. Sometimes you need to determine if the
1. The more confidence you want that you have the
means of two samples are “different” (as opposed to
correct answer, the larger tcritcal becomes, and the
one sample and a chosen μ). Sample means are “point
larger tobs must be to provide evidence that the
estimates.” Just because they are not exactly the same
sample mean is significantly different from μ.
does not mean that the populations are significantly dif-
2. The absolute value of tobs is compared vs. tcritical,
ferent. You need a statistical basis for determining if the
as the table only lists positive t-scores.
sample means are far enough apart such that you can
3. You select μ to compare to the sample mean.
say they are different with some level of confidence (or
You typically do not know the population
not). Given two sample means (x–1 and x–2), standard
mean, but you may have a “target value” that
deviations (SD1 and SD2), and sample sizes (n1 and n2),
you are trying to reach or think the population
first we decide on a confidence level (C), and we calcu-
should have, so you use that as μ.
late α/2 as before. Then, we determine df. For two sam-
ple means, df is calculated as follows:
Example G1 Suppose that your company makes a
df n1 n2 2 (4.27)
multivitamin with a label value of 35 mg vitamin E/
capsule. Per FDA requirements, the label value needs Then you find the critical t-value as before, using the
to be within a certain amount of the actual value. You α/2 and df values:
sample eight capsules and measure the vitamin E
t
content. The observed sample mean is 31.7 mg/cap- , df n1 n2 2
2
sule, and the sample standard deviation is 3.1 mg/
capsule. Can you say that the sample mean includes Once you have tcritical, you calculate tobs as follows:
the label value with 99 % confidence?
x1 x2 (4.28)
Determine α/2 and df: tobs
1 1
1 C 1 0.99 0.01 sp
2
0.005 and n
1 n 2
2 2 2 2
df n 1 8 1 7 This formula differs from the one-sample t-test in that
both n1 and n2 are used, and you employ a value
From these values, find the tcritical value (tα/2, df=n – 1): known as the pooled variance (sp2) instead of the sam-
t0.005 , df =7 = 3.499 ple SD. The pooled variance is a weighted average of
two sample SD values. The pooled variance is calcu-
Calculate tobs from the sample data:
lated as follows:
x 31.7 35
tobs 3.01 and tobs 3.01 n1 1 SD12 n2 1 SD 22 (4.29)
SD 3.1 Pooled variance sp2
n 8 n1 n2 2
Since│tobs│(3.01) < tα/2, df=n − 1 (3.499), you can say Variance is simply standard deviation squared.
the sample mean is not significantly different from Therefore, although you will not use this value, it may
the label value (35 mg/capsule) with 99 % confi- be useful to understand that the “pooled standard
www.dbooks.org
deviation” (sp), or the weighted average of the two

sample SD values, is simply the square root of the Decision:
pooled variance: tobs 2.23 and
t 1.796 tobs t
Pooledstandard deviation = pooled variance , df n1 n2 2 , df n1 n2 2
2 2
= s 2p =
( n1 − 1) SD12 + ( n2 − 1) SD22 (4.30) Thus, Lines 1 and 2 produce sauces with signifi-
n1 + n2 − 2 cantly different acidities (with 90 % confidence).
Once you have obtained the tcritical and tobs values, you
compare them as for the one-sample t-test:
4.8 PRACTICAL CONSIDERATIONS
tobs t means are significantly different
, df n1 n2 2
2 with specified confidence C Here are some practical considerations to help you use
statistics in a food analysis course and in a career in
which decisions are based on analytical data:
tobs t means are not significantly different
, df n1 n2 2
2 with specified confidence C 4.8.1 Sample Size
1. A larger sample size enables more accurate
approximation of the true population values.
Example G2 Suppose your company is concerned 2. Larger sample sizes decrease the sample SD.
that two production lines are producing spaghetti 3. Use t for n < 25–30; use Z for n > 25–30.
sauces with different acid contents. You measure 4. Sample size determines df, which determines
the titratable acidity in samples from both lines tcritical for a specified α/2 value.
(Line 1, 2.1; 2.0; 2.1; 2.2; 2.3; and 2.4 %; Line 2, 2.7; 5. Sampling more units is often useful but not
2.3; 2.2; 2.2; 2.4; 2.6; and 2.5 %). Do the two produc- always practical or cost-effective.
tion lines appear to be producing significantly dif- 6. A “happy medium” needs to be reached that
ferent acidity levels with 90 % confidence? provides “adequate” approximation of the mean
First, calculate the n, x–, and SD value for each sample: with a fixed amount of confidence. This is done by
Line 1: n = 6, x–=2.183 and SD = 0.147 calculating the minimum needed sample size (this
Line 2: n = 7, x–=2.4, and SD = 0.195 is covered in the textbook chapter on sampling).
What is the confidence level (C): 90 % or 0.90? 4.8.2 Confidence

1 C 1 0.90 0.1 1. Confidence values are chosen based on the
Next, calculate α/2: 0.05 acceptable risk to consumers (safety, poor qual-
2 2 2 2
ity, etc.), the company (quality, profit margin,
For the t-table, df: df n1 n2 2 6 7 2 11 formulation accuracy, etc.), or government reg-
Find the critical t-value on the table: ulatory agencies (e.g., labeling accuracy).
2. Confidence determines α/2, which determines
t t0.05 , 11 1.796
, df n1 n2 2 tcritical within a specified df value.
2
3. The more confidence is required (or desired):
Calculate a pooled variance (sp2):
(a) The wider a CI will be.
s 2 n 1 SD12 n2 1 SD 22
1
(b) The larger tcritical will be.
p
n1 n2 2 (c) The larger tobs must be to be > tcritical.
2 2 (d) The larger the n size must be to show

6 1 0.147
7 1 0.195 significance.
67 2
0.1083 0.2286 4. Increasing desired confidence increases the sta-
0.03063
11 tistical burden required to shown significant
Calculate tobs: differences; 90–99 % confidence is typically
used (95 % is fairly standard).
x1 x2 2.183 2.4 5. Increasing the desired confidence is not always
tobs
1 1 1 1 practical or cost-effective.
sp 2 0.03063
n1 n2 6 7
4.8.3 What Test to Use?
0.2217
2.23 Table 4.1 gives “rules of thumb” for what types of tests
0.09737
to use for various scenarios:
4.1
table Examples of when specific statistical tools should be used for food analysis
Question Test/calculation Examples

Is an observed sample mean One-sample Is the actual value in agreement with the label value?
statistically similar to or t -test Does the permitted level differ from the allowed level?
different from a chosen value? Does the raw material meet company specifications?
Is the composition of the finished product acceptable based on company
specifications?
Is a pipette delivering the desired volume?
Is a packaging machine filling packages to the desired level?
What is the actual population One-sample CI What is the actual concentration of a compound of interest?
value, based on the sample?
Based on samples from two Two-sample Are two lots different?
populations, are these t -test (μ = 0) Are two lines, plants, etc. producing products with different composition?
populations different? Is your product the same or different from a competitor’s product?
4.9 PRACTICE PROBLEMS Normal (N) A population distribution whose shape is

defined by a mean and standard
(Note: Answers are at end of laboratory manual.) deviation.
1. As QA manager for a canned soup manufacturer, Pooled variance (sp2) A measure of variability for two
you need to make sure that chicken noodle soup has individual samples, incorporating both
the sodium content indicated on the label (343 mg/ the sample sizes and sample standard
cup). You sample six cans of soup and measure the deviations of both.
sodium content: 322.8, 320.7, 339.1, 340.9, 319.2, and Population All individuals of interest.
324.4 mg/cup. What is the mean of the observa- Population mean (μx) The average value of all the mem-
tions (mg/cup)? What is the standard deviation of bers of a population.
the observations (mg/cup)? Calculate the 96 % con- Population standard deviation (σ) The average differ-
fidence interval for the true population mean, and ence between the value of each popula-
determine both the upper and lower limits of the tion member and the population mean.
confidence interval. Determine if the sample mean Probability (P) Statistical likelihood of an event or state
provides strong enough evidence that the popula- occurring by chance alone.
tion is “out of spec” with 99 % confidence. Sample Selected members of a population, from which
2. You perform moisture analysis on sweetened con- inferences are made about the entire
densed milk using a forced-draft oven. The follow- population.
ing data are obtained. Lot A: 86.7, 86.2, 87.9, 86.3, Sample mean (μx–) The average value of all the mem-
and 87.8 % solids; Lot B: 89.1, 88.9, 89.3, 88.8, and bers of a sample.
89.0 %. Determine if the lots are statistically different Sample standard deviation (SD, σx–) The average differ-
with 95 % confidence ence between the value of each sample
member and the sample mean.
Single point estimate A single value (mean or single
data point) used to predict information
4.10 TERMS AND SYMBOLS
about a population.
Standard deviation (σ) The average difference between
Confidence (C) Statistical probability of being correct
the value of each population member and
based on chance alone.
the population mean.
Confidence interval (CI) A range of values (based on a
Standard error of the mean (SEM) A measure of variabil-
point estimate plus a statistically deter-
ity of a sample, incorporating the sample
mined margin of error) used to predict
standard deviation and the sample size.
information about a population.
T-score (t) A normalized value describing how many
Degrees of freedom (df) The number of values that is free
standard deviations a value is from the
to vary independently.
mean but more conservative than the
Distribution All values in a population and the relative
Z-score.
or absolute occurrence of each value.
Z-score (Z) A normalized value describing how many
Histogram A plot of all values in a population and the
standard deviations a value is from the
relative occurrence of each value.
mean.
Mean (μ) Average value.
www.dbooks.org
2
part
Laboratory Exercises
5
chapter
Nutrition Labeling
Using a Computer
Program
Lloyd E. Metzger (*)
Department of Dairy Science, South Dakota State University,
Brookings, SD, USA
Ann M. Roland
Owl Software,
Columbia, MO, USA
www.dbooks.org
5.1 Introduction 5.3 Adding New Ingredients to a Formula
5.1.1 Background and Determining How They Influence
5.1.2 Reading Assignment The Nutrition Label
5.1.3 Objective 5.3.1 Procedure
5.1.4 Materials 5.4 An Example of Reverse Engineering in
5.1.5 Notes Product Development
5.2 Preparing Nutrition 5.4.1 Procedure
Labels for Sample Yogurt 5.5 Questions
Formulas
5.2.1 Procedure
Chapter 5 • Nutrition Labeling Using a Computer Program 67
5.1 INTRODUCTION heading. It is possible that the TechWizard™ program has

been updated since the publication of this laboratory manual
5.1.1 Background and any changes in the procedures described below will also
be found on this web page.
The 1990 Nutrition Labeling and Education Act man- *Install the software prior to the laboratory ses-
dated nutritional labeling of most foods. As a result, a sion to ensure that it works properly with your PC.
large portion of food analysis is performed for nutri-
tional labeling purposes. Interpretation of these regu-
lations (21 CFR 101.9) and the appropriate usage of
rounding rules, available nutrient content claims, ref-
5.2 PREPARING NUTRITION LABELS
erence amounts, and serving size can be challenging.
FOR SAMPLE YOGURT FORMULAS
Additionally, during the product development pro-
5.2.1 Procedure
cess, the effect of formulation changes on the nutrition
label may be important. As an example, a small change 1. Start the TechWizard™ program. (Start the pro-
in the amount of an ingredient may determine if a gram, the message appears “TechWizard success-
product can be labeled low fat. As a result, the ability fully opened the necessary files…” and click
to immediately approximate how a formulation OK. Click on the Enter Program button. Select User
change will impact the nutritional label can be valu- Name “Administrator” and click OK.)
able. In some cases, the opposite situation may occur 2. Enter the Nutrition Labeling section (From the
and a concept called reverse engineering is used. In Labeling tab select Labeling Section. You should see
reverse engineering the information from the nutrition “TechWizard™ – Nutrition Info & Labeling with
label is used to determine a formula for the product. New Formats” in the top left corner of the screen.)
Caution must be used during reverse engineering. In 3. Enter the ingredients for Formula #1 listed in
most cases, only an approximate formula can be Table 5.1. (Click on the Add Ingredients button,
obtained and additional information not provided by then select an ingredient from the ingredient list
the nutrition label may be necessary. window, and click the Add button. Repeat for each
The use of nutrient databases and computer pro- ingredient. After all the ingredients have been added,
grams designed for preparing and analyzing nutrition close the window.)
labels can be valuable in all of the situations described 4. Enter the amounts of the ingredients. (Enter the
earlier. In this laboratory you will use a computer pro- percentage of each ingredient for Formula #1 in the
gram to prepare a nutrition label from a product for- % (wt/wt) column. Selecting the Sort button above
mula, determine how changes in the formula affect the that column and then Sort Descending will sort the
nutrition label, and observe an example of reverse ingredients by the % (wt/wt) in the formula.)
engineering. 5. Enter the serving size (common household unit
and the equivalent metric quantity) and number
5.1.2 Reading Assignment of servings. (First click on the Serving Size button
under Common Household unit, enter 2/3 in the
Metzger, L.E. and Nielsen, S.S., 2017. Nutrition label-
window, click on OK, and select cup from the units
ing. Ch. 3, in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
drop down list; next click on the Serving Size button
Springer, New York.
under Equivalent Metric Quantity, enter 170 g in
the window, click on OK, and select g from the units
5.1.3 Objective
Prepare a nutrition label for a yogurt formula, deter- 5.1
mine how formulation changes will affect the nutrition table Sample yogurt formulas
label, and observe an example of reverse engineering.
Formula #1 (%) Formula #2 (%)
5.1.4 Materials
Milk (3.7 % fat) 38.201 48.201
TechWizard™ – Formulation and Nutrition Labeling Milk (skim), no vit. A 35.706 25.706
Software for Microsoft Excel for Windows. add
Owl Software. TechWizard™ Software Manual, Milk (skim), condensed 12.888 12.888
Columbia, MO. www.owlsoft.com (35 % TS)
Sweetener, sugar 11.905 11.905
(Liquid)
5.1.5 Notes Modified starch 0.800 0.800
Instructions on how to receive and install the software Stabilizer, gelatin, 0.500 0.500
used for this laboratory are located online at www. dry powder,
unsweetened
owlsoft.com, under the Academic link located in the
www.dbooks.org
68 L.E. Metzger and A.M. Roland
drop down list; and finally click on the Number of tion can now be pasted into your Microsoft Word
Servings button, enter 1 in the window, and click on Document).
OK.) 11. Return to the Nutrition Info & Labeling section
6. Save Formula #1. (Click on the File tab, then of the TechWizard™ program. (Go to the
Formula, and then Save Formula As. Type Student TechWizard™ program and click on the Return
Name_Formula Name in the Formula Name win- button.)
dow, click the Save button, then OK, and close the 12. Repeat the process for Formula #2 (Table 5.1).
window.) (Repeat Steps 4–10)
7. View the nutrition label and select label
options. (Click on the View Label button, click on
the Label Options button, and select Standard; 5.3 ADDING NEW INGREDIENTS
select Protein -- Show ADV for voluntary nutri- TO A FORMULA AND
ents since yogurt is high in protein, and enter 1 DETERMINING HOW THEY
for the PDCAAS; when you have finished selecting INFLUENCE THE NUTRITION
the label options, select Apply, then OK, and then LABEL
Close to view the label.)
8. Copy and paste the nutrition label for formula #1 Sometimes it may be necessary to add additional ingre-
to a Microsoft Word file (Click on the Copy button dients to a formula. As an example, let us say you
on the Labeling tab, select Standard Label, Enter 1 for decided to add an additional source of calcium to yogurt
the new label format. Click OK. When prompted that formula #1. After contacting several suppliers, you
the label has been copied, click OK. Open a Word doc- decided to add Fieldgate Natural Dairy Calcium 1000, a
ument, and type the name of the formula and paste the calcium phosphate product produced by First District
label. Return to the TechWizard™ program.) Association (Litchfield, MN), to the yogurt formula. This
9. Edit the ingredient declarations list (Click on product is a natural dairy-based whey mineral concen-
the View/Edit Declaration button, and click Yes trate and contains 25 % calcium. You want to determine
when asked – Do you wish to generate a simple how much Dairy Calcium 1000 you need to add to have
formula declaration using individual ingredi- 50 and 100 % of the Daily Value (DV) of calcium in one
ent declarations? – Each ingredient used in the serving of your yogurt. The composition of the Dairy
formula can be selected in the top window and the Calcium 1000 you will add is shown in Table 5.2.
ingredient declaration can be edited in the middle
window.)
5.3.1 Procedure
*Note the rules for ingredient declaration are found in 1. Add the name of the new ingredient to the data-
21 CFR 101.4. base. (From the Edit Ingredient File tab, select Edit
Current File; Click on Edit Ingredient File tab again
10. Copy and paste the ingredient declaration for in the Ingredients group; Click Add, type the ingre-
Formula #1 to a Microsoft Word file. (Select Edit dient name “Dairy Calcium 100_Student Name” in
Formula declaration, and click the Copy button then the Enter Ingredient Name box, and click Add.
OK to the pop-up and close the window. The declara- Answer Yes to the question, and click OK. Close the
window.)
2. Enter the new ingredient composition
(Table 5.2). (Select the ingredient name in the col-
umn named “Ingredients and Properties.” Click
5.2 Composition of Fieldgate Natural Dairy Edit Selected in the Ingredients group under the Edit
table Calcium 1000 (First District Association) Ingredient File tab, the row will turn blue, and enter
the amount of each component/nutrient in the appro-
Component Amount priate column.)
Ash (%) 75 3. Edit the ingredient declaration (which will
Calcium (mg/100 g) 25,000 appear on the ingredient list) for the new ingre-
Calories (cal/100 g) 40 dient. (Type “Whey mineral concentrate” in the col-
Lactose (%) 10 umn named “Default, Spec TEXT, zzzIngredient
Phosphorus (mg/100 g) 13,000 Declaration.”)
Protein (g/100 g) 4.0 4. Save the changes to the ingredient file. (Click
Sugars (g/100 g) 10 on the Finish Edit button, and answer Yes to the
Total carbohydrates (g/100 g) 10 question. Select Close Ingred. File from the Edit
Total solids (%) 92
Ingredient File tab.)
Water (%) 8.0
Chapter 5 • Nutrition Labeling Using a Computer Program 69
5. Open food analysis formula #1 in the Formula Labeling Section, click the File tab then Open
Development Section of the program. (Click Formula, and select your formula “Student Name_
the Formula Dev and Batching tab, and then Formula #1 added calcium 50 % DV,” and click
Formula Dev. From the File menu, select Open Open then click on No for the first question, and
Formula and select your Formula #1 file “Student click on Yes for the remaining questions).
Name_Formula Name,” click on the Open button, 11. Make sure you have the correct serving size
then click on No for the first question, and click on information (see Sect. 5.2, Step 5).
Yes for the remaining questions). 12. View and print the nutrition label for the new
6. Add the new Dairy Calcium 1000 ingredient to formula with 50 % of the calcium DV. Follow
your food analysis formula #1. (Click on the Add the instructions described in Sect. 5.2, Steps
Ingredients button, then select your ingredient 6–10.
“Dairy Calcium 1000_Student Name” from the 13. Produce a formula and label that has 100 % of
ingredient list, click on the Add button, and close the the calcium DV. (Repeat Steps 8–12 except using
window.) the calculated amount of calcium required to meet
7. Calculate the amount of calcium (mg/100g) 100 % of the calcium DV. You will have to perform
required to meet 50 and 100 % of the DV (see this calculation yourself following the example in
example below): Step 7.)
DV for calcium
Calcium required
serving size 5.4 AN EXAMPLE OF REVERSE
100 g % of DV required ENGINEERING IN PRODUCT
DEVELOPMENT
 1300 mg  5.4.1 Procedure
Calcium required for 50% of the DV   

 170 g  In this example the program will automatically go
 100 g  0.50 through the reverse engineering process. (Start the
example by selecting Cultured Products Automated
 mg  Examples from the Help menu and clicking on example #4.
Calcium required for 50% of the DV  382  
 During this example you proceed to the next step by clicking
 100 g  on the Next button.) Each step is described below:
1. The information from the nutrition label for the
8. Enter the amount of calcium required in the for-
product you want to reverse engineer is entered
mula and restrict all ingredients in the formula
into the program. (Comment: In this example serv-
except skim milk and Dairy Calcium 1000. (Find
ing size, calories, calories from fat, total fat, satu-
Calcium in the Property column and enter 382 in the
rated fat, cholesterol, sodium, total carbohydrate,
Min and Max columns for Calcium. This lets the
sugars, protein, vitamin A, vitamin C, calcium, and
program know that you want to have 382 mg of cal-
iron are entered.)
cium per 100 g. In both the Min and Max columns of
2. The minimum and maximum levels of each
the Ingredients, enter 38.201 for Milk (3.7 % fat);
nutrient are calculated on a 100-g basis.
12.888 for Milk (skim), condensed (35 % TS);
(Comment: The program uses the rounding rules to
11.905 for Swtnr, sugar (Liquid); 0.800 for
determine the possible range of each nutrient on a
Modified starch; and 0.500 for Stblzr, Gelatin, dry
100-g basis.)
powder, unsweetened. This lets the program
3. The information about nutrient minimum and
adjust the amount of skim milk and Dairy Calcium
maximums is transferred into the Formula
1000 (calcium phosphate) and keeps the amount of all
Development section of the program. (Comment:
the other ingredients constant. Click on the Formulate
The program has now converted nutrient range
button, click OK.). Record the % wt/wt for Dairy
information into a form it can use during the formu-
Calcium to answer Question 2 in Sect. 5.5.
lation process.)
9. Save the modified formula with your name.
4. Ingredients used in the formula are then
(Click on the File tab, then Formula, and then Save
selected based on the ingredient declaration
Formula As. Type “Student Name_Formula #1
statement on the nutrition label. (Comment:
added calcium 50 % DV” in the Formula Name
Selecting the right ingredients can be difficult and
window, click the Save button, then OK to close the
an extensive understanding of the ingredient decla-
window.)
ration rules is necessary. Additionally some of the
10. Open the new formula into the nutrition label-
required ingredients may not be in the database and
ing section. (Click on the Labeling tab, select
will need to be added.)
www.dbooks.org
70 L.E. Metzger and A.M. Roland
5.3 formula (see 21 CFR 101.13, Nutrient content

table
claims – general principles – and 21 CFR 101-54-
Recipe for chocolate chip cookies 101.67, Specific requirements for nutrient con-
Ingredients Amount Grams tent claims or summary tables from the FDA or
other sources based on these CFR sections)?
Wheat flour, white, 2.25 cups 281.25 2. How much Dairy Calcium 1000 did you have to
all-purpose, enriched, add to the yogurt formula to have 50 and 100 %
unbleached
of the DV of calcium in the formula?
Swtnr, sugar, granulated 0.75 cup
Candies, semisweet 100 g 100
3. If Dairy Calcium 1000 costs $2.50/lb and you
chocolate are going to have 100 % of the DV for calcium in
Sugars, brown 0.75 cup (packed) your yogurt, how much extra will you have to
Butter (salted) 1 cup 227 charge for a serving of yogurt to cover the cost
Egg, whole, fresh 2 units 112 of this ingredient?
Salt 0.75 tsp 4. Assume you added enough Dairy Calcium 1000
Source for ingredients: TechWizard, USDA ingredients as
to claim 100 % of the DV of calcium, would you
source expect the added calcium to cause any texture
Conversion data source: USDA web page changes in the yogurt?
5. Make a nutrition label using the chocolate chip
cookie recipe and other information in Table 5.3
5. Restrictions on the amount of each ingredient in (for simplicity assume 0 % loss of water during
the formula are imposed whenever possible. baking, number of servings = 1, serving size = 30
(Comment: This is a critical step that requires g). Conversion factors to get the weight of sugars
knowledge about the typical levels of ingredients and salt can be found in the US Department of
used in the product. Additionally, based on the order Agriculture Nutrient Database for Standard
of ingredients in the ingredient declaration, approxi- Reference website: http://ndb.nal.usda.gov/.
mate ranges can be determined. In this example, the When adding ingredients to the formula, you may
amount of modified starch is limited to 0.80 %, the have to select the tab “USDA Ingredients as Source”
amount of gelatin is limited to 0.50 %, and the in the Add Ingredients window to find the ingredient
amount of culture is limited to 0.002 %.) you need. Enter the grams of each ingredient in the
6. The program calculates an approximate for- %(wt/wt) column. Select Normalize from the Labeling
mula. (Comment: The program uses the information tab to convert the values so the total is 100. The ingre-
on nutrient ranges and composition of the ingredi- dient, Candies, semisweet chocolate, does not
ents to calculate the amount of each ingredient in the have Added Sugars data. You can enter a tempo-
formula.) rary value to the ingredient information to calcu-
7. The program compares the nutrition informa- late the Nutrition Facts. Click on Composition from
tion for the developed formula to the original the Labeling tab. In the row for that ingredient, scroll
nutrition label. (Comment: This information is to the right until you find the column for Added
viewed in the Nutrition Label to Formula Spec sec- Sugars with the units g. Enter the value you determine
tion of the program accessed by selecting Reverse for Added Sugars (hint: look at the Sugars value) and
Engineering Section from the Reverse Engineering click the Enter key. The value will be used for the
tab. Click the X to close the Cultured Products label but is not saved in the ingredient file.
Example window when finished.)
RESOURCE MATERIALS
5.5 QUESTIONS
Metzger LE, Nielsen SS (2017) Nutrition labeling. Ch. 3. In:
1. Based on the labels you produced for yogurt Nielsen SS edn. Food analysis, 5th edn. Springer, New York
Formula #1 and Formula #2 in Sect. 5.2, what Owl Software TechWizard™ Manual, Columbia, MO. www.
nutrient content claims could be made for each owlsoft.com
6
Chapter
Accuracy and Precision

Assessment
S.Suzanne Nielsen (*)
Charles E. Carpenter
Department of Nutrition, Dietetics and Food Sciences, Utah State University,
Logan, UT, USA
6.1 Introduction 6.1.6 Equipment

6.1.1 Background 6.1.7 Notes
6.1.2 Reading Assignment 6.2 Procedure
6.1.3 Objective 6.3 Data and Calculations
6.1.4 Principle of Method 6.4 Questions
6.1.5 Supplies
www.dbooks.org
72 S.S. Nielsen and C.E. Carpenter
6.1 INTRODUCTION It is generally required that reported values mini-

mally include the mean, a measure of precision, and
6.1.1 Background the number of replicates (Smith 2017). The number of
significant figures used to report the mean reflects the
Volumetric glassware, mechanical pipettes, and bal-
inherent uncertainty of the value, and it needs to be
ances are used in many analytical laboratories. If the
justified based on the largest uncertainty in making
basic skills in the use of this glassware and equipment
the measurements of relative precision of the assay.
are mastered, laboratory exercises are easier and more
The mean value is often expressed as part of a confi-
enjoyable, and the results obtained are more accurate
dence interval (CI) to indicate the range within which
and precise. Measures of accuracy and precision can be
the true mean is expected to be found. Comparison of
calculated based on data generated, given the glassware
the mean value or the CI to a standard or true value is
and equipment used, to evaluate both the skill of the
the first approximation of accuracy. A procedure or
user and the reliability of the instrument and glassware.
instrument is generally not deemed inaccurate if the
Determining mass using an analytical balance is
CI overlaps the standard value. Additionally, a CI that
the most basic measurement made in an analytical
is considerably greater than the readability indicates
laboratory. Determining and comparing mass is fun-
that the technician’s technique needs improvement. In
damental to assays such as moisture and fat determi-
the case of testing the accuracy of an analytical balance
nation. Accurately weighing reagents is the first step
with a standard weight, if the CI does not include the
in preparing solutions for use in various assays.
standard weight value, it would suggest that either the
Accuracy and precision of the analytical balance are
balance needs calibration or that the standard weight
better than for any other instruments commonly used to
is not as originally issued. Accuracy is sometimes esti-
make analytical measurements, provided the balance is
mated by the relative error (%Erel) between the mean
properly calibrated and the laboratory personnel use analysis value and the true value. However, %Erel only
proper technique. With proper calibration and technique, reflects tendencies and in practice is often calculated
accuracy and precision are limited only by the readability even when there is no statistical justification that the
of the balance. Repeatedly weighing a standard weight mean and true value differ. Also, note that there is no
can yield valuable information about the calibration of consideration of the number of replicates in the calcu-
the balance and the technician’s technique. lation of %Erel, suggesting that the number of repli-
Once the performance of the analytical balance cates will not affect this estimation of accuracy to any
and the technician using it has been proven accept- large extent. Absolute precision is reflected by the
able, determination of mass can be used to assess the standard deviation (SD), while relative precision is cal-
accuracy and precision of other analytical instruments. culated as the coefficient of variation (CV). Calculations
All analytical laboratories use volumetric glassware of precision are largely independent of the number of
and mechanical pipettes. Mastering their use is neces- replicates, except that more replicates may give a bet-
sary to obtain reliable analytical results. To report ana- ter estimate of the population variance.
lytical results from the laboratory in a scientifically Validation of a procedure or measurement tech-
justifiable manner, it is necessary to understand accu- nique can be performed, at the most basic level, as a
racy and precision. single-trial validation, as is described in this labora-
A procedure or measurement technique is vali- tory that includes estimating the accuracy and preci-
dated by generating numbers that estimate their accu- sion of commonly used laboratory equipment.
racy and precision. This laboratory includes However, for more general acceptance of procedures,
assessment of the accuracy and precision of automatic they are validated by collaborative studies involving
pipettors. An example application is determining the several laboratories. Collaborative evaluations are
accuracy of automatic pipettors in a research or qual- sanctioned by groups such as AOAC International,
ity assurance laboratory, to help assess their reliability AACC International, and the American Oil Chemists’
and determine if repair of the pipettors is necessary. Society (AOCS) (Nielsen 2017). Such collaborative
Laboratory personnel should periodically check the studies are prerequisite to procedures appearing as
pipettors to determine if they accurately dispense the approved methods in manuals published by these
intended volume of water. To do this, water dispensed organizations.
by the pipettor is weighed, and the weight is con-
verted to a volume measurement using the appropri-
6.1.2 Reading Assignment
ate density of water based on the temperature of the
water. If replicated volume data indicate a problem Neilson A.P., Lonergan D.A., and Nielsen S.S. 2017.
with the accuracy and/or precision of the pipettor, Laboratory standard operating procedures. Ch.1, in
repair is necessary before the pipettor can be reliably Food Analysis Laboratory Manual, 3rd ed., Nielsen S.S.
used again. (Ed.), Springer, New York.
Chapter 6 • Accuracy and Precision Assessment 73
Nielsen, S.S. 2017. Introduction to food analysis. (a) Tare a 100-mL beaker, deliver 10 mL of water
Ch. 1, in Food Analysis, 5th ed. S.S, Nielsen (Ed.), from a volumetric pipette into the beaker, and
Springer, New York. record the weight. Repeat this procedure of tar-
Smith, J.S. 2017. Evaluation of analytical data. Ch. ing the beaker, adding 10 mL, and recording the
5, in Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, weight, to get six determinations on the same
New York. pipette. (Note that the total volume will be
60 mL.) (It is not necessary to empty the beaker
6.1.3 Objective after each pipetting.)
(b) Repeat the procedure as outlined in Part 2a but
Familiarize, or refamiliarize, oneself with the use of
use a 20- or 30-mL beaker and a 1.0-mL volu-
balances, mechanical pipettes, and volumetric glass-
metric pipette. Do six determinations.
ware, and assess accuracy and precision of data
generated. 3. Analytical balance and buret.
(a) Repeat the procedure as outlined in Part 2a, but
6.1.4 Principle of Method
use a 100-mL beaker and a 50-mL (or 25-mL)
Proper use of equipment and glassware in analytical buret filled with water, and dispense 10 mL of
tests helps ensure more accurate and precise results. water (i.e., tare a 100 mL beaker, deliver 10 mL
of water from the buret into the beaker, and
6.1.5 Supplies record the weight). (Handle the beaker wearing
gloves, to keep oils from your hands off the bea-
• Beaker, 100 mL
ker.) Repeat this procedure of taring the beaker,
• Beaker, 20 or 30 mL
adding 10 mL, and recording the weight, to get
• Beaker, 250 mL
six determinations on the buret. (Note that the
• Buret, 25 or 50 mL
total volume will be 60 mL.) (It is not necessary
• Erlenmeyer flask, 500 mL
to empty the beaker after each addition.)
• Funnel, approximately 2 cm diameter (to fill buret)
(b) Repeat the procedure as outlined in Part 3a but
• Mechanical pipettor, 1000 μL, with plastic tips
use a 20- or 30-mL beaker and a 1.0-mL volume
• Plastic gloves
from the buret. Do six determinations.
• Ring stand and clamps (to hold buret)
• Rubber bulb or pipette pull-up 4. Analytical balance and mechanical pipette. Repeat
• Standard weight, 50 or 100 g the procedure as outlined in Part 2a but use a 20- or
• Thermometer, to read near room temperature 30-mL beaker and a 1.0-mL mechanical pipette
• Volumetric flask, 100 mL (i.e., tare a 20- or 30-mL beaker, deliver 1 mL of
• 2 Volumetric pipettes, one each of 1 and 10 mL water from a mechanical pipettor into the beaker,
and record the weight). Repeat this procedure of
6.1.6 Equipment taring the beaker, adding 1 mL, and recording the
weight to get six determinations on the same pipet-
• Analytical balance
tor. (Note that the total volume will be 6 mL.) (It is
• Top loading balance
not necessary to empty the beaker after each
pipetting.)
6.1.7 Notes 5. Total content (TC) versus total delivery (TD). Tare
Before or during the laboratory exercise, the instructor a 100-mL volumetric flask on a top loading bal-
is encouraged to discuss the following: (1) difference ance. Fill the flask to the mark with water. Weigh
between dispensing from a volumetric pipette and a the water in the flask. Now tare a 250-mL beaker
graduated pipette and (2) difference between mark- and pour the water from the volumetric flask into
ings on a 10-mL versus a 25- or 50-mL buret. the beaker. Weigh the water delivered from the
volumetric flask.
6. Readability versus accuracy. Zero a top loading
6.2 PROCEDURE balance and weigh a 100-g (or 50-g) standard
weight. Record the observed weight. Use gloves or
(Record data in tables that follow.) finger cots as you handle the standard weight to
keep oils from your hands off the weight. Repeat
1. Obtain ~400 mL deionized distilled (dd) H2O in a
with the same standard weight on at least two
500-mL Erlenmeyer flask for use during this labo-
other top loading balances, recording the observed
ratory session. Check the temperature of the water
weight and the type and model (e.g., Mettler,
with a thermometer.
Sartorius) of balance used.
2. Analytical balance and volumetric pipettes.
www.dbooks.org
6.3 DATA AND CALCULATIONS 6.1

table Viscosity and density of water at various
Calculate the exact volume delivered in Sect. 6.2, Parts temperatures
2–5, using each weight measurement and the known
density of water (see Table 6.1). Using volume data, cal- t
Temp. Density Viscosity Temp. Density Viscosity
culate the following indicators of accuracy and preci- (°C) (g/mL) (cps) (°C) (g/mL) (cps)
sion: mean, standard deviation, coefficient of variation,
20 0.99823 1.002 24 0.99733 0.9111
percent relative error, and 95 % confidence interval. 21 0.99802 0.9779 25 0.99707 0.8904
Use your first three measurements for n = 3 values 22 0.99780 0.9548 26 0.99681 0.8705
requested and all six measurements for n = 6 values. 23 0.99757 0.9325 27 0.99654 0.8513
Data for Sect. 6.2, Parts 2, 3, and 4:

Volumetric pipette Buret Mechanical 6.4 QUESTIONS
pipettor
1 mL 10 mL 1 mL 10 mL 1 mL (Questions refer to parts of Sect. 6.2)
Rep Wt. Vol. Wt. Vol. Wt. Vol. Wt. Vol. Wt. Vol. 1. Theoretically, how are standard deviation, coeffi-
1 cient of variation, mean, percent relative error,
2 and 95 % confidence interval affected by (1) more
3 replicates and (2) a larger size of the measure-
4 ment? Was this evident in looking at the actual
5 results obtained using the volumetric pipettes
6 and the buret, with n = 3 versus n = 6 and with
n=3 1 mL versus 10 mL? (See table below)
Mean – – – – –
SD – – – – –
Theoretical Actual, with results
CV – – – – –
obtained
% Erel – – – – –
CI95% – – – – – More Larger More Larger
replicates mesurement replicates mesurement
n=6
Standard
Mean – – – – –
deviation
SD – – – – –
Coefficient
CV – – – – –
of variation
% Erel – – – – –
Mean
CI95% – – – – –
Percent
relative error
Data for Sect. 6.2, Part 5: 95 %
confidence
Wt. Vol. interval
Water in flask =
Water in beaker =
2. Why are percent relative error and coefficient of
variation used to compare the accuracy and preci-
Data for Sect. 6.2, Part 6: sion, respectively, of the volumes from pipetting/
dispensing 1 and 10 mL with the volumetric pipettes
Type/model Standard and buret in Parts 2 and 3, rather than simply the
Balance of balance weight (g)
mean and standard deviation, respectively?
1 3. Compare and discuss the accuracy and the preci-
2 sion of the volumes from the 1 mL pipetted/dis-
3 pensed using a volumetric pipette, buret, and
Chapter 6 • Accuracy and Precision Assessment 75
mechanical pipettor (Parts 2, 3, and 4). Are these 9. You are considering adopting a new analytical
results consistent with what would be expected? method in your lab to measure the moisture con-
4. If accuracy and precision using the mechanical tent of cereal products, how would you determine
pipettor are less than should be expected, what the precision of the new method and compare it to
could you do to improve its accuracy and the old method? How would you determine (or
precision? estimate) the accuracy of the new method?
5. In a titration experiment using a buret, if you
expect to use much less than a 10-mL volume in
each titration, would you expect your accuracy
RESOURCE MATERIALS
and precision to be better using a 10-mL buret or a
50-mL buret? Why? Neilson AP, Lonergan DA, and Nielsen SS (2017) Laboratory
6. How do your results from Part 5 of this lab differen- standard operating procedures, Ch. 1. In: Food analysis
tiate “to contain” from “to deliver”? Is a volumetric laboratory manual, 3rd ed., Nielsen SS (ed.), Springer,
flask “to content” or “to deliver”? Which is a volu- New York
metric pipette? Nielsen SS (2017) Introduction to food analysis, Ch. 1. In:
7. From your results from Part 6 of this lab, would Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
you now assume that since a balance reads to 0.01 Smith JS (2017) Evaluation of analytical data, Ch. 4. In:
g that it is accurate to 0.01 g? Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
8. What sources of error (human and instrumental)
were evident or possible in Parts 2–4, and how
could these be reduced or eliminated? Explain.
www.dbooks.org
7
chapter
High-Performance
Liquid Chromatography
Stephen T. Talcott
Department of Nutrition and Food Science, Texas A&M University,
College Station, TX, USA
7.1 Introduction 7.2.3 Chemicals

7.1.1 Background 7.2.4 Hazards, Precautions,
7.1.2 Reading Assignment and Waste Disposal
7.2 Determination of Caffeine 7.2.5 Reagents
in Beverages By HPLC 7.2.6 Supplies
7.2.1 Introduction 7.2.7 Equipment
7.2.2 Objective 7.2.8 HPLC Conditions
7.2.9 Procedure 7.3.4 Hazards, Precautions,
7.2.10 Data and Calculations and Waste Disposal
7.2.11 Questions 7.3.5 Reagents
7.3 Solid-Phase Extraction and 7.3.6 Supplies
HPLC Analysis of Anthocyanidins 7.3.7 Equipment
from Fruits and Vegetables 7.3.8 HPLC Conditions
7.3.1 Introduction 7.3.9 Procedure
7.3.2 Objective 7.3.10 Data and Calculations
7.3.3 Chemicals 7.3.11 Questions
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Chapter 7 • High-Performance Liquid Chromatography 79
7.1 INTRODUCTION separation from other beverage components using

reversed-phase HPLC. An isocratic mobile phase gen-
7.1.1 Background erally provides for sufficient separation of the caffeine
from other beverage components. However, separation
High-performance liquid chromatography (HPLC) has and quantitation are much easier for soft drinks than
many applications in food chemistry. Food components for a beverage such as coffee, which has many more
that have been analyzed with HPLC include organic components. Commercially available caffeine can be
acids, vitamins, amino acids, sugars, nitrosamines, cer- used as an external standard to quantitate the caffeine
tain pesticides, metabolites, fatty acids, aflatoxins, pig- in the beverages by peak height or area.
ments, and certain food additives. Unlike gas
chromatography, it is not necessary for the compound
7.2.2 Objective
being analyzed to be volatile. It is necessary, however,
for the compounds to have some solubility in the mobile To determine the caffeine content of soft drinks
phase. It is important that the solubilized samples for by reversed-phase HPLC with ultraviolet (UV)
injection be free from all particulate matter, so centrifu- detection, using peak height and area to determine
gation and filtration are common procedures. Also, concentrations
solid-phase extraction is used commonly in sample
preparation to remove interfering compounds from the 7.2.3 Chemicals
sample matrix prior to HPLC analysis.
Many food-related HPLC analyses utilize reversed- CAS no. Hazards
phase chromatography in which the mobile phase is Acetic acid 64-19-7 Corrosive
relatively polar, such as water, dilute buffer, methanol, (CH3COOH)
or acetonitrile. The stationary phase (column packing) is Caffeine 58-08-2 Harmful
relatively nonpolar, usually silica particles coated with a Methanol, HPLC 67-56-1 Extremely flammable,
C8 or C18 hydrocarbon. As compounds travel through grade (CH3OH) toxic
the column, they partition between the hydrocarbon sta-
tionary phase and the mobile phase. The mobile phase 7.2.4 Hazards, Precautions, and Waste
may be constant during the chromatographic separation Disposal
(i.e., isocratic) or changed stepwise or continuously (i.e.,
gradient). When the compounds elute separated from Adhere to normal laboratory safety procedures. Wear
each other at the end of the column, they must be safety glasses at all times. Methanol waste must be
detected for identification and quantitation. handled as hazardous waste. Other waste likely may
Identification often is accomplished by comparing the be put down the drain using a water rinse, but follow
volume of liquid required to elute a compound from a good laboratory practices outlined by environmental
column (expressed as retention volume or retention health and safety protocols at your institution.
time) to that of standards chromatographed under the
same conditions. Quantitation generally involves com- 7.2.5 Reagents
paring the peak height or area of the sample peak of (** It is recommended that these solutions be prepared
interest with the peak height or area of a standard (at the by the laboratory assistant before class.)
same retention time). The results are usually expressed
in milligrams per gram or milliliters of food sample. • Mobile phase**
Deionized distilled (dd) water: HPLC-grade
7.1.2 Reading Assignment methanol: acetic acid, 65:35:1 (v/v/v), filtered
through a Millipore filtration assembly with 0.45-
Ismail, B.P. 2017. Basic principles of chromatography.
μm nylon membranes and degassed
Ch. 12, in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
Springer, New York. • Caffeine solutions of varying concentration for
Reuhs, B.L. 2017. High performance liquid chro- standard curve**
matography. Ch. 13, in Food Analysis, 5th ed. Prepare a stock solution of 20 mg caffeine/100 mL
S.S. Nielsen (Ed.), Springer, New York. dd water (0.20 mg/mL). Make standard solutions
containing 0.05, 0.10, 0.15, and 0.20 mg/mL, by
combining 2.5, 5.0, 7.5, and 10 mL of stock solution
7.2 DETERMINATION OF CAFFEINE with 7.5, 5.0, 2.5, and 0 mL dd water, respectively.
IN BEVERAGES BY HPLC
7.2.6 Supplies
7.2.1 Introduction (Used by students)
The caffeine content of beverages can be determined
readily by simple filtration of the beverage prior to • Disposable plastic syringe, 3 mL (for filtering sample)
80 S.S. Nielsen and S.T. Talcott
• Hamilton glass HPLC syringe, 25 μL (for injecting 3. With HPLC injector valve in LOAD position,
sample if using manual sample loading) insert syringe needle into the needle port all
• Pasteur pipettes and bulb the way.
• Sample vials for autosampler (if using 4. Gently depress syringe plunger to completely
autosampler) fill the 10-μL injector loop with sample.
• Soft drinks, with caffeine 5. Leaving the syringe in position, simultaneously
• Syringe filter assembly, (Syringe and 0.45 μm turn valve to INJECT position (mobile phase
filters) now pushes sample onto the column) and
• Test tubes, e.g., 13 × 100-mm disposable culture depress chart marker button on the detector (to
tubes (for filtering sample) mark start of run on chart recorder paper).
6. Remove syringe. (Leave valve in the INJECT
7.2.7 Equipment position so that the loop will be continuously
flushed with mobile phase, thereby preventing
cross contamination.)
• HPLC system, with UV–vis detector
7. After caffeine peak has eluted, return valve to
• Membrane filtering and degassing system
LOAD position in preparation for next injection.
8. Repeat Steps 3–7, injecting each caffeine stan-
7.2.8 HPLC Conditions
dard solution in duplicate or triplicate. (Note:
Column Waters μBondapak C18 (Waters, The laboratory assistant can inject all standard
Milford, MA) or equivalent reversed- solutions prior to the laboratory session).
phase column
Guard column Waters Guard-Pak Precolumn Module 7.2.10 Data and Calculations
with C18 Guard-Pak inserts or
equivalent
1. Chromatograms and peak area reports will be
Mobile phase dd H2O: HPLC-grade methanol: acetic printed out for you by the laboratory assistant.
acid, 65:35:1 (v/v/v) (combine Use peak areas of the standards to construct
and then filter and degas) your standard curve. Use the equation of the
Flow rate 1 mL/min line for your standard curve to calculate the
Sample loop size 10 μL concentration of caffeine in your sample. Use
Detector Absorbance at 254 nm or 280 nm the appropriate dilution factor when calcula-
Sensitivity Full-scale absorbance = 0.2 tion the caffeine concentration in your sample.
Chart speed 1 cm/min Complete the tables below with all data.
7.2.9 Procedure Standard curve:

(Instructions are given for manual injection and for Caffeine conc. (mg/ml) Rep Peak area (cm2)
analysis in triplicate)
0.05 1
1. Filter beverage sample. 2
(a) Remove plunger from a plastic 3-mL syringe, 3
and connect syringe filter assembly (with a 0.10 1
membrane in place) to the syringe barrel. 2
(b) Use a Pasteur pipette to transfer a portion of 3
0.15 1
beverage sample to the syringe barrel. Insert
2
and depress syringe plunger to force sam- 3
ple through the membrane filter and into a 0.20 1
small test tube. 2
2. Flush Hamilton HPLC syringe with filtered 3
sample, and then take up 15–20 μL of filtered
sample (try to avoid taking up air bubbles).
Sample: (Complete this table for each type of sample)
Conc. based on area after

Retention Peak area Conc. based on area from considering dilution factor Conc. based on area
Rep time (min) (cm2) std. curve (mg/ml) (mg/mL) (mg/100 mL)
1
2
3
X
SD =
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2. Using the mean values, calculate the concentra- linked organic acids. However, most foods contain
tion of caffeine in your sample expressed in up to six anthocyanin aglycones (without sugar or
terms of milligrams caffeine in a 12-oz. can organic acid substituents, referred to as anthocyani-
(1 mL = 0.0338 oz). dins) that include delphinidin, cyanidin, petunidin,
pelargonidin, peonidin, and malvidin (Fig. 7.1).
7.2.11 Questions Sample preparation for anthocyanin analysis gener-
ally involves solid-phase extraction of these com-
1. Based on the triplicate values and the linearity
pounds from the food matrix followed by acid
of your standard curves, which of the two meth-
hydrolysis to remove sugar and/or organic acid link-
ods used to calculate concentration seemed to
ages. Anthocyanidins are then easily separated by
work best in this case? Is this what you would
reversed-phase HPLC.
have expected, based on the potential sources of
The use of solid-phase extraction (SPE) is a com-
error for each method?
mon chromatographic sample preparation technique
2. Why was it important to filter and degas the
used to remove interfering compounds from a biologi-
mobile phase and the samples?
cal matrix prior to HPLC analysis. This physical
3. How is the “reversed-phase” HPLC used here
extraction technique is similar to an actual separation
different from “normal phase” with regard to
on a reversed-phase HPLC column. Although many
stationary and mobile phases and order of
SPE stationary phases exist, the use of reversed-phase
elution?
C18 is commonly employed for food analysis. On a
4. Mobile phase composition:
relative basis, anthocyanins are less polar than other
(a) If the mobile phase composition was
chemical constituents in fruits and vegetables and will
changed from 65:35:1 (v/v/v) to 75:25:1
readily bind to a reversed-phase C18 SPE cartridge.
(v/v/v) water to methanol to acetic acid,
Other compounds such as sugars, organic acids,
how would the time of elution (expressed
water-soluble vitamins, or metal ions have little or no
as retention time) for caffeine be changed,
affinity to the cartridge. After the removal of these
and why would it be changed?
interferences, anthocyanins can then be efficiently
(b) What if it was changed from 65:35:1 (water:
eluted with alcohol, thus obtaining a semipurified
methanol: acetic acid) to 55:45:1? How
extract for HPLC analysis.
would that change the retention time and
Separation of compounds by HPLC involves the
why?
use of a solid support (column) over which a liquid
mobile phase flows on a continuous basis. Chemical
interactions with an injected sample and the stationary
7.3 SOLID-PHASE EXTRACTION
and mobile phases will influence rates of compound
AND HPLC ANALYSIS
elution from a column. For compounds with similar
OF ANTHOCYANIDINS FROM FRUITS
polarities, the use of mixtures of mobile phases (gradi-
AND VEGETABLES
ent elution) is often employed. Reversed-phase sta-
tionary phases are most common for anthocyanin
7.3.1 Introduction
separations, and are based on column hydrophobicity
Anthocyanins are naturally occurring plant pigments of a silica-based column with varying chain lengths of
known for their diverse colors depending on solution n-alkanes such as C8 or C18. By setting initial chromato-
pH. Analysis for anthocyanins is often difficult due to graphic conditions to elute with a polar (water) mobile
their similar molecular structure and polarity and phase followed by an organic (alcohol) mobile phase,
their diversity of sugar and/or organic acid substitu- anthocyanins will generally elute in order of their
ents. Color intensity is a common means of analyzing polarity.
for anthocyanins since monomeric anthocyanins are You will be analyzing anthocyanins isolated from
colored bright red at low pH values from 1 to 3 (oxo- fruits or vegetables for anthocyanidins (aglycones) fol-
nium or flavylium forms) and are nearly colorless at lowing SPE and acid hydrolysis to remove sugar gly-
higher pH values from 4 to 6 (carbinol or pseudobase cosides. After sugar hydrolysis, samples will be
forms). A pure anthocyanin in solution generally fol- injected into an HPLC for compound separation.
lows Beer’s law; therefore concentration can be esti- Depending on plant source, you will obtain between
mated from an extinction coefficient when an authentic one and six chromatographic peaks representing com-
standard is not available. However, many standards mon anthocyanidins found in edible plants.
are commercially available with cyanidin-3-glucoside
used most often for quantification purposes.
7.3.2 Objective
Red-fleshed fruits and vegetables contain many
different anthocyanin forms due to their diverse Isolate and quantify anthocyanidin concentration
array of esterified sugar substituents and/or acyl- from common fruits and vegetables by reversed-phase
R1
R2
B
HO O
R3
A C
Glu
OH
OH OH OMe OMe OMe

OH OH OH OH OH OH
B B B B B B
OH OH OMe
Dp Cy Pt Pg Pn Mv
7. 1 Anthocyanin structures. Common substitutions on the B-ring include delphinidin (Dp), cyanidin (Cy), petunidin
figure
(Pt), pelargonidin (Pg), peonidin (Pn), and malvidin (Mv)
HPLC with Vis detection, using spectrophotometric [Each mobile phase should be filtered through a
absorbance readings and extinction coefficients of 0.45-μm nylon membrane (Millipore) and degassed
anthocyanidins to determine standard concentrations. while stirring using either a nitrogen sparge, under
vacuum (ca. 20 min/l of solvent), or by sonication.]
7.3.3 Chemicals 7.3.6 Supplies

CAS no. Hazards • Beaker, Pyrex, 500 mL (for boiling water for
hydrolysis)
Hydrochloric acid 7647-01-0 Corrosive • Blender, kitchen scale, for sample homogenization
(HCl)
• Disposable plastic syringe, 3–5 mL (for filtering
Methanol (CH3OH) 67-56-1 Extremely
flammable,
sample)
toxic • Filter paper (Whatman #4) and funnels
o-Phosphoric acid 7664-38-2 Corrosive • Fruit or vegetable that contains anthocyanins (blue-
(H3PO4) berries, grapes, strawberries, red cabbage, black-
berries, cherries, or commercial juices that contain
7.3.4 Hazards, Precautions, and Waste anthocyanins)
Disposal • Hamilton glass HPLC syringe, 25 μl (for injecting
sample)
Adhere to normal laboratory safety procedures. Wear • Reversed-phase C18 cartridge (for SPE, e.g., Waters
safety glasses at all times. Use hydrochloric acid under C18 Sep-Pak, WAT051910)
a fume hood. Methanol waste must be handled as haz- • Syringe filter (0.45-μm PTFE, polytetrafluo-
ardous waste. Other waste likely may be put down the roethylene)
drain using a water rinse, but follow good laboratory • Test tubes, screw cap, with lids (for anthocyanin
practices outlined by your environmental health and hydrolysis)
safety protocols.
7.3.7 Equipment
7.3.5 Reagents
(** It is recommended that these solutions be prepared • Hot plate
by a laboratory assistant before class.) • HPLC system, gradient, with Vis detector (520 nm)
• Membrane filtering and degassing system
• 4 N HCl in water (for anthocyanin hydrolysis)** • Spectrophotometer and cuvettes (1-cm path length)
• 0.01 % HCl in water (for sample extraction)**
• 0.01 % HCl in methanol (for elution from C18 7.3.8 HPLC Conditions
cartridge)**
• Mobile Phase A: 100 % water (pH 2.4 with o-phos- Column Waters Nova-Pak C18 (WAT044375)
phoric acid)** or equivalent reversed-phase column
• Mobile Phase B: 60 % methanol and 40 % water Guard column Waters Guard-Pak Precolumn Module
(pH 2.4 with o-phosphoric acid)** with C18 Guard-Pak inserts
www.dbooks.org
Mobile phase Phase A, 100 % water; Phase B, 60 % 7.3.9.3 Acid Hydrolysis

methanol and 40 % water (both [Note to the instructor: It is recommended that a previ-
adjusted to pH 2.4 with ously extracted sample be acid hydrolyzed before
o-phosphoric acid) class to save time].
Flow rate 1 mL/min
Sample loop size Variable: 10–100 μl 1. Pipette 2 mL of anthocyanins, dissolved in
Detector Visible at 520 nm methanol, into a screw-cap test tube and add an
Gradient Linear ramp. Hold time at 100 % Phase equal volume of aqueous 4 N HCl (final acid
conditions B after 15 min may vary with column concentration = 2 N) for a twofold dilution fac-
length and/or column packing material tor (see calculations below).
2. Under a fume hood, tightly cap the screw-cap
Time (min) % Phase A % Phase B vial and place in boiling water for ca. 90 min.
3. Remove test tubes and cool to room tempera-
0 100 0
ture before opening the vial. Filter an aliquot
5 50 50
10 50 50 through a 0.45-μm PTFE syringe filter for analy-
15 0 100 sis by HPLC.
35 0 100 (end) 4. Inject the filtered extract into the HPLC column
37 100 0 (equilibration) and record peak areas for quantification of each
compound (see Fig. 7.2).
7.3.9 Procedure
7.3.10 Data and Calculations
7.3.9.1 Sample Extraction
(Note to the instructor: Several different commodities Authentic standards for select anthocyanins can be
can be evaluated or the experiment replicated as obtained from several sources and should be used
needed.) according to manufacturer’s suggestions. If using
1. Weigh ca. 10 g of fruit or vegetable containing anthocyanin glycosides, then the acid hydrolysis pro-
anthocyanins (record exact weight) and place in cedure should be conducted prior to HPLC analysis.
blender. Add ca. 50 mL of water containing Some anthocyanin suppliers include: Polyphenols
0.01 % HCl and blend thoroughly (acidified ace- Laboratories, Sandnes, Norway; INDOFINE Chemical
tone, methanol, or ethanol are also suitable sub- Company, Somerville, NJ.
stitutions for water). Fruit juices that contain Cyanidin is a common anthocyanin present in
anthocyanins can be used without further large concentrations in many fruits and vegetables and
preparation. will be used for sample calculations. A standard solu-
2. Filter homogenate through filter paper and col- tion of cyanidin should be prepared in Mobile Phase A
lect aqueous filtrate. (water at pH 2.4) to establish a standard curve. Unless
3. Keep refrigerated until needed. the actual concentration is known from the manufac-
turer, the standard should be quantified by determin-
7.3.9.2 Solid-Phase Extraction ing its absorbance on a spectrophotometer at 520 nm
1. Condition a reversed-phase SPE cartridge by against a blank of the same solvent. Using the molar
first washing with 4 mL of 100 % methanol fol- extinction coefficient for cyanidin (obtained from
lowed by 4 mL of water acidified with 0.01 % manufacturer or expressed as cyanidin-3-glucoside
HCl. equivalents, ε = 29,600 for a 1-M solution and 1-cm
2. Slowly pass 1–2 mL of juice or filtrate (record light path), the concentration is calculated using Beer’s
exact volume) through the SPE cartridge being law, A = εbc, using the following calculation:
careful not to lose visible pigment. Anthocyanins (Absorbance at max ) 1000 MW
will adhere to the SPE support and less polar mg / L Cyanidin
compounds such as sugars, organic acids, and
ascorbic acid will be removed. where:
3. Slowly pass an additional 4 mL of water (acidi- MW ~ 457 g/mol
fied with 0.01 % HCl) through the cartridge to ε ~ 29,600
remove residual water-soluble components. 1. Inject a series of standard concentrations into
Remove residual moisture from the cartridge the HPLC to generate a standard curve. (Note:
by pushing air through the cartridge with an These procedures can be performed by a labo-
empty syringe or by flushing the cartridge with ratory assistant prior to the laboratory session.)
nitrogen gas until dry. 2. Chromatograms and peak area reports will be
4. Elute anthocyanins with 4 mL of 0.01 % HCl in printed out for you by the laboratory assistant.
methanol and collect for subsequent hydrolysis. Use peak areas of the standards to construct
7. 2 Typical 0.040
Pn
figure
reversed-phase
HPLC
Absorbance units
chromato- 0.030 Cy
graph of Mv
anthocyani-
dins (grape) 0.020
Pt
0.010
Dp Pg
0.000
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00

Time (minutes)
Typical Cyanidin standard curve

7. 3
figure
standard curve 1600000
for cyanidin 1400000
1200000
Peak area
1000000
y = 45108x – 6162.8
800000
R 2 = 0.9999
600000
400000
200000
0
0 10 20 30 40
Cyanidin concentration (mg/L)
your standard curve. Use the equation of the dilution factors are calculated based on weight of
line for your standard curve to calculate the fruit/vegetable per volume of extraction solvent (sam-
anthocyanin concentration of your acid hydro- ple weight + solvent volume/sample weight). Single-
lyzed samples (Fig 7.3). Use appropriate dilu- strength fruit juices would have a sample dilution
tion factors. factor of 1.
3. Express relative concentrations of each identifi-
able compound as cyanidin equivalents (mg/L)
based on their peak area (unless commercial 7.3.11 Questions
standards are available for each peak in the
chromatograph). 1. Based on chemical structure, why do anthocy-
anidins elute in their respective order?
2. Predict how each compound would elute from
Peak Peak area Relative concentration (mg/L) a normal-phase column.
1 3. If the retention time of a compound that had
2 absolutely no affinity to the column was
3 1.5 min and the flow rate was 1 mL/min,
4 what is the total volume of mobile phase con-
5 tained in the column, tubing, and pumps? Are
6 you surprised at this number? Why or why
not?
mg / L Cyanidin in unknown 4. What would the chromatograph look like if
Peak area 2 you injected 40 μl of a sample as compared to
Sample dilution factors 20 μl?
Slope of standard curve
5. What would the chromatograph look like if
Mobile Phases A and B were reversed (i.e.,
Peak area multiplied by 2 will compensate for the two- beginning with 100 % Phase B and increasing
fold dilution incurred during acid hydrolysis. Sample Phase A over time)?
www.dbooks.org
RESOURCE MATERIALS Hong V, Wrolstad RE (1990) Use of HPLC separation/photo-

diode array detection for characterization of anthocyanins.
AOAC International (2016) Official methods of analysis, 20th J Agr Food Chem 38:708–715
edn., (On-line) Method 979.08. Benzoate, caffeine, and Markakis P (ed) (1982) Anthocyanins as food colors.
saccharin in soda beverages. AOAC International, Academic Press, New York
Rockville, MD Ismail BP (2017) Basic principles of chromatography. Ch. 12.
Bridle P, Timberlake F (1997) Anthocyanins as natural food In: Nielsen SS (ed) Food analysis, 5th edn. Springer,
colours – selected aspects. Food Chem 58: 103–109 New York
Reuhs BL (2017) High-performance liquid chromatography.
Ch. 13. In: Nielsen SS (ed) Food analysis, 5th edn. Springer,
New York
8
chapter
Gas Chromatography
Michael C. Qian
Department of Food Science and Technology, Oregon State University,
Corvallis, OR, USA
8.1 Introduction 8.2.6 Hazards, Precautions,

8.1.1 Background and Waste Disposal
8.1.2 Reading Assignment 8.2.7 Supplies
8.2.8 Equipment
8.2 Determination of Methanol
8.2.9 Procedure
and Higher Alcohols in Wine by Gas
Chromatography
8.2.11 Questions
8.2.1 Introduction
8.2.2 Objective 8.3 Preparation of Fatty Acid Methyl
8.2.3 Principle of Method Esters (FAMEs) and Determination
8.2.4 Chemicals of Fatty Acid Profile of Oils by Gas
8.2.5 Reagents Chromatography
www.dbooks.org
8.3.1 Introduction 8.3.6 Supplies
8.3.2 Objective 8.3.7 Equipment
8.3.3 Chemicals 8.3.8 Procedure
8.3.4 Reagents and Samples 8.3.9 Data and Calculations
8.3.5 Hazards, Precautions, and Waste 8.3.10 Questions
Disposal
Chapter 8 • Gas Chromatography 89
8.1 INTRODUCTION 8.2 DETERMINATION OF METHANOL

AND HIGHER ALCOHOLS IN WINE
8.1.1 Background BY GAS CHROMATOGRAPHY
Gas chromatography (GC) has many applications in
8.2.1 Introduction
the analysis of food products. GC has been used for
the determination of fatty acids, triglycerides, choles- The quantification of higher alcohols, also known as
terol, gases, water, alcohols, pesticides, flavor com- fusel oils, in wine and distilled spirits is important
pounds, and many more. While GC has been used for because of the potential flavor impact of these com-
other food components such as sugars, oligosaccha- pounds. These higher alcohols include n-propyl alco-
rides, amino acids, peptides, and vitamins, these sub- hol, isobutyl alcohol, and isoamyl alcohol. Some
stances are more suited to analysis by high countries have regulations that specify the maximum
performance liquid chromatography. GC is ideally and/or minimum amounts of total higher alcohols in
suited to the analysis of volatile substances that are certain alcoholic beverages. Table wine typically con-
thermally stable. Substances such as pesticides and tains only low levels of higher alcohols, but dessert
flavor compounds that meet these criteria can be iso- wines contain higher levels, especially if the wine is
lated from a food and directly injected into the GC. For fortified with brandy.
compounds that are thermally unstable, too low in Methanol is produced enzymatically during the
volatility, or yield poor chromatographic separation production of wine. Pectin methyl esterase hydrolyzes
due to polarity, a derivatization step must be done the methyl ester of α-1,4-D-galacturonopyranose. The
before GC analysis. The two parts of the experiment action of this enzyme, which is naturally present in
described here include the analysis of alcohols that grapes and may also be added during vinification, is
requires no derivatization step and the analysis of necessary for proper clarification of the wine. White
fatty acids which requires derivatization. The experi- wines produced in the United States contain less
ments specify the use of capillary columns, but the methanol (4–107 mg/L) when compared with red and
first experiment includes conditions for a packed rosé wines (48–227 mg/L). Methanol has a lower boil-
column. ing point than the higher alcohols (Table 8.1), so it is
more readily volatilized and elutes earlier from a gas
8.1.2 Reading Assignment chromatography (GC) column.
Ellefson, W.C. 2017. Fat analysis. Ch. 17, in Food Methanol and higher alcohols in distilled liquors
Analysis, 5th ed. S.S. Nielsen (ed.), Springer, New York. are readily quantitated by gas chromatography, using
Pike O.A., and O’Keefe, S.F. 2017. Fat characteriza- an internal standard such as benzyl alcohol, 3-penta-
tion. Ch. 23 in Food Analysis, 5th ed. S.S. Nielsen (Ed.), nol, or n-butyl alcohol. The method outlined below is
Springer, New York. similar to AOAC Methods 968.09 and 972.10 [Alcohols
Qian, M.C., Peterson, D.G., and Reineccius, G.A. (Higher) and Ethyl Acetate in Distilled Liquors].
2017. Gas chromatography. Ch. 14, in Food Analysis,
5th ed. S.S. Nielsen (Ed.), Springer, New York. 8.2.2 Objective
Determine the content of methanol, n-propyl alcohol,
and isobutyl alcohol in wine by gas chromatography,
8.1 using benzyl alcohol as the internal standard.
table Alcohol structure and boiling point
Alcohol Structure b.p.(°C)
Gas chromatography uses high temperatures to vola-
Methanol CH3OH 64.5 tilize compounds that are separated as they pass
Ethanol CH3-CH2OH 78.3
through the stationary phase of a column and are
n-Propanol CH3-CH2-CH2OH 97
Isobutyl alcohol CH3-CH-CH2OH 108
detected for quantitation.
(2-methyl-1-propanol) |
CH3 8.2.4 Chemicals
Isoamyl alcohol CH3-CH-CH2CH2OH
(3-methyl-1-butanol) | CAS no. Hazards
CH3
Benzyl alcohol 100-51-6 Harmful
Active amyl alcohol CH3-CH2-CH-CH2OH 128
Ethanol 64-17-5 Highly flammable
(2-methyl- 1-butanol) |
Isobutyl alcohol 78-83-1 Irritant
CH3
Methanol 67-56-1 Extremely flammable
Benzyl alcohol 205
O CH2OH n-Propyl alcohol 71-23-8 Irritant, highly
flammable
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90 S.S. Nielsen and M.C. Qian
8.2.5 Reagents • 6 Volumetric flasks, 100 mL

• 4 Volumetric flasks, 1000 mL
(**It is recommended that these solutions be prepared
by the laboratory assistant before class.)
8.2.8 Equipment
• Ethanol, 16 % (vol/vol) with deionized distilled • Analytical balance
(dd) water, 500 mL** • Distillation unit (heating element to fit 500-mL
• Ethanol, 50 % (vol/vol) with dd water, 3200 mL** round bottom flask; cold water condenser)
• Ethanol, 95 % (vol/vol) with dd water, 100 mL**
• Stock solutions** • Gas chromatography unit:
Prepared with known amounts of ethanol and
fusel alcohols or methanol: Column DB-Wax (30 m, 0.32 nm ID,
0.5-μm film thickness) (Agilent
1. 10.0 g of methanol and 50 % (vol/vol) ethanol to Technologies, Santa Clara,
1000 mL CA) or equivalent (capillary
2. 5.0 g of n-propyl alcohol and 50 % (vol/vol) column) or 80/120
ethanol to 1000 mL Carbopack BAW/5 %
3. 5.0 g of isobutyl alcohol and 50 % (vol/vol) eth- Carbowax 20 M, 6 ft × 1/4 in
anol to 1000 mL OD × 2 mm ID glass column
4. 5.0 g of benzyl alcohol in 95 % (vol/vol) ethanol (packed column)
to 100 mL Injector temperature 200 °C
• Working Standard Solutions** Column temperature 70–170 °C at 5 °C/min
Prepared from stock solutions, to contain different Carrier gas He at 2 mL/min (N2 at 20 mL/min
amounts of each of the fusel alcohols; aliquots of for packed column)
these are used to get standard curves. Prepare Detector Flame ionization
four working standards by combining: Attenuation 8 (for all runs)
1. 0.5 mL of stock solutions 1, 2, and 3 with 4.5 mL ID inner diameter, OD outer diameter, BAW base and acid
washed
of 50 % (vol/vol) ethanol plus 16 % (vol/vol)
ethanol to 100 mL
2. 1.0 mL of stock solutions 1, 2, and 3 with 3 mL of 8.2.9 Procedure
50 % (vol/vol) ethanol plus 16 % (vol/vol) etha- (Instructions are given for single standard and sample
nol to 100 mL analysis, but injections can be replicated.)
3. 1.5 mL of stock solutions 1, 2, and 3 with 1.5 mL
of 50 % (vol/vol) ethanol plus 16 % (vol/vol) 8.2.9.1 Sample Preparation
ethanol to 100 mL 1. Fill a 100-mL volumetric flask to volume with
4. 2.0 mL of stock solutions 1, 2, and 3 with 16 % the wine sample to be analyzed.
(vol/vol) ethanol to 100 mL 2. Pour the wine into a 500-mL round bottom flask
and rinse the volumetric flask several times
(Note: The final concentration of ethanol in each of with dd water to complete the transfer. Add
these working standard solutions is 18 % (vol/vol) additional water if necessary to bring the vol-
ethanol.) ume of sample plus dd water to ca. 150 mL.
3. Distil the sample and recover the distillate in a
8.2.6 Hazards, Precautions, and Waste clean 100-mL volumetric flask. Continue the dis-
Disposal tillation until the 100-mL volumetric is filled to
the mark.
The alcohols are fire hazards; avoid open flames, breath-
4. Add 1.0 mL of the stock benzyl alcohol solution
ing vapors, and contact with skin. Otherwise, adhere to
to 100 mL of each working standard solution
normal laboratory safety procedures. Wear safety
and wine sample to be analyzed.
glasses at all times. Aqueous waste can go down the
drain with a water flush. 8.2.9.2 Analysis of Sample and Working
Standard Solutions
8.2.7 Supplies 1. Inject 1 μL of each sample and working stan-
dard solution in separate runs on the GC col-
(Used by students)
umn (split ratio 1:20) (For packed column, inject
5.0 μL.)
• Mechanical pipettor, 1000 μL, with tips
2. Obtain chromatograms and data from integra-
• Round bottom flask, 500 mL
tion of peaks.
• Syringe (for GC)
8.2.10 Data and Calculations Peak height ratioa

1. Calculate the concentration (mg/L) of metha- n-Propyl Isobutyl
nol, n-propyl alcohol, and isobutyl alcohol in Methanol alcohol alcohol
each of the four Working Standard Solutions Alcohol Conc. Benzyl Benzyl Benzyl
(see sample calculation below). (mg/l) alcohol alcohol alcohol
25
Alcohol concentration (mg/l): 50
75
Working N-Propyl Isobutyl 100
standard Methanol alcohol alcohol 150
1 200
2 Wine sample
3 a
Give individual values and the ratio
4
3. Construct standard curves for methanol, n-pro-
Example calculations: pyl alcohol, and isobutyl alcohol using the peak
Working Standard Solution #1 contains height ratios. All lines can be shown on one
methanol + n-propyl alcohol + isobutyl graph. Determine the equations for the lines.
alcohol, all in ethanol. 4. Calculate the peak ratios for methanol, n-pro-
pyl alcohol, and isobutyl alcohol in the wine
Methanol in Stock Solution #1: sample and their concentrations in mg/L.
10 g methanol 1g 0.01 g
= = 8.2.11 Questions
1000 mL 100 mL mL
1. Explain how this experiment would have differed
Working Standard Solution #1 contains in standard solutions used, measurements taken,
0.5 mL of Stock Solution #1. and standard curves used if you had used exter-
nal standards rather than an internal standard.
=0.5 mL of 0.01 g methanol/mL
2. What are the advantages of using an internal
=0.005 g methanol = 5 mg methanol
standard rather than external standards for this
That 5 mg methanol is contained in 100 application, and what were the appropriate cri-
mL volume. teria to use in selecting the internal standard?
=5 mg/100 mL = 50 mg/1000 mL
= 50 mg methanol/L 8.3 PREPARATION OF FATTY ACID
Repeat procedure for each alcohol in METHYL ESTERS (FAMEs)
each working standard solution. AND DETERMINATION OF FATTY
ACID PROFILE OF OILS BY GAS
2. Calculate the peak height or peak area ratios for CHROMATOGRAPHY
methanol, n-propyl alcohol, and isobutyl alco-
hol, compared to the internal standard, for each 8.3.1 Introduction
of the Working Standard Solutions and the wine
sample. To identify which is the methanol, Information about fatty acid profile on food is important
n-propyl alcohol, and isobutyl peak, see the for nutrition labeling, which involves the measurement
example chromatogram that follows. Note that of not only total fat but also saturated, unsaturated,
data from automatic integration of the peaks and monounsaturated fat. Gas chromatography is an
can be used for these calculations. Report the ideal instrument to determine (qualitatively and
ratios in a table as shown below. Show an exam- quantitatively) fatty acid profile or fatty acid compo-
ple calculation of concentration for each type of sition of a food product. This usually involves
alcohol. extracting the lipids and analyzing them using capil-
lary gas chromatography. Before such analysis, triac-
ylglycerols and phospholipids are saponified, and
Peak hseight ratios for alcohol peaks at various
the fatty acids liberated are esterified to form fatty
concentrations of methanol, n-propyl alcohol,
acid methyl esters (FAMEs) so that the volatility is
and isobutyl alcohol, with benzyl alcohol as
increased.
internal standard:
www.dbooks.org
Two methods of sample preparation for FAME 8.3.4 Reagents and Samples
determination will be used in this experiment: (1)
• Boron trifluoride (BF3) – in methanol, 12–14 % solution
sodium methoxide method and (2) boron trifluoride
• Hexane (GC grade. If fatty acids contain 20 C atoms
(BF3) method. In the sodium methoxide method,
or more, heptane is recommended.)
sodium methoxide is used as a catalyst to interester-
• Methanolic sodium hydroxide 0.5 N (Dissolve 2 g
ify fatty acid. This method is applicable to saturated
of NaOH in 100 mL of methanol.)
and unsaturated fatty acids containing from 4 to 24
• Oils: pure olive oil, safflower oil, salmon oil
carbon atoms. In the BF3 method, lipids are saponi-
• Reference standard [Gas–liquid chromatography
fied, and fatty acids are liberated and esterified in
(GLC)-60 Reference Standard FAME 25 mg is dis-
the presence of a BF3 catalyst for further analysis.
solved in 10 mL hexane, (Table 8.2) (Nu-Chek Prep,
This method is applicable to common animal and
Inc. Elysian, MN)]
vegetable oils and fats and fatty acids. Lipids that
• Sodium methoxide, 0.5 M solution in methanol
cannot be saponified are not derivatized and, if
(Aldrich)
present in large amount, may interfere with subse-
• Sodium chloride, saturated
quent analysis. This method is not suitable for prep-
• Sodium sulfate, anhydrous granular
aration of methyl esters of fatty acids containing
large amounts of epoxy, hydroperoxy, aldehyde,
ketone, cyclopropyl, and cyclopentyl groups and 8.3.5 Hazards, Precautions, and Waste
conjugated polyunsaturated and acetylenic com- Disposal
pounds because of partial or complete destruction Do all work with the boron trifluoride in the hood; avoid
of these groups. contact with the skin, eyes, and respiratory tract. Wash
It should be noted that AOAC Method 969.33 is all glassware in contact with boron trifluoride immedi-
used in this laboratory exercise, rather than AOAC ately after use. Otherwise, adhere to normal laboratory
Methods 996.06, which is the method for nutrition safety procedures. Wear safety glasses at all times. Boron
labeling, with a focus on trans fats. Compared to trifluoride, hexane, and sodium methoxide must be dis-
AOAC Method 969.33, Method 996.06 used a longer posed of as hazardous wastes. Other wastes likely may
and more expensive capillary column, requires a lon- be put down the drain using a water rinse, but follow
ger analysis time per sample, and involves more com- good laboratory practices outlined by environmental
plicated calculations. health and safety protocols at your institution.
8.3.2 Objective 8.3.6 Supplies

Utilize two methods to prepare methyl esters from (Used by students)
fatty acids in food oils and then determine the fatty
acid profile and their concentration in the oils by gas • Boiling flask, 100 mL, with water-cooled condenser
chromatography. for saponification and esterification
8.3.3 Chemicals
8.2
table FAME GLC-60 reference standard
CAS no. Hazards
Boron trifluoride 7637-07-2 Toxic, highly No. Chain Item Weight %
(BF3) flammable
Hexane 110-54-3 Harmful, highly 1 C4:0 Methyl butyrate 4.0
flammable, dangerous 2 C6:0 Methyl caproate 2.0
for the environment 3 C8:0 Methyl caprylate 1.0
Methanol 67-56-1 Extremely flammable 4 C10:0 Methyl caprate 3.0
Sodium chloride 7647-14-5 Irritant 5 C12:0 Methyl laurate 4.0
(NaCl) 6 C14:0 Methyl myristate 10.0
Sodium 1310-73-2 Corrosive 7 C14:1 Methyl myristoleate 2.0
hydroxide 8 C16:0 Methyl palmitate 25.0
(NaOH) 9 C16:1 Methyl palmitoleate 5.0
Sodium sulfate 7757-82-6 Harmful 10 C18:0 Methyl stearate 10.0
(Na2SO4) 11 C18:1 Methyl oleate 25.0
Sodium 124-41-4 Toxic, highly 12 C18:2 Methyl linoleate 3.0
methoxide flammable 13 C18:3 Methyl linolenate 4.0
14 C20:0 Methyl arachidate 2.0
• Pasteur pipette
• Syringe 8.3
• Vials or sample bottle with tight-seal cap Determination of flask size and amount of
table reagent from approximate sample size
8.3.7 Equipment 0.5 N NaOH BF3 reagent

Sample (mg) Flask (mL) (mL) (mL)
• Centrifuge 100–250 50 4 5
• Vortex mixer 250–500 50 6 7
500–750 100 8 9
• Gas chromatography unit (with running conditions):
750–1000 100 10 12
Instrument Gas chromatograph (Agilent 2. Attach condenser and reflux until fat globules
6890 or similar) disappear (about 5–10 min).
Detector Flame ionization detector
3. Add 9 mL BF3 solution through condenser and
Capillary column DB-Wax or equivalent
Length 30 m
continue boiling for 2 min.
Internal diameter (ID) 0.32 mm 4. Add 5 mL hexane through condenser and boil
Df 1.0 μm for 1 more min.
Carrier gas He 5. Remove the boiling flask and add ca. 15 mL
Makeup gas Nitrogen saturated NaCl solution.
Sample injection 1 μl 6. Stopper flask and shake vigorously for 15 s
Split ratio 1:20 while solution is still tepid.
Flow rate 2 mL/min (measured at room 7. Add additional saturated NaCl solution to float
temperature) hexane solution into neck of flask.
Injector temperature 250 °C
8. Transfer 1 mL upper hexane solution into a
Detector temperature 250 °C
small bottle and add anhydrous Na2SO4 to
Temperature program
Initial oven temperature 100 °C remove H2O.
Initial time 2 min
Rate 5 °C/min Method B Preparation of Methyl Esters by
Final temperature 230 °C Sodium Methoxide Method
Final time 10 min 1. Using a Pasteur pipette to transfer, weigh
100 mg (±5 mg) of sample oil to the nearest
8.3.8 Procedure 0.1 mg into a vial or small bottle with a tight-
sealing cap.
(Instructions are given for single sample preparation
2. Add 5 mL of hexane to the vial and vortex
and injection, but injections of samples and standards
briefly to dissolve lipid.
can be replicated.)
3. Add 250 μl of sodium methoxide reagent, cap
the vial tightly, and vortex for 1 min, pausing
8.3.8.1 Preparation of Methyl Esters
every 10 s to allow the vortex to collapse.
4. Add 5 mL of saturated NaCl solution to the vial,
Method A. Preparation of Methyl Esters by cap the vial, and shake vigorously for 15 s. Let
Boron Trifluoride (Adapted from AOAC Method stand for 10 min.
969.33) 5. Remove the hexane layer and transfer to a vial
Notes Methyl ester should be analyzed as soon possi- containing a small amount of Na2SO4. Do not
ble or sealed in an ampule and stored in a freezer. You transfer any interfacial precipitate (if present) or
might also add equivalent 0.005 % 2, 6-di-tert-butyl-4- any aqueous phase.
methylphenol (BHT). Sample size needs to be known 6. Allow the hexane phase containing the methyl
to determine the size of the flask and the amount of esters to be in contact with Na2SO4 for at least
reagents, according to Table 8.3. 15 min prior to analysis.
1. Add 500 mg sample (see Table 8.3) to 100-mL 7. Transfer the hexane phase to a vial or small bot-
boiling flask. Add 8 mL methanolic NaOH solu- tle for subsequent GC analysis. (Hexane solu-
tion and boiling chip. tion can be stored in the freezer.)
www.dbooks.org
8.3.8.2 Injection of Standards and Samples Results from chromatograms using boron trifluoride
into GC method to prepare methyl esters:
1. Rinse the syringe three times with hexane and
three times with the reference standard mix- Safflower oil Pure olive oil Salmon oil
ture (25 mg of 20A GLC Reference Standard Retention Identity Retention Identity Retention
FAME dissolved in 10 mL hexane). Inject 1 μl Peak time time time Identity
of standard solution, remove syringe from 1
injection port, and then press start button. 2
Rinse syringe again three times with solvent. 3
Use the chromatogram obtained as described 4
below. 5
2. Rinse the syringe three times with hexane and 6
7
three times with the sample solution prepared
8
by Method A. Inject 1 μl of sample solution,
9
remove syringe from injection port, and then 10
press start button. Rinse syringe again three 11
times with solvent. Use the chromatogram 12
obtained as described below. 13
3. Repeat Step 3 for sample solution prepared by 14
Method B.
Results from chromatograms using sodium methoxide
8.3.9 Data and Calculations method to prepare methyl esters:
1. Report retention times and relative peak areas Safflower oil Pure olive oil Salmon oil
for the peaks in the chromatogram from the Retention Identity Retention Identity Retention
FAME reference standard mixture. Use this Peak time time time Identity
information to identify the 14 peaks in the 1
chromatogram. 2
3
Retention Peak Identity of 4
Peak time area peak 5
6
1 7
2 8
3 9
4 10
5 11
6 12
7 13
8 14
9
10
3. For the one oil analyzed by your group, prepare
11
12
a table (with appropriate units) comparing your
13 experimentally determined fatty acid profile to
14 that found in your cited literature source.
2. Using the retention times for peaks in the chro- Quantity determined
matogram from the FAME reference standard Boron Sodium
mixture, and your knowledge of the profile of Quantity in trifluoride methoxide
the oil, identify the peaks in the chromatograms literature method method
for each type of oil analyzed, [Cite your C4:0
source(s) of information on the fatty acid profile C6:0
of each oil.] Report results for samples from C8:0
both methods of derivatization. C10:0
Quantity determined size of the oil). To make this procedure suffi-

Boron Sodium ciently quantitative for a purpose like that just
Quantity in trifluoride methoxide described, an internal standard must be used.
literature method method (a) Why is the fatty acid profiling method used in
C12:0 this lab inadequate to quantify the fatty acids?
C14:0 (b) What are the characteristics required of a
C14:1 suitable internal standard for FAME quanti-
C16:0
fication by GC and how does this overcome
C16:1
the problem(s) identified in (a)?
C18:0
C18:1 (c) Would the internal standard be added to the
C18:2 reference standard mixture and the sample
C18:3 or only to one of these?
C20:0 (d) When would the internal standard be added?
Type of oil tested:
8.3.10 Questions RESOURCE MATERIALS
1. Comment on the similarities and differences in Amerine MA, Ough CS (1980) Methods for analysis of musts
the fatty acid profiles in question #3 of Data and and wine. Wiley, New York.
Calculations, comparing experimental data to AOAC International (2016) Official methods of analysis, 20th
literature reports. From the results, compare edn., (On-line). Methods 968.09, 969.33, 972.10, 996.06.
AOAC International, Rockville, MD
and decide which method of esterification to
Martin GE, Burggraff JM, Randohl DH, Buscemi PC (1981)
obtain FAMEs was better for your sample. Gas–liquid chromatographic determination of congeners
2. The approach taken in this lab provides a fatty in alcoholic products with confirmation by gas chromatog-
acid profile for the oils analyzed. This is suffi- raphy/mass spectrometry. J Assoc Anal Chem 64:186
cient for most analytical questions regarding Ellefson WC (2017) Fat analysis. Ch. 17. In: Nielsen SS (ed)
fatty acids. However, determining the fatty acid Food analysis, 5th edn. Springer, New York
profile is not quite the same thing as quantifying Pike OA, O’Keefe SF (2017) Fat characterization. Ch. 23. In:
the fatty acids in the oil. (Imagine that you Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
wanted to use the results of your GC analysis to Qian MC, Peterson DG, Reineccius GA (2017) Gas chroma-
calculate the amount of mono- and polyunsatu- tography. Ch. 14. In: Nielsen SS (ed) Food analysis, 5th
edn. Springer, New York
rated fatty acids as grams per a specified serving
www.dbooks.org
9
chapter
Mass Spectrometry
with High-Performance
Liquid Chromatography
Baraem P. Ismail
St. Paul, MN, USA
9.1 Introduction 9.2 Procedure

9.1.1 Background 9.2.1 Sample Preparation
9.1.2 Reading Assignment 9.2.2 LC–MS Procedure
9.1.3 Objectives 9.2.3 MS Conditions
9.1.4 Chemicals 9.3 Data and Calculations
9.1.5 Hazards, Precautions, 9.4 Questions
and Waste Disposal 9.4.1 General Questions
9.1.6 Reagents 9.4.2 Questions Specific to
9.1.7 Supplies Isoflavone Analysis
9.1.8 Equipment
9.5 Case Study
98 B.P. Ismail
9.1 INTRODUCTION understand how to analyze a mass spectrum to obtain

mass and structural information of a compound.
9.1.1 Background
9.1.4 Chemicals
Mass spectrometry (MS) is an analytical technique that
provides information about the molecular weight and
CAS No. Hazards
chemical characteristics of a compound. This tech-
nique is commonly linked to high-performance liquid HPLC grade 1334547-72-6 Highly flammable,
chromatography (LC–MS), gas chromatography (GC– acetonitrile acute toxicity, irritant
MS), inductively coupled plasma (ICP–MS), and other HPLC grade 67-56-1 Highly flammable,
methanol acute toxicity, serious
methods.
health hazard
A mass spectrometer has three essential functions:
sample ionization, ion separation, and ion detection.
Components in a sample can be ionized by various 9.1.5 Hazards, Precautions, and Waste
ionization techniques; regardless of the source, the Disposal
charged particles that are generated are separated Adhere to normal laboratory safety procedures. Wear
based on their mass-to-charge ratio. Separation occurs safety glasses at all times. Methanol and acetonitrile
in the mass analyzer which sorts the different ion waste must be handled as hazardous waste. Other
masses by applying electric and magnetic fields. These wastes likely may be put down the drain using a water
ions are then detected which provide data on the rinse, but follow good laboratory practices outlined by
abundance of each ion fragment present. environmental health and safety protocols at your
For LC–MS analysis, the compounds are first sep- institution.
arated on a LC system. After detection in the LC sys-
tem (by ultraviolet–visible spectroscopy (UV–VIS)), 9.1.6 Reagents
the eluent enters the ion source of a mass spectrometer
where the compounds get ionized and subsequently • Deionized distilled water
separated based on “mass-to-charge” (m/z) ratio in • HPLC grade water
the analyzer. The separated ions are then detected • Methanol/water mixture, 80/20
using an electron multiplier. Ionization using electro-
spray ionization (ESI) often produces precursor ions 9.1.7 Supplies
with some fragmentation. The ion which undergoes • Aluminum foil
fragmentation is the precursor ion, and the ions that • Automatic pipettors, 1000 μL, 200 μL, 100 μL, and
are produced upon fragmentation of the precursor ion 10 μL
are called product ions. To obtain further structural • Autosampler vials, 1.5 mL, with caps
information about the compound, tandem mass spec- • 3 Beakers, 125 mL, each containing either 80/20
trometry (MS/MS) can be employed. After ionization, methanol, deionized distilled water, or acetonitrile
the ions of interest are separated and fragmented by • 1 Beaker, 125 mL, for balancing the centrifuge tubes
collision-induced dissociation (CID) by using an inert • Buchner funnel
gas (helium, argon, etc.) as the collision gas. The • 2 Centrifuge tubes, 50 mL centrifuge tubes
energy given to the collision gas is varied depending • Defatted soy flour
on the desired extent of fragmentation, which will pro- • Erlenmeyer flask, 25 mL (rinsed prior with 80/20
vide valuable structural information. The mass spec- methanol)
trum obtained by tandem MS contains only the • Erlenmeyer flask, 10 mL (rinsed prior with 80/20
product ions. A case study is presented to better under- methanol)
stand the advantages of MS in tandem. • 3 Graduated cylinders, 10 mL
• Filter paper, Whatman 42, 90 mm diameter
9.1.2 Reading Assignment • Glass rods
Reuhs, B.L. 2017. High-performance liquid chroma- • Markers
tography. Ch. 13 in Food Analysis, 5th ed. S.S. Nielsen • Parafilm
(Ed.), Springer, New York. • Rotary evaporator (rotovap) collection flasks
Smith, J.S., and Thakur, R.A. 2017. Mass spectrom- (125 mL or smaller) (rinsed prior with 80/20
etry. Ch. 11 in Food Analysis, 5th ed. S.S. Nielsen (Ed.), methanol)
Springer, New York. • Sidearm flask, 125 mL, with a nozzle to plug the
vacuum pump (rinsed prior with 80/20 methanol)
9.1.3 Objectives • Stir bars and stirrer
• Syringes, 3 mL
Determine and identify various phytochemicals (mainly • Syringe filters, non-sterile, nylon; pore size 0.45 μm;
isoflavones, Table 9.1 and Fig. 9.1) in soy flour, and diameter 25 mm
www.dbooks.org
Chapter 9 • Mass Spectrometry with High-Performance Liquid Chromatography 99
9.1
table Twelve known isoflavones found in soybean and their molecular weight (MW)
Isoflavone MW Isoflavone MW Isoflavone MW

Daidzein 254 Genistein 270 Glycitein 284
Daidzin 416 Genistin 432 Glycitin 446
Acetyldaidzin 458 Acetylgenistin 474 Acetylglycitin 488
Malonyldaidzin 502 Malonylgenistin 518 Malonylglycitin 532
OH
a 1
8
HO 7 8a O O
2 HO 1
8
A C 2' HO O 7 8a O
3 1' 2
3' OH
R1 6 4a A C 2'
5 4 3
B 4a 1'
4' R1 6 4 3'
R2 O 5 B
6' OH 4'
5' R2 O
6' OH
5'
Aglycone
Non-conjugated glucoside
O O
O
O O
4" 6" 6"
5" O 4"
2" 1 5" O
HO 8 HO 2" 1
HO 1" O 7 8a O 2' 8
2 HO O 7 8a O
3" OH 2
A C 2' 3" OH 1"
3 A C 2'
1' 3
R1 6 4a 3' 4a 1'
5 4 R1 6 4 3'
B 5 B
R2 O 4' 4'
6' R2 O 6'
OH OH
5' 5'
6"-O-Acetylglucoside 6"-O-Malonylglucoside
OH
b O O
4" 6"
5" O
O 2" 2 1
8
HO O' 7 8a O 2
3" OH 1"
A C 2'
3
6 4a 1' 3'
R1 5 4
B
R2 O 4'
6'
OH
5'
4"-O-Malonylglucoside
9.1 (a) Structures and numbering of the 12 known isoflavones categorized as aglycone, nonconjugated glucoside,
figure
acetylglucoside, and malonylglucoside. R1 can be -H in the case of daidzin and genistin or -OCH3 in the case of
glycitin, while R2 can be -H in the case of daidzin and glycitin or -OH in the case of genistin. (b) Structures and
numbering system of 4”-O-malonylglucosides (malonylglucoside isomers)
100 B.P. Ismail
9.2
table Example combinations of ionization sources and mass analyzers coupled with chromatographic separation
Name Ionization source Mass analyzer Tandem MS Separation mode

LTQ Orbitrap Electrospray ionization (ESI) Linear ion trap and Yes Liquid chromatography
orbital trap
5500 QTRAP® ESI Triple quadrupole with Yes Liquid chromatography
Q3 linear trap
4000 QTRAP® ESI Triple quadrupole with Yes Liquid chromatography
Q3 linear trap
LTQ ESI Linear ion trap Yes Liquid chromatography
LCT Premier ESI Time of flight (TOF) No Liquid chromatography
4800 AB Sciex Matrix-associated laser desorption TOF–TOF Yes Liquid chromatography
ionization (MALDI)
Pegasus 4D Electron impact (EI) TOF No Gas chromatography
Agilent 6130 Dual ESI/atmospheric pressure Quadrupole Yes Liquid chromatography
chemical ionization (APCI)
LCQ (2) ESI Ion trap Yes Liquid chromatography
LCQ(1), Dual ESI/APCI Ion trap Yes Liquid chromatography
Biflex III MALDI TOF No Liquid chromatography
QSTAR XL ESI Quadrupole TOF Yes Liquid chromatography
Saturn 3 EI/chemical ionization (CI) Ion trap Yes Gas chromatography
• Tape (There will be a sample on the stirrer prepared

• Vacuum pump by the TA and ready for you to take to the next
• Vortex step.)
5. Quantitatively transfer the sample solution
9.1.8 Equipment from the Erlenmeyer flask to a labeled centri-
fuge tube. Rinse the flask three times with 1 mL
acetonitrile and add the rinse to the centrifuge
• Centrifuge, for 15 mL tubes
tube. Use the scale to balance the tube with a
• High-performance liquid chromatography system
tube containing water.
• Mass-selective detector (quadrupole), equipped
6. Centrifuge the solution in a centrifuge. Example
with electrospray ionization source
conditions with a Marathon 3200 centrifuge are:
• Rotary evaporator
Speed = 4000 rpm
Various combinations of ionization sources and Time = 15 min
mass analyzers can be used for LC–MS and GC–MS Temperature = 15 °C
analysis (see Table 9.2). This laboratory exercise as
7. Take the centrifuge tube carefully out of the
describes utilizes a LC–MS system coupled with elec-
rotor so that the precipitate does not get
trospray ionization (ESI) and a quadrupole mass
disturbed.
analyzer.
8. Filter the permeate using a Buchner funnel,
labeled 125 mL filter flask with a nozzle to plug
the vacuum pump, Whatman 42 filter paper,
9.2 PROCEDURE and vacuum pump. The setup should look like
in Fig. 9.2.
9.2.1 Sample Preparation 9. Quantitatively transfer the filtered solution into
a labeled rotary evaporator (rotovap) collection
1. Weigh 0.05 gm of defatted soy into a labeled
flask. Again rinse three times with 1 mL acetoni-
25 mL Erlenmeyer flask.
trile and add the rinse to the rotovap collection
2. Add 10 mL of HPLC grade acetonitrile to the
flask.
sample and put in a stir bar.
10. Evaporate to dryness using a rotovap. Example
3. Stir on a magnetic stir plate until the sample is
conditions with a Buchi R-II Rotavapor are:
thoroughly mixed (stirrer speed = 7, time = 5 min).
4. Add 9 mL DDW and stir for 2 h (stirrer Cooler temperature = 4 °C.
speed = 7). Occasionally, scrape the surface of Water bath temperature = 38 °C.
the plastic bottle with a glass rod to make the Rotovap RPM = 120.
sample sticking to the sides go into the solution. Time = 15 min (may take up to 30 min or so).
www.dbooks.org
9.3
Buchner funnel table HPLC gradient time program
Whatman 42
filter paper
Solvent B concentration (5 %) Time (min)
125 mL filter flasks with a
nozzle to plug vacuum 11 0 (Initial)
14 30
14 35
Vacuum pump 30 40
30 50
11 52
11 60
9.2.3 MS Conditions
9.2 Setup to filter sample permeate
Mass spectrometry is performed on a Waters ZQ quad-
figure
rupole instrument. Positive ionization will be
Pressure: since it is a two-step evaporation, employed. Masses are scanned from 200 to 600 m/z
evaporating of acetonitrile will be done at over 60 min, at a scan rate of one scan/s.
115 mbar, and evaporation of water will be Eight selected ions of the following m/z are moni-
done at 30 mbar. tored, [M + H+]: 255, 271, 417, 433, 459, 475, 503, and 519.
Note: If the available pump is not adequate The injection volume is 20 μL; the split ratio is 1:3
enough to use for evaporating water from (so ¼ of the flow goes into the mass spectrometer, ¾
the sample, shell freezing is used to freeze flow through the UV detector). The source tempera-
the sample so it can be dried using a lyophi- ture is 150 °C; the desolvation temperature is
lizer (freeze dryer). 450 °C. The desolvation gas flow is 600 L/h; the cone
gas flow is 75 L/h. The cone voltage is 30 eV; the capil-
11. There will be a sample already dried out for you lary voltage is 3 kV.
and ready for the next step. Add 10 mL of 80/20
methanol/water and quantitatively transfer the 1. Turn on the gas flow by pressing the button
solution into a labeled 25 or 10 mL Erlenmeyer “API gas” on the tune page.
flask. This time do not rinse; just transfer using 2. Click the button “Press to Operate” on the tune
a pipette. page.
12. Vortex for 2 min (Speed = 5). 3. Turn on the oven and the degasser.
13. Attach a 0.45 μm filter onto the end of a 3 mL 4. Load the HPLC conditions on the inlet page
syringe. and turn on the flow.
14. Pour some of the sample into the filter 5. Wait until the HPLC column is conditioned and
assembly. the oven has reached the desired temperature.
15. Push sample through the filter into an autosam- 6. Enter the file name, MS file, inlet file, and MS
pler vial. tune file name, and save the data set.
16. Label the sample vial (include group name, 7. Before you inject sample, make sure that the
date, sample name). mass spectrometer is in the “Load” mode.
17. Seal the flask with parafilm, wrap with silver 8. Press the “Start” button, inject the sample, click
foil, and label adequately. on “Start” again, and press the “Inject” button
on the mass spectrometer.
9.2.2 LC–MS Procedure
A Shimadzu LC-10 AD HPLC equipped with two sol- 9.3 DATA AND CALCULATIONS
vent pumps and a CT-10A column oven will be used.
The chromatographic column is a YMC AM-303 (ODS, Analyze the total ion chromatogram and mass spectra
250 mm × 4.6 mm i.d.) comprising C18 reversed-phase of the selected isoflavones provided to you at the end
packing (5 μm average pore size), equipped with a C18 of the lab exercise. To help you understand how to
guard column (4 mm × 20 mm i.d.). analyze a mass spectrum to obtain mass and struc-
The chosen flow rate is 1 mL/min and oven temp tural information of a compound, see the Case
of 45 °C. A linear HPLC gradient will be used: Solvent Study, Sect. 9.5. Use the resource materials provided
A will be 0.1 % (v/v) formic acid, and solvent B will be to answer the general questions below, and analyze
acetonitrile. The gradient time program is described in the ion chromatogram and mass spectra of the
Table 9.3. isoflavones.
102 B.P. Ismail
9.4 QUESTIONS 9.4 Identified isoflavones, precursor, and product

table ions m/z values
9.4.1 General Questions
Retention Isoflavone Precursor ion List of product ions
Time m/z (name and m/z)
1. What is a molecular ion? Does electron impact
(EI) ionization produce a single precursor ion?
How about ESI? Answer by explaining the dif-
ference between soft ionization to hard
ionization.
2. List the ionization interfaces that are capable of
delivering soft ionization.
3. Which ion travels farthest in unit time (low m/z
or high m/z) in time-of-flight mass analyzer?
How does the length of time-of-flight mass ana-
lyzer affect the resolution?
4. Explain the different between a quadrupole and
an ion trap.
5. What more information can MS/MS (i.e., MS in
tandem) provide versus just one MS?
9.4.2 Questions Specific to Isoflavone

Analysis Recent studies have shown the formation of a new
derivative upon heat treatment of malonylgenistin. The
1. Discuss the peaks and how you identify them. wavescans obtained using a photodiode array detector
2. Indicate if there were any co-elutions and how (PDA) of the new derivative and malonylgenistin were
one can determine that. similar. Hence, LC/UV failed to provide any informa-
3. Discuss the presence of isomers, if any, and tion about the structural differences between the two
how useful the MS was in identifying their compounds. MS analysis of the two compounds
presence, pointing to advantages and revealed that the compounds had same mass (m/z = 519
limitations. Dalton) indicating that the new derivative is an isomer
4. Were you able to see precursor ions? Were you of malonylgenistin (Fig. 9.3). Tandem mass spectrome-
able to see product ions? Use a table format try was employed to probe structural differences
(Table 9.4) to list the precursor ions identified between the two isomers. There were two noticeable
and their product ions as in the example table differences in the product ion spectra (Fig. 9.4).
below.
1. Isomer fragmented more at a lower collision
level as compared to malonylgenistin.
2. Malonylgenistin product ion spectra had an ion
9.5 CASE STUDY with m/z = 433, which was absent in the isomer
product ion spectra.
Malonylgenistin is a type of isoflavone abundantly These differences highlight the structural differ-
found in soybeans. Epidemiological studies have ences between the two compounds. Nuclear magnetic
shown the association of isoflavones with many health resonance has to be employed to obtain further struc-
benefits such as reducing the risk of cancer, alleviation tural details.
of postmenopausal symptoms, etc. Processing of soy-
beans into various soy products results in the conver-
sion of malonylgenistin to various compounds. RESOURCE MATERIALS
Monitoring the conversions of malonylgenistin to dif-
ferent compounds is important to determine if the Smith JS, Thakur RA (2017) Mass spectrometry. Ch. 11. In:
resultant compounds have any biological significance. Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
www.dbooks.org
a
RT: 0.00 - 70.00 SM: 11B
100 Isomer
Malonylgenistin NL: 9.87E7
80 Genistin TIC MS ESI(+) of 20 ppm
Relative Malonylgenistin treated at
60 pH 8 & 100C for 15min
40
20
Abundance
0
NL: 5.74E4
b Total Scan PDA ESI(+) of
40000 20 ppm Malonylgenistin
Malonylgenistin
uAU treated at pH 8 & 100C for
Genistin Isomer 15min
20000
0
100 NL: 6.66E6
c m/z= 518.5-519.5 MS
80 Malonylgenistin
Relative ESI(+) of 20 ppm
60 Malonylgenistin treated at
Abundance Isomer pH 8 & 100C for 15min
40
20
0
0 10 20 30 40 50 60
Time (min)
9.3 ESI–MS analysis malonylgenistin and its isomer: (a) total ion chromatogram, (b) PDA view of malonylgenistin
figure
solution, and (c) reconstructed single ion chromatogram of m/z 519 ion (protonated molecule of a
malonylgenistin)
104 B.P. Ismail
Isomer # 705 RT: 32.01 AV: 1 Malonylgenistin RT: 33.60 AV:

a b
271.3 271.3
100 100
518.9
90 [M + H – 248]+ 90 [M + H – 248]+
80 80
70 70 [M + H ] +
60 60
50 50
[M + H – 86]+
40 40
Relative 30 [M + H ] + 30
abundance 519.9
20 20 433.0
10 476.5 519.0 10
272.3 349.2 272.4 474.9
432.8 536.5 313.2 432.3 548.0
0 0
300 400
# RT: 500 AV: 1 300 400
# RT: 500 AV: 1
m/z m/z
c d
271.3 518.9
100 100
90 90
80 80
70 Isomer 70 Malonylgenistin
60 60
50 50
40 40 271.3
518.8
30 30 520.0
20 272.1 20
432.9
10 451.1 476.7 10 272.3
322.8 357.9 537.4 379.4 501.1 528.3
0 0
300 400 500 300 400 500
m/z m/z
Relative
abundance
9.4 ESI–MS/MS analysis of the protonated forms of isomer and malonylgenistin at various collision levels: (a)
figure
isomer at 20 %, (b) malonylgenistin at 20 %, (c) isomer at 17 %, and (d) malonylgenistin at 17 % collision
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10
chapter
Moisture Content
Determination
S.Suzanne Nielsen
10.1 Introduction 10.3 Vacuum Oven

10.1.1 Background 10.3.1 Objective
10.1.2 Reading Assignment 10.3.2 Principle
10.1.3 Overall Objective 10.3.3 Supplies
10.2 Forced Draft Oven 10.3.4 Equipment
10.2.1 Objective 10.3.5 Cautions and Hazards
10.2.2 Principle of Method 10.3.6 Procedure
10.2.3 Supplies 10.3.7 Data and Calculations
10.2.4 Equipment 10.4 Microwave Drying Oven
10.2.5 Note 10.4.1 Objective
10.2.6 Cautions and Hazards 10.4.2 Principle
10.2.7 Procedure 10.4.3 Supplies
10.2.8 Data and Calculations 10.4.4 Equipment
10.4.5 Procedure 106 10.7 Karl Fischer Method
10.4.6 Data and Calculations 10.7.1 Objective
10.5 Rapid Moisture Analyzer 10.7.2 Principle
10.5.1 Objective 10.7.3 Chemicals
10.5.2 Principle 10.7.4 Reagents
10.5.3 Supplies 10.7.5 Hazards, Cautions, and Waste
10.5.4 Equipment Disposal
10.5.5 Procedure 10.7.6 Supplies
10.5.6 Data and Calculations 10.7.7 Equipment
10.6 Toluene Distillation 10.7.8 Procedure
10.6.1 Objective 10.7.9 Data and Calculations
10.6.2 Principle 10.8 Near-Infrared Analyzer
10.6.3 Chemicals 10.8.1 Objective
10.6.4 Hazards, Cautions, and Waste 10.8.2 Principle
Disposal 10.8.3 Supplies
10.6.5 Supplies 10.8.4 Equipment
10.6.6 Equipment 10.8.5 Procedure
10.6.7 Procedure 10.8.6 Data and Calculations
10.6.8 Notes 10.9 Questions
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Chapter 10 • Moisture Content Determination 107
10.1 INTRODUCTION 10.1.2 Reading Assignment

Mauer, L.J., and Bradley, R.L., Jr. 2017. Moisture and
10.1.1 Background total solids analysis, Ch. 15, in Food Analysis, 5th ed.
The moisture (or total solids) content of foods is impor- S.S. Nielsen (Ed.), Springer, New York.
tant to food manufacturers for a variety of reasons.
Moisture is an important factor in food quality, preser- 10.1.3 Overall Objective
vation, and resistance to deterioration. Determination
of moisture content also is necessary to calculate the The objective of this experiment is to determine and
content of other food constituents on a uniform basis compare the moisture contents of foods by various
(i.e., dry weight basis). The dry matter that remains methods of analysis.
after moisture analysis is commonly referred to as
total solids.
While moisture content is not given on a nutri- 10.2 FORCED DRAFT OVEN
tion label, it must be determined to calculate total
carbohydrate content. Moisture content of foods can 10.2.1 Objective
be determined by a variety of methods, but obtain- Determine the moisture content of corn syrup and
ing accurate and precise data is commonly a chal- corn flour using a forced draft oven method.
lenge. The various methods of analysis have different
applications, advantages, and disadvantages (see 10.2.2 Principle of Method
Sect. 10.1.2). If the ash content also is to be deter-
mined, it is often convenient to combine the mois- The sample is heated under specified conditions, and
ture and ash determinations. In this experiment the loss of weight is used to calculate the moisture con-
several methods to determine the moisture content tent of the sample.
of foods will be used and the results compared. Note
that in some cases, the method is not ideal for the 10.2.3 Supplies
sample type (e.g., moisture content of corn syrup or (Note: Samples for moisture analysis could be the same
basil by forced draft oven), but the intent is to com- products tested also for ash, fat, and protein analysis,
pare results to those obtained using more appropri- by procedures in respective chapters. Content of ash,
ate methods (i.e., vacuum oven for corn syrup, fat, and protein could then be expressed on both a wet
toluene distillation for basil). Summarized below are weight basis and dry weight basis.)
the food samples proposed for analysis and the
methods used. However, note that other types of
• Beaker, 20–30 mL (for corn syrup)
food samples could be analyzed, and groups of stu-
• Basil (fresh), 15 g (ground)
dents could analyze different types of food samples.
• Beaker, 25–50 mL (to pour corn syrup into pans)
It is recommended that all analyses be performed in
• Corn flour, 10 g
triplicate, as time permits.
• Corn syrup, 15 g
• 3 Crucibles (preheated at 550 °C for 24 h)
• 2 Desiccators (with dried desiccant)
Corn Corn Milk Nonfat
• Liquid milk, 20 mL
syrup flour (liquid) dry milk Basil
• Nonfat dry milk (NFDM), 10 g
Forced X X X X X • Plastic gloves (or tongs)
draft oven • 2 Spatulas
Vacuum X • 5 Trays (to hold/transfer samples)
oven • 2 Volumetric pipettes, 5 mL
Microwave X X • 6 Weighing pans – disposable aluminum open
drying
pans (for use with corn syrup) (predried at 100 °C
Rapid X
moisture
for 24 h)
analyzer • 6 Weighing pans – metal pans with lids [for use
Toluene X X with corn flour and nonfat dry milk (NFDM)]
distillation (predried at 100 °C for 24 h)
Karl Fischer X X
method 10.2.4 Equipment
Near- X
infrared • Analytical balance, 0.1 mg sensitivity
analyzer • Forced draft oven
• Hot plate
108 S.S. Nielsen
10.2.5 Note 2. Place 5 g of sample in the crucible and weigh

accurately.
Glass microfiber filters (e.g., GF/A, Whatman,
3. Evaporate a majority of water on a hot plate; do
Newton, MA), predried for 1 h at 100 °C, can be used
not dry the sample completely. (Gently heat the
to cover samples to prevent splattering in the forced
milk in the crucibles. Wear gloves as you handle
draft oven and the vacuum oven. Instructors may
the crucibles, swirling the milk to coat the sides
want to have students compare results with and with-
of the crucible. Try to avoid development of a
out these fiberglass covers.
film on the surface, until most of the water has
been evaporated.)
10.2.6 Cautions and Hazards 4. Place in a forced draft oven at 100 °C for 3 h.
Be sure to label all containers used with complete 5. Store in a desiccator until samples are weighed.
information, or record container information linker to 6. Calculate percentage moisture (wt/wt) as
each sample. Use gloves or tongs when handling sam- described below.
ple pans and crucibles. These pans and crucibles have
been dried and stored in desiccators prior to weighing. Note: Ash content of this milk sample could be
They will pick up moisture by sitting on the counter, determined by placing the milk sample, dried at
so remove them from the desiccator only just before 100 °C for 3 h, in a muffle furnace at 550 °C for
use. Open desiccators slowly to avoid damage and 18–24 h. After cooling in a desiccator, the crucibles
danger from broken glass. containing ashed milk would be weighed and the
ash content calculated. See Ash Analysis laboratory
10.2.7 Procedure in Chap. 11.
Instructions are given for analysis in triplicate.
10.2.7.4 Moisture of Nonfat Dry Milk
1. Weigh accurately the dried pan with lid. (Note
10.2.7.1 Moisture in Corn Syrup
identifier number on pan and lid.)
1. Label dried pans (disposable aluminum open
2. Place 3 g of sample in the pan and weigh
pans) and weigh accurately.
accurately.
2. Place 5 g of sample in the pan and weigh accu-
3. Place pan in a forced draft oven at 100 °C for
rately. (Because corn syrup is very hygroscopic,
24 h.
work quickly, using a plastic transfer pipette, as
4. Store in a desiccator until samples are weighed.
you weigh the corn syrup.)
5. Calculate percentage moisture (wt/wt) as
3. Place in a forced draft oven at 98–100 °C for
described below.
24 h.
10.2.7.5 Moisture in Fresh Basil
1. Label dried pans (disposable aluminum open
described below.
pans) and weigh accurately.
2. Place 3 g of ground sample in the pan and
10.2.7.2 Moisture in Corn Flour (Method
weigh accurately.
44–15A of AACC International,
3. Place in a forced draft oven at 98–100 °C for
One-Stage Procedure)
24 h.
1. Weigh accurately the dried pan with lid. (Note
identifier number on pan and lid.)
2. Place 2–3 g of sample in the pan and weigh
described below.
accurately.
3. Place in a forced draft oven at 130 °C for 1 h. Be
sure metal covers are ajar, to allow water loss.
4. Remove from oven, realign covers to close, cool, Calculate percentage moisture (wt/wt):
and store in desiccator until samples are
weighed. wt of H 2 O in sample
% moisture 100
5. Calculate percentage moisture (wt/wt) as wt of wet sample
described below.
10.2.7.3 Moisture in Liquid Milk (AOAC

 wt of wet   wt of dried 
 sample + pan  −  sample + pan 
Method 990.19, 990.20)
% moisture =
    × 100
1. Label and weigh accurately the predried cruci-  wt of wet 
bles (550 °C for 24 h). (Note identified number  sample + pan  − ( wt of pan )
on crucible.)  
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Pan + wet sample Pan + dried sample Wet sample Moisture content
Sample Rep. Pan (g) (g) (g) (g) Water (g) (%)
Corn syrup 1
2
3
X =
SD =
Corn flour 1
2
3
X =
SD =
Liquid milk 1
2
3
X =
SD =
Nonfat dry 1
milk 2
3
X =
SD =
Fresh basil 1
2
3
X =
SD =
10.3 VACUUM OVEN • Volumetric pipette, 5 mL

• 6 Weighing pans – disposable aluminum open pans
10.3.1 Objective (predried at 100 °C for 3 h)
Determine the moisture content of corn syrup by the
10.3.4 Equipment
vacuum oven method, with and without the addition
of sand to the sample. • Analytical balance, 0.1 mg sensitivity
• Vacuum oven (capable of pulling vacuum to <100 mm
10.3.2 Principle of mercury)
The sample is heated under conditions of reduced
10.3.5 Cautions and Hazards
pressure to remove water, and the loss of weight
is used to calculate the moisture content of the See same information in Sect. 10.2.6.
sample.
10.3.6 Procedure
10.3.3 Supplies 10.3.6.1 Moisture of Corn Syrup,
• Corn syrup, 30 g Without the Use of Drying Sand
• Desiccator (with dried desiccant) 1. Label weighing pans (i.e., etch identifier into tab of
• 3 Glass stirring rods (ca. 2–3 cm long, predried at disposable aluminum pan) and weigh accurately.
100 °C for 3 h) 2. Place 5 g of sample in the weighing pan and
• Plastic gloves (or tongs) weigh accurately.
• Pipette bulb or pump 3. Dry at 70 °C and a vacuum of at least 26 in. for
• Sand, 30 g (predried at 100 °C for 24 h) 24 h, but pull and release the vacuum slowly.
• 2 Spatulas (Note that samples without drying sand will
110 S.S. Nielsen
bubble up and mix with adjoining samples if stirring rod being careful not to spill any of the
pans are too close together.) Bleed dried air into samples. Leave the stirring bar in the pan.
the oven as vacuum is released. 3. Dry at 70 °C and a vacuum of <100 mm mercury
4. Store in a desiccator until samples are cooled to for 24 h. Bleed dried air into the oven as vac-
ambient temperature. Weigh. uum is released.
4. Store in a desiccator until samples are cooled to
10.3.6.2 Moisture of Corn Syrup, with the Use ambient temperature. Weigh.
of Drying Sand
1. Label weighing pan, add 10 g dried sand and
stirring rod, then weigh accurately.
2. Add 5 g of sample and weigh accurately. Add Calculate percentage moisture (wt/wt) as in
5 mL of deionized distilled (dd) water. Mix with Sect. 10.2.8.
Pan + wet Pan + dried Moisture content

Sample Rep. Pan (g) sample (g) sample (g) Wet sample (g) Water (g) (%)
Corn syrup 1
without 2
sand
3
X =
SD =
Corn syrup 1
with sand 2
3
X =
SD =
10.4 MICROWAVE DRYING OVEN 10.4.5 Procedure

Follow instructions from the manufacturer for the use of
10.4.1 Objective the microwave drying oven, regarding the following:
Determine the moisture content of corn syrup and
milk (liquid) using a microwave drying oven. • Turning on instrument and warming up
• Loading method for specific application (i.e., sets
time, power, etc.)
10.4.2 Principle • Taring instrument
The sample is heated using microwave energy, and the • Testing sample
loss of weight is used to calculate the moisture content • Obtaining results
of the sample.
10.4.3 Supplies Moisture content Water/dry
• Corn syrup, 4 g Sample Rep. (%) matter (g/g)
• Glass stirring rod (to spread corn syrup) Corn syrup 1
• Milk (liquid), 4 g 2
• 6 Paper pads (for use in microwave oven) 3
• Pasteur pipette and bulb (to spread milk sample) X = X =
• Plastic gloves SD = SD =
Milk (liquid) 1
2
10.4.4 Equipment 3
• Microwave drying oven (e.g., from CEM X = X =
SD = SD =
Corporation, Matthew, NC)
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10.5 RAPID MOISTURE ANALYZER 10.6.3 Chemicals
10.5.1 Objective CAS no. Hazards

Determine the moisture content of corn flour using a Toluene 108-88-3 Harmful, highly
rapid moisture analyzer. flammable
10.5.2 Principle 10.6.4 Hazards, Cautions, and Waste

Disposal
The sample placed on a digital balance is heated under
controlled high-heat conditions, and the instrument Toluene is highly flammable and is harmful if inhaled.
automatically measures the loss of weight to calculate Use adequate ventilation. Wear safety glasses and
the percentage moisture or solids. gloves at all times. For disposal of toluene waste, fol-
low good laboratory practices outlined by environ-
10.5.3 Supplies mental health and safety protocols at your institution.
• Corn flour, 10 g 10.6.5 Supplies

• Plastic gloves
• Spatula • Fresh basil, 40–50 g
• NFDM, 40–50 g
• Toluene, ACS grade
10.5.4 Equipment
• Rapid moisture analyzer (e.g., from Computrac®, 10.6.6 Equipment
Arizona Instrument LLC., Chandler, AZ)
• Analytical balance, 0.1 mg sensitivity.
• Glass distillation apparatus with ground glass
10.5.5 Procedure joints: (1) boiling flask, 250 mL or 300 mL, round-
bottom, short-neck flask with a TS 24/40 joint; (2)
Follow instructions from the manufacturer for the use
West condenser with drip tip, 400 mm in length
of the rapid moisture analyzer, regarding the
with a TS 24/40 joint; (3) Bidwell-Sterling trap, TS
following:
24/40 joint, 3-mL capacity graduated in 0.1-mL
intervals.
• Turning on instrument and warming up • Heat source, capable of refluxing toluene in the
• Select test material apparatus above (e.g., heating mantle connected to
• Taring instrument voltage controller). No open flame!
• Testing sample • Nylon bristle buret brush, ½ in. in diameter, and a
• Obtaining results wire loop. (It should be long enough to extend
through the condenser, ca. 450 mm. Flatten the loop
10.5.6 Data and Calculations on the buret brush and use this brush, inverted, as a
wire to dislodge moisture drops in the moisture
Moisture content (%)
trap.)
Sample 1 2 3 Mean
10.6.7 Procedure
Corn flour
1. Grind the fresh basil with a small tabletop food
grinder. Pulse grind the sample in 5–10 s inter-
10.6 TOLUENE DISTILLATION vals. Avoid long pulses and excessive grinding
to prevent frictional heat.
10.6.1 Objective 2. Weigh approximately 40 g of sample (basil or
NFDM) accurately (amount chosen to yield
Determine the moisture content of basil by the toluene
2–5 mL water).
distillation method.
3. Transfer sample quantitatively to distilling
flask. Add sufficient toluene to cover the sam-
10.6.2 Principle ple completely (not less than 75 mL).
The moisture in the sample is codistilled with toluene, 4. Assemble the apparatus appropriate. Fill the
which is immiscible in water. The mixture that distills trap with toluene by pouring it through the con-
off is collected, and the volume of water removed is denser until it just fills the trap and begins to
measured. flow into the flask. Insert a loose nonabsorbing
112 S.S. Nielsen
cotton plug into the top of the condenser to pre- 10.7.2 Principle
vent condensation of atmospheric moisture in
When the sample is titrated with the KF reagent, which
the condenser.
contains iodine and sulfur dioxide, the iodine is
5. Bring to boil and reflux at about two drops per
reduced by sulfur dioxide in the presence of water
second until most of the water has been col-
from the sample. The water reacts stoichiometrically
lected in the trap and then increase the reflux
with the KF reagent. The volume of KF reagent required
rate to ca. four drops per second.
to reach the endpoint of the titration (visual, conducto-
6. Continue refluxing until two consecutive read-
metric, or coulometric) is directly related to the amount
ings 15 min apart show no change. Dislodge
of water in the sample.
any water held up in the condenser with a brush
or wire loop. Rinse the condenser carefully with
ca. 5 mL toluene. Dislodge any moisture drop- 10.7.3 Chemicals
lets adhering to the Bidwell-Sterling trap or
toluene trapped under the collected moisture. CAS no. Hazards
For this, use the wire. Rinse wire with a small Karl Fischer reagent Toxic
amount (10 mL) of toluene before removing 2-Methoxyethanol 109-86-4
from the apparatus. Pyridine 110-86-1
7. Continue refluxing for 3–5 min, remove the Sulfur dioxide 7446-09-5
heat, and cool the trap to 20 °C in a suitable Iodine 7553-56-2 Harmful,
water bath. dangerous to
8. Calculate the moisture content of the sample: the environment
Methanol, anhydrous 67-56-1 Extremely
flammable
% Moisture vol.of water mL /wt of sample g 100 Sodium tartrate 868-18-8
dihydrate
(Na2C4H4O6 · 2H2O)
10.6.8 Notes
1. Flask, condenser, and receiver must be scrupu- 10.7.4 Reagents
lously clean and dry. For example, the appara-
• KF reagent
tus, including the condenser, could be cleaned
• Methanol, anhydrous
with potassium dichromate-sulfuric acid clean-
• Sodium tartrate dihydrate, 1 g, dried at 150 °C for 2 h
ing solution, rinsed with water, rinsed with
0.05 N potassium hydroxide solution, rinsed
with alcohol, and then allowed to drain for 10.7.5 Hazards, Cautions, and Waste
10 min. This procedure will minimize the adher- Disposal
ence of water droplets to the surfaces of the con- Use the anhydrous methanol in an operating hood,
denser and the Bidwell-Sterling trap. since the vapors are harmful and are toxic. Otherwise,
2. A correction blank for toluene must be con- adhere to normal laboratory safety procedures. Use
ducted periodically by adding 2–3 mL of dis- appropriate eye and skin protection. The KF reagent
tilled water to 100 mL of toluene in the and anhydrous methanol should be disposed of as
distillation flask and then following the proce- hazardous wastes.
dure in Steps 2–6 in Sect. 10.6.7.
10.6.9 Data and Calculations 10.7.6 Supplies

• Corn flour
Wt. sample (g) Vol. water Moisture
removed (mL) content (%)
• Graduated cylinder, 50 mL
• NFDM
• 2 Spatulas
• Weighing paper
10.7 KARL FISCHER METHOD 10.7.7 Equipment

• Analytical balance, 0.1 g sensitivity
10.7.1 Objective
• KF titration unit, nonautomated unit (e.g., from
Determine the moisture content of NFDM and corn Barnsted Themaline, Berkeley, CA, Aquametry
flour by the Karl Fischer (KF) method. Apparatus) or automated unit
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10.7.8 Procedure 7. Calculate the KF reagent water (moisture)

equivalence (KFReq) in mg H2O/mL:
Instructions are given as for a nonautomated unit and
for analysis in triplicate. If using an automated unit, 36 g / mol S 1000
follow instructions of the manufacturer. KFR eq
230.08 g / mol A
10.7.8.1 Apparatus Setup where:

Assemble titration apparatus and follow instructions
of the manufacturer. The titration apparatus includes S = weight of sodium tartrate dihydrate (g)
the following: buret, reservoir for reagent, magnetic A = mL of KF reagent required for titration of
stirring device, reaction/titration vessel, electrodes, sodium tartrate dihydrate
and circuitry for dead stop endpoint determination.
Note that the reaction/titration vessel of the KF 10.7.8.3 Titration of Sample
apparatus (and the anhydrous methanol within the 1. Prepare samples for analysis and place in reaction
vessel) must be changed after analyzing several sam- vessel as described below.
ples (exact number depends on type of sample).
Remember that this entire apparatus is very fragile. If samples are in powder form:
To prevent contamination from atmospheric mois-
ture, all openings must be closed and protected with • Use an analytical balance to weigh out approxi-
drying tubes. mately 0.3 g of sample, and record the exact sample
weight (S) to the nearest milligram.
10.7.8.2 Standardizing Karl Fischer Reagent • Remove the conductance meter from the reaction
The KF reagent is standardized to determine its vessel, and then transfer your sample to the reac-
water equivalence. Normally, this needs to be done tion vessel through the sample port immediately.
only once a day, or when changing the KF reagent (Use an extra piece of weighing paper to form a
supply. cone-shaped funnel in the sample port, and then
pour your sample through the funnel into the reac-
1. Add approximately 50 mL of anhydrous metha- tion vessel.)
nol to reaction vessel through the sample port.
• Put the conductance meter and stopper back in the
2. Put the magnetic stir bar in the vessel and turn
on the magnetic stirrer. reaction vessel. The color of the solution in the ves-
3. Remove the caps (if any) from drying tube. sel should change to light yellow, and the meter
Turn the buret stopcock to the filling position. will register below the KF zone on the meter.
Hold one finger on the air-release hold in the
rubber bulb and pump the bulb to fill the buret. If any samples analyzed are in liquid form:
Close the stopcock when the KF reagent reaches
the desired level (at position 0.00 mL) in the • Use a 1-mL syringe to draw up about 0.1 mL of
buret. sample. Weigh the syringe with sample on an ana-
4. Titrate the water in the solvent (anhydrous lytical balance and record the exact weight (S1) to
methanol) by adding enough KF reagent to just the nearest milligram.
change the color of the solution from clear or • Inject 1–2 drops of sample into the reaction vessel
yellow to dark brown. This is known as the KF through the sample port and then weigh the syringe
endpoint. Note and record the conductance again (S0), to the nearest milligram.
meter reading. (You may titrate to any point in • Sample weight (S) is the difference of S1 and S0.
the brown KF zone on the meter, but make sure
that you always titrate to that same endpoint S S1 S0
for all subsequent samples in the series.) Allow
the solution to stabilize at the endpoint on the 1. Put the stopper back in the sample port of the
meter for at least 1 min before proceeding to the reaction vessel. The color of the solution in
next step. the vessel should change to light yellow, and
5. Weigh, to the nearest milligram, approximately the meter will register below the KF zone on the
0.3 g of sodium tartrate dihydrate, previously meter.
dried at 150 °C for 2 h. 2. Fill the buret, then titrate the water in the sam-
6. Fill the buret with the KF reagent and then ple as in Sect. 10.7.8.2, Step 4. Record the vol-
titrate the water in the sodium tartrate dihy- ume (mL) of KF reagent used.
drate sample as in Sect. 10.7.8.2, Step 4. Record 3. To titrate another sample, repeat Steps 5–7 above
the volume (mL) of KF reagent used. (Sect. 10.7.8.2) with the new sample. After
114 S.S. Nielsen
titrating several samples (exact number depends by the sample, which is inversely proportional to the
on the nature of the sample), it is necessary to energy absorbed.
start with fresh methanol in a clean reaction ves-
sel. Record the volume (mL) of KF reagent used 10.8.3 Supplies
for each titration.
• Corn flour
• Pans and sample preparation tools for near-infra-
red analyzer
Calculate the moisture content of the sample as follows:
KFR eq K s 10.8.4 Equipment
% H2O 100
S • Near-infrared analyzer
where: 10.8.5 Procedure

KFReq = water equivalence of KF reagent Follow instructions from the manufacturer for the use
(mg H20/mL) of the near-infrared analyzer, regarding the following:
Ks = mL of KF reagent required for titration of
sample • Turning on instrument and warming up
S = weight of sample (mg) • Calibrating instrument
• Testing sample
Karl Fischer reagent water equivalence (KFReq): • Obtaining results
Wt. 10.8.6 Data and Calculations

sodium
tartrate Buret Volume Corn flour % moisture
dihydrate Buret start end titrant Calculated
Rep. (g) (mL) (mL) (mL) KFReq 1 2 3 Mean
1
2
3 10.9 QUESTIONS
X =
1. In separate tables, summarize the results from
Moisture content of samples by Karl Fischer method: the various methods used to determine the mois-
ture content of each type of food sample ana-
Buret Buret Volume Moisture lyzed: (a) corn syrup, (b) liquid milk, (c) corn
Sample Wt. sample start end titrant content flour, (d) NFDM, and (e) basil. Include in each
Rep (g) (mL) (mL) (mL) (%)
table the following for each method: (a) data
from individual determinations, (b) mean value,
(c) standard deviation, (d) observed appearance
of samples, (e) relative advantages of method,
and (f) relative disadvantages of method.
2. Calculate the moisture content of the liquid
milk samples as determined by the forced draft
oven and microwave drying oven methods in
terms of g H2O/g dry matter and include this in
10.8 NEAR-INFRARED ANALYZER a table of results.
10.8.1 Objective Liquid milk moisture content
Determine the moisture content of corn flour using a Mean % Mean g water/g dry
near-infrared (NIR) analyzer. Method moisture matter
Forced draft oven
Microwave drying
10.8.2 Principle
oven
Specific frequencies of infrared radiation are absorbed
by the functional groups characteristic of water (i.e., 3. For the liquid milk sample analyzed for mois-
the –OH stretch of the water molecule). The concen- ture content using a forced draft oven, why was
tration of moisture in the sample is determined by the milk sample partially evaporated on a hot
measuring the energy that is reflected or transmitted plate before being dried in the oven?
www.dbooks.org
4. Of the various methods used to measure the (c) You are considering the use of a toluene distilla-
moisture content of corn syrup, based on con- tion procedure or Karl Fischer titration method
cerns for accuracy and precision, what method for some of your products that are very low in
would you choose if you needed to measure moisture. What are the advantages of each of
moisture content again? Explain your answer. these methods over the hot air oven method in
5. What is the difference between moisture con- the proposed use? What disadvantages or
tent and water activity measurements? potential problems might you encounter with
6. What method would you use to measure the the other two methods?
moisture content of cornflakes for a) rapid qual-
ity control and b) a research project? Explain Acknowledgments This experiment was developed in
your answers. For each method, what would part with materials provided by Dr. Charles E. Carpenter,
you have to do to the cornflakes before measur- Department of Nutrition, Dietetics and Food Sciences,
ing the moisture content? Utah State University, Logan, UT, and by Dr. Joseph
7. Explain the theory/principles involved in pre- Montecalvo, Jr., Department of Food Science and Nutrition,
California Polytechnic State University, San Luis Obispo,
dicting the concentrations of various constitu- CA. Arizona Instrument Corp., Tempe, AZ, is acknowl-
ents in a food sample by NIR analysis. Why do edged for its partial contribution of a Computrac moisture
we say “predict” and not “measure”? What analyzer for use in developing a section of this laboratory
assumptions are being made? exercise.
8. Your quality control lab has been using a hot air
oven method to make moisture determinations
RESOURCE MATERIALS
on various products produced in your plant.
You have been asked to evaluate the feasibility AACC International (2010) Approved methods of analysis,
of switching to new methods (the specific one 11th edn. (On-line). AACC International, St. Paul, MN
would depend on the product) for measuring AOAC International (2016) Official methods of analysis, 20th
moisture content. edn. (On-line). AOAC International, Rockville, MD
Mauer LJ, Bradley RL Jr (2017) Moisture and total solids anal-
(a) Describe how you would evaluate the accuracy ysis, Ch. 15. In: Nielsen SS (ed) Food analysis, 5th edn.
and precision of any new method. Springer, New York
(b) What common problems or disadvantages with Wehr HM, Frank JF (eds.) (2004) Standard methods for the
the hot air oven method would you seek to examination of dairy products, 17th edn. American Public
reduce or eliminate using any new method? Health Association, Washington, DC
11
chapter
Ash Content
Determination
Baraem P. Ismail
St. Paul, MN, USA
11.1 Introduction 11.1.7 Supplies

11.1.1 Background 11.1.8 Equipment
11.1.3 Objective 11.3 Data and Calculations
11.1.4 Principle of Method 11.4 Questions
11.1.5 Chemical
11.1.6 Hazards, Cautions,
and Waste Disposal
www.dbooks.org
118 B.P. Ismail
11.1 INTRODUCTION so remove them from the desiccators only just before
use. Open desiccators slowly to avoid damage and
11.1.1 Background danger from broken glass.
Ash refers to the inorganic residue remaining after
11.1.7 Supplies
either ignition or complete oxidation of organic matter
in a food sample. The inorganic residue consists • Ashed crucibles (numbered) (prewashed with 0.2 N
mainly of the minerals present in the food sample. HCl, heated in a muffle furnace at 550 °C for 24 h,
Determining the ash content is part of the proximate and stored in a desiccator prior to use)
analysis for nutritional evaluation. Also, ashing is the • Variety of food products, e.g., Cheddar cheese,
first step in the preparation of a sample for specific Parmesan cheese, pasteurized processed cheese,
elemental analysis. Two major types of ashing proce- dry baby cereal (rice), whole wheat flour, all-pur-
dures are commonly used, dry ashing and wet ashing. pose flour, quinoa, and dry breakfast cereal
Dry ashing is heating food at elevated temperatures
(500–600 °C) in a muffle furnace. Water and volatiles 11.1.8 Equipment
will evaporate, and organic matter will burn in the
presence of oxygen and convert to CO2 and oxides of
• Desiccator
N2. In contrast, wet ashing is based on oxidizing
• Electric muffle furnace
organic matter using acids and oxidizing agents or
their combination. Minerals are thus solubilized with-
out oxidation. Food with high-moisture content, such
11.2 PROCEDURE
as vegetables, is often dried prior to ashing. Food with
high-fat content, such as meat, may need to be dried Note: Food products such as cheese will need to be
and their fat extracted prior to ashing. The ash content dried before ashing (i.e., also determine the moisture
can be expressed on a wet basis or a dry basis. content). For dry food products such as those listed
above, drying is not needed before ashing. However,
11.1.2 Reading Assignment moisture content must be determined to calculate ash
Harris, G.K., and Marshall, M. R. 2017. Ash analysis. content on a dry weight basis. Follow standard proce-
Ch. 16, in Food Analysis, 5th ed. S.S. Nielsen (Ed.), dures such as those described in the moisture determi-
Springer, New York. nation experiment to obtain the moisture content of all
samples to be ashed.
11.1.3 Objective
Determine the ash content of a variety of food prod- 1. Remove ashed crucibles from the desiccator
ucts by the dry ashing technique and express on a wet and record weight and number of crucible in
weight basis and dry weight basis. the table.
2. Accurately weigh ca. 2 g of sample (note that
11.1.4 Principle of Method cheese samples are pre-dried and placed in des-
iccators) into the crucible, and record weight on
Organic materials are incinerated at elevated tempera- the spreadsheet. Prepare triplicate samples for
tures (550 °C) in a muffle furnace, and inorganic mat- each type of food product analyzed.
ter (ash) remains. Ash content is measured by weight 3. Place crucibles in muffle oven at 550 °C for 24 h.
of inorganic matter remaining. 4. Turn off the muffle furnace and allow it to cool
(might take a few hours).
11.1.5 Chemical 5. Remove crucibles from the muffle furnace and
place into a desiccator to cool (note that this
CAS No. Hazards
may need to be done by a teaching assistant).
Hydrochloric acid (HCl) 7647-01-0 Corrosive Return the following day to weigh the ashed
sample and record weight of crucible plus
11.1.6 Hazards, Cautions, and Waste ashed sample in the table.
Disposal
Concentrated hydrochloric acid is corrosive; avoid 11.3 DATA AND CALCULATIONS
breathing vapors and contact with skin and clothes.
The muffle furnace is extremely hot. Use gloves and
tongs when handling crucibles. The crucibles have Weight of ash weight of crucible andash
been dried and stored in desiccators prior to weighing.
– weight of crucible
They will pick up moisture by sitting on the counter,
Chapter 11 • Ash Content Determination 119
% Ash weight of ash / originalsampleweight 100 weight basis (wwb), using the moisture % obtained in
the moisture analysis experiment.
Report the average ash %, standard deviation, and
coefficient of variation for the food product analyzed. Converting wet basis to dry basis:
Also calculate average ash % on a dry basis, using the
average % moisture value determined in the moisture % ash on dry basis % ash on wet basis 100
analysis experiment. /100 % moisture content
Converting dry basis to wet basis:
Note: For cheese samples, the % ash obtained is on a
dry weight basis (dwb), since samples to be ashed % ash on wet basis % ash on dry basis
need to be pre-dried. Also, calculate % ash on a wet 100 % moisture content / 100
Crucible + Crucible + Moisture (%)

Crucible Crucible wt. un-ashed Un-ashed ashed (previously Ash Ash
Rep number (g) sample (g) sample (g) sample (g) Ash (g) determined) (% wwb) (% dwb)
Sample A
1
2
3
X = X =
SD = SD =
CV = CV =
Sample B
1
2
3
X = X =
SD = SD =
CV = CV =
11.4 QUESTIONS 4. Why was it necessary to pre-acid wash, pre-ash,

and dry in a desiccator the crucibles prior to use?
1. For this laboratory we used a dry ashing tech- 5. Were your results comparable to USDA reported
nique, in what instance would you want to use values? Explain any discrepancies.
a wet ashing technique?
2. What are the advantages and disadvantages to RESOURCES
using a dry ashing technique?
3. What are the disadvantages of using a wet ash- Harris GK, Marshall MR (2017) Ash analysis, Ch. 16. In:
ing technique? Nielsen SS (ed) Food Analysis, 5th edn. Springer, New York
www.dbooks.org
12
chapter
Fat Content
Determination
S.Suzanne Nielsen ( ) *

Charles E.Carpenter
Logan, UT, USA
12.1 Introduction 12.2.3 Hazards, Precautions, and

12.1.1 Background Waste Disposal
12.2 Soxhlet Method 12.2.6 Procedure
12.2.1 Principle of Method 12.2.7 Data and Calculations
12.3 Goldfish Method 12.4.5 Equipment
12.3.1 Principle 12.4.6 Notes
12.3.3 Hazards, Precautions, and Waste 12.4.8 Data and Calculations
Disposal 12.4.9 Questions
12.3.4 Supplies 12.5 Babcock Method
12.3.5 Equipment 12.5.1 Principle
12.3.6 Procedure 12.5.2 Note
12.3.7 Data and Calculations 12.5.3 Chemicals
12.3.8 Questions 12.5.4 Hazards, Precautions, and Waste
12.4 Mojonnier Method Disposal
12.4.1 Principle 12.5.5 Supplies
12.4.2 Chemicals 12.5.6 Equipment
12.4.3 Hazards, Precautions, and Waste 12.5.7 Procedure
Disposal 12.5.8 Data and Calculations
12.4.4 Supplies 12.5.9 Questions
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Chapter 12 • Fat Content Determination 123
12.1 INTRODUCTION 12.2 SOXHLET METHOD
12.1.1 Background 12.2.1 Principle of Method

The term “lipid” refers to a group of compounds that are Fat is extracted, semicontinuously, with an organic sol-
sparingly soluble in water but show variable solubility vent. Solvent is heated and volatilized, then is con-
in a number of organic solvents (e.g., ethyl ether, petro- densed above the sample. Solvent drips onto the
leum ether, acetone, ethanol, methanol, benzene). The sample and soaks it to extract the fat. At 15–20 min
lipid content of a food determined by extraction with intervals, the solvent is siphoned to the heating flask,
one solvent may be quite different from the lipid content to start the process again. Fat content is measured by
as determined with another solvent of different polar- the weight loss of sample or weight of fat removed.
ity. Fat content is determined often by solvent extraction
methods (e.g., Soxhlet, Goldfish, Mojonnier), but it also 12.2.2 Chemicals
can be determined by nonsolvent wet extraction meth-
ods (e.g., Babcock, Gerber) and by instrumental meth- CAS no. Hazards
ods that rely on the physical and chemical properties
of lipids (e.g., infrared, density, X-ray absorption). The Petroleum 8032-32-4 Harmful, highly flammable,
ether dangerous for environment
method of choice depends on a variety of factors, includ-
ing the nature of the sample (e.g., dry versus moist), the (or ethyl 60-29-7 Harmful, extremely flammable
purpose of the analysis (e.g., official nutrition labeling ether)
or rapid quality control), and instrumentation available
(e.g., Babcock uses simple glassware and equipment; 12.2.3 Hazards, Precautions, and Waste
infrared requires an expensive instrument). Disposal
This experiment includes the Soxhlet, Goldfish,
Mojonnier, and Babcock methods. If samples ana- Petroleum ether and ethyl ether are fire hazards; avoid
lyzed by these methods can be tested by an instrumen- open flames, breathing vapors, and contact with skin.
tal method for which equipment is available in your Ether is extremely flammable, is hygroscopic, and may
laboratory, data from the analyses can be compared. form explosive peroxides. Otherwise, adhere to nor-
Low-moisture snack foods are suggested for analysis mal laboratory safety procedures. Wear gloves and
and comparison by the Soxhlet and Goldfish meth- safety glasses at all times. Petroleum ether and ether
ods, and milk by the Mojonnier and Babcock methods. liquid wastes must be disposed of in designated haz-
However, other appropriate foods could be substi- ardous waste receptacles.
tuted, and results compared between methods. Also,
the experiment specifies the use of petroleum ether as 12.2.4 Supplies
the solvent for the Soxhlet and Goldfish methods, for • 3 Aluminum weighing pans, predried in 70 °C vac-
reasons of cost and safety. Anhydrous ethyl ether could uum oven for 24 h
be used for both extraction methods, but appropriate • Beaker, 250 mL
precautions must be taken in handling the solvent, plus • Cellulose extraction thimbles, predried in 70 °C
the snack foods would need to be dried before extrac- vacuum oven for 24 h
tion. Using low-moisture content snack foods (versus • Desiccator
dried samples) admittedly introduces a low bias with • Glass boiling beads
petroleum ether extraction, and requires that separate • Glass wool, predried in 70 °C vacuum oven for 24 h
samples be analyzed for moisture content, to correct • Graduated cylinder, 500 mL
for moisture in the fat content calculations. In this labo- • Mortar and pestle
ratory exercise, the moisture content data are used to • Plastic gloves
express the fat content on a wet and dry weight basis. • Snack foods (need to be fairly dry and able to be
ground with a mortar and pestle)
12.1.2 Reading Assignment • Spatula
Ellefson, W.C. 2017. Fat analysis. Ch. 17, in Food • Tape (to label beaker)
Analysis, 5rd ed. S.S. Nielsen (Ed.), Springer, New York. • Tongs
• Weighing pan (to hold 30-g snack food)
12.1.3 Objective
12.2.5 Equipment
Determine the lipid contents of various snack foods by
the Soxhlet and Goldfish methods, and determine the • Analytical balance
lipid content of milk by the Mojonnier and Babcock • Soxhlet extractor, with glassware
methods. • Vacuum oven
12.2.6 Procedure dry in a vacuum oven at 70 °C, 25 in. mercury,

for 24 h. Cool dried samples in a desiccator then
(Instructions are given for analysis in triplicate.)
reweigh.
1. Record the fat content of your snack food product 7. Correct for moisture content of product as
as reported on the package label. Also record serv- follows:
ing size so you can calculate g fat/100-g product.
(a) Using the remainder of the ground sample
2. Slightly grind ~30-g sample with mortar and
and three dried, labeled, and weighed alu-
pestle (excessive grinding will lead to greater
minum sample pans, prepare triplicate
loss of fat in mortar).
2–3-g samples for moisture analysis.
3. Wearing plastic gloves, remove three predried
(b) Dry sample at 70 °C, 25 in. mercury, for 24 h
cellulose extraction thimbles from the desicca-
in a vacuum oven.
tor. Label the thimbles on the outside with your
(c) Reweigh after drying, and calculate mois-
initials and a number (use a lead pencil), then
ture content of the sample.
weigh accurately on an analytical balance.
4. Place ~2–3 g of sample in the thimble. Reweigh.
Place a small plug of dried glass wool in each
thimble. Reweigh. Using the weights recorded in the tables below, calcu-
5. Place the three samples in a Soxhlet extractor. late the percent fat (wt/wt) on a wet weight basis as
Put ~350-mL petroleum ether in the flask, add determined by the Soxhlet extraction method. If the fat
several glass boiling beads, and extract for 6 h or content of the food you analyzed was given on the
longer. Place a 250-mL beaker labeled with your label, report this theoretical value.
name below your samples on the Soxhlet extrac-
tion unit. Samples in thimbles will be placed in Name of snack food
the beaker after extraction and before drying. Label g fat/serving
6. Remove thimbles from the Soxhlet extractor Label serving size (g)
using tongs, air dry overnight in a hood, then Label g fat/100-g product
Data from Soxhlet extraction:
Dry, extracted
Wet Wet sample + sample + Fat + Fat +
Wet sample + sample thimble + thimble + moisture moisture % Fat % Fat
Rep Thimble (g) thimble (g) (g) glass wool (g) glass wool (g) (g) (%) (wwb) (dwb)
1
2
3
X =
SD =
Wet sample thimble glass wool , g Dry , extracted sample thimble glass wool , g
% Fat Moisture 100
Wet sample,g
Data from moisture analysis:
Rep Pan (g) Pan + wet sample (g) Pan + dried sample (g) % Moisture
1
2
3
X =
SD =
www.dbooks.org
Calculation of % moisture: 12.3.6 Procedure

Wt of wet sample Pan Wt of dried sample Pan 100 (Instructions are given for analysis in triplicate.)
Wt of wet sample Pan Wt of pan Note: Analyze samples in triplicate.
1. Follow Steps 1–4 in Sect. 12.2.6.
Calculation of % fat, wet weight basis: 2. Place the thimble in the Goldfish condenser
bracket. Push the thimble up so that only about
% Fat wt / wt , wet weight basis 1–2 cm is below the bracket. Fill the reclaiming
% Fat % Moisture % Moisture beaker with petroleum ether (50 mL) and trans-
fer to beaker. Seal beaker to apparatus using
(Note: Use average % moisture in this gasket and metal ring. Start the water flow
calculation.) through the condenser. Raise the hot plate up to
the beaker, turn on, and start the ether boiling.
Extract for 4 h at a condensation rate of 5–6
Calculate % fat on a dry weight basis:
drops per second.
% Fat on dry basis ( % Fat on wet basis 100) / 3. Follow Steps 6 and 7 in Sect. 12.2.6.
100 % Moisture content
12.2.8 Questions Record the data and do the calculations for the
Goldfish method using the same tables and equations
1. The Soxhlet extraction procedure utilized petro- given for the Soxhlet method in Sect. 12.2.7. Calculate
leum ether. What were the advantages of using the percent fat (wt/wt) on a wet weight basis. If the fat
it rather than ethyl ether? content of the food you analyzed was given on the
2. What were the advantages of using the Soxhlet label, report this theoretical value:
extraction method rather than the Goldfish
extraction method? Name of snack food
3. If the fat content measured here differed from Label g fat/serving
that reported on the nutrition label, how might Label serving size (g)
this be explained? Label g fat/100-g product
12.3 GOLDFISH METHOD 12.3.8 Questions

12.3.1 Principle 1. What would be the advantages and disadvan-
tages of using ethyl ether rather than petroleum
Fat is extracted, continuously, with an organic solvent.
ether in a solvent extraction method, such as the
Solvent is heated and volatilized, then is condensed
Goldfish method?
above the sample. Solvent continuously drips through
2. What were the advantages of using the Goldfish
the sample to extract the fat. Fat content is measured
extraction method rather than the Soxhlet
by the weight loss of sample or weight of fat removed.
extraction method?
3. If the fat content measured here differed from
12.3.2 Chemicals that reported on the nutrition label, how might
Same as for Sect. 12.2.2. this be explained?
12.3.3 Hazards, Precautions, and Waste

Disposal 12.4 MOJONNIER METHOD
Same as for Sect. 12.2.2. 12.4.1 Principle
12.3.4 Supplies Fat is extracted with a mixture of ethyl ether and petro-
leum ether. The extract containing the fat is dried and
Same as for Sect. 12.2.2. expressed as percent fat by weight.
The assay uses not only ethyl ether and petroleum
12.3.5 Equipment ether, but also ammonia and ethanol. Ammonia dis-
• Analytical balance solves the casein and neutralizes the acidity of the
• Goldfish extraction apparatus product to reduce its viscosity. Ethanol prevents gela-
• Hot plate tion of the milk and ether, and aids in the separation of
• Vacuum oven the ether-water phase. Ethyl ether and petroleum ether
serve as lipid solvents, and petroleum ether decreases 12.4.7 Procedure

the solubility of water in the ether phase.
12.4.2 Chemicals 1. Turn on power unit and temperature controls
for oven and hot plate on the fat side of the
CAS no. Hazards Mojonnier unit.
2. Warm milk samples to room temperature and
Ammonium 1336-21-6 Corrosive, dangerous for the
hydroxide environment
mix well.
Ethanol 64-17-5 Highly flammable 3. When oven is at 135 °C, heat cleaned fat dishes
Petroleum 8032-32-4 Harmful, highly flammable, in oven under a vacuum of 20-in. mercury for
ether dangerous for environment 5 min. Handle dishes from this point on with
(Ethyl 60-29-7 Harmful, extremely tongs or gloves. Use three dishes for each type
ether) flammable of milk samples, and two dishes for the reagent
blank.
12.4.3 Hazards, Precautions, and Waste 4. Cool dishes in cooling desiccator for 7 min.
Disposal 5. Weigh dishes, record weight of each dish and its
identity, and place dishes in desiccator until
Ethanol, ethyl ether, and petroleum ether are fire haz- use.
ards; avoid open flames, breathing vapors, and contact 6. Weigh samples accurately (ca. 10 g) into
with skin. Ether is extremely flammable, is hygroscopic, Mojonnier flasks. If weighing rack is used, fill
and may form explosive peroxides. Ammonia is a cor- curved pipettes and place in rack on the bal-
rosive; avoid contact and breathing vapors. Otherwise, ance. Weigh each sample by difference.
adhere to normal laboratory safety procedures. Wear 7. Add chemicals for the first extraction in the
gloves and safety glasses at all times. Petroleum ether order and amounts given below. After each
and ether liquid wastes must be disposed of in desig- addition of chemicals, stopper the flask and
nated hazardous waste receptacles. The aqueous waste shake by inverting for 20 s.
can go down the drain with a water rinse.
12.4.4 Supplies First extraction Second extraction

Chemicals Step Amount (mL) Step Amount (mL)
• Milk, whole and 2 % fat
Ammonia 1 1.5 – None
• Mojonnier extraction flasks, with stoppers
Ethanol 2 10 1 5
• Mojonnier fat dishes with lids Ethyl ether 3 25 2 15
• Plastic gloves Petroleum ether 4 25 3 15
• Tongs
12.4.5 Equipment 8. Place the extraction flasks in the holder of the

centrifuge. Place both flask holders in the cen-
trifuge. Operate the centrifuge to run at 30 turns
• Hot plate
in 30 s, to give a speed of 600 rpm (revolutions
• Mojonnier apparatus (with centrifuge, vacuum
per minute). [In lieu of centrifuging, the flasks
oven, and cooling desiccator)
can be allowed to stand 30 min until a clear sep-
aration line forms, or three drops of phenol-
12.4.6 Notes
phthalein indicator (0.5% w/v ethanol) can be
Reagents must be added to the extraction flask in the added during the first extraction to aid in deter-
following order: water, ammonia, alcohol, ethyl ether, mining the interface.]
and petroleum ether. The burets on the dispensing 9. Carefully pour off the ether solution of each
cans or tilting pipets are graduated for measuring the sample into a previously dried, weighed, and
proper amount. Make triplicate determinations on cooled fat dish. Most or all of the ether layer
both the sample and reagent blanks. The procedure should be poured into the dish, but none of the
given here is for fresh milk. Other samples may need remaining liquid must be poured into the dish.
to be diluted with distilled water in Step 2 and require 10. Place dishes with ether extract on hot plate
different quantities of reagents in subsequent steps. under glass hood of Mojonnier unit, with power
Consult the instruction manual or AOAC International unit running. (If this hot plate is not available,
Official Methods of Analysis for samples other than fresh use a hot plate placed in a hood, with the hot
milk. plate at 100 °C.)
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11. Repeat the extraction procedure a second time 12.4.9 Questions

for the samples in the Mojonnier flasks, follow-
1. List possible causes for high and low results in
ing the sequence and amount given in the table
a Mojonnier fat test.
above. Again, after each addition of chemicals,
2. How would you expect the elimination of alco-
stopper the flask and shake by inverting for
hol from the Mojonnier procedure to affect the
20 s. Centrifuge the flasks again, as described
results? Why?
above. Distilled water may be added now to the
3. How would you propose to modify the
flask to bring the dividing line between ether
Mojonnier procedure to test a solid, nondairy
and water layers to the center of the neck of
product? Explain your answer.
flask. If this is done, repeat the centrifugation.
12. Pour ether extract into the respective fat dish
(i.e., the ether for a specific sample should be
12.5 BABCOCK METHOD
poured into the same fat dish used for that sam-
ple from the first extraction), taking care to 12.5.1 Principle
remove all the ether but none of the other liquid
in the flask. Sulfuric acid is added to a known amount of milk sam-
13. With lids ajar, complete the evaporation of ether, ple in a Babcock bottle. The acid digests the protein,
either very carefully on the hot plate (this can generates heat, and releases the fat. Centrifugation
be problematic and a fire hazard) or open in a and hot water addition isolate the fat into the gradu-
hood. In using a hot plate, the ether should boil ated neck of the bottle. The Babcock fat test uses a
slowly; not fast enough to cause splattering. If volumetric measurement to express the percent of fat
the plate appears to be too hot and boiling is in milk or meat by weight.
too fast, only part of the dish should be placed
on the hot plate. If instead using an operating 12.5.2 Note
hood, leave collection containers with lids ajar The fat column in the Babcock test should be at
to have them evaporated by the next day. 57–60 °C when read. The specific gravity of liquid fat
14. When all the ether has been evaporated from at that temperature is approximately 0.90 g per
the dishes, place the dishes in the vacuum oven mL. The calibration on the graduated column of the
70–75 °C for 10 min with a vacuum of at least test bottle reflects this fact and enables one to make a
20 in. volumetric measurement, which expresses the fat con-
15. Cool the dishes in the desiccator for 7 min. tent as percent by weight.
16. Accurately weigh each dish with fat. Record
weight.
12.5.3 Chemicals
12.4.8 Data and Calculations CAS No. Hazards

Glymol (red reader) 8042-47-5 Toxic, irritant
Calculate the fat content of each sample. Subtract the
Sulfuric acid 7664-93-9 Corrosive
average weight of the reagent blank from the weight of
each fat residue in the calculation.
Rep Milk start (g) Milk end (g) Milk tested (g) Dish (g) Dish + fat (g) Calculated % fat
Reagent blanks:
1 –
2 –
Samples: X =
1
2
3
X =
SD =

% Fat 100 wt dish fat wt dish avg wt blank residue / wt sample
12.5.4 Hazards, Precautions, and Waste horizontal configuration. Be sure that the heater
Disposal of the centrifuge is on.
4. Centrifuge the bottles for 5 min after reaching
Concentrated sulfuric acid is extremely corrosive;
the proper speed (speed will vary depending
avoid contact with skin and clothes and breathing
upon the diameter of the centrifuge head).
vapors. Wear gloves and safety glasses at all times.
5. Stop the centrifuge and add soft hot water
Otherwise, adhere to normal laboratory safety proce-
(60 °C) until the liquid level is within 0.6 cm of
dures. Sulfuric acid and glymol wastes must be dis-
the neck of the bottle. Carefully permit the
posed in a designated hazardous waste receptacle.
water to flow down the side of the bottle. Again,
For safety and accuracy reasons, dispense the
centrifuge the bottles for 2 min.
concentrated sulfuric acid from a bottle fitted with a
6. Stop the centrifuge and add enough soft hot
repipettor (i.e., automatic bottle dispenser). Fit the dis-
water (60 °C) to bring the liquid column near
penser with a thin, semirigid tube to dispense directly
the top graduation of the scale. Again, centri-
and deep into the Babcock bottle while mixing con-
fuge the bottles for 1 min.
tents. Set the bottle with dispenser on a tray to collect
7. Remove the bottles from the centrifuge and place
spills. Wear corrosive- and heat-resistant gloves when
in a heated (55–60 °C, preferably 57 °C) water
mixing the sulfuric acid with samples.
bath deep enough to permit the fat column to be
below the water level of the water bath. Allow
12.5.5 Supplies
bottles to remain at least 5 min before reading.
• 3 Babcock bottles 8. Remove the samples from the water bath one at
• Babcock caliber (or shrimp divider) a time, and quickly dry the outside of the bottle.
• Measuring pipette, 10 mL Add glymol (red reader) to the top of fat layer.
• Pipette bulb or pump Immediately use a divider or caliper to measure
• Plastic gloves the fat column to the nearest 0.05%, holding the
• Standard milk pipette (17.6 mL) bottle in a vertical position at eye level. Measure
• Thermometer from the highest point of the upper meniscus to
the bottom of the lower meniscus.
12.5.6 Equipment 9. Reject all tests in which the fat column is milky
or shows the presence of curd or charred matter,
• Babcock centrifuge
or in which the reading is indistinct or uncer-
• Water bath
tain. The fat should be clear and sparkling, the
upper and lower meniscus clearly defined, and
12.5.7 Procedure the water below the fat column should be clear.
(Instructions are given for analysis in triplicate.) 10. Record the readings of each test and determine
the mean % fat and the standard deviation.
1. Adjust milk sample to ca. 38 °C and mix until
homogenous. Using a standard milk pipette,
pipette 17.6 mL of milk into each of three Babcock
bottles. After the pipette has emptied, blow out
Rep Measured % fat
the last drops of milk from the pipette tip into the
bottle. Allow milk samples to adjust to ca. 22 °C. 1
2. Dispense ca. 17.5 mL of sulfuric acid (specific 2
gravity 1.82–1.83) and carefully add into the test 3
bottle, with mixing during and between addi- X =
tions, taking care to wash all traces of milk into the SD =
bulb of the bottle. Time for complete acid addition
should not exceed 20 s. Mix the milk and acid
12.5.9 Questions
thoroughly. Be careful not to get any of the mix-
ture into the column of the bottle while shaking. 1. What are the possible causes of charred parti-
Heat generated behind any such lodged mixture cles in the fat column of the Babcock bottle?
may cause a violent expulsion from the bottle. 2. What are the possible causes of undigested curd
3. Place bottles in centrifuge heated to 60 °C. Be in the Babcock fat test?
sure bottles are counterbalanced. Position bot- 3. Why is sulfuric acid preferred over other acids
tles so that bottlenecks will not be broken in for use in the Babcock fat test?
www.dbooks.org
RESOURCE MATERIALS Ellefson WC (2017) Fat analysis. Ch. 17. In: Nielsen SS (ed)
Food analysis, 5th edn. Springer, New York
AOAC International (2016) Official methods of analysis, 20th Wehr HM, and Frank JF (eds) (2004) Standard methods for
edn, (On-line). AOAC International, Rockville, MD the examination of dairy products. 17th edn. American
Public Health Administration, Washington, DC
13
Chapter
Protein Nitrogen
Determination
S.Suzanne Nielsen
13.1 Introduction 13.2.9 Data and Calculations

13.1.1 Background 13.2.10 Questions
13.1.2 Reading Assignment 13.3 Nitrogen Combustion Method
13.1.3 Notes 13.3.1 Objective
13.2 Kjeldahl Nitrogen Method 13.3.2 Principle of Method
13.2.2 Principle of Method 13.3.4 Hazards, Cautions,
13.2.3 Chemicals and Waste Disposal
13.2.4 Reagents 13.3.5 Supplies
13.2.5 Hazards, Cautions, 13.3.6 Equipment
and Waste Disposal 13.3.7 Procedure
13.2.7 Equipment 13.3.9 Questions
13.2.8 Procedure
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132 S.S. Nielsen
13.1 INTRODUCTION 13.2.3 Chemicals
13.1.1 Background CAS No. Hazards
The protein content of foods can be determined by Boric acid (H3BO3) 10043-35-3
numerous methods. The Kjeldahl method and the Bromocresol green 76-60-8
nitrogen combustion (Dumas) method for protein Ethanol, 95 % 64-17-5 Highly
flammable
analysis are based on nitrogen determination. Both
Hydrochloric acid, 7647-01-0 Corrosive
methods are official for the purposes of nutrition
conc. (HCl)
labeling of foods. While the Kjeldahl method has Methyl red 493-52-7
been used widely for over 100 years, the recent avail- Sodium hydroxide 1310-73-2 Corrosive
ability of automated instrumentation for the Dumas (NaOH)
method in many cases is replacing use of the Kjeldahl Sulfuric acid, conc. 7664-93-9 Corrosive
method. (H2SO4)
Kjeldahl digestion Irritant
13.1.2 Reading Assignment tablets
Potassium sulfate 7778-80-5
Chang, S.K.C., Zhang, Y. 2017. Protein analysis. Ch. 18, (K2SO4)
in Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, Cupric sulfate 7758-98-7
New York. Titanium dioxide 13463-67-7
(TiO2)
13.1.3 Notes Tris (hydroxymethyl) 77-86-1 Irritant
aminomethane
Both the Kjeldahl and nitrogen combustion methods (THAM)
can be done without automated instrumentation, but
they are commonly done with automated instruments.
The descriptions below are based on the availability of 13.2.4 Reagents
such automated instrumentation. If protein content of (** It is recommended that these solutions be prepared
samples analyzed by Kjeldahl and/or nitrogen com- by the laboratory assistant before class.)
bustion has been estimated in a previous experiment
by near infrared analysis, values can be compared • Sulfuric acid (concentrated, N-free)
between methods. • Catalyst/salt mixture (Kjeldahl digestion tablets)
Contains potassium sulfate, cupric sulfate, and
titanium dioxide
13.2 KJELDAHL NITROGEN METHOD Note: There are several types of Kjeldahl digestion
tablets that contain somewhat different chemicals.
13.2.1 Objective • Sodium hydroxide solution, 50 %, w/v, NaOH in
deionized distilled (dd) water **
Determine the protein content of corn flour using the
Dissolve 2000 g sodium hydroxide (NaOH) pel-
Kjeldahl method.
lets in ~3.5 L dd water. Cool. Add dd water to
make up to 4.0 L.
13.2.2 Principle of Method • Boric acid solution **
The Kjeldahl procedure measures the nitrogen con- In a 4-L flask, dissolve 160-g boric acid in ca. 2 L
tent of a sample. The protein content then can be cal- boiled, and still very hot, dd water. Mix and then
culated assuming a ratio of protein to nitrogen for the add an additional 1.5 L of boiled, hot dd water.
specific food being analyzed. The Kjeldahl procedure Cool to room temperature under tap water (cau-
can be basically divided into three parts: (1) digestion: glassware may break due to sudden cool-
tion, (2) distillation, and (3) titration. In the digestion ing) or leave overnight. When using the rapid
step, organic nitrogen is converted to an ammonium procedure, the flask must be shaken occasionally
in the presence of a catalyst at approximately to prevent recrystallization of the boric acid.
370 °C. In the distillation step, the digested sample is Add 40 mL of bromocresol green solution (100-
made alkaline with NaOH, and the nitrogen is dis- mg bromocresol green/100-mL ethanol) and
tilled off as NH3. This NH3 is “trapped” in a boric 28 mL of methyl red solution (100-mg methyl
acid solution. The amount of ammonia nitrogen in red/100-mL ethanol). Dilute to 4 L of water and
this solution is quantified by titration with a standard mix carefully. Transfer 25 mL of the boric acid
HCl solution. solution to a receiver flask and distill a digested
A reagent blank is carried through the analysis blank (a digested catalyst/salt/acid mixture).
and the volume of HCl titrant required for this blank is The contents of the flask should then be a neutral
subtracted from each determination. gray. If not, titrate with 0.1 N NaOH solution
Chapter 13 • Protein Nitrogen Determination 133
until this color is obtained. Calculate the amount ric acid and sodium hydroxide has been largely
of NaOH solution necessary to adjust the boric neutralized (check pH to ensure it is pH 3–9), so it can
acid solution in the 4-L flask with the formula: be discarded down the drain with a water rinse.
However, for disposing any chemical wastes, follow
mL 0.1 N NaOH
mL titer 4000 mL good laboratory practices outlined by environmental
25 mL health and safety protocols at your institution.
For safety and accuracy reasons, dispense the con-
Add the calculated amount of 0.1 N NaOH solu- centrated sulfuric acid from a bottle fitted with a repi-
tion to the boric acid solution. Mix well. Verify pettor (i.e., automatic dispenser). Fit the dispenser with
the adjustment results by distilling a new blank a thin, semirigid tube to dispense directly into the
sample. Place adjusted solution into a bottle Kjeldahl tube. Set the bottle with dispenser on a tray to
equipped with a 50-mL repipettor. collect spills.
• Standardized HCl solution**
Dilute 3.33-mL conc. HCl to 4 L with dd water. 13.2.6 Supplies
Empty old HCl solution from the titrator reser-
voir and rinse three times with a small portion of (Used by students)
the new HCl solution. Fill the titrator with the
new HCl solution to be standardized. Using a • Corn flour (not dried)
volumetric pipet, dispense 10-mL aliquots of the • 5 Digestion tubes
THAM solution prepared as described below • 5 Erlenmeyer flasks, 250 mL
into three Erlenmeyer flasks (50 mL). Add 3–5 • Spatula
drops indicator (3 parts 0.1 % bromocresol green • Weighing paper
in ethanol to 1 part of 0.2 % methyl red in etha-
nol) to each flask and swirl. Titrate each solution 13.2.7 Equipment
with the HCl solution to a light pink endpoint. • Analytical balance
Note the acid volume used and calculate the nor- • Automatic titrator
mality as described below. • Kjeldahl digestion and distillation system
Calculation to standardize HCl solution:
13.2.8 Procedure
mL THAM THAM Normality (Instructions are given for analysis in triplicate. Follow
Normality
average acid volume AAV manufacturer’s instructions for specific Kjeldahl
digestion and distillation system used. Some instruc-
20 mL 0.01 N
tions given here may be specific for one type of
AAV Kjeldahl system.)
Write the normality of the standardized HCl
solution on the stock container.
13.2.8.1 Digestion
1. Turn on digestion block and heat to appropriate
• Tris (hydroxymethyl) aminomethane (THAM)
temperature.
Solution – (0.01 N) **
2. Accurately weigh approximately 0.1 g of corn
Place 2 g of THAM in a crucible. Leave in a dry-
flour. Record the weight. Place corn flour in
ing oven (95 °C) overnight. Let cool in a desicca-
digestion tube. Repeat for two more samples.
tor. In a 1-L volumetric flask, dissolve 1.2114 g of
3. Add one catalyst tablet and appropriate volume
oven dried THAM in distilled water. Dilute to
(e.g., 7 mL) of concentrated sulfuric acid to each
volume.
tube with corn flour. Prepare duplicate blanks:
one catalyst tablet + volume of sulfuric acid
13.2.5 Hazards, Cautions, and Waste
used in the sample + weigh paper (if weigh
Disposal
paper was added with the corn flour samples).
Concentrated sulfuric acid is extremely corrosive; 4. Place rack of digestion tubes on digestion block.
avoid breathing vapors and contact with skin and Cover digestion block with exhaust system
clothes. Concentrated sodium hydroxide is a corro- turned on.
sive. Wear corrosion-resistant gloves and safety glasses 5. Let samples digest until digestion is complete.
at all times. Perform the digestions in an operating The samples should be clear (but neon green),
hood with an aspirating fume trap attached to the with no charred material remaining.
digestion unit. Allow samples to cool in the hood 6. Take samples off the digestion block and allow
before removing the aspirating fume trap from the to cool with the exhaust system still turned on.
digestion unit. Otherwise, adhere to normal labora- 7. Carefully dilute digest with an appropriate vol-
tory safety procedures. The waste of combined sulfu- ume of dd water. Swirl each tube.
www.dbooks.org
134 S.S. Nielsen
13.2.8.2 Distillation * Normality is in mol/1000 mL.

1. Follow appropriate procedure to start up distil- ** Corrected acid vol = (mL std. acid for sample)
lation system. – (mL std. for blank)
2. Dispense appropriate volume of boric acid
% Protein = % N × Protein Factor
solution into the receiving flask. Place receiving
flask on distillation system. Make sure the tube
% Protein, wwb
coming from the distillation of the sample is = % Protein, dwb
[% Solids (wet)/100 %]
submerged in the boric acid solution.
3. Put sample tube from Sect. 13.2.8.1 in place,
making sure it is seated securely, and proceed
with the distillation until completed. In this dis- Vol. HCl % %
tillation process, a set volume of NaOH solution Sample titrant % Protein, Protein,
will be delivered to the tube and, a steam gen- Rep wt. (g) (mL) Nitrogen wwb dwb
erator will distill the sample for a set period of Blank 1 – – – –
time. 2 – – – –
4. Upon completing distillation of one sample, pro- X =
ceed with a new sample tube and receiving flask. Sample 1
5. After completing distillation of all samples, fol- 2
low manufacturer’s instructions to shut down 3
the distillation unit. X = X =
SD = SD =
13.2.8.3 Titration
1. Record the normality of the standardized HCl
solution, as determined by the teaching 13.2.10 Questions
assistant.
2. If using an automated pH meter titration system, 1. If the alkali pump timer on the distillation sys-
follow manufacturer’s instructions to calibrate the tem was set to deliver 25 mL of 50 % NaOH and
instrument. Put a magnetic stir bar in the receiver 7 mL of concentrated H2SO4 was used to digest
flask from Sect. 13.2.8.2 and place it on a stir plate. the sample, how many milliliters of the 50 %
Keep the solution stirring briskly while titrating, NaOH is actually required to neutralize the
but do not let the stir bar hit the electrode. Titrate amount of sulfuric acid used in the digestion?
each sample and blank to an endpoint pH of 4.2. How would your results have been changed if
Record volume of HCl titrant used. the alkali pump timer malfunctioned and deliv-
3. If using a colorimetric endpoint, put a magnetic ered only 15 mL of the 50 % NaOH? (Molarity of
stir bar in the receiver flask, place it on a stir conc. H2SO4 = 18.)
plate, and keep the solution stirring briskly while 2. Could phenolphthalein be used as an indicator
titrating. Titrate each sample and blank with the in the Kjeldahl titration? Why or why not?
standardized HCl solution to the first faint gray 3. Describe the function of the following chemi-
color. Record volume of HCl titrant used. cals used in this determination:
(a) Catalyst pellet
13.2.9 Data and Calculations (b) Borate
Calculate the percent nitrogen and the percent protein (c) H2SO4
for each of your duplicate or triplicate corn flour sam- (d) NaOH
ples, then determine average values. The corn flour
sample you analyzed was not a dried sample. Report 4. Why was it not necessary to standardize the
percent protein results on a wet weight basis (wwb) boric acid solution?
and on a dry weight basis (dwb). Assume a moisture 5. Explain how the factor used to calculate the per-
content of 10 % (or use the actual moisture content if cent protein for your product was obtained, and
previously determined on this corn flour sample). Use why the protein factors for some other cereal
6.25 for the nitrogen to protein conversion factor. grains (e.g., wheat, oats) differ from that for
corn.
6. For each of the disadvantages of the Kjeldahl
corrected acid method, give another protein analysis method
vol. mL ** 14 g N that overcomes (at least partially) that
% N Normality HCl * 100 disadvantage.
g of sample mol
Chapter 13 • Protein Nitrogen Determination 135
13.3 NITROGEN COMBUSTION METHOD Weigh appropriate amount of sample into a tared
sample cup on an analytical balance. (Sample weight
13.3.1 Objective will be coordinated with sample number in autosam-
pler, if autosampler is used.) Remove sample from
Determine the protein content of corn flour using the balance and prepare for insertion following manu-
nitrogen combustion method. facturer’s instructions. If an autosampler is used, the
weighed sample must be placed into autosampler in
13.3.2 Principle of Method the appropriate slot for the sample number. Repeat
this procedure for EDTA standard. Sample and stan-
The nitrogen combustion method measures the nitro- dard should be run in duplicate or triplicate.
gen content of a sample. The protein content then is
calculated assuming a ratio of protein to nitrogen for 13.3.8 Data and Calculations
the specific food being analyzed. In the assay, the sam-
ple is combusted at a high temperature (900–1110 °C) Record the percent nitrogen content for each of your
to release nitrogen gas and other products (i.e., water, duplicate or triplicate corn flour samples. Calculate
other gases). The other products are removed, and the protein content from percent nitrogen data, and deter-
nitrogen is quantitated by gas chromatography using mine average percent protein. The corn flour sample
a thermal conductivity detector. you analyzed was not a dried sample. Report percent
protein results on a wet weight basis (wwb) and on a
dry weight basis (dwb). Assume a moisture content of
13.3.3 Chemicals 10 % (or use the actual moisture content if previously
determined on this corn flour sample). Use 6.25 for the
CAS No. Hazards nitrogen to protein conversion factor.
Ethylenediaminetetraacetic acid 60-00-4 Irritant
(EDTA), disodium salt Sample % Nitrogen % Protein, wwb % Protein, dwb
(Na2EDTA · 2H2O)
1
2
(The other chemicals used are specific to each 3
manufacturer for the columns within the instrument.) X = X =
SD = SD =
13.3.4 Hazards, Cautions, and Waste
Disposal 13.3.9 Questions
During operation, the front panel of the instrument 1. What are the advantages of the nitrogen com-
gets very hot. Check instructions of manufacturer for bustion method compared to the Kjeldahl
any other hazards, especially those associated with method?
maintenance of instrument. 2. Explain why ethylenediaminetetraacetic acid
(EDTA) can be used as a standard to check the
calibration of the nitrogen analyzer.
13.3.5 Supplies
3. If you analyzed the corn flour sample by both
(Used by students) the Kjeldahl and nitrogen combustion methods,
compare the results. What might explain any
Corn flour differences?
Sample cup
13.3.6 Equipment RESOURCE MATERIALS

Nitrogen combustion unit Chang SKC, Zhang Y (2017) Protein analysis. Ch. 18. In:
Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
13.3.7 Procedure AOAC International (2016) Official methods of analysis, 20th
edn, (On-line). Method 960.52 (Micro-Kjeldahl method)
Follow manufacturer’s instructions for start-up, ana- and Method 992.23 (Generic combustion method). AOAC
lyzing samples, and shutdown. International, Rockville, MD
www.dbooks.org
14
chapter
Total Carbohydrate
by Phenol-Sulfuric Acid
Method
S.Suzanne Nielsen
14.1 Introduction 14.1.7 Hazards, Cautions,

14.1.4 Principle of Method 14.2 Procedure
14.1.6 Reagents 14.4 Questions
138 S.S. Nielsen
14.1 INTRODUCTION 14.1.5 Chemicals
14.1.1 Background CAS No. Hazards
The phenol-sulfuric acid method is a simple and D-Glucose (C6H12O6) 50-99-7

rapid colorimetric method to determine total carbo- Phenol (C6H6O) 108-95-2 Toxic
hydrates in a sample. The method detects virtually Sulfuric acid (H2SO4) 7664-93-9 Corrosive
all classes of carbohydrates, including mono-, di-,
oligo-, and polysaccharides. Although the method 14.1.6 Reagents
detects almost all carbohydrates, the absorptivity of
the different carbohydrates varies. Thus, unless a (** It is recommended that these solutions be prepared
sample is known to contain only one carbohydrate, by the laboratory assistant before class.)
the results must be expressed arbitrarily in terms of
one carbohydrate. • Glucose std. solution, 100 mg/L **
In this method, the concentrated sulfuric acid • Phenol, 80 % wt/wt in H2O, 1 mL **
breaks down any polysaccharides, oligosaccharides, Prepare by adding 20-g deionized distilled (dd)
and disaccharides to monosaccharides. Pentoses water to 80 g of redistilled reagent grade phenol
(5-carbon compounds) then are dehydrated to furfu- (crystals).
ral, and hexoses (6-carbon compounds) to hydroxy- • Sulfuric acid, concentrated
methyl furfural. These compounds then react with
phenol to produce a yellow-gold color. For products 14.1.7 Hazards, Cautions, and Waste
that are very high in xylose (a pentose), such as wheat Disposal
bran or corn bran, xylose should be used to construct Use concentrated H2SO4 and the 80 % phenol solution
the standard curve for the assay and measure the with caution. Wear gloves and safety glasses at all
absorption at 480 nm. For products that are high in times, and use good lab technique. The concentrated
hexose sugars, glucose is commonly used to create the H2SO4 is very corrosive (e.g., to clothes, shoes, skin).
standard curve, and the absorption is measured at The phenol is toxic and must be discarded as hazard-
490 nm. The color for this reaction is stable for several ous waste. Other waste not containing phenol likely
hours, and the accuracy of the method is within ±2 % may be put down the drain using a water rinse, but
under proper conditions. follow good laboratory practices outlined by environ-
Carbohydrates are the major source of calories in mental health and safety protocols at your institution.
soft drinks, beer, and fruit juices, supplying 4 Cal/
gram carbohydrate. In this experiment, you will create
14.1.8 Supplies
a standard curve with a glucose standard solution, use
it to determine the carbohydrate concentration of soft (Used by students)
drinks and beer, then calculate the caloric content of
those beverages. • Beer (lite and regular, of same brand)
• Bottle to collect waste
14.1.2 Reading Assignment • Cuvettes (tubes) for spectrophotometer
• Erlenmeyer flask, 100 mL, for dd water
BeMiller, J.N. 2017. Carbohydrate analysis. Ch. 19, in • 2 Erlenmeyer flasks, 500 mL, for beverages
Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, • Gloves
New York. • Mechanical, adjustable volume pipettors, 1000 μL
and 100 μL (or 200 μL), with plastic tips
14.1.3 Objective • Pasteur pipettes and bulb
Determine the total carbohydrate content of soft drinks • Parafilm®
and beers. • Pipette bulb or pump
• Repipettor (for fast delivery of 5-mL conc. H2SO4)
• Soft drinks (clear-colored, diet and regular, of same
14.1.4 Principle of Method brand)
Carbohydrates (simple sugars, oligosaccharides, poly- • 20 test tubes, 16–20-mm internal diameter
saccharides, and their derivatives) react in the pres- • Test tube rack
ence of strong acid and heat to generate furan • 4 Volumetric flasks, 100 mL or 2 Volumetric flasks,
derivatives that condense with phenol to form stable 1000 mL
yellow-gold compounds that can be measured • Volumetric pipette, 5 mL
spectrophotometrically. • 2 Volumetric pipettes, 10 mL
www.dbooks.org
Chapter 14 • Total Carbohydrate by Phenol-Sulfuric Acid Method 139
14.1.9 Equipment Recommended dilution scheme for 1:2000 dilution:

• Spectrophotometer (a) Pipette 5 mL of beverage into a 100-mL volu-
• Vortex mixer metric flask, and dilute to volume with dd
• Water bath, maintained at 25 °C water. Seal flask with Parafilm® and mix well
(this is a 1:20 dilution). Then, pipette 1.0 mL
of this 1:20 diluted beverage into another 100-
14.2 PROCEDURE mL volumetric flask. Dilute to volume with
dd water. Seal flask with Parafilm® and mix
(Instructions are given for analysis in duplicate.) well.
1. Standard curve tubes: Using the glucose stan- OR
dard solution (100 mg glucose/L) and dd water (b) Pipette 1.0 mL of beverage into a 1000-mL vol-
as indicated in the table below, pipette aliquots umetric flask, and dilute to volume with dd
of the glucose standard into clean test tubes water. Seal flask with Parafilm® and mix well.
(duplicates for each concentration) such that Then, in a test tube, combine 1 mL of the
the tubes contain 0–100 μL of glucose (use 1000- 1:1000 diluted beverage and 1 mL dd water.
μl mechanical pipettor to pipette samples), in a Mix well.
total volume of 2 mL. These tubes will be used
to create a standard curve, with values of 0–100- Recommended dilution scheme for 1:1000 dilution:
μg glucose/2 mL. The 0-μg glucose/2 mL sam- (a) Pipette 10 mL of beverage into a 100-mL volu-
ple will be used to prepare the reagent blank. metric flask, and dilute to volume with dd
water. Seal flask with Parafilm® and mix well
μg Glucose/2 mL (this is a 1:10 dilution). Then, pipette 1.0 mL
0 20 40 60 80 100 of this 1:10 diluted beverage into another 100-
mL glucose stock 0 0.2 0.4 0.6 0.8 1.0 mL volumetric flask. Dilute to volume with
solution dd water. Seal flask with Parafilm® and mix
mL dd water 2.0 1.8 1.6 1.4 1.2 1.0 well.
OR
2. Record caloric content from label: You will ana- (b) Pipette 1.0 mL of beverage into a 1000-mL volu-
lyze for total carbohydrate content: (1) a regular metric flask, and dilute to volume with dd
and diet soft drink of the same brand, and/or (2) water. Seal flask with Parafilm® and mix well.
a regular and lite beer of the same brand. Before 5. After dilution as indicated, pipette 1.0 mL
you proceed with the sample preparation and of sample into a test tube and add 1.0 mL of
analysis, record the caloric content on the nutri- dd water. Analyze each diluted sample in
tion label of the samples you will analyze. duplicate.
3. Decarbonate the beverages: With the beverages 6. Phenol addition: To each tube from Parts 1 and
at room temperature, pour approximately 4 containing a total volume of 2 mL, add 0.05-
100 mL into a 500-mL Erlenmeyer flask. Shake mL 80 % phenol (use 100 or 200-μl mechanical
gently at first (try not to foam the sample if it is pipettor). Mix on a Vortex test tube mixer.
beer) and continue gentle shaking until no 7. H2SO4 addition: To each tube from Part 6, add
observable carbon dioxide bubbles appear. If 5.0-mL H2SO4. The sulfuric acid reagent should
there is any noticeable suspended material in be added rapidly to the test tube. Direct the
the beverage, filter the sample before analysis. stream of acid against the liquid surface rather
4. Sample tubes: So the sample tested will contain than against the side of the test tube in order to
20–100-μg glucose/2 mL, the dilution procedure obtain good mixing. (These reactions are driven
and volumes to be assayed are given below. by the heat produced upon the addition of
H2SO4 to an aqueous sample. Thus, the rate of
Dilution Volume assayed (mL) addition of sulfuric acid must be standardized.)
Mix on a Vortex test tube mixer. Let tubes stand
Soft drink for 10 min and then place in a 25 °C bath for
Regular 1:2000 1 10 min (i.e., to cool them to room temperature).
Diet 0 1
Vortex the test tubes again before reading the
Beer
Regular 1:2000 1
absorbance.
Lite 1:1000 1 8. Reading absorbance: Wear gloves to pour sam-
ples from test tubes into cuvettes. Do not
140 S.S. Nielsen
rinse cuvettes with water between samples. Glucose

Zero the spectrophotometer with the stan- equivalent
dard curve sample that contains 0-μg glu- A490 ug Dilution μg/mL, g/L,
cose/2 mL (i.e., blank). Retain this blank Sample glucose/ scheme original original
sample in one cuvette for later use. Read identity 2 mL sample sample
absorbances of all other samples at 490 nm. Soft drink,
Read your standard curve tubes from low to diet
high concentration (i.e., 20 μg/2 mL up to Beer, reg.
100 μg/2 mL), and then read your beverage
Beer, reg.
samples. To be sure that the outside of the
cuvettes are free of moisture and smudges, Beer, lite
wipe the outside of the cuvette with a clean Beer, lite
paper wipe prior to inserting it into the spec-
trophotometer for a reading. Sample calculation for soft drink, regular:
9. Absorbance spectra: Use one of the duplicate Equation of the line : y 0.011x 0.1027
tubes from a standard curve sample with an
absorbance reading of 0.5-0.8. Determine the
y = 0.648
absorbance spectra from 450–550 nm by read-
ing the tube at 10-nm intervals. Zero the spec-
trophotometer with the blank at each 10-nm x 49.57 g / 2 mL
interval.
Ci C f V2 / V1 V4 / V3
14.3 DATA AND CALCULATIONS (See Chap. 3 in this laboratory manual, Ci = initial
concentration; Cf = final concentration)
1. Summarize your procedures and results for all
standards and samples in the tables immedi- Ci  49.57 g glucose / 2 mL2000 mL / 1mL
ately below. Use the data for the standard curve 2 mL / 1mL  99140 g / mL
samples in the first table to calculate the equa-  99 .14 mg / mL
tion for the line, which is used to calculate the
concentrations in the original samples reported  99.14 g / L
in the second table.
2. Construct a standard curve for your total carbo-
Standard Curve: hydrate determinations, expressed in terms of
glucose (A490 versus μg glucose/2 mL).
A490 A490
Sample identity msmt 1 msmt 2 Avg.
Determine the equation of the line for the stan-
dard curve.
Blank 3. Calculate the concentration of glucose in your
Std. 20 μg soft drink samples and beer samples, in terms
Std. 40 μg of (a) grams/liter and (b) g/12 fl. oz. (Note:
Std. 60 μg
29.56 mL/fl. oz.)
Std. 80 μg
4. Calculate the caloric content (based only on
Std. 100 μg
carbohydrate content) of your soft drink
samples and beer samples in term of
Samples: Cal/12 fl. oz.
Glucose
equivalent g Glucose/ Measured Nutrition label
A490 ug Dilution μg/mL, g/L, Sample 12 fl. oz. Cal/12 fl. oz. Cal/12 fl. oz.
Sample glucose/ scheme original original Soft drink
identity 2 mL sample sample
Regular
Soft drink,
reg. Diet
Soft drink, Beer

reg. Regular
Soft drink, Lite
diet
www.dbooks.org
Chapter 14 • Total Carbohydrate by Phenol-Sulfuric Acid Method 141
5. Plot the absorbance spectra obtained by mea- soft drinks (US Department of Agriculture
suring the absorbance between 450 and 550 nm. Nutrient Database for Standard Reference indi-
cates ca. 3 g carbohydrate/fl. oz.), how could
wave- 450 460 470 480 490 500 510 520 530 540 550 you have calculated the 2000-fold dilution was
length appropriate if you wanted to use 1 mL of diluted
soft drink in the assay. Show all calculations.
Abs.
5. How does your calculated value compare to the
caloric content on the food label? Do the round-
ing rules for Calories explain any differences?
14.4 QUESTIONS (See Metzger and Nielsen, 2017, Table 3.3). Does
the alcohol content (assume 4–5% alcohol at
1. What are the advantages, disadvantages, and
7 Cal/g) of beer explain any differences?
sources of error for this method to determine
6. Was it best to have read the absorbance for the
total carbohydrates?
standard curve and other samples at 490 nm?
2. Your lab technician performed the phenol-
Explain why a wavelength in this region is
H2SO4 analysis on food samples for total carbo-
appropriate for this reaction.
hydrates but the results showed low precision,
and the values seemed a little high. The techni-
Acknowledgment This laboratory was developed with
cian had used new test tubes (they had never
input from Dr Joseph Montecalvo, Jr., Department of Food
been used, and were taken right from the card- Science & Nutrition, California Polytechnic State University,
board box). What most likely caused these San Luis Obispo, California.
results? Why? Describe what happened.
3. If you started with a glucose standard solution
of 10-g glucose/liter, what dilution of this solu- RESOURCE MATERIALS
tion would be necessary such that you could
pipette 0.20, 0.40, 0.60, 0.80, and 1.0 mL of the BeMiller JN (2017) Carbohydrate analysis, Ch. 19. In: Nielsen
diluted glucose standard solution into test tubes SS (ed) Food analysis, 5th edn. Springer, New York
and add water to 2 mL for the standard curve Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)
tubes (20–100 μg/2 mL)? Show all calculations. Colorimetric method for determination of sugars and
4. If you had not been told to do a 2000-fold dilu- related substances. Anal Chem 28: 350–356
Metzger LE, Nielsen SS (2017) Nutrition labeling. Ch. 3. In:
tion of a soft drink sample, and if you know the
Nielsen SS edn. Food analysis, 5th edn. Springer,
approximate carbohydrate content of regular
New York
15
chapter
Vitamin C
Determination
by Indophenol Method
S.Suzanne Nielsen
15.1 Introduction 15.1.9 Equipment

15.1.1 Background 15.1.10 Notes
15.1.3 Objective 15.2.1 Standardization of Dye
15.1.4 Principle of Method 15.2.2 Analysis of Juice Samples
15.1.6 Reagents 15.3.1 Data
15.1.7 Hazards, Precautions, 15.3.2 Calculations
and Waste Disposal 15.4 Questions
15.1.8 Supplies
www.dbooks.org
144 S.S. Nielsen
15.1 INTRODUCTION 15.1.6 Reagents

(**It is recommended that samples be prepared by the
15.1.1 Background laboratory assistant before class.)
Vitamin C is an essential nutrient in the diet, but is eas-
ily reduced or destroyed by exposure to heat and oxy- • Ascorbic acid standard solution (prepare only at
gen during processing, packaging, and storage of time of use)
food. The instability of vitamin C makes it more diffi- Accurately weigh (on an analytical balance)
cult to ensure an accurate listing of vitamin C content approximately 50 mg ascorbic acid [preferably
on the nutrition label. US Pharmacopeia (USP) Ascorbic Acid Reference
The official method of analysis for vitamin C Standard]. Record this weight. Transfer to a
determination of juices is the 2,6-dichloroindophenol 50-mL volumetric flask. Dilute to volume imme-
titrimetric method (AOAC Method 967.21). While this diately before use with the metaphosphoric acid-
method is not official for other types of food products, acetic acid solution (see below for preparation of
it is sometimes used as a rapid, quality control test for this solution).
a variety of food products, rather than the more time- • Indophenol solution – dye
consuming microfluorometric method (AOAC To 50 mL deionized distilled (dd) water in a 150-
Method 984.26). The procedure outlined below is from mL beaker, add and stir to dissolve 42 mg sodium
AOAC Method 967.21. bicarbonate, and then add and stir to dissolve
50 mg 2,6-dichloroindophenol sodium salt.
15.1.2 Reading Assignment Dilute mixture to 200 mL with dd water. Filter
through fluted filter paper into an amber bottle.
AOAC International. 2016. Official Methods of Analysis, Close the bottle with a stopper or lid and store
20th ed., (On-line). Method 976.21, AOAC refrigerated until used.
International, Rockville, MD. • Metaphosphoric acid-acetic acid solution
Pegg, R.B., and Eitenmiller, R.R. 2017. Vitamin To a 250-mL beaker, add 100 mL dd water then
analysis. Ch. 20, in Food Analysis, 5th ed. S.S. Nielsen 20 mL acetic acid. Add and stir to dissolve 7.5 g
(Ed.), Springer, New York. metaphosphoric acid. Dilute mixture to 250 mL
with distilled water. Filter through fluted filter
15.1.3 Objective paper into a bottle. Close the bottle with a stop-
per or lid and store refrigerated until used.
Determine the vitamin C content of various orange • Orange juice samples**
juice products using the indicator dye 2,6-dichloroin- Use products processed and packaged in various
dophenol in a titration method. ways (e.g., canned, reconstituted frozen concen-
trate, fresh squeezed, not-from-concentrate).
15.1.4 Principle of Method Filter juices through cheesecloth to avoid prob-
Ascorbic acid reduces the indicator dye to a color- lems with pulp when pipetting. Record from the
less solution. At the endpoint of titrating an ascor- nutrition label for each product the percent of
bic acid-containing sample with dye, excess the Daily Value for vitamin C.
unreduced dye is a rose-pink color in the acid solu-
tion. The titer of the dye can be determined using a 15.1.7 Hazards, Precautions, and Waste
standard ascorbic acid solution. Food samples in Disposal
solution then can be titrated with the dye and the Preparation of reagents involves corrosives. Use appro-
volume for the titration used to calculate the ascor- priate eye and skin protection. Otherwise, adhere to
bic acid content. normal laboratory safety procedures. Waste likely may
be put down the drain using a water rinse, but follow
15.1.5 Chemicals good laboratory practices outlined by environmental
health and safety protocols at your institution.
CAS no. Hazards
15.1.8 Supplies
Acetic acid (CH3COOH) 64-19-7 Corrosive
Ascorbic acid 50-81-7 (Used by students)
2,6-Dichloroindophenol 620-45-1
(DCIP) (sodium salt) • Beaker, 250 mL
Metaphosphoric acid (HPO3) 37267-86-0 Corrosive • 2 Bottles, glass, 200–250 mL, one amber and one
Sodium bicarbonate 144-55-8
clear, both with lids or stoppers
(NaHCO3)
• Buret, 50 or 25 mL
Chapter 15 • Vitamin C Determination by Indophenol Method 145
• 9 Erlenmeyer flasks, 50 mL (or 125 mL) readings and calculate the volume of dye used
• Fluted filter paper, two pieces for each sample.
• Funnel, approx. 6–9 cm diameter (to hold filter 7. Prepare blanks: Pipette 7.0 mL metaphosphoric
paper) acid-acetic acid solution into each of three
• Funnel, approx. 2–3 cm diameter (to fill buret) 50-mL Erlenmeyer flasks. Add to each flask a
• 2 Glass stirring rods volume of distilled water approximately equal
• Graduated cylinder, 25 mL to the volume of dye used above (i.e., average
• Graduated cylinder, 100 mL volume of dye used to titrate three standard
• Pipette bulb or pump samples).
• Ring stand 8. Titrate the blanks in the same way as Steps 3–5
• 3 Spatulas above. Record initial and final buret readings
• Volumetric flask, 50 mL for each titration of the blank, and then calcu-
• Volumetric flask, 200 mL late the volume of dye used.
• Volumetric flask, 250 mL
• 2 Volumetric pipettes, 2 mL 15.2.2 Analysis of Juice Samples
• Volumetric pipette, 5 mL
1. Pipet into each of three 50-mL Erlenmeyer flasks
5 mL metaphosphoric acid-acetic acid solution
• Volumetric pipette, 10 or 20 mL
and 2 mL orange juice.
• Weighing boats or paper
2. Titrate each sample with the indophenol dye
solution (as you did in Sect. 15.2.1, Steps 3–5)
15.1.9 Equipment
until a light but distinct rose-pink color persists
• Analytical balance for >5 s.
3. Record the initial and final readings and calcu-
15.1.10 Notes late the difference to determine the amount of
dye used for each titration.
The instructor may want to assign one or two types of
orange juice samples to each student (or lab group) for
analysis, rather than having all students analyze all
15.3 DATA AND CALCULATIONS
types of orange juice samples. Quantities of supplies
and reagents specified are adequate for each student 15.3.1 Data
(or lab group) to standardize the dye and analyze one
type of orange juice sample in triplicate. Buret start Buret end
Rep (mL) (mL) Vol. titrant (mL)
15.2 PROCEDURE Ascorbic 1

acid 2
(Instructions are given for analysis in triplicate.) standards 3
X =
15.2.1 Standardization of Dye Blank 1
1. Pipette 5 mL metaphosphoric acid-acetic acid 2
solution into each of three 50-mL Erlenmeyer 3
flasks. X =
Sample 1
2. Add 2.0 mL ascorbic acid standard solution to
2
each flask. 3
3. Using a funnel, fill the buret with the indophe-
nol solution (dye) and record the initial buret
reading.
15.3.2 Calculations
4. Place the Erlenmeyer flask under the tip of the 1. Using the data obtained in standardization of
buret. Slowly add indophenol solution to stan- the dye, calculate the titer using the following
dard ascorbic acid solution until a light but dis- formula:
tinct rose-pink color persists for >5 s (takes
about 15–17 mL). Swirl the flask as you add the mg ascorbic acid in volume
indophenol solution. of standard solution titrated ***
Titer F
5. Note final buret reading and calculate the average mL average mL
volume of dye used.
6. Repeat Steps 3–5 for the other two standard dye used to dye used to
titrate standards titrate blank
samples. Record the initial and final buret
www.dbooks.org
146 S.S. Nielsen
**mg ascorbic acid in volume of standard

solution titrated: Titer  F 
(50.2mg / 50 mL) 2 mL
(15.5 mL  0.10 mL )
mg of ascorbic acid / 50 mL 2 mL
 0.130 mg / mL
2. Calculate the ascorbic acid content of the juice
sample in mg/mL, using the equation that fol- mg ascorbic acid / mL = (7.1mL  0.10 mL)
lows and the volume of titrant for each of your
replicates. Calculate the mean and standard  (0.130mg / 2mL)  ( 7 mL / 7 mL)
deviation of the ascorbic acid content for your = 0.455mg / mL
juice (in mg/mL). Obtain from other lab mem-
bers the mean ascorbic acid content (in mg/ 0.455mg / mL  45.4 mg / 100 mL
mL) for other types of juice. Use these mean
values for each type of juice to express the vita- 0.455mg ascorbic acid / mL juice  29.56 mL / fl.oz.8fl.oz.
min C content of the juice samples as milli-  107.6 mL ascorbic acid / 8fl.oz.
grams ascorbic acid/100 mL and as milligrams
ascorbic acid/8 fl. oz. (29.56 mL/fl. oz.).
mg ascorbic acid / mL X B F / E V / Y 15.4 QUESTIONS
where: 1. By comparing results obtained for various

orange juice products, did heat and/or oxygen
X = mL for sample titration exposure during processing and storage of the
B = average mL for sample blank titration samples analyzed seem to affect the vitamin C
F = titer of dye content?
(= mg ascorbic acid equivalent to 2. How do results you have available for the juice
1.0 mL indophenol standard solution) samples analyzed compare to (1) values listed on
E = mL assayed (=2 mL) the nutrition label for the same juice product and
V = volume of initial assay solution (=7 mL) (2) values in the US Department of Agriculture
Y = volume of sample aliquot titrated Nutrient Database for Standard Reference (web
(=7 mL) address: http://ndb.nal.usda.gov/)? For the
Ascorbic acid (AA) content for replicates of orange nutrition label values, convert percent of Daily
juice sample: Value to mg/8 fl. oz., given that the Daily Value
for vitamin C is 90 mg. Why might the vitamin C
Replicate mg AA/mL
content determined for a specific orange juice
1 product not match the value as calculated from
2 percent of Daily Value on the nutrition label?
3
X = Ascorbic acid content of orange juices (mg AA/8 fl. oz):
SD =
Sample
Summary of ascorbic acid (AA) content of orange juice identity Lab values USDA database Nutrition label
samples: 1
Sample 2
identity mg AA/mL mg AA/100 mL mg AA/8 fl. oz. 3
4
1
2 3. Why was it necessary to standardize the indo-
3 phenol solution?
4 4. Why was it necessary to titrate blank samples?
5. Why might the vitamin C content as deter-
Example calculation: mined by this method be underestimated in the
case of the heat-processed juice samples?
Weight of ascorbic acid used = 50.2 mg
Average volume of titrant used: RESOURCE MATERIALS
Ascorbic acid standards = 15.5 mL
AOAC International (2016) Official methods of analysis, 20th
Blanks = 0.10 mL
edn. (On-line). AOAC International, Rockville, MD
Volume of titrant used for orange juice Pegg RB, Eitenmiller RR (2017) Vitamin analysis. Ch. 20. In:
sample = 7.1 mL Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
16chapter
Water Hardness Testing

by Complexometric
Determination
of Calcium
S.Suzanne Nielsen
16.1 Introduction 16.2.4 Notes

16.1.1 Background 16.2.5 Hazards, Precautions,
16.1.2 Reading Assignment and Waste Disposal
16.2 EDTA Titrimetric Method for Testing 16.2.7 Equipment
Hardness of Water 16.2.8 Procedure
16.2.1 Principle of Method 16.2.9 Data and Calculations
16.2.3 Reagents 16.3 Test Strips for Water Hardness
www.dbooks.org
16.3.1 Note 16.3.6 Supplies
16.3.2 Principle of Method 16.3.7 Procedure
16.3.3 Chemicals 16.3.8 Data and Calculations
16.3.4 Reagents 16.3.9 Question
16.3.5 Hazards, Precautions,
and Waste Disposal
149
16.1 INTRODUCTION appears blue. This blue color is the endpoint of the
titration. The volume and concentration of the EDTA
16.1.1 Background in the titration are used to calculate the concentration
of calcium in the sample, which is expressed as mg cal-
Ethylenediaminetetraacetate (EDTA) complexes with
cium carbonate/l. Stoichiometry of the reaction is
numerous mineral ions, including calcium and mag-
1 mol of calcium complexing with 1 mol of EDTA.
nesium. This reaction can be used to determine the
amount of these minerals in a sample by a complexo-
16.2.2 Chemicals
metric titration. Endpoints in the titration are detected
using indicators that change color when they complex
CAS no. Hazards
with mineral ions. Calmagite and eriochrome black T
(EBT) are such indicators that change from blue to Ammonium chloride 12125-02-9 Harmful
pink when they complex with calcium and magne- (NH4Cl)
sium. In the titration of a mineral-containing solution Ammonium hydroxide 1336-21-6 Corrosive,
(NH4OH) dangerous
with EDTA, the solution turns from pink to blue at the
for the
endpoint with either indicator. The pH affects a com-
environment
plexometric EDTA titration in several ways and must Calcium carbonate 471-34-1
be carefully controlled. A major application of EDTA (CaCO3)
titration is testing the hardness of water, for which the Calmagite 3147-14-6
method described is an official one (Standard Methods [3-Hydroxy-4-(6-hydroxy-
for the Examination of Water and Wastewater, Method m-tolylazo)
2340C; AOAC Method 920.196). naphthalene-1-sulfonic
Hardness of water also can be tested by a more acid]
rapid test strip method. Such test strips are available Ethylenediaminetetraacetic 60-00-4 Irritant
from various companies. The strips contain EDTA and acid, disodium salt
(Na2EDTA · 2H2O)
an indicator chemical to cause a color change when the
calcium and magnesium in water react with the EDTA. concentrated (HCl)
Magnesium chloride, 7791-18-6
16.1.2 Reading Assignment hexahydrate (MgCl2 .
6H2O)
Ward, R.E., and Legako, J.F. 2017. Traditional methods
Magnesium sulfate, 10034-99-8
for mineral analysis. Ch. 21, in Food Analysis, 5th ed.
heptahydrate (MgSO4
S.S. Nielsen (Ed.), Springer, New York. . 7H2O)
16.1.3 Objective
16.2.3 Reagents
Determine the hardness of water by EDTA titration
and with Quantab® test strips.
16.2 EDTA TITRIMETRIC METHOD • Buffer solution**

FOR TESTING HARDNESS Dissolve 16.9 g NH4Cl in 143 mL concentrated
OF WATER NH4OH. In 50 mL deionized distilled (dd) water,
dissolve 1.179 g Na2EDTA · 2H2O (analytical
16.2.1 Principle of Method reagent grade) and either 780 mg MgSO4 . 7H2O
or 644 mg MgCl2 . 6H2O. Combine these two solu-
Ethylenediaminetetraacetic acid (EDTA) forms a tions with mixing and dilute to 250 mL with dd
Stable 1:1 complex with calcium or magnesium at water. Store in tightly stoppered Pyrex or plastic
pH 10. The metal ion indicators, calmagite and erio- bottle to prevent loss of ammonia (NH3) or
chrome black T (EBT), are pink when complexed to pickup of carbon dioxide (CO2). Dispense this
metal ions but blue when no metal ions are complexed buffer solution with a repipette system. Discard
to them. The indicators bind to metal ions less strongly buffer when 1–2 mL added to a sample fails to
than does EDTA. When the indicator is added to a give pH 10.0 ± 0.1 at the endpoint of the titration.
solution containing metal ions, the solution becomes • Calcium standard solution, 1.00 mg CaCO3/mL**
pink. When EDTA is added as titrant to the mineral- (modified from official method; omit use of methyl
containing sample, metal ions preferentially complex red indicator)
with the EDTA, leaving the indicator without a metal Use primary standard or special reagent that is
ion to complex. When enough EDTA has been titrated low in heavy metals, alkalis, and magnesium.
to complex with all the metal ions present, the indicator Dry CaCO3 at 100 °C for 24 h. Accurately weigh
www.dbooks.org
150
ca. 1.0 g CaCO3. Transfer to a 500-mL Erlenmeyer tion, which contains ammonium hydroxide, should
flask. Place a funnel in the neck of the flask and be disposed of as hazardous waste. Other wastes
add HCl (1:1, conc. HCl:H2O) a little at a time, likely may be put down the drain using a water rinse,
until all the CaCO3 has dissolved (make sure all but follow good laboratory practices outlined by envi-
the CaCO3 in the neck of the flask has been ronmental health and safety protocols at your
washed down with HCl). Add 200 mL dd water institution.
and boil a few minutes to expel CO2. Cool. Adjust
to pH 3.8 with 3 M NH4OH or HCl (1:1, conc. 16.2.6 Supplies
HCl : H2O), as required. Transfer quantitatively
to a 1-L volumetric flask and dilute to volume (Used by students)
with dd water (1 mL = 1.00 mg CaCO3).
• EDTA standard solution, 0.01 M • Buret, 25 or 50 mL
Weigh 3.723 g Na2EDTA · 2H2O. Dilute to 1 L • 9 Erlenmeyer flasks, 125 mL
with dd water. Store in polyethylene (preferable) • Funnel (to fill buret)
or borosilicate glass bottles. Standardize this • Graduated cylinder, 50 mL
solution using the calcium standard solution as • 3 Graduated cylinders, 25 mL
described in the Procedure. • (Graduated cylinder of larger volumes may be nec-
• Hydrochloric acid, 1:1 with water** essary, for example, 100 mL or larger; size to be
To 10 mL of dd water, add 10 mL concentrated determined by trial in Sect. 16.2.8.2)
HCl. Mix carefully. • Mechanical pipettor, 1000 μL, with plastic tips
• Calmagite** • Pasteur pipette and bulb
Dissolve 0.10 g calmagite in 100 mL dd water. • Spatula
Use 1 mL per 30 mL solution to be titrated. Put in • Volumetric flask, 1000 mL
bottle with eye dropper. • Volumetric pipette, 10 mL
• Weighing paper/boat
16.2.4 Notes
16.2.7 Equipment
In this experiment, calmagite will be used as the indi-
cator dye rather than EBT. Unlike EBT, calmagite is • Analytical balance
stable in aqueous solution. Calmagite gives the same • Drying oven, 100 °C
color change as EBT, but with a sharper endpoint. • Hot plate
To give a satisfactory endpoint, magnesium ions • pH meter
must be present. To ensure this, a small amount of
neutral magnesium salt is added to the buffer. 16.2.8 Procedure
The specified pH of 10.0 + 0.1 is a compromise sit- (Modified from Method 2340 Hardness, Standard
uation. With increasing pH, the sharpness of the end- Methods for the Examination of Water and Wastewater,
point increases. However, at high pH, the indicator 22nd ed.) (Instructions are given for analysis in
dye changes color and there is risk of precipitating cal- triplicate.)
cium carbonate (CaCO3) or magnesium hydroxide.
The tendency toward CaCO3 precipitation is the rea- Standardization of EDTA Solution
son for the titration duration time limit of 5 min. 1. Pipette 10 mL of calcium standard solution into
Fading or indistinct endpoints can be caused by each of three 125-mL Erlenmeyer flasks.
interference from some metal ions. Certain inhibitors 2. Adjust to pH 10.0 ± 0.05 with buffer solution. (If
can be added before titration to reduce this interfer- possible, do this pH adjustment with the buffer in
ence, but the inhibitors specified are toxic (i.e., sodium an operating hood, due to its odor.) As necessary,
cyanide) or malodorous. Magnesium salt of 1,2-cycloh use the HCl solution (1:1) in pH adjustment.
exanediaminetetraacetic acid (MgCDTA), which selec- 3. Add 1 mL of calmagite to each flask, and then
tively complexes heavy metals, may be substituted for titrate each flask with EDTA solution slowly,
these inhibitors. However, for samples with high con- with continuous stirring, until last reddish tinge
centrations of heavy metals, a non-EDTA method is disappears, adding last few drops at 3–5 s inter-
recommended. In this experiment, inhibitors or vals. Color at endpoint is blue in daylight and
MgCDTA will not be used. under daylight fluorescent lamp. Color may
first appear lavender or purple, but will then
16.2.5 Hazards, Precautions, and Waste turn to blue. Complete titration within 5 min
Disposal from time of buffer addition.
Adhere to normal laboratory safety procedures. Wear 4. Record the volume of EDTA solution used for
gloves and safety glasses at all times. The buffer solu- each titration.
151
Titration of Water Sample Calcium content of water sample (g Ca/L and g

1. Dilute 25 mL tap water sample (or such vol- CaCO3/L):
ume as to require <15 mL titrant) to ca. 50 mL
with dd water in 125-mL Erlenmeyer flask. mol calcium = mol EDTA
For tap distilled water, test 50 mL, without
M1 V1 = M2V2
dilution. Prepare samples in triplicate
[Official method recommends the following: M Ca in sample V
sample , liter
For water of low hardness (<5 mg/L), use
100–1000 mL specimen, proportionately M EDTA solution VEDTA solution used in titration , L
larger amounts of reagents, micro-buret, and Solve for M Ca in sample:
blank of distilled water equal to specimen
volume.] MCa in sample 40.085 g Ca / mol g Ca / L
2. Adjust pH to 10 ± 0.05 as described in
Sect. 16.2.8.1, Step 2. g Ca / L 100.09 g CaCO3 / 40.085 g Ca
3. Titrate each sample with EDTA standard solu- 1000 mg / g mg CaCO3 / L
tion slowly, as described in Sect. 16.2.8.1, Step 3,
for standardization of EDTA solution. 16.2.10 Questions
4. Record the volume of EDTA solution used for
each titration. 1. If a sample of water is thought to have a hard-
ness of approximately 250 mg/L CaCO3, what
16.2.9 Data and Calculations size sample (i.e., how many mL) would you use
so that you would use approximately 10 mL of
Calculate molarity of calcium standard solution: your EDTA solution?
Molarity of calcium solution = 2. Why were you asked to prepare the CaCl2 solu-
tion by using CaCO3 and HCl rather than just
g CaCO3
= molcalcium / L weighing out CaCl2?
(100 . 09 g / mol ) ( liter solution ) 3. In this EDTA titration method, would over-
Standardization of EDTA solution: shooting the endpoint in the titration cause an
over- or underestimation of calcium in the sam-
Buret start Buret end Volume titrant ple? Explain your answer.
Rep (mL) (mL) (mL) Molarity
1
2 16.3 TEST STRIPS FOR WATER
3 HARDNESS
X =
SD = 16.3.1 Note
All information given is for AquaChek test strips,
Calculate molarity of EDTA solution: from Environmental Test Systems, Inc., a HACH
mol calcium = mol EDTA Company, Elkhart, IN. Other similar test strips could
be used. Any anion (e.g., magnesium, iron, copper)
M1 V1 = M2V2 that will bind the EDTA may interfere with the
(M )(V ) AquaChek test. Very strong bases and acids also may
Ca solution Ca solution , L
interfere.
= ( M EDTA solution ) (VEDTA solution , L )
Solve for M EDTA solution
The test strips have a paper, impregnated with chemi-
Titration of water sample with EDTA solution: cals, that is adhered to polystyrene for ease of han-
dling. The major chemicals in the paper matrix are
Buret Buret Volume
start end titrant g mg calmagite and EDTA, and minor chemicals are added
Rep Dilution (mL) (mL) (mL) Ca/L CaCO3/L to minimize reaction time, give long-term stability,
and maximize color distinction between levels of
1 water hardness. The strips are dipped into the water to
2
test for total hardness caused by calcium and magne-
3
sium. The calcium displaces the magnesium bound to
X = X =
SD = SD = EDTA, and the released magnesium binds to calmag-
ite, causing the test strip to change color.
www.dbooks.org
152
16.3.3 Chemicals 1. Dip the test strip into a beaker filled with water
or the standard calcium solution. Follow
CAS no. Hazards instructions on strip about how to read it, relat-
ing color to ppm CaCO3.
Calcium carbonate 471-34-1 Harmful
(CaCO3)
2. Convert ppm CaCO3 as determined with the
Calmagite 3147-14-6 test strips to mg CaCO3/L and g Ca/L.
Ethylenediaminetetraacetic 60-00-4 Irritant
acid, disodium salt 16.3.8 Data and Calculations
(Na2EDTA · 2H20)
Hydrochloric acid, 7647-01-0 Corrosive Rep (ppm Rep (mg
concentrated (HCl) CaCO3) CaCO3/L) Rep (g Ca/L)
Other proprietary Sample 1 2 3 1 2 3 1 2 3
chemicals in test strip
Tap water
Tap
16.3.4 Reagents distilled
(**It is recommended that this solution be prepared by water
the laboratory assistant before class.) Standard
Ca
• Calcium standard solution, 1.000 mg CaCO3/mL** solution
Prepare as described in Sect.16.2.3, using CaCO3
and concentrated HCl. 16.3.9 Question
1. Compare and discuss the accuracy and preci-
16.3.5 Hazards, Precautions, and Waste sion of the EDTA titration and test strip meth-
Disposal ods to measure calcium carbonate contents of
No precautions are needed in use of the test strip. Adhere the water samples and the calcium standard
to normal laboratory safety procedures. Wastes likely solution.
may be put down the drain using a water rinse, but fol-
low good laboratory practices outlined by environmen-
tal health and safety protocols at your institution. RESOURCE MATERIALS
16.3.6 Supplies Rice, EW, Baird RB, Eaton AD, Clesceri LS (eds) (2012)
Standard methods for the examination of water and waste-
• AquaChek® Test Strips (Environmental Test water, 22st edn, Method 2340. American Public Health
Systems, Inc., a HACH Company, Elkhart, IN) Association, American Water Works Association, Water
• 2 Beakers, 100 mL Environment Federation, Washington, DC, pp. 2–37 to
2–39
16.3.7 Procedure Ward RE, Legako JF (2017) Traditional methods for mineral
analysis. Ch. 21. In: Nielsen SS (ed) Food analysis, 5th edn.
(Note: Test the same standard calcium solution as used Springer, New York
in Sect. 16.2.8.1 and the same tap water and tap dis-
tilled water as used in Sect.16.2.8.2.)
17
chapter
Phosphorus
Determination
by Murphy-Riley
Method
Young-Hee Cho (*) • S.Suzanne Nielsen

17.1.3 Objective 17.2.1 Ashing
17.1.4 Principle of Method 17.2.2 Phosphorus Measurement
and Waste Disposal
www.dbooks.org
154 Y.-H. Cho and S.S. Nielsen
17.1 INTRODUCTION distilled or distilled deionized water must be used in

all dilutions and preparation of all reagents.)
17.1.1 Background
• Ammonium molybdate**
Phosphorus is one of the important minerals found in
Dissolve 48 g of ammonium molybdate to
foods. Murphy-Riley method, a dry ashing colorimetric
1000 mL in a volumetric flask.
method, has been widely used to measure the phospho-
• Potassium antimonyl tartrate**
rus content in natural waters as well as in foods. In this
Dissolve 1.10 g of potassium antimonyl tartrate
procedure, it is necessary to ash the sample prior to
to 1000 mL in a volumetric flask.
analysis. The method described here is applicable to
• Ascorbic acid**
most foods following ashing. If the food sample has a
Prepare fresh daily. Dissolve 2.117 g to 50 mL in
low magnesium content, several milliliters of saturated
a volumetric flask.
Mg(NO3)2 · 6H2O in ethanol should be added to the sam-
• Phosphorus stock solution and working standard
ple prior to ashing to prevent volatilization and loss of
solution**
phosphorus at the high temperatures used in ashing.
Dissolved 0.6590 g of dried (105 °C, 2 h) mono-
potassium phosphate (KH2PO4) to 1000 mL in a
17.1.2 Reading Assignment volumetric flask. This solution contains 0.150 mg
Ward, R.E., and Legako, J.F. 2017. Traditional methods phosphorus/mL (i.e., 150 μg/mL). To prepare a
for mineral analysis. Ch. 21, in Food Analysis, 5th ed. working standard solution, dilute 10 mL of this
S.S. Nielsen (Ed.), Springer, New York. solution to 100 mL in a volumetric flask. The
solution now contains 15 μg phosphorus/mL.
17.1.3 Objective • 2.88 N sulfuric acid (H2SO4)**
Prepare 200 mL of 2.88 N sulfuric acid (H2SO4)
Determine the phosphorus content of milk using a col- using concentrated sulfuric acid (H2SO4) and
orimetric method, with the Murphy and Riley reagent. double-distilled or distilled deionized water.
Always add concentrated acid to water, not
17.1.4 Principle of Method water to concentrated acid. Do not use a
Ammonium molybdate reacts with phosphorus to mechanical pipette to pipette concentrated sul-
form phosphomolybdate. This complex is then furic acid and 2.88 N sulfuric acid, since this
reduced by ascorbic acid with antimony serving as a would corrode the pipettor.
catalyst. The reduced phosphomolybdate complex has • Murphy and Riley reagent
an intense blue color. The maximum absorptivity of Combine 14 mL of 2.88 N H2SO4, 2 mL of ammo-
this complex is at approximately 880 nm. This wave- nium molybdate solution, and 2 mL of potas-
length is above the operating range of most spectro- sium antimonyl tartrate solution in a 50 mL
photometers. The absorptivity of the molybdate Erlenmeyer flask.
complex is great enough in the region of 600–700 nm,
however, to allow determination of the phosphorus 17.1.7 Hazards, Precautions, and Waste
content of most foods at any wavelength within this Disposal
region. Most spectrophotometers have a maximum Adhere to normal laboratory safety procedures. Wear
usable wavelength of 600–700 nm. gloves, lab coat, and safety glasses at all time. Care
should be exercised in pipetting any solution that con-
17.1.5 Chemicals tains antimony since it is a toxic compound. Waste
containing antimony, sulfuric acid, and molybdate
CAS no. Hazard(s) must be discarded as hazardous waste. Other wastes
Ammonium molybdate 13106-76-8 Irritant likely may be put down the drain using a water rinse,
Potassium antimonyl 28300-74-5 Harmful but follow good laboratory practices outlined by envi-
tartrate ronmental health and safety protocols at your
Ascorbic acid 50-81-7 attention.
Monopotassium 7778-77-0
phosphate 17.1.8 Supplies
(Used by students)
17.1.6 Reagents
• 1 Crucible (preheated at 550 °C for 24 h)
(** It is recommended that these solutions be prepared • Glass funnel
by the laboratory assistant before class. Glassware • Glass stirring rods
cleaned by phosphate-free detergents and double- • Kimwipes
Chapter 17 • Phosphorus Determination by Murphy-Riley Method 155
• Mechanical adjustable volume pipettes, 1000 μL 3. Sample analysis: (Note: Do not use an automatic
with pipette tips pipettor to obtain 2 mL of the sulfuric acid. This
• Nonfat liquid milk, 5 g would corrode the pipettor.) Analyze one ashed
• Repipettor (for fast delivery of 2 mL H2SO4) sample. Carefully moisten ash in crucible with
• Test tubes (13 × 100 mm) H2O and then add 2 mL of 2.88 N H2SO4. Filter
• 1 Volumetric flask, 250 mL through ashless filter paper into a 250 mL volu-
• Whatman No. 41 ashless filter paper metric flask. Thoroughly rinse crucible, ash, and
filter paper. Dilute to volume with distilled water
17.1.9 Equipment and mix. Analyze in duplicate by combining
0.2 mL of sample, 3.8 mL of distilled water, and
• Analytical balance, 0.1 mg sensitivity
1 mL of Murphy and Riley reagent. Mix and allow
• Forced draft oven
to react for 10–20 min. Absorbance is read at
• Hot plate
700 nm as per the phosphorus standard solutions.
• Muffle furnace
• Spectrophotometer
• Vortex mixer 17.3 DATA AND CALCULATIONS
1. Report data obtained, giving A700 of the dupli-

17.2 PROCEDURE cate standards and sample and the average A700.
(Instructions are given for analysis in duplicate.) Absorbance (700 nm)

μg P/tube (5 mL) 1 2 Average
17.2.1 Ashing 1.5
3.0
(Based on note in Chap. 10, Sect. 10.2.7.3.) 4.5
1. Predry crucible at 550 °C for 24 h and weigh 6.0
Sample
accurately.
2. Accurately weigh ca. 5 g of sample in the
2. Construct a standard curve for your phosphorus
crucible.
determination, expressed in terms of phospho-
3. Heat on the hot place until a majority of water
rus (A700 vs. μg phosphorus/5 mL). Determine
has been evaporated.
the equation of the line for the phosphorus stan-
4. Dry in a forced draft oven at 100 °C for 3 h.
dard curve.
5. Ash in a muffle furnace at 550 °C for 18–24 h.
3. Calculate the concentration of phosphorus in
your milk sample expressed in terms of mg phos-
17.2.2 Phosphorus Measurement phorus/100 g sample. Show all calculations.
1. Preparation of Murphy and Riley reagent: Add Sample calculation for milk sample:
2 mL of ascorbic acid solution to pre-prepared
18 mL of Murphy and Riley reagent. Swirl to mix. Weight of milk sample: 5.0150 g
2. Standards: Prepare phosphorus standards using A700=0.394
working standard solution (15 μg phosphorus/ Equation of the line:
mL) and water as indicated in the table below. y = 0.12x + 0.0066
Pipet aliquots of the phosphorus standard into y = 0.394
clean test tubes (duplicated for each concentra- x = 3.3 μg P/5 mL
tion) and add water so each test tube contains
4 mL. Add 1 mL of Murphy and Riley (M&R) P content of milk sample
reagent to each tube. The final volume of each 3.2 g P 5 mL 250 mL
tube should be 5 mL. Mix with a vortex mixer.
5 mL 0.2 mL 5.0150g
Allow the color to develop at room temperature
for 10–20 min. Read the absorbance at 700 nm. 820 g P 82 mg P

g 100g
μg P/5 mL 15 μg P/mL Vol. water (mL) Vol. M&R (mL)

0 (blank) 0 4.0 1.0 17.4 QUESTIONS
1.5 0.1 3.9 1.0
3.0 0.2 3.8 1.0 1. How does your value compare to literature
4.5 0.3 3.7 1.0 value (US Department of Agriculture Nutrient
6.0 0.4 3.6 1.0 Database for Standard Reference) for phospho-
rus content of milk?
www.dbooks.org
156 Y.-H. Cho and S.S. Nielsen
2. If you had not been told to use a 250 mL volu- 4. What was the function of ascorbic acid in the
metric flask to prepare your ashed milk sam- assay?
ple, how could you have calculated the
dilution scheme was appropriate if you
wanted to use 0.2 mL ashed milk sample in RESOURCE MATERIALS
the assay. US Department of Agriculture
Murphy J, Riley JP (1962) A modified single solution method
Nutrient Database for Standard Reference for the determination of phosphate in natural waters.
indicates ca. 101 mg phosphorus/100 g. Show Anal. Chim. Acta 27:31–36
all calculations. Ward RE, Legako JF (2017) Traditional methods for mineral
3. What are the possible sources of error using this analysis. Ch. 21, In: Nielsen SS (ed) Food Analysis, 5th edn.
method to determine the phosphorus content of Springer, New York
milk?
18
chapter
Iron Determination
by Ferrozine Method
Charles E. Carpenter (*) • Robert E. Ward
Logan, UT, USA

18.1.3 Objective 18.2.1 Ashing
18.1.4 Principle of Method 18.2.2 Iron Measurement
18.1.6 Reagents 18.4 Question
and Waste Disposal
www.dbooks.org
158 C.E. Carpenter and R.E. Ward
18.1 INTRODUCTION 18.1.6 Reagents

• Ferrozine reagent, 1 mM in water
18.1.1 Background • Dissolve 0.493 g ferrozine reagent in water and
Chromogens are chemicals that react with compounds dilute to 1 l in a volumetric flask.
of interest and form colored products that can be quan- • Ascorbic acid; 0.02 % in 0.2 N HCl, made fresh daily
tified using spectroscopy. Several chromogens that • Ammonium acetate, 30 % w/v
selectively react with minerals are available. In this lab • Iron stock solution (10 μg iron/mL/0.1 N HCl)
ferrozine is used to measure ferrous iron in an ashed • Solutions of 1.0 N, 0.1 N, and 0.2 N HCl
food sample. The relationship between the absorbance
of the chromogen-mineral complex is described by 18.1.7 Hazards, Precautions, and Waste
Beer’s Law. In this procedure, a standard curve is gen- Disposal
erated with a stock iron solution to quantify the min- Adhere to normal laboratory safety procedures. Wear
eral in beef samples. safety glasses at all times! Waste may be put down the
In this experiment, meat samples are first ashed to drain using water rinse.
dissociate the iron bound to proteins, and the ash resi-
due is solubilized in dilute HCl. The acid is necessary 18.1.8 Supplies
to keep the mineral in solution. Ferrozine complexes
only with ferrous iron and not with ferric iron. Prior to • 16 Test tubes, 18 × 150 mm
the reaction with ferrozine, the solubilized ash is first • Meat sample
treated with ascorbic acid to reduce iron to the ferrous • Pipettes
form. This step is necessary with ashed samples, as this • Porcelain crucible
procedure would be expected to oxidize all the iron • Volumetric flask
present in the meat. However, when other treatments
are used to liberate iron, for example, trichloroacetic 18.1.9 Equipment
acid precipitation, comparison of samples treated with • Analytical balance
ascorbic acid and untreated samples could be done to • Hot plate
determine the ratio of ferrous to ferric iron in foods. • Muffle furnace
• Spectrophotometer
Ward, R.E., and Legako, J.F. 2017. Traditional methods
for mineral analysis. Ch. 21, in Food Analysis, 5th ed. 18.2 PROCEDURE
S.S. Nielsen (Ed.), Springer, New York.
(Instructions are given for analysis in duplicate.)
18.1.3 Objective
18.2.1 Ashing
Determine the iron content of food samples using the
ferrozine method. 1. In duplicate, place a ~5 g sample into the cruci-
ble and weigh accurately.
2. Heat on the hot plate until the sample is well
charred and has stopped smoking.
Ferrous iron in extracts or ashed samples reacts with 3. Ash in muffle furnace at ca 550 °C until the ash
ferrozine reagent to form a stable colored product is white.
which is measured spectrophotometrically at 562 nm.
Iron is quantified by converting absorbance to concen- 18.2.2 Iron Measurement
tration using a standard curve.
1. Prepare standards of 10, 8, 6, 4, 2, and 0 μg iron/
mL from a stock solution of 10 μg iron/
18.1.5 Chemicals mL. Make dilutions using ca 0.1 N HCl.
2. Dissolve ash in small amount of 1 N HCl, and
CAS no. Hazard(s)
dilute to 50 mL in volumetric flask with 0.1 N HCl.
3-(2-Pyridyl)-5,6-bis(4- 69898-45-9 3. In duplicate, put 0.500 mL of appropriately
phenylsulfonic acid)-1,2, diluted samples and standards into 10 mL test
-triazine (ferrozine; Sigma tubes.
P-9762) 4. Add 1.250 mL ascorbic acid (0.02 % in 0.2 N HCl,
Ascorbic acid (Sigma 255564) 50-81-7 made fresh daily). Vortex and let set for 10 min.
Ammonium acetate (Aldrich 631-61-8
5. Add 2.000 mL 30 % ammonium acetate. Vortex
372331)
(pH needs to be >3 for color development).
Iron stock solution (e.g., 4200-4205
200 ppm) (Aldrich 372331) 6. Add 1.250 mL ferrozine (1 mM in water). Vortex
and let set in dark for 15 min.
Chapter 18 Iron Determination by Ferrozine Method 159
7. Use water to zero the spectrophotometer at Calculation of total iron in sample:

562 nm (single-beam instrument) or place in the
1. Plot absorbance of standards on the y-axis ver-
reference position (dual-beam instrument).
sus μg iron/mL on the x-axis.
Take two readings (repeated measures, msmt)
2. Calculate the iron concentration in the ash solu-
for each tube at 562 nm.
tion from the standard curve: (abs – y inter-
cept)/slope = μg iron/mL ash solution.
3. Calculate iron in the sample using measured
18.3 DATA AND CALCULATIONS
iron value from standard curve and meat
Weight of original samples: sample:
(1)___________________ g (2)__________________ g Csample = (μg iron/mL ash solution) x (50 mL ash
solution/g meat) = μg iron/g meat
Absorbance of standards and samples:
Absorbance 18.4 QUESTION
Standards
(μg iron/ml) Rep 1 Rep 2 Mean 1. How else could iron be determined using the
0 ash digest? What would be the advantages and
2 disadvantages of the ferrozine method versus
4 the other method you identified?
6
8
10 RESOURCE MATERIALS
Ward RE and Legako, JF (2017) Traditional methods for min-

Meat sample
eral analysis. Ch. 21. In: Nielsen SS (ed) Food analysis, 5th
1
2
www.dbooks.org
19
chapter
Sodium Determination
Using Ion-Selective
Electrodes, Mohr
Titration, and Test
Strips
S. Suzanne Nielsen
19.1 Introduction 19.2.3 Chemicals

19.1.1 Background 19.2.4 Reagents
19.1.2 Reading Assignment 19.2.5 Hazards, Precautions,
19.2 Ion-Selective Electrodes and Waste Disposal
19.2.2 Principle of Method 19.2.7 Equipment
19.2.8 Procedure 19.3.9 Data and Calculations
19.2.9 Data and Calculations 19.3.10 Questions
19.2.10 Question 19.4 Quantab® Test Strips
19.3 Mohr Titration 19.4.1 Objective
19.3.1 Objective 19.4.2 Principle of Method
19.3.2 Principle of Method 19.4.3 Chemicals
19.3.3 Chemicals 19.4.4 Reagents
19.3.4 Reagents 19.4.5 Supplies
19.3.5 Hazards, Precautions, 19.4.6 Equipment
and Waste Disposal 19.4.7 Procedure
19.3.7 Equipment 19.5 Summary of Results
19.3.8 Procedure 19.6 Questions
www.dbooks.org
Chapter 19 • Sodium Determination Using Ion-Selective Electrodes, Mohr Titration, and Test Strips 163
19.1 INTRODUCTION CAS No. Hazards
19.1.1 Background Sodium nitrate (NaNO3) 7631-99-4 Harmful, oxidizing
Sodium content of foods can be determined by various

methods, including an ion-selective electrode (ISE), 19.2.4 Reagents
the Mohr or Volhard titration procedure, or indicator (**If these solutions are not purchased, it is recom-
test strips. These methods are official methods of anal- mended that these solutions be prepared by the labo-
ysis for numerous specific products. All these methods ratory assistant before class.)
are faster and less expensive procedures than analysis Note: You can use a chloride and/or sodium ion-
by atomic absorption spectroscopy or inductively cou- selective electrode, with the appropriate associated
pled plasma-optical emission spectroscopy. This solutions (commercially available from companies
experiment allows one to compare sodium analysis of that sell the electrodes): electrode rinse solution, ionic
several food products by ISE, Mohr titration, and strength adjuster, reference electrode fill solution,
Quantab® Chloride Titrators. standard solution, and electrode storage solution.
19.1.2 Reading Assignment • Electrode rinse solution**

Ward, R.E., and Legako, J.F. 2017. Traditional methods For sodium electrode, dilute 20-mL ionic strength
for mineral analysis. Ch. 21, in Food Analysis, 5th ed. adjuster to 1 L with deionized distilled (dd) water.
S.S. Nielsen (Ed.), Springer, New York. For chloride electrode, deionized distilled water.
• Ionic strength adjuster (ISA)
For sodium electrode, 4 M NH4Cl and 4 M
19.2 ION-SELECTIVE ELECTRODES NH4OH. For chloride electrode, 5 M NaNO3.
• Nitric acid, 0.1 N
19.2.1 Objective Dilute 6.3-mL conc. HNO3 to 1 L with dd water.
• Reference electrode fill solution**
Determine the sodium content of various foods with For sodium electrode, 0.1 M NH4Cl. For chloride
sodium and/or chloride ion-selective electrodes. electrode, 10 % KNO3.
• Standard solutions**: 1000 ppm, sodium and/or
19.2.2 Principle of Method chloride
The principle of ISE is the same as for measuring pH, but Use the 1000 ppm sodium or chloride solution to
by varying the composition of the glass in the sensing prepare 50 mL for each of the following concen-
electrode, the electrode can be made sensitive to sodium trations: 10, 20, 100, 500, and 1000 ppm sodium
or chloride ions. Sensing and reference electrodes are or chloride.
immersed in a solution that contains the element of
interest. The electrical potential that develops at the sur- 19.2.5 Hazards, Precautions, and Waste
face of the sensing electrode is measured by comparing Disposal
the reference electrode with a fixed potential. The volt-
Adhere to normal laboratory safety procedures. Wear
age between the sensing and reference electrodes relates
gloves and safety glasses at all times. Ammonium
to the activity of the reactive species. Activity (A) is
hydroxide waste should be discarded as hazardous
related to concentration (C) by A = γC, where γ is the
waste. Other waste likely can be put down the drain
activity coefficient, which is a function of ionic strength.
using a water rinse, but follow good laboratory prac-
By adjusting the ionic strength of all test samples and
tices outlined by environmental health and safety pro-
standards to a nearly constant (high) level, the Nernst
tocols at your institution.
equation can be used to relate electrode response to con-
centration of the species being measured.
19.2.6 Supplies
19.2.3 Chemicals • 16–18 Beakers, 250 mL (or sample cups to hold 100 mL)
• Food products: catsup, cottage cheese, potato chips,
CAS No. Hazards and sports drink (e.g., Gatorade, white or clear)
Ammonium chloride 12125-02-9 Harmful
(NH4Cl) • Magnetic stir bars
Ammonium hydroxide 1336-21-6 Corrosive, • Pipette bulb or pump
(NH4OH) dangerous for • 3 Spatulas
environment • 16–18 Volumetric flasks, 100 mL
Nitric acid (HNO3) 7697-37-2 Corrosive • 2 Volumetric flasks, 50 mL
Potassium nitrate (KNO3) 7757-79-1 • Volumetric pipette, 2 mL
Sodium chloride (NaCl) 7647-14-5 Irritant • 9 Volumetric pipettes, 5 mL
164 S.S. Nielsen
• Watch glass 1000 ppm sodium or chloride) to a 100-mL

• Weighing paper volumetric flask. Add 2-mL ISA, then dilute to
volume with dd water.
19.2.7 Equipment Note: Sample/standard preparation calls
for identical 1:20 dilution of each (i.e., 5 mL
diluted to 100 mL). Therefore, since sam-
• Direct concentration readout ISE meter (i.e., suit-
ples and standards are treated the same, no
able meter with millivolt accuracy to 0.1 mV)
correction for this dilution needs to be
• Heating plate with stirrer
made in calibration or calculation of results.
• Magnetic stirrer
• Chloride electrode (e.g., Van London-pHoenix
Company, Houston, TX, Chloride Ion Electrode, 19.2.8.2 Sample Analysis by ISE
Cat. # CL01502) 1. Condition sodium electrode as specified by the
• Sodium electrode (e.g., Van London-pHoenix manufacturer.
Company, Houston, TX, Sodium Ion Electrode, Cat. 2. Assemble, prepare, and check sodium and ref-
# NA71502) erence electrodes as described in electrode
instruction manuals.
19.2.8 Procedure 3. Connect electrodes to meter according to meter
instruction manual.
(Replicate the preparation and analysis of standards 4. For instruments with direct concentration read-
and samples as specified by an instructor.) out capability, consult meter manual for correct
direct measurement procedures.
19.2.8.1 Sample Preparation (General 5. Using the pH meter set on mV scale, determine
Instructions) the potential (mV) of each standard solution
1. Treat specific samples as described below (i.e., (10, 20, 100, 500, and 1000 ppm), starting with
prehomogenized and/or diluted if necessary, as the most dilute standard. Use a uniform stirring
per Technical Services of ISE manufacturer), rate, with a magnetic stir bar in each solution,
and then add 5 g or 5 mL of prepared sample to placed on a magnetic stir plate.
a 100-mL volumetric flask, then add dd 2-mL 6. Rinse electrodes with electrode rinse solution
ISA, and dilute to volume with dd water. (See between standards.
instructions specific for each type of food prod- 7. Measure samples and record the mV reading.
uct below. Samples with high fat levels may As you rinse electrodes with electrode rinse
require fat removal. Consult technical services solution between measurements, be careful not
of the company that manufactures the ISE.) to get rinse solutions into the hole for outer fill
Specific Samples: solution in the reference electrode (or ensure
that the hole is covered).
Sports drink: No dilution is required 8. After use, store sodium electrode and reference
before use. electrode as specified by manufacturer.
Catsup: Accurately weigh ca. 1 g catsup
into 50-mL volumetric flask, and dilute to 19.2.9 Data and Calculations
volume with dd water. Mix well. 1. Prepare a standard curve, with electrode
Cottage cheese: Accurately weigh ca. 1 g response plotted against concentration on a log
of finely grated cheese into a 250-mL bea- scale. (Plot actual concentration values on the
ker containing a stir bar. Add 100-mL 0.1 N log scale, not log values.) Concentrations may
HNO3. Cover beaker with a watch glass be determined by reading directly off the stan-
and boil gently for 20 min on stirrer/hot dard curve or using a calculated equation of the
plate in a hood. Remove from hot plate line.
and cool to room temperature in the hood. 2. Use the standard curve and the mV readings for
the samples to determine the sodium and/or
Potato chips: Accurately weigh ca. 5 g of chloride concentrations in ppm for the food
potato chips into a 250-mL beaker. Crush samples as analyzed.
chips with a glass stirring rod. Add 95-mL 3. Convert the ppm sodium and/or chloride val-
boiling dd water and stir. Filter water ues for the food samples to mg/mL for the
extract into a 100-mL volumetric flask, sports drink, catsup, cheese, and potato chips.
using a funnel with glass wool. Let cool to 4. Taking into account the dilution of the samples,
room temperature and dilute to volume. calculate the sodium and/or chloride content
2. Prepare standards by adding 5-mL standard of for catsup, cheese, and potato chips (in mg/g)
proper dilution (e.g., 10, 20, 100, 500, and (on a wet weight basis). Summarize the data
www.dbooks.org
and calculated results in one table. Show all • Silver nitrate solution, ca. 0.1 M **
sample calculations below each table. Prepare approximately 400 mL of the ca. 0.1 M
5. Calculate sodium chloride content of each food, AgNO3 (molecular weight (MW) 169.89) for each
based on the (a) chloride content and/or (b) student or lab group. Students should accurately
sodium content. standardize the solution, as described in the
6. Calculate the sodium content of each food, Sect. 19.3.8.1.
based on the sodium chloride content.
7. Compare the sodium/sodium chloride contents 19.3.5 Hazards, Precautions, and Waste
of the foods you analyzed to those reported in Disposal
the US Department of Agriculture (USDA)
Wear gloves and safety glasses at all times, and use
Nutrient Database for Standard Reference
good lab technique. Potassium chromate may cause
(http://ndb.nal.usda.gov).
serious skin sensitivity reactions. The use of crystal-
line AgNO3 or solutions of the silver salt can result in
19.2.10 Question
dark brown stains caused by photodecomposition of
1. If you used both a sodium and chloride ISE, the salt to metallic silver. These stains are the result
which electrode worked better, concerning accu- of poor technique on the part of the analyst, with
racy, precision, and time to response? Explain spilled AgNO3 causing discoloration of the floor. If
your answer, with appropriate justification. you do spill this solution, immediately sponge up
the excess solution and thoroughly rinse out the
sponge at a sink. Then come back with the clean,
19.3 MOHR TITRATION rinsed sponge and mop up the area at least 3–4 times
to remove all of the silver nitrate. Also, be sure to
19.3.1 Objective rinse all pipettes, burets, beakers, flasks, etc. to
Determine the sodium content of various foods using remove residual AgNO3 when you are finished with
the Mohr titration method to measure chloride this experiment. Otherwise these items also will
content. stain, and drip stains are likely to appear on the floor.
Potassium chromate and silver nitrate must be dis-
posed of as a hazardous waste. Other waste likely
can be put down the drain using a water rinse, but
The Mohr titration is a direct titration method to quan- follow good laboratory practices outlined by envi-
titate chloride ions and then to calculate sodium ions. ronmental health and safety protocols at your
The chloride-containing sample solution is titrated institution.
with a standard solution of silver nitrate. After the sil-
ver from silver nitrate has complexed with all the 19.3.6 Supplies
available chloride in the sample, the silver reacts with
chromate that has been added to the sample, to form • 6 Beakers, 250 mL
an orange-colored solid, silver chromate. The volume • Brown bottle, 500 mL
of silver used to react with the chloride is used to cal- • Buret, 25 mL
culate the sodium content of the sample. • 3 Erlenmeyer flasks, 125 mL
• 4 Erlenmeyer flasks, 250 mL
19.3.3 Chemicals • Food products: cottage cheese (30 g), potato chips
(15 g), and sports drink (15 mL) (e.g., Gatorade,
CAS No. Hazards white or clear)
• Funnel
Potassium 7447-40-7 Irritant • Glass wool
chloride (KCl) • Graduated cylinder, 25 mL
Potassium chromate 7789-00-6 Toxic, dangerous for
• Magnetic stir bars (to fit 125 or 250-mL flasks)
(K2CrO4) environment
• Pipette bulb or pump
Silver nitrate 7761-88-8 Corrosive, dangerous
(AgNO3) for environment • Spatulas
• Weighing paper and boats
19.3.4 Reagents
(**It is recommended that these solutions be prepared 19.3.7 Equipment
by laboratory assistant before class.)
• Hot plate
• Potassium chloride
• Magnetic stir plate
• Potassium chromate, 10 % solution**
166 S.S. Nielsen
19.3.8 Procedure 5. Add 1 mL of potassium chromate indicator to

each 50 mL of filtrate.
6. Titrate each solution with standardized ca.
0.1 M AgNO3, to the first visible pale red-brown
19.3.8.1 Standardization of ca. 0.1 M AgNO3
color that persists for 30 s. Record the volume of
1. Transfer 400 mL of the 0.1 M AgNO3 solution to
titrant used.
a brown bottle. This solution will be standard-
ized and then used to titrate the food samples.
Fill a buret with this AgNO3 solution. Potato Chips
2. Prepare the primary standard (KCl, MW = 74.55) 1. Weigh accurately approximately 5 g of potato
solution in triplicate. Accurately weigh to four chips in duplicate into 250-mL beakers, then
decimal places about 100 mg KCl into three 125- add 95-mL boiling dd water to each beaker.
mL Erlenmeyer flasks. Dissolve in dd water 2. Stir the mixture vigorously for 30 s, wait for
(about 25 mL), and add 2–3 drops of K2CrO4 1 min, stir again for 30 s, then let cool to room
solution. (Caution: potassium chromate may temperature.
cause serious skin sensitivity reactions!) 3. Filter each solution through glass wool. Transfer
3. Put a magnetic stir bar in each flask with the KCl 50 mL of each solution to 250-mL Erlenmeyer
solution, and place the beaker on a magnetic stir flasks.
plate below the buret for titration. Using the 4. Add 1 mL of potassium chromate indicator to
AgNO3 solution in the buret, titrate the KCl solu- each 50 mL of filtrate.
tions to the appearance of the first permanent, 5. Titrate each solution with standardized ca.
pale, pink-orange color. (Note: you will first get 0.1 M AgNO3, to the first visible pale red-brown
a white precipitate, then green color, and then color that persists for 30 s. Record the volume of
the pink-orange color.) This endpoint is due to titrant used.
the formation of Ag2CrO4. The solution must
be vigorously stirred during the addition of the
AgNO3 solution to avoid erroneous results. Sports Drink (Clear or White)
4. Record volume of AgNO3. 1. Pipette accurately 5 mL of sports drink in dupli-
5. Calculate and record molarity of AgNO3. cate into 250-mL beakers, then add 95-mL boil-
g KC1 1 molKCl 1000 mL ing dd water to each beaker.
2. Stir the mixture vigorously for 30 s, wait 1 min,
mL AgNO 3 74.555 g 1L
and stir again for 30 s.
M of AgNO 3 / L M AgNO 3 3. Transfer 50 mL of each solution to 250-mL
6. Label bottle of AgNO3 with your name and the Erlenmeyer flasks.
molarity of the solution. 4. Add 1 mL of potassium chromate indicator to
each 50 mL of prepared sample.
19.3.8.2 Sample Analysis by Mohr Titration 5. Titrate each solution with standardized ca.
0.1 M AgNO3, to the first visible pale red-brown
Cottage Cheese color that persists for 30 s. Record the volume of
titrant used.
1. Accurately weigh 10 g of cottage cheese in trip-
licate into 250-mL beakers.
2. Add about 15 mL of warm dd water (50–55 °C) 19.3.9 Data and Calculations
to each beaker. Mix to a thin paste using a glass
stirring rod or spatula. Add another ca. 25-mL dd 1. Calculate the chloride content and the sodium
water to each beaker until the sample is dispersed. chloride content of each replicated sample,
3. Quantitatively transfer each solution to a 100- then calculate the mean and standard devia-
mL volumetric flask, rinsing beaker, and mag- tion for each type of sample. Express the val-
netic stir bar with dd water several times. Dilute ues in terms of percent, wt/vol, for the
to volume with dd water. cottage cheese and potato chips, and percent,
4. Filter each solution through glass wool. Transfer vol/vol, for the sports drink. Note that
50 mL of each solution to 250-mL Erlenmeyer answers must be multiplied by the dilution
flasks. factor.
mL of AgNO 3 mol AgNO 3 35.5 g Cl 1L

% chloride 100 dilution factor
g or mL sample L mol NaCl 1000 mL
mL of AgNO 3 mol AgNO 3 58.5 g 1L
% sodium chloride salt 100 dilution factor
g or mL sample liter mol NaCl 1000 mL
www.dbooks.org
Sample Rep Buret start (mL) Buret end (mL) Vol. AgNO3 (mL) % Cl % NaCl
Cottage cheese 1
2
3
X =
SD =
Potato chips 1
2
3
X =
SD =
Sports drink 1
2
3
X =
SD =
19.3.10 Questions the white color change is proportional to the chloride

concentration of the liquid being tested. The value on
1. Show the calculations of how to prepare 400 mL
the numbered scale is read at the tip of the color change
of an approximately 0.1 M solution of AgNO3
and then is converted to percent salt using a calibra-
(MW = 169.89).
tion table.
2. Would this Mohr titration procedure as
described above work well to determine the salt
19.4.3 Chemicals
content of grape juice or catsup? Why or why
not?
CAS No. Hazards
3. How did this method differ from what would
be done using a Volhard titration procedure? Sodium chloride (NaCl) 7647-14-5 Irritant
Include in your answer what additional
reagents would be needed.
19.4.4 Reagents
4. Would overshooting the endpoint result in an
over- or underestimation of the salt content • Sodium chloride stock solution
using the (a) Mohr titration or (b) Volhard Accurately weigh 5.00 g of dried sodium chlo-
titration? ride and quantitatively transfer to a 100-mL vol-
umetric flask. Dilute to volume with dd water
and mix thoroughly.
19.4 QUANTAB® TEST STRIPS • Sodium chloride standard solutions
Dilute 2 mL of the stock solution to 1000 mL
19.4.1 Objective with dd water in a volumetric flask to create a
0.010 % sodium chloride solution to use as a
To measure the chloride content of foods using
standard solution with the low range Quantab®
Quantab® Chloride Titrators, then calculate the sodium
Chloride Titrators.
chloride content.
Dilute 5 mL of the stock solution to 100 mL with
dd water in a volumetric flask to create a 0.25 %
sodium chloride solution to use as a standard
Quantab® Chloride Titrators are thin, chemically inert solution with the high range Quantab® Chloride
plastic strips. These strips are laminated with an absor- Titrators.
bent paper impregnated with silver nitrate and potas-
sium dichromate, which together form brown-silver
19.4.5 Supplies
dichromate. When the strip is placed in an aqueous
solution that contains chlorine, the liquid rises up the • 5 Beakers, 200 mL
strip by capillary action. The reaction of silver dichro- • Filter paper (when folded as a cone, should fit into
mate with chloride ions produces a white column of a 200-mL beaker)
silver chloride in the strip. When the strip is com- • Funnels
pletely saturated with the liquid, a moisture-sensitive • Glass wool
signal across the top of the titrator turns dark blue to • Glass stirring rod
indicate the completion of the titration. The length of • Graduated cylinder, 100 mL
168 S.S. Nielsen
• Quantab® Chloride Titrators, range: 0.05–1.0 % 2. Stir mixture vigorously for 30 s, wait for 1 min,
NaCl; 300–6000 ppm Cl (High Range, HR) and stir again for 30 s, and then let cool to room
0.005–0.1 % NaCl; 30–600 ppm Cl (Low Range, LR) temperature.
(Environmental Test Systems/Hach Company, 3. Fold a piece of filter paper into a cone-shaped
Elkhart, IN, 1-800-548-4381). cup and place it point end down the beaker.
• Spatulas This will allow liquid from the beaker to seep
• Sports drink, 10 mL (i.e., same one used in Sects. through the filter paper at the pointed end.
19.2 and 19.3) 4. Testing with both the Low Range and the High
• 2 Volumetric flasks, 100 mL Range Quantab® Test Strips, place the lower
end of the Quantab® into the filtrate within the
19.4.6 Equipment pointed end of the filter paper cone, being sure
not to submerge the titrator more than 2.5 cm.
• Hot plate 5. Thirty seconds after the moisture-sensitive sig-
• Top loading balance nal string at the top of the titrator turns dark
blue or a light brown, record the Quantab®
19.4.7 Procedure reading at the tip of the yellow-white peak, to
(Instructions are given for analysis in triplicate.) the nearest 0.1 units on the titrator scale.
6. Using the calibration chart included with the
19.4.7.1 Standard Solutions of Sodium Quantab® package, convert the Quantab® read-
Chloride ing to percent sodium chloride (NaCl) and to
1. Transfer 50 mL of the 0.25 % standard sodium ppm chloride (Cl−). Note that each lot of
chloride solution to a 200-mL beaker. Quantab® has been individually calibrated. Be
2. Fold a piece of filter paper into a cone-shaped sure to use the correct calibration chart (i.e., the
cup and place it point end down into the beaker. control number on the product being used must
This will allow liquid from the beaker to seep match the control number on the bottle).
through the filter paper at the pointed end. 7. Multiply the result by the dilution factor 20 to
3. Using the 0.25 % sodium chloride standard obtain the actual salt concentration in the
solution, place the lower end of the High Range sample.
Quantab® Strip (0.05–1.0 %) into the filtrate
Potato Chips
within the pointed end of the filter paper cone,
being sure not to submerge the titrator more 1. Weigh accurately approximately 5 g of potato
than 1.0 in. chips into a 200-mL beaker. Crush chips with a
4. Thirty seconds after the moisture-sensitive sig- glass stirring rod. Add 95-mL boiling dd water
nal string at the top of the titrator turns dark and stir.
blue or a light brown, record the Quantab® 2. Filter water extract into a 100-mL volumetric
reading at the tip of the yellow-white peak, to flask, using a funnel with glass wool. Let cool to
the nearest 0.1 units on the titrator scale. room temperature and dilute to volume.
5. Using the calibration chart included with the Transfer to a 200-mL beaker.
Quantab® package, convert the Quantab® 3. Follow Steps 3–7 from the procedure for cottage
reading to percent sodium chloride (NaCl) cheese, Sect. 19.4.7.2.
and to ppm chloride (Cl−). Note that each lot
of Quantab® has been individually calibrated. Catsup
Be sure to use the correct calibration chart (i.e., 1. Weigh accurately approximately 5 g of catsup
the control number on the product being used into a 200-mL beaker. Add 95-mL boiling dd
must match the control number on the water and stir.
bottle). 2. Filter water extract into a 100-mL volumetric
6. Repeat Steps 1–5 given above (Sect. 19.4.7.1) flask. Let cool to room temperature and dilute
using the 0.01 % sodium chloride standard solu- to volume. Transfer to a 200-mL beaker.
tion with the Low Range Quantab® Strip. 3. Follow Steps 3–7 from the procedure for cottage
cheese, Sect. 19.4.7.2.
19.4.7.2 Sample Analysis with Quantab® Test
Strips Sports Drink
Cottage Cheese 1. Weigh accurately approximately 5 mL of sports

drink into a 200-mL beaker. Add 95-mL boiling
1. Weigh accurately approximately 5 g of cottage dd water and stir.
cheese into a 200-mL beaker, then add 95-mL 2. Follow Steps 3–7 from procedure for cottage
boiling dd water. cheese, Sect. 19.4.7.2.
www.dbooks.org
From calibration chart Corrected for dilution factor

% NaCl ppm Cl % NaCl ppm Cl
Rep LR HR LR HR LR HR LR HR
Catsup
1
2
3
X = X = X = X =
SD = SD = SD = SD =
Cottage cheese
1
2
3
X = X = X = X =
SD = SD = SD = SD =
Potato chips
1
2
3
X = X = X = X =
SD = SD = SD = SD =
Sports drink
1
2
3
X = X = X = X =
SD = SD = SD = SD =
19.5 SUMMARY OF RESULTS described in this experiment. Include in the table the
sodium chloride contents of the foods from the nutri-
Summarize in a table the sodium chloride content tion label and those published in the USDA Nutrient
(mean and standard deviation) of the various food Database for Standard Reference (web address: http://
products as determined by the three methods ndb.nal.usda.gov/).
Sodium chloride content (%) of foods by various methods:
Food Product Ion-selective electrode Mohr titration Quantab® titrator Nutrition label USDA Database
Catsup X =
SD =
Cottage cheese X =
SD =
Potato chips X =
SD =
Sports drink X =
SD =
170 S.S. Nielsen
19.6 QUESTIONS AOAC International (2016) Official methods of analysis, 20th

edn. (On-line). Method 976.25, Sodium in foods for special
1. Based on the results and characteristics of the dietary use, ion selective electrode method. AOAC
International, Rockville, MD
methods, discuss the relative advantages and
disadvantages of each method of analysis for edn. (On-line). Method 971.19, Salt (chlorine as sodium
these applications. chloride) in meat, fish, and cheese; Indicating strip method.
2. Comparing your results to data from the nutri- AOAC International, Rockville, MD
tion label and USDA Nutrient Database, what Ward RE, Legako JF (2017) Traditional methods for mineral
factors might explain any differences observed? analysis. Ch. 21. In: Nielsen SS (ed) Food analysis, 5th edn.
Springer, New York
Acknowledgments Van London-pHoenix Company, Environmental Test Systems (2016) Quantab® Technical
Houston, TX, is acknowledged for its contribution of the Bulletin. Chloride analysis for cottage cheese.
sodium and chloride ion-selective electrodes, and related Environmental Test Systems, Elkhart, IN
supplies, for use in developing a section of this laboratory Van London-pHoenix Company, Houston, TX. Product
exercise. Environmental Test Systems/HACH Company, literature.
Elkhart, IN, is acknowledged for contributing the Quantab® Wehr HM, Frank JF (eds) (2004) Standard methods for the
Chloride Titrators for use in developing a section of this labo- examination of dairy products, 17th edn., Part 15.053
ratory exercise. Chloride (Salt). American Public Health Association,
Washington, DC
RESOURCE MATERIALS

edn. (On-line). Method 941.18, Standard solution of silver
nitrate; Method 983.14, Chloride (total) in cheese. AOAC
International, Rockville, MD
www.dbooks.org
20
Chapter
Sodium and Potassium

Determinations
by Atomic Absorption
Spectroscopy
and Inductively
Coupled Plasma-
Optical Emission
Spectroscopy
20.1 Introduction
20.1.1 Background
20.1.3 Note
20.1.4 Objective
20.1.6 Chemicals
20.1.7 Reagents
20.1.8 Hazards, Precautions, and Waste Disposal
20.1.9 Supplies
20.1.10 Equipment
20.2 Procedure
20.2.1 Sample Preparation: Liquid Samples
20.2.2 Sample Preparation: Solid Samples
20.2.3 Analysis
20.3 Data and Calculations
20.3.1 Standard Curve Data
20.3.2 Sample Data
20.3.3 Data Handling
20.4 Questions
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Chapter 20 • Sodium and Potassium Determinations by Atomic Absorption Spectroscopy 173
20.1 INTRODUCTION 20.1.4 Objective

The objective of this experiment is to determine the
20.1.1 Background sodium and potassium contents of food products
The concentration of specific minerals in foods can using atomic absorption spectroscopy and inductively
be determined by a variety of methods. The aim of coupled plasma-atomic emission spectroscopy.
this lab is to acquaint you with the use of atomic
absorption spectroscopy (AAS) and atomic emission 20.1.5 Principle of Method
spectroscopy (AES) for mineral analysis. As
Atomic absorption is based on atoms absorbing
described in Yeung et al., 2017, AES is also com-
energy, once heat energy from a flame has converted
monly called optical emission spectroscopy (OES).
molecules to atoms. By absorbing the energy, atoms
The OES term is usually used in place of AES when
go from ground state to an excited state. The energy
it is combined with inductively coupled plasma
absorbed is of a specific wavelength from a hollow
(ICP). Hence, this chapter will refer to inductively
cathode lamp. One measures absorption as the dif-
coupled plasma-optical emission spectroscopy as
ference between the amount of energy emitted from
ICP-OES.
the hollow cathode lamp and that reaching the detec-
This experiment specifies the preparation of stan-
tor. Absorption is linearly related to concentration.
dards and samples for determining the sodium (Na)
Atomic emission is based on atoms emitting
and potassium (K) contents by AAS and ICP-OES. The
energy, after heat energy from a flame has converted
samples suggested for analysis include two solid food
molecules to atoms and then raised the atoms from
products that require wet and/or dry ashing prior to
ground state to an excited state. The atoms emit energy
analysis (catsup and potato chips) and one liquid food
of a specific wavelength as they drop from an excited
product that does not (a clear sports drink or a clear
state back to ground state. One measures the amount
fruit juice).
of emitted energy of a wavelength specific for the ele-
Procedures for both wet ashing and dry ashing of
ment of interest. Emission is linearly related to
the solid samples are described. Experience can be
concentration.
gained with both types of ashing and results can be
compared between the two methods of ashing. Sodium
20.1.6 Chemicals
results from this experiment can be compared with
sodium results from analysis of the same products in
CAS no. Hazards
the experiment that uses the more rapid methods of
analysis of ion selective electrodes, the Mohr titration, Hydrochloric acid (HCl) 7647-01-1 Corrosive
and Quantab® test strips. Hydrogen peroxide, 30 % 7722-84-1 Corrosive
The limit of detection for sodium is 0.3 parts per (H2O2)
billion (ppb) for flame AAS, 3 ppb by radial ICP-OES, Lanthanum chloride (LaCl3) 10025-84-0 Irritant
Nitric acid (HNO3) 7697-37-2 Corrosive
and 0.5 ppb by axial ICP-OES. The limit of detection
Potassium chloride (KCl) (for 7447-40-7 Irritant
for potassium is 3 ppb for flame AAS, 0.2–20 ppb K std. solution)
(depending on the model) by radial ICP-OES, and Sodium chloride (NaCl) (for 7647-14-5 Irritant
1 ppb by axial ICP-OES. Other comparative character- Na std. solution)
istics of AAS and ICP-OES are described in Yeung
et al., 2017.
20.1.7 Reagents
20.1.2 Reading Assignment (**It is recommended that these solutions be prepared
Harris, G.K., and Marshall, M.R. 2017. Ash analysis.
• Potassium and sodium standard solutions,
Ch. 16, in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
1000 ppm**
Springer, New York.
Used to prepare 100-mL solutions of each of the
Yeung, C.K., Miller, D.D., and M.A. Rutzke. 2017.
concentrations listed in Table 20.1. Each stan-
Atomic absorption spectroscopy, atomic emission
dard solution must contain 10 mL conc.
spectroscopy, and inductively coupled plasma mass
HCl/100 mL final volume.
spectrometry. Ch. 9, in Food Analysis, 5th ed. S.S. Nielsen
(Ed.), Springer, New York.
Disposal
20.1.3 Note
If there is no access to an ICP-OES, a simple AES unit Adhere to normal laboratory safety procedures. Wear
can be used, likely with the same standard solutions safety glasses and gloves during sample preparation.
and samples prepared as described below. Use acids in a hood.
174 S.S. Nielsen
20.1 4. Shake well. (If there is any particulate matter

table
present, the sample will need to be filtered
Concentrations (ppm) of Na and K stan- through ashless filter paper.)
dard solutions for AAS and ICP-OES
5. Make appropriate dilution and analyze (to sam-
AAS ICP-OES ple for AAS, add LaCl3 to final conc. of 0.1 %).
Na K Na K
0.20 0.10 50 50 Liquid Blank:
0.40 0.50 100 100
0.60 1.00 200 200 Prepare a liquid blank sample to be assayed,
0.80 1.50 300 300 following the sample preparation procedure
1.00 2.00 400 400 but excluding the sample.
20.1.9 Supplies 20.2.2 Sample Preparation: Solid Samples
(Used by students) Wet Ashing
• Two crucibles, previously cleaned and heated at Note: Digestion procedure described is a wet digestion
550 °C in a muffle furnace for 18 h (for dry ashing) with nitric acid and hydrogen peroxide. Other types of
• Desiccator, with dry desiccant digestion can be used instead.
• Digestion tubes (for wet ashing; size to fit digestion 1. Label one digestion tube per sample plus one
block) tube for the reagent blank (control).
• Filter paper, ashless 2. Accurately weigh out 300–400 mg of each sam-
• Funnels, small (to filter samples) ple and place in a digestion tube. Prepare sam-
• Plastic bottles, with lids, to hold 50 mL (or plastic ples in duplicate or triplicate.
sample tubes with lids, to hold 50 mL, to fit autos- 3. Pipette 5-mL concentrated nitric acid into each
ampler, if one is available) tube, washing the sides of the tube as you add
• Eight volumetric flasks, 25 mL the acid.
• Four volumetric flasks, 50 mL 4. Set tubes with samples and reagent blank in
• Volumetric flask, 100 mL digestion block. Turn on the digestion block
• Volumetric pipettes, 2 mL, 4 mL, 5 mL, and 10 mL and set to 175 °C to start the predigestion.
(2) 5. Swirl the samples gently once or twice during
• Weigh boats/paper the nitric acid predigestion, using tongs and
protective gloves.
20.1.10 Equipment 6. Remove tubes from digestion block when
brown gas starts to elute (or when solution
• Analytical balance begins to steam, if there is no brown gas) and
• Atomic absorption spectroscopy unit set in the cooling rack. Turn off the digestion
• Digestion block (for wet ashing; set to 175 °C) block.
• Inductively coupled plasma-atomic absorption 7. Let the samples cool for at least 30 min. (Samples
spectroscopy unit (or simple atomic absorption can be stored at this point for up to 24 h.)
spectroscopy unit) 8. Add 4 mL of 30 % hydrogen peroxide to each
• Muffle furnace (for dry ashing; set to 550 °C) tube, doing only a few tubes at one time. Gently
• Water bath, heated to boil water (for dry ashing) swirl the tubes. Put the tubes back in the diges-
tion block. Turn on the digestion block still set
to 175 °C.
20.2 PROCEDURE 9. Watch tubes closely for the start of the reaction,
indicated by the appearance of rapidly rolling
20.2.1 Sample Preparation: Liquid Samples bubbles. As soon as the reaction starts, remove
1. Put an appropriate volume of liquid sample in a the tubes from the block and let the reaction
100-mL volumetric flask. For a sports drink, use continue in the cooling rack. (Caution: Some
0.2 mL for both Na and K analysis by AAS. Use sample types will have a vigorous reaction, and
50 mL for Na analysis and 80 mL for K analysis for some the sample is lifted to the top of the
by ICP-OES. tube, with the risk of boiling over.)
2. Add 10 mL conc. HCl. 10. Repeat Steps 8 and 9 for all the samples and the
3. Add deionized distilled (dd) water to volume. reagent blank.
www.dbooks.org
20.2
table Dilution of samples for Na and K analysis by AAS and ICP-OES, using weta or dry ashingb
Na K
Sample AAS ICP-OES AAS ICP-OES
Catsup
Wet ashing Ashed sample diluted to Ashed sample diluted Ashed sample diluted to Ashed sample diluted
25 mL, then 0.2 mL to 25 mL 25 mL, then 0.4 mL to 10 mL
diluted to 100 mL diluted to 100 mL
Dry ashing Ashed sample diluted to Ashed sample diluted Ashed sample diluted to Ashed sample diluted
Cottage cheese
Potato chips
a
For wet ashing, use ca. 300–400 mg sample
b
For dry ashing, use ca. 1-g sample, dry matter (calculate based on moisture content)
11. Put all tubes in the digestion block, and leave 2. Pre-dry sample over boiling water bath.
until ca. 1–1.5 mL remains, and then remove 3. Complete drying of sample in vacuum oven at
each tube from the digestion block. Check the 100 °C, 26-in. Hg, for 16 h.
tubes every 10–15 min during this digestion. (If 4. Dry ash sample for 18 h. at 550 °C and then let
the tubes are left on the digestion block too long cool in desiccator.
and they become dry, remove, cool, and carefully 5. Dissolve ash in 10-mL HCl solution (1:1,
add ca. 2-mL concentrated nitric acid and con- HCl:H2O).
tinue heating.) Turn off the digestion block when 6. Make appropriate dilution of samples with dd
all the tubes have been digested and removed. water in a volumetric flask as indicated in
12. Make appropriate dilution of samples with dd Table 20.2. (To sample for AAS, add LaCl3 to
water in a volumetric flask as indicated in final conc. of 0.1 %.)
Table 20.2. (To sample for AAS, add LaCl3 to 7. If necessary, filter samples using Whatman
final conc. of 0.1 %.) hardened ashless #540 filter paper into con-
13. If necessary, filter samples using Whatman tainer appropriate for analysis by AAS or
hardened ashless #540 filter paper into con- ICP-OES.
tainer appropriate for analysis by AAS or
ICP-OES. 20.2.3 Analysis
Dry Ashing Follow manufacturer’s instructions for start-up, use,
and shutdown of the AAS and ICP-OES. Take appro-
1. Accurately weigh out blended or ground ca. l-g priate caution with the acetylene and flame in using
sample dry matter into crucible (i.e., take mois- AAS and the liquid or gas argon and the plasma in
ture content into account, so you have ca. 1-g using the ICP-OES. Analyze standards, reagent blanks,
dry product). and samples.
176 S.S. Nielsen
20.3 DATA AND CALCULATIONS parison to AAS standard curves. If ICP-OES emission
data are available for samples, they should be con-
Note: Because of the nature of the differences between verted to concentration data in ppm using the appro-
printouts for various ICP-OES manufacturers, the ICP priate standard curve. If ICP-OES emission data are
operator should assist with interpretation of ICP-OES not available, report concentration in ppm.
results. As specified under data handling instructions Do all calculations for each duplicate sample indi-
below, if ICP-OES emission data are available for stan- vidually, before determining a mean value on the final
dards, they should be recorded and plotted, for com- answer.
20.3.1 Standard Curve Data

Potassium standard curves Sodium standard curves
AAS ICP-OES AAS ICP-OES
ppm Absorption ppm Emission ppm Absorption ppm Emission
50 1 50 1
100 5 100 5
200 10 200 10
300 20 300 20
20.3.2 Sample Data

Atomic absorption spectroscopy:
Rep Sample size Absorption ppm Dilution Diluted (mg/mL Original (mg/mL or
Sample (g or mL) or g/g) mg/g)
Liquid blank 1 – – –
2 – – –
Sports drink 1
2
Solid blank 1 – – –
2 – – –
Catsup 1
2
Cottage cheese 1
2
Potato chips 1
2
Inductively coupled plasma – optical emission spectroscopy:

Rep Sample size Emission ppm Dilution Diluted (mg/mL Original (mg/mL or
Sample (g or mL) or g/g) mg/g)
Liquid blank 1 – – –
2 – – –
Sports drink 1
2
Solid blank 1 – – –
2 – – –
Catsup 1
2
Cottage cheese 1
2
Potato chips 1
2
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20.3.3 Data Handling 20.4 QUESTIONS

1. Prepare standard curves for sodium and potas-
sium as measured by AAS. 1. Compare the sodium and potassium values for
2. Use the standard curves from AAS and the catsup and potato chips to those reported in the
absorption readings of the samples to deter- US Department of Agriculture Nutrient
mine the concentrations in ppm of sodium and Database for Standard Reference (http://ndb.
potassium for the food samples as analyzed nal.usda.gov/). Which method of analysis gives
(i.e., ashed and/or diluted). a value closer to that reported in the database
Note: For the AAS samples, you need to subtract for sodium and for potassium?
the liquid blank absorbance from the sports drink 2. Describe how you would prepare the Na and K
sample values, and the solid blank absorbance standard solutions for AES, using the 1000-ppm
from the catsup and potato chip sample values. solutions of each, which are commercially avail-
3. Prepare standard curves for sodium and potas- able. If possible, all solutions for points in the
sium as measured by ICP-OES (if emission data standard curve should be made using different
are available). volumes of the same stock solution. Do not use
4. Use the standard curves from ICP-OES and the volumes of less than 0.2 mL. Make all standards
emission readings of the samples to determine to the same volume of 100 mL. Note that each
the concentrations in ppm of sodium and potas- standard solution must contain 10 mL conc.
sium for the food samples as analyzed (i.e., ashed HCl/100 mL final volume, as described under
and/or diluted). If emission data are not avail- Reagents.
able for the samples, record the concentrations in 3. Describe how you would prepare a 1000-ppm
ppm of sodium and potassium for the food sam- Na solution, starting with commercially avail-
ples as analyzed (i.e., ashed and/or diluted). able solid NaCl.
5. Convert the AAS and ICP-OES values for sam-
ples in ppm to mg/mL for the sports drink and
to mg/g for the catsup and potato chips. RESOURCE MATERIALS
6. Calculate the sodium and potassium contents
Harris, GK, Marshall MR (2017) Ash analysis. Ch. 16. In:
by AAS and ICP-OES for the original samples Nielsen SS (ed) Food analysis, 5th edn. Springer,
of sports drink (in mg/mL), catsup (in mg/g), New York
and potato chips (mg/g) (on a wet weight Yeung CK, Miller DD, Rutzke MA (2017) Atomic absorption
basis). Summarize the data and calculated spectroscopy, atomic emission spectroscopy, and induc-
results in one table for AAS and ICP-OES. Show tively coupled plasma mass spectrometry. Ch. 9. In:
examples of all calculations below the table. Nielsen (SS) Food analysis, 5th edn. Springer, New York
21
chapter
Standard Solutions
and Titratable Acidity
S.Suzanne Nielsen
21.1 Introduction 21.2.9 Procedure

21.1.1 Background 21.2.10 Data and Calculations
21.1.2 Reading Assignment 21.2.11 Questions
21.1.3 Notes 21.3 Titratable Acidity and pH
21.2 Preparation and Standardization of 21.3.1 Objective
Base and Acid Solutions 21.3.2 Principle of Method
21.2.2 Principle of Method 21.3.4 Reagents
21.2.3 Chemicals 21.3.5 Hazards, Precautions,
21.2.4 Reagents and Waste Disposal
21.2.5 Hazards, Precautions, 21.3.6 Supplies
and Waste Disposal 21.3.7 Equipment
21.2.6 Supplies 21.3.8 Procedure
21.2.7 Equipment 21.3.9 Data and Calculations
21.2.8 Calculations Required 21.3.10 Questions
Before Lab
www.dbooks.org
180 S.S. Nielsen
21.1 INTRODUCTION 21.2 PREPARATION AND STANDARDIZATION

OF BASE AND ACID SOLUTIONS
21.1.1 Background
21.2.1 Objective
Many types of chemical analyses are made using a
method in which a constituent is titrated with a solu- Prepare and standardize solutions of sodium hydrox-
tion of known strength to an indicator endpoint. Such a ide and hydrochloric acid.
solution is referred to as a standard solution. From the
volume and concentration of standard solution used in 21.2.2 Principle of Method
the titration, and the sample size, the concentration of
the constituent in the sample can be calculated. A standard acid can be used to determine the exact
The assay for titratable acidity is a volumetric normality of a standard base and vice versa.
method that uses a standard solution and, most com-
monly, the indicator phenolphthalein. In the titration, 21.2.3 Chemicals
a standard solution of sodium hydroxide reacts with
the organic acids present in the sample. The normality CAS No. Hazards
of the sodium hydroxide solution, the volume used,
Ascarite 81133-20-2 Corrosive
and the volume of the test sample are used to calculate
Ethanol (CH3CH2OH) 64-17-5 Highly
titratable acidity, expressing it in terms of the predomi- flammable
nant acid present in the sample. A standard acid such Hydrochloric acid (HCl) 7647-01-0 Corrosive
as potassium acid phthalate can be used to determine Phenolphthalein 77-09-8 Irritant
the exact normality of the standard sodium hydroxide Potassium acid phthalate 877-24-7 Irritant
used in the titration. (HOOCC6H4COOK)
The phenolphthalein endpoint in the assay for Sodium hydroxide 1310-73-2 Corrosive
titratable acidity is pH 8.2, where there is a significant (NaOH)
color change from clear to pink. When colored solu-
tions obscure the pink endpoint, a potentiometric 21.2.4 Reagents
method is commonly used. A pH meter is used to
titrate such a sample to pH 8.2. (** It is recommended that these solutions be prepared
21.1.2 Reading Assignment (Note: Preparation of NaOH and HCl solutions is
described under Procedure.)
Tyl, C., and Sadler, G. D. 2017. pH and titratable acid-
ity. Ch. 22, in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
• Ascarite trap**
Springer, New York.
Put the Ascarite® in a syringe that is attached to
the flask of CO2-free water (see note about CO2-
21.1.3 Notes
free water).
1. Carbon dioxide (CO2) acts as an interfering sub- • Carbon dioxide-free water**
stance in determining titratable acidity, by the Prepare 1.5 L of CO2-free water (per person or
following reactions: group) by boiling deionized, distilled (dd) water
for 15 min in a 2-L Erlenmeyer flask. After boil-
H2O + CO2 ↔ H2CO3 (carbonic acid)
ing, stopper the flask with a rubber stopper
H2CO3 ↔ H+ + HCO3− (bicarbonate)
through which is inserted in a tube attached to
HCO3− ↔ H+ + CO3−2 (carbonate)
an Ascarite® trap. Allow the water to cool with
In these reactions, buffering compounds Ascarite® protection.
and hydrogen ions are generated. Therefore, • Ethanol, 100 mL
CO2-free water is prepared and used for stan- • Hydrochloric acid, concentrated
dardizing acids and base and for determining • Phenolphthalein indicator solution, 1 % **
titratable acidity. An Ascarite® trap is attached Dissolve 1.0 g in 100 mL ethanol. Put in bottle
to bottles of CO2-free water, so that as air with eyedropper.
enters the bottle when water is siphoned out, • Potassium acid phthalate (KHP)**
the CO2 is removed from the air. 3–4 g, dried in an oven at 120 °C for 2 h cooled
2. Ascarite® is a silica base coated with NaOH, and it and stored in a closed bottle inside a desiccator
removes CO2 from the air by the following reaction: until use
• Sodium hydroxide, pellets
2NaOH + CO2 → Na2CO3 + H2O
Chapter 21 • Standard Solutions and Titratable Acidity 181
21.2.5 Hazards, Precautions, and Waste adding about 40 mL of dd water, stir the NaOH
Disposal pellets with a glass stirring rod. Continue stir-
ring until all pellets are dissolved. Quantitatively
Use appropriate precautions in handling concentrated
transfer the NaOH solution into a 50-mL volu-
acid and base. Otherwise, adhere to normal laboratory
metric flask. Dilute to volume with dd water.
safety procedures. Wear gloves and safety glasses at all
The solution must be cooled to room tempera-
times. Waste likely may be put down the drain using a
ture before final preparation. Store this solution
water rinse, but follow good laboratory practices out-
in a plastic bottle and label appropriately.
lined by environmental health and safety protocols at
2. Prepare ca. 0.1 N HCl solution: Prepare 100 mL
your institution.
of ca. 0.1 N HCl using concentrated HCl (12.1 N)
21.2.6 Supplies and dd water. (Note: Do not use a mechanical
pipettor to prepare this, since the acid can easily
(Used by students) get into the shaft of the pipettor and cause dam-
age.) To prepare this solution, place a small
• Beaker, 50 mL (for waste NaOH from buret) amount of dd water in a 100-mL volumetric
• Beaker, 100 mL flask, pipette in the appropriate amount of con-
• Buret, 25 or 50 mL centrated HCl, then dilute to volume with dd
• 5 Erlenmeyer flasks, 250 mL water. Mix well, and transfer into a glass bottle,
• Erlenmeyer flask, 1 L seal bottle, and label appropriately.
• Funnel, small, to fit top of 25 or 50 mL buret 3. Prepare ca. 0.1 N NaOH solution: Transfer
• Glass stirring rod 750 mL CO2-free water to a 1-L plastic storage
• Glass storage bottle, 100 mL bottle. Add ca. 12.0 mL of well-mixed 25 % (wt/
• Graduated cylinder, 50 mL vol) NaOH solution prepared in Step 1. Mix thor-
• Graduated cylinder, 1 L oughly. This will give an approximately 0.1 N
• Graduated pipette, 1 mL solution. Fill the buret with this solution using a
• Graduated pipette, 10 mL funnel. Discard the first volume of the buret and
• Parafilm® then refill the buret with the NaOH solution.
• Pipette bulb or pump 4. Standardize ca. 0.1 N NaOH solution: Accurately
• Plastic bottle, with lid, 50 or 100 mL weigh about 0.8 g of dried potassium acid
• Plastic bottle, with lid, 1 L phthalate (KHP) into each of three 250-mL
• Spatula Erlenmeyer flasks. Record the exact weights.
• Squirt bottle, with dd water Add ca. 50 mL of cool CO2-free water to each
• Volumetric flask, 50 mL flask. Seal the flasks with Parafilm® and swirl
• Volumetric flask, 100 mL gently until the sample is dissolved. Add three
• Weighing paper/boat drops of phenolphthalein indicator and titrate,
• White piece of paper against a white background, with the NaOH
solution being standardized. Record the begin-
21.2.7 Equipment ning and ending volume on the buret. Titration
• Analytical balance should proceed to the faintest tinge of pink that
• Forced draft oven (heated to 120 °C) persists for 15 s. after swirling. The color will
• Hot plate fade with time. Record the total volume of
NaOH used to titrate each sample. Data from
21.2.8 Calculations Required Before Lab this part will be used to calculate the mean nor-
1. Calculate how much NaOH to use to prepare mality of the diluted NaOH solution.
50 mL of 25 % NaOH (wt/vol) in water (see 5. Standardize ca. 0.1 N HCl solution: Devise a
Table 2.1, in Chap. 2 for definition of wt%). scheme to standardize (i.e., determine the exact
2. Calculate how much concentrated HCl to use to N) the ca. 0.1 N HCl solution that you prepared
prepare 100 mL of ca. 0.1 N HCl in water (con- in Step 2. Remember that you have your stan-
centrated HCl = 12.1 N). dardized NaOH to use. Do analyses in at least
duplicate. Record the volumes used.
21.2.9 Procedure
1. Prepare 25 % (wt/vol) NaOH solution: Prepare
50 mL of 25 % NaOH (wt/vol) in dd water. To Using the weight of KHP and the volume of NaOH
do this, weigh out the appropriate amount of titrated in Sect. 21.2.9, Step 4, calculate the normality
NaOH and place it in a 100-mL beaker. While of the diluted NaOH solution as determined by each
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182 S.S. Nielsen
titration and then calculate the mean normality 21.3.2 Principle of Method
(molecular weight (MW) potassium acid phthal-
The volume of a standard base used to titrate the
ate = 204.228). The range of triplicate determinations
organic acids in foods to a phenolphthalein endpoint
for normality should be less than 0.2 % with good
can be used to determine the titratable acidity.
technique.
Weight 21.3.3 Chemicals

of KHP Buret start Buret end Vol. NaOH N
Rep (g) (mL) (mL) titrated (mL) NaOH CAS No. Hazards
1 Ascarite 81133-20-2 Corrosive

2 Ethanol (CH3CH2OH) 64-17-5 Highly
3 flammable
X = Phenolphthalein 77-09-8 Irritant
SD= Sodium hydroxide 1310-73-2 Corrosive
(NaOH)
Sample calculation:
Weight of KHP = 0.8115 g 21.3.4 Reagents

MW of KHP = 204.228 g/mol (** It is recommended that these items/solutions be
Vol. of ca. 0.10 N NaOH used in titration = 39 mL prepared by the laboratory assistant before class:)
Mol KHP = 0.8115 g/204.228 g/mol
= 0.003974 mol • Ascarite trap**
Mol KHP = mol NaOH Put the Ascarite® in a syringe that is attached to
= 0.003974 mol = N NaOH × L NaOH the flask of CO2-free water:
0.003978 mol NaOH/0.039 L NaOH = 0.1019 N • Carbon dioxide-free water**
Prepared and stored as described in Method A
With the volumes of HCl and NaOH used in • Phenolphthalein indicator solution, 1 %**
Sect. 21.2.9, Step 5, calculate the exact normality of the Prepared as described in Method A
HCl solution as determined by each titration and then • Sodium hydroxide, ca. 0.1 N
calculate the mean normality. From Sect. 21.2.9, Step 4; exact N calculated
• Standard buffers, pH 4.0 and 7.0
Rep Vol. HCl (mL) Vol. NaOH (mL) N HCl
1 21.3.5 Hazards, Precautions, and Waste
2 Disposal
X =
safety glasses at all times. Waste likely may be put
21.2.11 Questions down the drain using a water rinse, but follow good
1. What does 25 % NaOH (wt/vol) mean? How laboratory practices outlined by environmental health
would you prepare 500 mL of a 25 % NaOH and safety protocols at your institution.
(wt/vol) solution?
2. Describe how you prepared the 100 mL of ca. 21.3.6 Supplies
0.1 N HCl. Show your calculations.
3. If you had not been told to use 12 mL of 25 % • Apple juice, 60 mL
NaOH (wt/vol) to make 0.75 L of ca. 0.1 N NaOH, • 3 Beakers, 250 mL
how could you have determined this was the • 2 Burets, 25 or 50 mL
appropriate amount? Show all calculations. • 4 Erlenmeyer flasks, 250 mL
4. Describe in detail how you standardized your • Funnel, small, to fit top of 25 or 50 mL buret
ca. 0.1 N HCl solution. • Graduated cylinder, 50 mL
• Soda, clear, 80 mL
• 2 Volumetric pipettes, 10 or 20 mL
21.3 TITRATABLE ACIDITY AND pH
21.3.7 Equipment
21.3.1 Objective
• Hot plate
Determine the titratable acidity and pH of food samples. • pH meter
Chapter 21 • Standard Solutions and Titratable Acidity 183
21.3.8 Procedure color of the apple juice. Sample B (without phe-

nolphthalein) does not need to be followed with
21.3.8.1 Soda
the pH meter but is to be titrated along with the
Do at least duplicate determinations for unboiled soda other beakers to aid in observing color changes.
and for boiled soda sample; open soda well before use
to allow escape of carbon dioxide, so that the sample 21.3.9 Data and Calculations
can be pipetted.
21.3.9.1 Soda
1. Unboiled soda: Pipette 20 mL of soda into a 250-
Using the volume of NaOH used, calculate the titrat-
mL Erlenmeyer flask. Add ca. 50 mL CO2-free
able acidity (TA) of each soda sample as percentage
dd water. Add three drops of a 1 % phenol-
citric acid, and then calculate the mean TA of each type
phthalein solution and titrate with standard-
of sample (MW citric acid = 192.14; equivalent
ized NaOH (ca. 0.1 N) to a faint pink color
weight = 64.04) (Note: See Eq. 22.20 in Tyl and Sadler,
(NaOH in buret, from Method A). Record the
2017).
beginning and ending volumes on the buret to
determine the total volume of NaOH solution
Buret Buret Vol. NaOH Color
used in each titration. Observe the endpoint.
Rep start end titrant fades? TA
Note whether the color fades.
2. Boiled soda: Pipette 20 mL of soda into a 250-mL Unboiled
Erlenmeyer flask. Bring the sample to boiling on a soda:
hot plate, swirling the flask often. Boil the sample 1
only 30–60 s. Cool to room temperature. Add ca. 2
X =
50 mL CO2-free dd water. Add three drops of the
Boiled
phenolphthalein solution and titrate as described soda:
above. Record the beginning and ending volumes 1
on the buret to determine the total volume of 2
NaOH solution used in each titration. Observe the X =
endpoint. Note whether the color fades.
Sample calculation:
21.3.8.2 Apple Juice % Acid =
1. Standardize the pH meter with pH 7.0 and 4.0 ( mL base titrant ) × ( N of basein mol / liter ) × ( Eq. Wt.of acid )
buffers, using instructions for the pH meter
( sample volumein mL ) ×10
available.
2. Prepare as described below three apple juice
samples that will be compared: N NaOH = 0.1019 N
mL base = 7 mL
A – Apple juice (set aside, to recall original Eq. wt. citric acid = 64.04
color) Vol. sample = 20 mL
B – Apple juice; titrate with std. NaOH
C – Apple juice; add phenolphthalein; titrate % Acid 7 ml 0.1019 64.04 / 20 ml 10
with std. NaOH; follow pH during titration 0.276% citric acid
Procedure: Into each of three (A, B, C) 250-

mL beakers, pipette 20 mL of apple juice. Add 21.3.9.2 Apple Juice
ca. 50 mL CO2-free water to each. To sample C,
Sample B
add three drops of a 1 % phenolphthalein solu-
Color change during titration:
tion. Using two burets filled with the standard-
Color at end of titration:
ized NaOH solution (ca. 0.1 N), titrate Samples B
and C simultaneously. Follow the pH during
Sample C (titrate to >pH 9.0)
titration of Sample C containing phenolphtha-
lein. (Note: If only one buret is available, titrate
mL NaOH 1 2 3 4 5 6 7 8 9 10
Samples B and C sequentially, i.e., add 1 mL to B
and then 1 mL to C.) Record the initial pH and pH
the pH at ca. 1.0 mL intervals until a pH of 9.0 is
reached. Also, observe any color changes that mL
occur during the titration to determine when the NaOH 11 12 13 14 15 16 17 18 19 20
phenolphthalein endpoint is reached. Sample A
pH
is intended to help you remember the original
www.dbooks.org
184 S.S. Nielsen
Plot pH versus mL of 0.1 N NaOH (but use the 3. You are determining the titratable acidity of a
normality of your own NaOH solution) (pH on the large number of samples. You ran out of freshly
y-axis) for the sample that contained phenolphthalein boiled dd H2O with an Ascarite trap on the water
(Sample C). Interpolate to find the volume of titrant at container, so you switch to using tap distilled
pH 8.2 (the phenolphthalein endpoint). H2O. Would this likely affect your results? Explain.
Calculate the titratable acidity of the apple juice as 4. The electrode of your pH meter has a slow
percentage malic acid (MW malic acid = 134.09; equiv- response time and seems to need cleaning, since
alent weight = 67.04). it is heavily used for a variety of solutions high
in proteins, lipids, and minerals. You would
21.3.10 Questions ideally check the electrode instructions for spe-
cific recommendations on cleaning, but the
1. Soda samples. (a) Did any color changes occur
instructions were thrown away. (As the new lab
in either the boiled or the unboiled sample
supervisor, you have since started a policy of
within several minutes of the phenolphthalein
filing all instrument/equipment instructions.)
endpoint being reached? (b) How did boiling
What solutions would you use to try to clean
the sample affect the determination of titratable
the electrode?
acidity? (c) Explain the differences in color
changes and titratable acidity between the two
samples.
RESOURCE MATERIALS
2. What caused the color changes in the apple
juice titrated without any phenolphthalein AOAC International (2016) Official methods of analysis, 20th
present? (Hint: Consider the pigments in edn. (On-line). AOAC International, Rockville, MD
apples.) How would you recommend determin- Tyl C, Sadler GD (2017) pH and titratable acidity. Ch. 22. In:
ing the endpoint in the titration of tomato juice? Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
22
chapter
Fat Characterization
S.Suzanne Nielsen ( ) *

Michael C. Qian
Oregon State University, Corvallis, OR, USA
Oscar A. Pike
Department of Nutrition, Dietetics, and Food Science,
Brigham Young University, Provo, UT, USA
22.1 Introduction 22.2 Saponification Value

22.1.1 Background 22.2.1 Objective
22.1.2 Reading Assignment 22.2.2 Principle of Method
22.1.3 Overall Objective 22.2.3 Chemicals
www.dbooks.org
22.2.4 Reagents 22.4.8 Procedure
22.2.5 Hazards, Precautions, 22.4.9 Data and Calculations
and Waste Disposal 22.4.10 Questions
22.2.6 Supplies 22.5 Peroxide Value
22.2.7 Equipment 22.5.1 Objective
22.2.8 Procedure 22.5.2 Principle of Method
22.2.9 Data and Calculations 22.5.3 Chemicals
22.2.10 Questions 22.5.4 Reagents
22.3 Iodine Value 22.5.5 Hazards, Precautions,
22.3.1 Objective and Waste Disposal
22.3.2 Principle of Method 22.5.6 Supplies
22.3.3 Chemicals 22.5.7 Equipment
22.3.4 Reagents 22.5.8 Procedure
22.3.5 Hazards, Precautions, 22.5.9 Data and Calculations
and Waste Disposal 22.5.10 Questions
22.3.6 Supplies 22.6 Thin-Layer Chromatography
22.3.7 Equipment Separation of Simple Lipids
22.3.8 Procedure 22.6.1 Objective
22.3.9 Data and Calculations 22.6.2 Principle of Method
22.3.10 Questions 22.6.3 Chemicals
22.4 Free Fatty Acid Value 22.6.4 Reagents
22.4.1 Objective 22.6.5 Hazards, Precautions,
22.4.2 Principle of Method and Waste Disposal
22.4.4 Reagents 22.6.7 Equipment
22.4.5 Hazards, Precautions, 22.6.8 Procedure
and Waste Disposal 22.6.9 Data and Calculations
22.4.6 Supplies 22.6.10 Questions
22.4.7 Equipment
Chapter 22 • Fat Characterization 187
22.1 INTRODUCTION • Alcoholic potassium hydroxide, ca. 0.7 N **

Dissolve 40 g KOH, low in carbonate, in 1 L of
22.1.1 Background distilled ethanol, keeping temperature below
15.5 °C while the alkali is being dissolved. The
Lipids in food are subjected to many chemical reac- solution should be clear.
tions during processing and storage. While some of • Hydrochloric acid, ca. 0.5 N, accurately standard-
these reactions are desirable, others are undesirable; ized **
so, efforts are made to minimize the reactions and their Prepare ca. 0.5 N HCl. Determine the exact nor-
effects. The laboratory deals with characterization of mality using solution of standard base.
fats and oils with respect to composition, structure, • Phenolphthalein indicator solution **
and reactivity. 1 %, in 95 % ethanol
Pike, O.A., and O’Keefe, S.F. 2017. Fat characterization, Disposal
Ch. 23 in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
Springer, New York. Use hydrochloric acid in a fume hood. Otherwise,
adhere to normal laboratory safety procedures. Wear
22.1.3 Overall Objective safety glasses at all times. Wastes likely may be put
down the drain using a water rinse, but follow good
The overall objective of this laboratory is to determine laboratory practices outlined by environmental health
aspects of the composition, structure, and reactivity of and safety protocols at your institution.
fats and oils by various methods.
22.2.6 Supplies
22.2 SAPONIFICATION VALUE (Used by students)
22.2.1 Objective • Air (reflux) condenser (650 mm long, minimum)

• Beaker, 250 mL (to melt fat)
Determine the saponification number of fats and oils.
• Buchner funnel (to fit side-arm flask)
• Boiling beads
• 2 Burets, 50 mL
Saponification is the process of treating a neutral fat • Fat and/or oil samples
with alkali, breaking it down to glycerol and fatty acids. • Filter paper (to fit Buchner funnel, to filter oil and
The saponification value (or number) is defined as the melted fat)
amount of alkali needed to saponify a given quantity of • 4 Flasks, 250–300 mL, to fit condenser
fat or oil, expressed as mg potassium hydroxide to • Mechanical pipettor, 1000 μL, with plastic tips (or
saponify 1 g sample. Excess alcoholic potassium 1 mL volumetric pipette)
hydroxide is added to the sample, the solution is heated • Side-arm flask
to saponify the fat, the unreacted potassium hydroxide
is back-titrated with standardized hydrochloric acid 22.2.7 Equipment
using a phenolphthalein indicator, and the calculated
amount of reacted potassium hydroxide is used to • Analytical balance
determine the saponification value. • Hot plate or water bath (with variable heat
control)
22.2.3 Chemicals
22.2.8 Procedure
CAS No. Hazards
Ethanol 64-17-5 Highly flammable
Hydrochloric acid (HCl) 7647-01-0 Corrosive 1. Melt any solid samples. Filter melted fat sample
Phenolphthalein 77-09-8 Irritant and oil sample through filter paper to remove
Potassium hydroxide 1310-58-3 Corrosive impurities.
(KOH) 2. Weigh accurately ca. 5 g melted fat or oil into
each of two 250–300 ml flasks that will connect
to a condenser. Record weight of sample.
22.2.4 Reagents Prepare sample in duplicate.
(**It is recommended that these solutions be prepared 3. Add accurately (from a buret) 50 mL of alco-
by the laboratory assistant before class.) holic KOH into the flask.
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188 S.S. Nielsen et al.
4. Prepare duplicate blank samples with just 22.2.10 Questions

50 mL of alcoholic KOH in a 250–300 mL flask.
1. What is meant by unsaponifiable matter in lipid
5. Add several boiling beads to the flasks with fat
samples? Give an example of such a type of
or oil sample.
compound.
6. Connect the flasks with the sample to a con-
2. What does a high versus a low saponification
denser. Boil gently but steadily on a hot plate
value tell you about the nature of a sample?
(or water bath) until the sample is clear and
homogenous, indicating complete saponifica-
tion (requires ca. 30–60 min). (Note: The fumes
22.3 IODINE VALUE
should condense as low as possible in the con-
denser; otherwise, a fire hazard will be 22.3.1 Objective
created.)
7. Allow samples to cool somewhat. Wash down Determine the iodine value of fats and oils.
the inside of the condenser with a little deion-
ized distilled (dd) water. Disconnect flask from 22.3.2 Principle of Method
condenser. Allow the samples to cool to room The iodine value (or number) is a measure of the
temperature. degree of unsaturation, defined as the grams of iodine
8. Add 1 mL phenolphthalein to samples and absorbed per 100 g sample. In the assay, a measured
titrate with 0.5 N HCl (from a buret) until the quantity of fat or oil dissolved in solvent is reacted
pink color just disappears. Record the volume with a measured excess amount of iodine or some
of titrant used. other halogen, which reacts with the carbon-carbon
9. Repeat Steps 5–8 with sample blanks. Reflux the blanks double bonds. After a solution of potassium iodide is
for the same time period as used for the sample. added to reduce excess ICl to free iodine, the liberated
iodine is titrated with a standardized solution of
22.2.9 Data and Calculations sodium thiosulfate using a starch indicator. The calcu-
lated amount of iodine reacted with the double bonds
Titrant Saponification is used to calculate the iodine value.
Sample Weight (g) volume (mL) value
1 22.3.3 Chemicals
2
X= CAS no. Hazards
Acetic acid (glacial) 64-19-7 Corrosive
Oil/fat sample type tested: Carbon tetrachloride 56-23-5 Toxic, dangerous for
(CCl4) the environment
Blank titration (mL): Chloroform 67-66-3 Harmful
Hydrochloric acid 7647-01-0 Corrosive
Sample 1 =
(HCl)
Sample 2 =
Iodine 7553-56-2 Harmful, dangerous
X= for the
environment
Calculate the saponification number (or value) of Potassium dichromate 7789-00-6 Toxic, dangerous for
(K2Cr2O7) the environment
each sample as follows:
Potassium iodide (KI) 7681-11-0
Sodium thiosulfate 7772-98-7
Saponification value
B S N 56.1 Soluble starch 9005-25-8
W
where:
22.3.4 Reagents
Saponification value = mg KOH per g of (**It is recommended that these solutions be prepared
sample by the laboratory assistant before class.)
B = volume of titrant (mL) for blank
S = volume of titrant (mL) for sample • Potassium iodide solution, 15 %
N = normality of HCl (mmol/mL) Dissolve 150 g KI in dd water and dilute to 1 l.
56.1 = molecular weight (MW) of KOH (mg/ • Sodium thiosulfate, 0.1 N standardized solution
mmol) (AOAC Method 942.27) **
W = sample mass (g) Dissolve ca. 25 g sodium thiosulfate in 1 L dd
water. Boil gently for 5 min. Transfer while hot to
a storage bottle (make sure bottle has been well • Mechanical pipettor, 1000 μL, with plastic tips (or 1
cleaned and is heat resistant). Store solution in mL volumetric pipette)
a dark, cool place. Use the following procedure • Side-arm flask
to standardize the sodium thiosulfate solu- • Volumetric pipette, 10 mL
tion: Accurately weigh 0.20–0.23 g potassium • Volumetric pipette, 20 mL
dichromate (K2Cr2O7) (previously dried for 2 h
at 100 °C) into a glass-stoppered flask. Dissolve 22.3.7 Equipment
2 g potassium iodide (KI) in 80 mL chlorine-
free water. Add this water to the potassium
• Hot plate
dichromate. To this solution, add, with swirl-
ing, 20 mL ca. 1 M HCl and immediately place
22.3.8 Procedure
in the dark for 10 min. Titrate a known volume
of this solution with the sodium thiosulfate (Instructions are given for analysis in duplicate.)
solution, adding starch solution after most of
1. Melt any samples that are solid at room tem-
the iodine has been consumed.
perature by heating to a maximum of 15 °C
• Starch indicator solution, 1 % (prepare fresh daily)**
above the melting point. Filter melted fat sam-
Mix ca. 1 g soluble starch with enough cold dd
ple and oil sample through filter paper to
water to make a thin paste. Add 100 mL boiling
remove impurities.
dd water. Boil ca. 1 min while stirring.
2. Weigh accurately 0.1–0.5 g sample (amount
• Wijs iodine solution **
used depends on expected iodine number) into
Dissolve 10 g ICl3 in 300 mL CCl4 and 700 mL
each of two dry 500 mL glass-stoppered flasks.
glacial acetic acid. Standardize this solution
Add 10 mL chloroform to dissolve the fat or oil.
against 0.1 N sodium thiosulfate (25 mL of Wijs
3. Prepare two blanks by adding only 10 mL chlo-
solution should consume 3.4–3.7 mEq of thio-
roform to 500 mL glass-stoppered flasks.
sulfate). Then, add enough iodine to the solu-
4. Pipette 25 mL Wijs iodine solution into the
tion such that 25 mL of the solution will require
flasks. (The amount of iodine must be 50–60 %
at least 1.5 times the milliequivalency of the
in excess of that absorbed by the fat.)
original titration. Place the solution in an amber
5. Let flasks stand for 30 min in the dark with
bottle. Store in the dark at less than 30 °C.
occasional shaking.
6. After incubation in the dark, add 20 mL potas-
sium iodide solution to each flask. Shake thor-
Disposal
oughly. Add 100 mL freshly boiled and cooled
Carbon tetrachloride and potassium chromate are water, washing down any free iodine on the
toxic and must be handled with caution. Use acetic stopper.
acid and hydrochloric acid in a fume hood. Otherwise, 7. Titrate the iodine in the flasks with standard
adhere to normal laboratory safety procedures. Wear sodium thiosulfate, adding it gradually with con-
safety glasses at all times. Carbon tetrachloride, chlo- stant and vigorous shaking until the yellow color
roform, iodine, and potassium chromate must be han- almost disappears. Then add 1–2 mL of starch
dled as hazardous wastes. Other wastes likely may be indicator and continue the titration until the blue
put down the drain using a water rinse, but follow color entirely disappears. Toward the end of the
good laboratory practices outlined by environmental titration, stopper the flask and shake violently so
health and safety protocols at your institution. that any iodine remaining in the chloroform can
be taken up by the potassium iodide solution.
22.3.6 Supplies Record the volume of titrant used.
(Used by students)
• 2 Beakers, 250 mL (one to melt fat; one to boil water)
Sample Weight (g) Titrant volume (mL) Iodine value
• Buret, 10 or 25 mL 1
• Fat and/or oil samples 2
• Filter paper (to fit Buchner funnel, to filter melted X=
fat and oil)
• 4 Flasks, 500 mL, glass stoppered Oil/fat sample type tested:
• Graduated cylinder, 100 mL Blank titration (mL):
www.dbooks.org
Sample 1 = 22.4.4 Reagents

Sample 2 =
X =
Calculate the iodine value of each sample as
• Ethanol, neutralized
follows:
Neutralize 95 % ethanol to a permanent pink
color with alkali and phenolphthalein.
Iodine value  
B  S N  126.9
 100 • Phenolphthalein indicator**
W  1000
In a 100 mL volumetric flask, dissolve 1 g phe-
where:
nolphthalein in 50 mL 95 % ethanol. Dilute to
Iodine value = g iodine absorbed per 100 g of volume with dd water.
sample • Sodium hydroxide, 0.1 N, standardized **
B = volume of titrant (mL) for blank Use commercial product, or prepare as described
S = volume of titrant (mL) for sample in other laboratory manual chapters (Chaps. 2
N = normality of Na2S2O3 (mol/1000 mL) and 21).
126.9 = MW of iodine (g/mol)
W = sample mass (g) 22.4.5 Hazards, Precautions, and Waste
Disposal
22.3.10 Questions Adhere to normal laboratory safety procedures. Wear
1. In the iodine value determination, why is the safety glasses at all times. Wastes likely may be put
blank volume higher than that of the sample? down the drain using a water rinse, but follow good
2. What does a high versus a low iodine value tell laboratory practices outlined by environmental health
you about the nature of the sample? and safety protocols at your institution.
22.4.6 Supplies
22.4 FREE FATTY ACID VALUE
(Used by students)
22.4.1 Objective
Determine the free fatty acid (FFA) value of fats and • Buchner funnel (to fit side-arm flask)
oils. • Buret, 10 mL
• 4 Erlenmeyer flasks, 250 mL
22.4.2 Principle of Method • Fat and/or oil samples
Free fatty acid value, or acid value, reflects the • Filter paper (to fit Buchner funnel, to filter melted
amount of fatty acids hydrolyzed from triacylglycer- fat and oil)
ols. Free fatty acid is the percentage by weight of a • Graduated cylinder, 100 mL
specific fatty acid. Acid value is defined as the milli- • Mechanical pipettor, 1000 μL, with plastic tips (or 1
grams of potassium hydroxide needed to neutralize mL volumetric pipette)
the free acids present in 1 g of fat or oil. A liquid fat • Side-arm flask
sample combined with neutralized 95 % ethanol is
titrated with standardized sodium hydroxide to a 22.4.7 Equipment
phenolphthalein endpoint. The volume and normal- • Analytical balance
ity of the sodium hydroxide are used, along with the • Hot plate
weight of the sample, to calculate the free fatty acid
value. 22.4.8 Procedure
22.4.3 Chemicals (Instructions are given for analysis in triplicate.)
1. Melt any samples that are solid at room temper-
CAS no. Hazards ature by heating to a maximum of 15 °C above
Ethanol 64-17-5 Highly flammable the melting point. Filter melted fat sample
Phenolphthalein 77-09-8 Irritant and oil sample through filter paper to remove
Sodium hydroxide 1310-73-2 Corrosive impurities.
(NaOH) 2. As a preliminary test, accurately weigh ca. 5 g
melted fat or oil into a 250 mL Erlenmeyer flask.
3. Add ca. 100 mL neutralized ethanol and 2 mL
phenolphthalein indicator.
4. Shake to dissolve the mixture completely. 3. In a crude fat extract, FFA are naturally present,
Titrate with standard base (ca. 0.1 N NaOH), but they are removed during processing to
shaking vigorously until the endpoint is enhance the stability of the fat. State and
reached. This is indicated by a slight pink color describe the processing step that removes the
that persists for 30 s. Record the volume of FFA naturally present.
titrant used. Use the information below to
determine if the sample weight you have used
is correct for the range of acid values under 22.5 PEROXIDE VALUE
which your sample falls. This will determine
the sample weight to be used for Step 5. 22.5.1 Objective
Determine the peroxide value of fats and oils, as an
The Official Methods and Recommended Practices of indicator of oxidative rancidity.
the AOCS (AOCS 2009) recommends the following
sample weights for ranges of expected acid values: 22.5.2 Principle of Method
Peroxide value is defined as the milliequivalents of
Strength of peroxide per kilogram of fat, as determined in a titra-
FFA range (%) Sample (g) Alcohol (mL) alkali tion procedure to measure the amount of peroxide or
0.00–0.2 56.4 ± 0.2 50 0.1 N hydroperoxide groups. To a known amount of fat or
0.2–1.0 28.2 ± 0.2 50 0.1 N oil, excess potassium iodide is added, which reacts
1.0–30.0 7.05 ± 0.05 75 0.25 N with the peroxides in the sample. The iodine liberated
is titrated with standardized sodium thiosulfate using
a starch indicator. The calculated amount of potassium
5. Repeat Steps 1–3 more carefully in triplicate, iodide required to react with the peroxide present is
recording each weight of the sample and the used to determine the peroxide value.
volume of titrant.
22.5.3 Chemicals
CAS no. Hazards
Sample Weight (g) Titrant volume (mL) FFA value
Acetic acid (glacial) 64-19-7 Corrosive
1 Chloroform 67-66-3 Harmful
2 Hydrochloric acid (HCl) 7647-01-0 Corrosive
3 Potassium chromate 7789-00-6 Toxic, dangerous
X = (K2Cr2O7) for environment
SD = Potassium iodide (KI) 7681-11-0
Sodium thiosulfate 7772-98-7
Oil/fat sample type tested: Soluble starch 9005-25-8
Calculate the FFA value of each sample as follows: 22.5.4 Reagents

V  N  282 (**It is recommended that these solutions be prepared
% FFA asoleic   100 by the laboratory assistant before class.)
W 1000
where: • Acetic acid-chloroform solution
Mix three volumes of concentrated acetic acid
% FFA = percent free fatty acid (g/100 g), with two volumes of chloroform.
expressed as oleic acid • Potassium iodide solution, saturated **
V = volume of NaOH titrant (mL) Dissolve excess KI in freshly boiled dd water.
N = normality of NaOH titrant (mol/1000 mL) Excess solid must remain. Store in the dark. Test
282 = MW of oleic acid (g/mol) before use by adding 0.5 mL acetic acid-chloro-
W = sample mass (g) form solution, and then add 2 drops 1 % starch
indicator solution. If solution turns blue, requir-
22.4.10 Questions ing >1 drop 0.1 N thiosulfate solution to discharge
1. What is a high FFA value indicative of relative color, prepare a fresh potassium iodide solution.
to product history? • Sodium thiosulfate, 0.1 N, standard solution (AOAC
2. Why is the FFA content of frying oil important? Method 942.27) **
Dissolve ca. 25 g sodium thiosulfate pentahy-
drate in 1 L dd water. Boil gently for 5 min.
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Transfer while hot to a storage bottle (make sure above the melting point. Filter melted fat sam-
bottle has been well cleaned and is heat resis- ple and oil sample through filter paper to
tant). Store solution in a dark, cool place. Use remove impurities.
the following procedure to standardize the 2. Accurately weigh ca. 5 g fat or oil (to the nearest
sodium thiosulfate solution: Accurately weigh 0.001 g) into each of two 250 mL glass-stoppered
0.20–0.23 g potassium chromate (K2Cr2O7) (pre- Erlenmeyer flasks.
viously dried for 2 h at 100 °C) into a glass-stop- 3. Add 30 mL acetic acid-chloroform solution and
pered flask. Dissolve 2 g potassium iodide (KI) swirl to dissolve.
in 80 mL chlorine-free water. Add this water to 4. Add 0.5 mL saturated KI solution. Let stand
the potassium chromate. To this solution, add, with occasional shaking for 1 min. Add 30 mL
with swirling, 20 mL ca. 1 M HCl, and immedi- dd water.
ately place in the dark for 10 min. Titrate a 5. Slowly titrate samples with 0.1 N sodium thio-
known volume of this solution with the sodium sulfate solution, with vigorous shaking until
thiosulfate solution, adding starch solution after yellow color is almost gone.
most of the iodine has been consumed. 6. Add ca. 0.5 mL 1 % starch solution, and con-
• Starch indicator solution, 1 % (prepare fresh daily)** tinue titration, shaking vigorously to release all
Mix ca. 1 g soluble starch with enough cold dd iodine from chloroform layer, until blue color
water to make a thin paste. Add 100 mL boiling just disappears. Record the volume of titrant
dd water. Boil ca. 1 min while stirring. used. (If <0.5 mL of the sodium thiosulfate solu-
tion is used, repeat determination.)
22.5.5 Hazards, Precautions, and Waste 7. Prepare (omitting only the oil) and titrate a blank
Disposal sample. Record the volume of titrant used.
Potassium chromate is toxic and must be handled with 22.5.9 Data and Calculations
caution. Use hydrochloric acid in a fume hood.
Otherwise, adhere to normal laboratory safety proce- Sample Weight (g) Titrant volume (mL) Peroxide value
dures. Wear gloves and safety glasses at all times.
Chloroform and potassium chromate must be handled 1
as hazardous wastes. Other wastes likely may be put 2
down the drain using a water rinse, but follow good X =
laboratory practices outlined by environmental health
and safety protocols at your institution. Oil/fat sample type tested:
22.5.6 Supplies Blank titration (mL):

(Used by students) Sample 1 =
Sample 2 =
X =
• Buret, 25 mL or 50 mL
• 4 Erlenmeyer flasks, 250 mL, glass stoppered Calculate the peroxide value of each sample as
• Fat and/or oil samples follows:
• Filter paper (to fit Buchner funnel, to filter melted S B N
fat and oil) Peroxide value 1000
W
• 2 Graduated cylinders, 50 mL
• Mechanical pipettor, 1000 μL, with plastic tips (or where:
1 mL volumetric pipette)
• Side-arm flask Peroxide value = mEq peroxide per kg of sample
S = volume of titrant (mL) for sample
22.5.7 Equipment B = volume of titrant (mL) for blank
N = normality of Na2S2O3 solution (mEq/mL)
• Analytical balance 1000 = conversion of units (g/kg)
• Hot plate W = sample mass (g)
22.5.8 Procedure
22.5.10 Questions
1. What are some cautions in using peroxide value
1. Melt any samples that are solid at room tem- to estimate the amount of autoxidation in
perature by heating to a maximum of 15 °C foods?
2. The peroxide value method was developed for 22.6.5 Hazards, Precautions, and Waste
fat or oil samples. What must be done to a food Disposal
sample before measuring its peroxide value
Use acetic acid and sulfuric acid in a fume hood.
using this method?
Diethyl ether is extremely flammable, is hygroscopic,
and may form explosive peroxides. Otherwise, adhere
to normal laboratory safety procedures. Wear safety
22.6 THIN-LAYER CHROMATOGRAPHY
glasses at all times. Diethyl ether and hexane must be
SEPARATION OF SIMPLE LIPIDS
handled as hazardous wastes. Other wastes likely may
22.6.1 Objective be put down the drain using a water rinse, but follow
good laboratory practices outlined by environmental
Separate and identify the lipids in some common health and safety protocols at your institution.
foods using thin-layer chromatography (TLC).
22.6.6 Supplies
• Capillary tubes (or syringes) (to apply samples to
Like all types of chromatography, TLC is a separation plates)
technique that allows for the distribution of com- • Developing tank, with lid
pounds between a mobile phase and a stationary • Filter paper, Whatman No. 1 (to line developing
phase. Most classes of lipids can be separated from tank)
each other by adsorption chromatography on thin lay- • Oil/fat food samples (e.g., hamburger, safflower
ers. In TLC, a thin layer of stationary phase is bound to oil) (prepare at a concentration of 20 μg/mL in
an inert support (i.e., glass plate, plastic, or aluminum 2:1 v/v chloroform-methanol solution)
sheet). The sample and standards are applied as spots • Pencil
near one end of the plate. For ascending chromatogra- • Thin-layer chromatography plates: Silica Gel 60,
phy, the plate is placed in a developing chamber, with 0.25 mm thick coating on glass backing, 20 × 20 cm
the end of the plate nearest the spots being placed in (EM Science)
the mobile phase at the bottom of the chamber. The
mobile phase migrates up the plate by capillary action, 22.6.7 Equipment
carrying and separating the sample components. The
separated bands can be visualized or detected and • Air blower (e.g., blow hair dryer)
compared to the separation of standard compounds. • Oven

Preparation of Silica Gel Plates
CAS no. Hazards
1. Place plates in oven at 110 °C for 15 min, then
Acetic acid 64-19-7 Corrosive cool to ambient temperature (5 min).
Diethyl ether 60-29-7 Harmful, extremely flammable 2. With a pencil, draw a line to mark the origin,
Hexane 110-54-3 Harmful, highly flammable, 2.5 cm from the bottom of the plate.
dangerous for the 3. Make marks with a pencil to divide the plate
environment into 10 “lanes” of equal width.
4. Use capillary tubes or syringes to apply approx-
imately 10 μl of each standard and sample to a
separate lane (use the middle eight lanes). The
22.6.4 Reagents
application should be done as a streak across
• Chloroform/methanol, 2:1, v/v the center of the lane origin. This is best accom-
• Mobile phase plished with four spots of 2.5 μl each.
Hexane/diethyl ether/acetic acid, 78:20:2 5. Below the origin line, write the identity of the
• Standards sample/standard in each lane.
Triacylglycerol, fatty acid, cholesteryl ester, and 6. Allow spots to dry. You may accelerate drying
cholesterol by using a low-temperature air blower.
• Sulfuric acid solution 7. Write your name in the top right corner of the
Concentrated H2SO4, in 50 % aqueous solution plate.
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Development of Plates
Standard Distance from origin Rf value
1. Line the developing tank with Whatman no. 1
or similar filter paper. Triacylglycerol
2. Pour the mobile phase gently over the filter Fatty acid
paper until the depth of solvent in the tank is Cholesteryl ester
approximately 0.5 cm. About 200 mL is required. Cholesterol
3. Place the lid on the tank and allow 15 min for
the atmosphere in the tank to become saturated
Sample Distance from Rf value Identity
with solvent vapor. spot number origin
4. Place the spotted TLC plate in the developing
tank and allow it to develop until the solvent
front reaches a point about 2 cm from the top of
the plate.
5. Remove the plate from the tank and immediately
mark the position of the solvent front. Evaporate Oil/fat sample type tested:
the solvent in the fume hood.
22.6.10 Questions
Visualization of Lipids
1. In a well-ventilated fume hood, spray lightly 1. Explain the chemical structure of an ester of
with 50 % aqueous H2SO4. Allow to dry. cholesterol.
2. Heat plate for 5–10 min at 100–120 °C. Remove 2. Besides the four fat constituents used as stan-
from oven, cool, and inspect. Handle the plate dards, what other fat constituents might be
with caution as the surface still contains sulfuric found using a TLC method such as this?
acid.
3. Mark all visible spots at their center, and note Acknowledgments This laboratory exercise was developed
the color of the spots. with input from Dr Arun Kilara with Arun Kilara Worldwide,
Northbrook, IL.
For the spots of each of the standards and the samples, RESOURCE MATERIALS
report the distance from the origin for the spot. Also
for each spot, calculate the Rf value, as the distance AOCS (2009) Official methods and recommended practices
from the origin to the spot divided by the distance of the AOCS, 6th edn. American Oil Chemists’ Society,
from the origin to the solvent front. Using the Rf value Champaign, IL
of the standards, identify as many of the spots (bands) Pike OA, O’Keefe SF (2017) Fat characterization. Ch. 23. In:
in the samples as possible. Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
23
chapter
Proteins: Extraction,
Quantitation,
and Electrophoresis
Denise M. Smith
School of Food Science, Washington State University,
Pullman, WA, USA
23.1 Introduction 23.3 Supplies

23.1.1 Background 23.3.1 Sample Extraction
23.1.2 Reading Assignment 23.3.2 Protein Determination
23.1.3 Objective (BCA Method)
23.1.4 Principle of Method 23.3.3 Electrophoresis
23.1.5 Notes 23.3.4 Equipment
23.1.6 Chemicals 23.4 Procedure
23.2 Reagents 23.4.1 Sample Preparation
23.2.1 Sample Extraction 23.4.2 BCA Protein Assay
23.2.2 Protein Determination 23.4.3 Electrophoresis
(BCA Method) 23.5 Data and Calculations
23.2.3 Electrophoresis 23.5.1 Protein Determination
23.2.4 Hazards, Precautions, 23.5.2 Electrophoresis
www.dbooks.org
196 D.M. Smith
23.1 INTRODUCTION reagent to form a purple color that can be quantified

spectrophotometrically and related to the protein con-
23.1.1 Background tent by comparison to a standard curve. Proteins in
the extract can be separated by SDS-PAGE. Proteins
Electrophoresis can be used to separate and visualize
become negatively charged when bound to SDS, so
protein banding patterns. In sodium dodecyl sulfate-
they move through the gel matrix toward the anode
polyacrylamide gel electrophoresis (SDS-PAGE), pro-
(pole with positive charge) at a rate based on size
teins are dissociated into subunits and then separated
alone. The molecular mass of a given protein subunit
based on size within a gel matrix by applying an elec-
can be estimated by comparing its electrophoretic
tric field. It is usually necessary to apply a sample vol-
mobility with proteins of known molecular weight
ume to the gel that contains a known amount of total
using a standard curve. A linear relationship is
protein to allow comparisons between samples. While
obtained by plotting the logarithm of the molecular
it is possible to use an official method (e.g., Kjeldahl, N
mass of standard proteins against their respective
combustion) to determine total protein, it is often con-
electrophoretic mobilities (Rf).
venient to use a rapid colorimetric method of protein
analysis that requires only a small amount of sample.
23.1.5 Notes
The bicinchoninic acid (BCA) assay method will be
used for this purpose. This experiment may be done over two laboratory ses-
In this experiment, sarcoplasmic muscle proteins sions. The protein can be extracted, quantified, and
are extracted with a 0.15 M salt solution, the protein prepared for electrophoresis in the first session. The
content of the extract is measured by the BCA colori- prepared protein samples can be frozen until electro-
metric assay, and the proteins in the fish extracts are phoresis is completed in the second laboratory ses-
separated and visualized by SDS-PAGE. Visualization sion. Alternatively, in a single laboratory session, one
of the protein banding patterns makes it possible to group of students could do the protein extraction and
distinguish among different types of fish since many quantitation, while a second group of students pre-
fish have a characteristic protein subunit pattern. For pares the electrophoresis gels. Also, different groups of
example, one might use this technique as part of a students could be assigned different fish species.
scheme to detect economic adulteration when an inex- Multiple groups could run their samples on a single
pensive fish species is substituted for a more expen- electrophoresis gel. The gels for electrophoresis can be
sive fish in the wholesale or retail marketplace. purchased commercially (e.g., Bio-Rad, Mini-
PROTEAN TGX precast gels, 12 % resolving gel) or
23.1.2 Reading Assignment made as described below.
Some fish species work better than others for pre-
Chang, S.K.C., and Zhang Y. 2017. Protein analysis.
paring the extracts and comparing differences in pro-
Ch.18, in Food Analysis, 5th ed. S.S. Nielsen (Ed.),
tein patterns. Catfish (freshwater) and tilapia
Springer, New York.
(saltwater) work well as extracts and show some dif-
Smith, D.M. 2017. Protein separation and charac-
ferences. Trout gives very thick extracts. Freshwater
terization. Ch. 24, in Food Analysis, 5th ed. S.S. Nielsen
and saltwater salmon show few differences.
Extract proteins from the muscles of freshwater and CAS No. Hazards
saltwater fish, measure the protein content of the
extracts, separate the proteins by electrophoresis, and Sample Extraction:
then compare the different protein banding patterns Sodium chloride (NaCl) 7647-14-5 Irritant
that result based on subunit size and relative Sodium phosphate, 7558-80-7 Irritant
quantity. monobasic (NaH2PO4 .
H2O)
23.1.4 Principle of Method Protein Determination (BCA method):
Sarcoplasmic proteins can be extracted from fish mus- Bicinchoninic acid

Bovine serum albumin (BSA) 9048-46-8
cle with 0.15 M salt. Protein content of the extract can
Copper sulfate (CuSO4) 7758-98-7 Irritant
be determined by the colorimetric BCA assay method. Sodium bicarbonate 144-55-8
In this assay, protein reduces cupric ions to cuprous (NaHCO3)
ions under alkaline conditions. The amount of Sodium carbonate (Na2CO3) 497-19-8 Irritant
cuprous ions formed is proportional to the amount of Sodium hydroxide (NaOH) 1310-73-2 Corrosive
protein present. The cuprous ions react with the BCA Sodium tartrate 868-18-8
Chapter 23 • Proteins: Extraction, Quantitation, and Electrophoresis 197
Electrophoresis: 23.2.3 Electrophoresis

Acetic acid (CH3COOH) 64-19-7 Corrosive (Note: Running buffers (Tris/glycine/SDS), sample
Acrylamide 79-06-1 Toxic buffers (Laemmli sample buffer), and precast gels can
Ammonium persulfate (APS) 7727-54-0 Harmful, be purchased commercially or made in the laboratory.
oxidizing β-Mercaptoethanol may need to be added to a com-
Bis-acrylamide 110-26-9 Harmful
mercial sample preparation buffer.)
Bromophenol blue 115-39-9
Butanol 71-36-3 Harmful
Coomassie Blue R-250 6104-59-2 • Acrylamide: bis-acrylamide solution**
Ethylenediaminetetraacetic 60-00-4 Irritant 29.2 g acrylamide and 2.4 g methylene bis-acryl-
acid, disodium salt amide, with dd water to 100 mL
(Na2EDTA · 2H2O) • Ammonium persulfate (APS), 7.5 %, in dd water,
Glycerol (C3H8O3) 56-81-5 1 mL, prepared fresh daily**
Glycine 56-40-6 • Bromophenol blue, 0.05 %
Hydrochloric acid (HCl) 7647-01-0 Corrosive
• Coomassie Brilliant Blue stain solution**
β-Mercaptoethanol 60-24-2 Toxic
Methanol (CH3OH) 67-56-1 Highly
Purchase premixed or prepare: 454 mL dd water,
flammable 454 mL methanol, 92 mL acetic acid, and 1.5 g
Protein molecular weight Coomassie Brilliant Blue R-250. Note: Bio-Rad
standards (e.g., Bio-Rad offers Coomassie G-250 stains that do not require
161–0374, Precision Plus traditional destaining with methanol or acetic
Protein Dual Color acid. A water wash stops the staining process.
Standards, 10–250 KD) • Destain solution**
Sodium dodecyl sulfate 151-21-3 Harmful 850 mL dd water, 75 mL methanol, and 75 mL
(SDS, dodecyl sulfate, acetic acid
sodium salt)
• EDTA, disodium salt, 0.2 M, 50 mL **
N, N, N′, N′-Tetramethylethyl- 110-18-9 Highly
enediamine (TEMED) flammable,
• Glycerol, 37 % (use directly)
corrosive • Electrophoresis sample preparation buffer **
Tris base 77-86-1 Purchase premixed or prepare: 1 mL of 0.5 M Tris
(pH 6.8), 0.8 mL glycerol, 1.6 mL 10 % SDS,
0.4 mL β-mercaptoethanol, and 0.5 mL 0.05 %
(wt/vol) bromophenol blue, diluted to 8 mL
23.2 REAGENTS
with dd water.
• Sodium dodecyl sulfate, 10 % solution in dd water,
(** It is recommended that these solutions be prepared
10 mL**
by laboratory assistant before class.)
• TEMED (use directly)
• Tray (running) buffer, 25 mM Tris, 192 mM glycine,
23.2.1 Sample Extraction
0.1 % SDS, pH 8.3**
• Extraction buffer, 300 mL/fish species ** Purchase premixed or prepare
Buffer of 0.15 M sodium chloride, 0.05 M sodium • Tris buffer, 1.5 M, pH 8.8, 50 mL (separating (resolv-
phosphate, pH 7.0 (Students asked to show the cal- ing) gel buffer)**
culations for this buffer later in Questions.) Purchase premixed or prepare
• Tris buffer, 0.5 M, pH 6.8, 50 mL (stacking gel
23.2.2 Protein Determination (BCA Method) buffer)**
Purchase premixed or prepare
Commercial BCA test kits can be purchased, such as
the Pierce BCA Protein Assay Kit (cat. No. 23225,
Rockford, IL) which includes procedures to complete Gel preparation: Use the formula that follows and
either test tube (2 mL working reagent/tube) or micro- the instructions in the procedure to prepare two
plate assays (200 uL working reagent/well). 8.4 × 5.0 cm SDS-PAGE slab gels, 15 % acrylamide,
This kit includes: 0.75 mm thick. Actual formulation will depend on size
• Bovine serum albumin (BSA) standard ampules, of slab gels.
2 mg/mL in 0.9 % saline and 0.05 % sodium azide as
Reagent Separating gel Stacking gel
a preservative
• BCA Reagent A: Contains sodium carbonate, 15 % gel 4.5 % gel
sodium bicarbonate, BCA detection reagent, and Acrylamide: 2.4 mL 0.72 mL
sodium tartrate in 0.1 M sodium hydroxide bis-acrylamide
• Reagent B: 4 % cupric sulfate solution 10 % SDS 80 μL 80 μL
www.dbooks.org
198 D.M. Smith
Reagent Separating gel Stacking gel

23.3.3 Electrophoresis
15 % gel 4.5 % gel • Beaker, 250 mL (for boiling samples)
1.5 M Tris, pH 8.8 2.0 mL – • Two Erlenmeyer flasks, 2 L (for stain and destain
0.5 M Tris, pH 6.8 – 2.0 mL solutions)
dd water 3.6 mL 5.3 mL • Glass boiling beads (for boiling samples)
37 % glycerol 0.15 mL – • Graduated cylinder, 100 mL
10 % APSa 40 μL 40 μL • Graduated cylinder, 500 mL
TEMED 10 μL 10 μL • Hamilton syringe (to load samples on gels)
a
APS added to separating and stacking gels after all other • Mechanical, adjustable volume pipettors, 1000 μL,
reagents are combined, solution is degassed, and each gel is 100 μL, and 20 μL, with plastic tips
ready to be poured • Pasteur pipettes, with bulbs
• Rubber stopper (to fit 25-mL sidearm flasks)
23.2.4 Hazards, Precautions, and Waste • Two sidearm flasks, 25 mL
Disposal • Test tubes or culture tubes, small size, with caps
• Tubing (to attach to vacuum system to degas gel
Acrylamide monomers may cause cancer and are
solution)
very toxic in contact with skin and if swallowed.
• Weighing paper/boats
β-Mercaptoethanol is harmful if swallowed, toxic in
contact with the skin, and irritating to eyes. Adhere
23.3.4 Equipment
to normal laboratory safety procedures. Wear
gloves and safety glasses at all times. Acrylamide • Analytical balance
and β-mercaptoethanol wastes must be disposed of • Aspirator system (for degassing solutions)
as hazardous wastes. Gloves and pipette tips in con- • Blender
tact with acrylamide and β-mercaptoethanol also • Centrifuge
should be handled as hazardous wastes. Other waste • Electrophoresis unit
likely can be washed down the drain with a water • pH meter
rinse, but follow good laboratory practices outlined • Power supply
by environmental health and safety protocols at your • Spectrophotometer
institution. • Top loading balance
• Vortex mixer
• Water bath
23.3 SUPPLIES
(Used by students) 23.4 PROCEDURE
(Single sample extracted)

23.3.1 Sample Extraction
• Beaker, 250 mL 23.4.1 Sample Preparation
• Centrifuge tubes, 50 mL
1. Coarsely cut up about 100-g fish muscle (repre-
• Cutting board
sentative sample) with a knife. Accurately
• Erlenmeyer flask, 125 mL
weigh out 90 g on a top loading balance.
2. Blend one part fish with three parts extraction
• Filter paper, Whatman No. 1
buffer (90-g fish and 270 mL extraction buffer)
• Fish, freshwater (e.g., catfish) and saltwater species
for 1.0 min in a blender. (Note: Smaller amounts
(e.g., tilapia)
of fish and buffer, but in the same 1:3 ratio, can
• Funnel
be used for a small blender.)
• Knife
3. Pour 30 mL of the muscle homogenate into a
• Pasteur pipettes and bulbs
50-mL centrifuge tube. Label tube with tape.
• Test tube with cap
Balance your tube against a classmate’s sample.
• Weighing boat
Use a spatula or Pasteur pipette to adjust tubes
to an equal weight.
23.3.2 Protein Determination (BCA Method)
4. Centrifuge the samples at 2000× g for 15 min at
• Beaker, 50 mL room temperature. Collect the supernatant.
• Graduated cylinder, 25 mL 5. Filter a portion of the supernatant by setting a
• Mechanical, adjustable volume pipettor, 1000 μL, small funnel in a test tube. Place a piece of
with plastic tips Whatman No. 1 filter paper in the funnel and
• Test tubes moisten it with the extraction buffer. Filter the
supernatant from the centrifuged sample. Collect tion used. (Note: Do not use the diluted samples
about 10 mL of filtrate in a test tube. Cap the tube. for electrophoresis. Use the original extract pre-
6. Determine protein content of filtrate using the pared as described below.)
BCA method and prepare sample for electro-
phoresis (see below). 23.4.3 Electrophoresis
1. Assemble the electrophoresis unit according to
23.4.2 BCA Protein Assay the manufacturer’s instructions.
(Instructions are given for duplicate analysis of each 2. Use the table that follows the list of electrophore-
concentration of standard and sample.) sis reagents to combine appropriate amounts of
all reagents for the separating gel, except APS, in
1. Prepare the Working Reagent for the BCA assay a sidearm flask. Degas the solution and then add
by combining Pierce Reagent A with Pierce APS. Proceed immediately to pour the solution
Reagent B, 50:1 (v/v), A:B. For example, use 50-mL between the plates to create the separating
Reagent A and 1.0-mL Reagent B to prepare 51.0- (resolving) gel. Pour the gel to a height approxi-
mL Working Reagent, which is enough for the mately 1 cm below the bottom of the sample well
BSA standard curve and testing the extract from comb. Immediately, add a layer of butanol across
one type of fish. (Note: This volume is adequate the top of the separating gel, adding it carefully
for assaying duplicates of five standard samples so as not to disturb the upper surface of the sepa-
and two dilutions of each of two types of fish.) rating gel. This butanol layer will prevent a film
2. Prepare the following dilutions of the superna- from forming and help obtain an even surface.
tant (filtrate from Procedure, Sample Allow the separating gel to polymerize for
Preparation, Step 5): dilutions of 1:5, 1:10, and 30 min and then remove the butanol layer just
1:20 in extraction buffer. Mix well. before the stacking gel is ready to be poured.
3. In test tubes, prepare duplicates of each reaction 3. Use the table that follows the list of electrophore-
mixture of diluted extracts and BSA standards sis reagents to combine appropriate amounts of
(using 2 mg BSA/mL solution) as indicated in all reagents for the stacking gel, except APS, in a
the table that follows: sidearm flask. Degas the solution for 15 min (as
per the manufacturer’s instructions) and then
Tube dd water BSA Std. Fish extract Working add APS. Proceed immediately to pour the solu-
identity (μL) (μL) (μL) reagent (mL)
tion between the plates to create the stacking gel.
Blank 100 0 – 2.0 Immediately, place the well comb between the
Std. 1 80 20 – 2.0 plates and into the stacking gel. Allow the stack-
Std. 2 60 40 – 2.0 ing gel to polymerize for 30 min before removing
Std. 3 40 60 – 2.0 the well comb. Before loading the samples into
Std. 4 20 80 – 2.0 the wells, wash the wells twice with dd water.
Std. 5 0 100 – 2.0
4. Mix the fish extract samples well (filtrate from
Sample 50 – 50 2.0
Sect. 23.4.1, Step 5). For each sample, combine
1:5
Sample 50 – 50 2.0 0.1-mL sample with 0.9-mL electrophoresis sam-
1:10 ple buffer in screw cap culture tube. Apply cap.
Sample 50 – 50 2.0 5. Heat capped tubes for 3 min in boiling water.
1:20 6. Apply 10 and 20 μg protein of each fish extract to
wells of the stacking gel using a syringe. Calculate
4. Mix each reaction mixture with a vortex mixer the volume to apply based on the protein content
and then incubate in a water bath at 37°C for of the extract and the dilution used when prepar-
30 min. ing the extract in electrophoresis sample buffer.
5. Read the absorbance of each tube at 562 nm 7. Apply 10 μl of molecular weight standards to
using a spectrophotometer. one sample well.
6. Use the data from the BSA samples to create a 8. Follow the manufacturer’s instructions to
standard curve of absorbance at 562 nm versus assemble and run the electrophoresis unit. When
μg protein/tube. Determine the equation of the the line of Bromophenol Blue tracking dye has
line for the standard curve. Calculate the pro- reached the bottom of the separating gel, shut
tein concentration (μg/mL) of the extract from off the power supply. Disassemble the electro-
each fish species using the equation of the line phoresis unit, and carefully remove the separat-
from the BSA standard curve and the absor- ing gel from between the plates. Place the gel in
bance value for a dilution of the fish extract that a flat dish with the Coomassie Brilliant Blue
had an absorbance near the middle point on the stain solution. Allow the gel to stain for at least
standard curve. Remember to correct for dilu- 30 min. (If possible, place the dish with the gel
www.dbooks.org
200 D.M. Smith
on a gentle shaker during staining and destain- Sample calculation to determine the volume of
ing.) Pour off the stain solution and then destain prepared extract to apply 20 ug protein to each sample
the gel for at least 2 h using the destain solution well:
with at least two changes of the solution.
9. Measure the migration distance (cm) from the top How many μL are needed to get 20 μg protein?
of the gel to the center of the protein band for the Remember the electrophoresis sample buffer dilution
molecular weight standards and for each of the is 1:10:
major protein bands in the fish extract samples.
Also measure the migration distance of the bromo- 24.99 mg protein / mL ´ Z mL ´ (1 mL / 10 mL) = 20 mg
phenol blue tracking dye from the top of the gel. Z = 8.00 uL
10. Observe and record the relative intensity of the
major protein bands for each fish extract.
23.5.2 Electrophoresis
23.5 DATA AND CALCULATIONS 1. Calculate the relative mobility of three major
protein bands and all the molecular weight
23.5.1 Protein Determination standards. To determine the relative mobility
(Rf) of a protein band, divide its migration dis-
μg protein/ μg/mL tance from the top of the gel to the center of the
Tube identity Absorbance tube sample protein band by the migration distance of the
bromophenol blue tracking dye from the top of
Std. 1, 20 μL BSA
the gel:
Std. 1, 20 μL BSA
Std. 2, 40 μL BSA distance of protein migration
Std. 2, 40 μL BSA Rf =
distance of tracking dye migration
Std. 3, 60 μL BSA
Std. 3, 60 μL BSA
Std. 4, 80 μL BSA
Std. 4, 80 μL BSA Distance
Std. 5, 100 μL BSA of
Std. 5, 100 μL BSA Distance tracking
Sample of protein dye Relative Molecular
identity migration migration mobility weight
Sample 1:5
Sample 1:5 Molecular
X = weight
Sample 1:10 standards
Sample 1:10 1
X = 2
3
Sample 1:20
4
Sample 1:20
5
X =
Fish species
Freshwater
Saltwater
Sample calculation for fish extract protein
concentration:
2. Prepare a standard curve by plotting relative
For fish extract diluted 1:20 and 50 μL analyzed mobility (x-axis) versus log molecular weight of
with absorbance of 0.677: standards (y-axis).
Equation of the line: y = 0.0108x + 0.0022 3. Using the standard curve, estimate the molecu-
If y = 0.677, x = 62.48 lar weight of the major protein subunits in the
freshwater and saltwater fish extracts.
Ci = Cf (V2 / V1 ) (V4 / V3 )
(See Chap. 3 in this laboratory manual; Ci = initial 23.6 QUESTIONS
concentration; Cf = final concentration)
1. Describe how you would prepare 1 L of the buf-
Ci = (62.48-ug protein/tube) × (20 ml/1 mL) × fer used to extract the fish muscle proteins
(tube/50 uL) (0.15 M sodium chloride, 0.05 M sodium phos-
= 24.99μg protein/uL fish extract phate, pH 7.0). Show all calculations.
2. Discuss the differences between the fish species, Laemmli UK (1970) Cleavage of structural proteins during
regarding the presence or absence of major pro- the assembly of the head of bacteriophage T4. Nature
tein bands identified by the molecular mass and 227:680–685
the relative amounts of these proteins. Olsen BJ and Markwell J (2007) Assays for the determination
of protein concentration. Unit 3.4 Basic Protocol 3. BCA
Assay. Current Protocols in Protein Science John Wiley and
Sons, New York
RESOURCE MATERIALS Pierce (2013) Instructions: Pierce BCA protein assay kit.
Pierce Biotechnology, Rockford, IL https://tools.lifetech-
Bio-Rad (2013) A Guide to Polyacrylamide Gel Electrophoresis nologies.com/content/sfs/manuals/MAN0011430_
and Detection. Bulletin 6040 Rev B. Bio-Rad Laboratories, Pierce_BCA_Protein_Asy_UG.pdf Accessed June 24, 2015
http://www.bio-rad.com/webroot/web/pdf/lsr/litera- Piñeiro C et al (1999) Development of a sodium dodecyl
ture/Bulletin_6040.pdf Accessed June 24, 2015 sulfate-polyacrylamide gel electrophoresis reference
Chang SKC, Zhang Y (2017) Protein analysis. Ch. 18. In: method for the analysis and identification of fish species in
Nielsen SS (ed) Food analysis, 5th edn. Springer, New York raw and heat-processed samples: a collaborative study.
Etienne M et al. (2000) Identification of fish species after Electrophoresis 20:1425–1432
cooking by SDS-PAGE and urea IEF: a collaborative study. Smith DM (2017) Protein separation and characterization.
J Agr Food Chem 48:2653–2658 Ch. 24. In: Nielsen SS (ed) Food analysis, 5th edn. Springer,
Etienne M et al. (2001) Species identification of formed fish- New York
ery products and high pressure-treated fish by electropho-
resis: a collaborative study. Food Chem 72:105–112
www.dbooks.org
24
Chapter
Glucose Determination
by Enzyme Analysis
Charles E. Carpenter (*) • Robert E. Ward
Logan, UT, USA
24.1 Introduction 24.1.7 Hazards, Precautions,

24.1.4 Principle of Method 24.2 Procedure
24.1 INTRODUCTION Glucose oxidase 9001-37-0

(Sigma G6641)
24.1.1 Background Horseradish 9003-99-0
peroxidase (Sigma
Enzyme analysis is used for many purposes in food sci- P6782)
ence and technology. Enzyme activity is used to indi- Sodium acetate 127-09-3
cate adequate processing, to assess enzyme preparations, (Sigma S2889)
and to measure constituents of foods that are enzyme Sulfuric acid (Aldrich 7664-93-9 Corrosive
substrates. In this experiment, the glucose content of 320501)
corn syrup solids is determined using the enzymes, glu-
cose oxidase and peroxidase. Glucose oxidase catalyzes
the oxidation of glucose to form hydrogen peroxide 24.1.6 Reagents
(H2O2), which then reacts with a dye in the presence of
peroxidase to give a stable-colored product.
As described, this experiment uses individual,
commercially available reagents, but enzyme test kits
• Acetate buffer**, 0.1 M, pH 5.5
that include all the reagents to quantitate glucose also
Dissolve 8 g sodium acetate in ca. 800 mL water
are available as a package. Enzyme test kits also are
in a 1-L beaker. Adjust pH to 5.5 using 1 M HCl.
available to quantitate various other components of
Dilute to 1 L in a volumetric flask.
foods. Companies that sell enzyme test kits usually
• Glucose test solution**
provide detailed instructions for the use of these kits,
In a 100-mL volumetric flask, dissolve 20 mg glu-
including information about the following: (1) princi-
cose oxidase (~300–1000 units), 40 mg horserad-
ple of the assay, (2) contents of the test kit, (3) prepara-
ish peroxidase, and 40 mg o-dianisidine.2HCl in
tion of solutions, (4) stability of solutions, (5) procedure
the 0.1 M acetate buffer. Dilute to volume with
to follow, (6) calculations, and (7) further instructions
the acetate buffer and filter as necessary.
regarding dilutions and recommendations for specific
• Glucose standard solution, 1 mg/mL
food samples.
Use commercial D-glucose solution (e.g., Sigma).
24.1.2 Reading Assignment • Sulfuric acid, diluted** (1 part H2SO4 + 3 parts
water)
BeMiller, J.N. 2017. Carbohydrate analysis. Ch. 19, in In a 500-mL beaker in the hood, add 150 mL
Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, water and then add 50 mL H2SO4. This will gen-
New York. erate a lot of heat.
Reyes-De-Coreuera, J.I., and Powers, J.R. 2017.
Application of enzymes in food analysis. Ch. 25, in Food
Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, New York. 24.1.7 Hazards, Precautions, and Waste
Disposal
24.1.3 Objective Concentrated sulfuric acid is extremely corrosive;
Determine the glucose content of food products using avoid contact with skin and clothes and breathing
the enzymes, glucose oxidase and peroxidase. vapors. Acetic acid is corrosive and flammable. Wear
safety glasses at all times and corrosive-resistant
24.1.4 Principle of Method gloves. Otherwise, adhere to normal laboratory
safety procedures. The o-dianisidine 2HCl must be
Glucose is oxidized by glucose oxidase to form hydro- disposed of as hazardous waste. Other waste likely
gen peroxide, which then reacts with a dye in the pres- may be put down the drain using a water rinse, but
ence of peroxidase to give a stable-colored product follow good laboratory practices outlined by envi-
that can be quantitated spectrophotometrically (cou- ronmental health and safety protocols at your
pled reaction). institution.
24.1.5 Chemicals
24.1.8 Supplies
CAS No. Hazards • Beaker, 1 L
• Corn syrup solids (or high fructose corn syrup),
Acetic acid (Sigma 64-19-7 Corrosive
A6283)
0.5 g
o-Dianisidine.2HCl 20325-40-0 Tumor causing, • 5 Spatulas
(Sigma D3252) carcinogenic • 14 Test tubes, 18 × 150 mm, heavy walled to keep
D-Glucose (Sigma 50-99-7 from floating in water bath
G3285) • Test-tube rack
www.dbooks.org
Chapter 24 • Glucose Determination by Enzyme Analysis 205
• 2 Volumetric flasks, 100 mL 24.3 DATA AND CALCULATIONS

• Volumetric flask, 250 mL
• Volumetric pipette, 10 mL Weight of original sample: _______ g
• 2 volumetric flasks, l L
• Weighing paper Absorbance of standard solutions:
24.1.9 Equipment (mg glucose/mL)

Tube Msmt 0 0.05 0.10 0.15 0.20
• Mechanical, adjustable volume pipettors, 200, 1000, 1 1
2
and 5000 μL, with tips
2 1
• pH meter 2
• Spectrophotometer Average
• Water bath, 30 °C absorbance:
Absorbance of samples:
24.2 PROCEDURE
Tube Msmt Sample A Sample B
1 1
1. Prepare dilutions for standard curve. Use the 2
adjustable pipettors to deliver aliquots of glu- 2 1
cose standard solution (1 mg/mL) and deion- 2
ized distilled (dd) water as indicated in the Average absorbance:
table below into clean test tubes. These dilu-
tions will be used to create a standard curve of Calculation of glucose concentration in sample:
0–0.2 mg glucose/mL. 1. Plot absorbance of standards on the y-axis ver-
sus mg glucose/mL on the x-axis.
mg glucose/mL
2. Calculate the concentration of glucose for the
0 0.05 0.10 0.15 0.20 sample dilution A or B that had an absorbance
mL glucose 0 0.150 0.300 0.450 0.600 within the working range of the standard curve:
std. (Abs – y-intercept)/slope = mg glucose/mL.
solution 3. Calculate the glucose concentration in the origi-
mL dd water 3.000 2.850 2.700 2.550 2.400
nal sample, as a percentage.
2. Prepare sample solution and dilutions.
Example calculations:
Accurately weigh ca. 0.50 g corn syrup solids
and dilute with water to volume in a 250-mL Original sample of 0.512 g
volumetric flask (Sample A). Using volumetric
pipettes and flasks, dilute 10.00 mL of Sample A Average measured absorbance sample dilution
to 100 mL with water (Sample B). These sample B: 0.200
dilutions will let you determine glucose con-
Calculation from standard curve: 0.200–0.003/
centrations in samples containing 1–100 %
(2.98 mL/mg glucose) = 0.066 mg glucose/mL B)
glucose.
3. Add 1.000 mL of water to each of 14 test tubes. Csample = (0.066 mg glucose/mL B)
In duplicate, add 1.000 mL of the individual × (100 mL B/10 mL A)
standard and sample dilutions to the test × (250 mL A/512 mg sample)
tubes. = 0.323 mg glucose/mg sample
4. Put all tubes in the water bath at 30 °C for 5 min. = 32.3 % glucose
Add 1.000 mL glucose test solution to each tube
at 30 s intervals. 24.4 QUESTIONS
5. After exactly 30 min, stop the reactions by add-
ing 10 mL of the diluted H2SO4. Cool to room 1. Explain why this experiment is said to involve a
temp. coupled reaction. Write in words the equations
6. Zero spectrophotometer with water in the ref- for the reactions. What conditions must be in
erence position using a double beam spectro- place to ensure accurate results for such a cou-
photometer. Take two readings (repeated pled reaction?
measures, msmt) using separate aliquots from 2. How do the results obtained compare to specifi-
each tube. cations for the commercial product analyzed?
RESOURCE MATERIALS SS (ed) Food analysis, 5th edn. Springer, New York
Reyes-De-Coreuera JI, and Powers JR (2017) Application of
BeMiller JN (2017) Carbohydrate analysis. Ch. 19. In: Nielsen enzymes in food analysis. Ch. 26. In: Nielsen SS (ed) Food
analysis, 5rd edn. Springer, New York
www.dbooks.org
25
chapter
Gliadin Detection
by Immunoassay
Y.H. Peggy Hsieh (*) • Qinchun Rao
Department of Nutrition, Food and Exercise Sciences, Florida State University,

25.1.3 Objective 25.2.1 Sample Preparation
25.1.4 Principle of Method 25.2.2 Standard Gliadin
25.1.5 Chemicals 25.2.3 Nitrocellulose Dot Blot
25.1.6 Reagents Immunoassay
25.1.7 Hazards, Precautions, 25.3 Data and Calculations
208 Y.H.P. Hsieh and Q. Rao
25.1 INTRODUCTION 25.1.3 Objective

Determine the presence of gliadin in various food
25.1.1 Background products using a rabbit anti-gliadin antibody horse-
Immunoassays are very sensitive and efficient tests radish peroxidase conjugate in a dot blot
that are commonly used to identify a specific protein. immunoassay.
Examples of applications in the food industry include
identification of proteins expressed in genetically 25.1.4 Principle of Method
modified foods, allergens, or proteins associated with
A simple dot blot immunoassay will be used in this lab
a disease, including celiac disease. This genetic disease
to detect gliadin in food samples. Dot blot assays use
is associated with up to 1 % of the world’s population
nitrocellulose (NC) paper for a solid phase. Initially,
and more than two million Americans. These individ-
gliadin proteins are isolated by differential centrifuga-
uals react immunologically to wheat proteins, and
tion, in which most of the non-gliadin proteins are
consequently their own immune systems attack and
washed away with water and sodium chloride (NaCl)
damage their intestines. This disease can be managed
solutions, and then the gliadin is extracted with a
if gluten is avoided in foods. Gluten consists of alcohol-
detergent solution. A drop of the food sample extract
insoluble glutenins and alcohol-soluble prolamins,
or standard antigen (gliadin) is applied to the NC
which are mainly found in wheat and also in oat, bar-
paper, where it adheres nonspecifically. The remaining
ley, rye, and other grain flours and related starch
binding sites on the NC paper are then “blocked”
derivatives. Rice and corn are two common grains that
using a protein unrelated to gliadin, such as bovine
do not contain significant gluten and are well tolerated
serum albumin (BSA) to minimize the nonspecific
by those with celiac disease. Wheat protein makes up
binding. The bound gliadin antigen in the food spot
7–15 % of a wheat grain. Prolamins in wheat are called
then can be reacted with an antigen-specific antibody-
gliadin. About 40 % of the wheat proteins are various
enzyme conjugate. Theoretically, this antibody probe
forms of gliadin protein.
then will bind only to gliadin antigen bound already
The immune system of animals can respond to
to the NC paper. Next, the strip is washed free of
many foreign substances by the development of spe-
unbound antibody-enzyme conjugate and then placed
cific antibodies. Antibodies bind strongly to and assist
in a substrate solution in which an enzymatically cata-
in the removal of a foreign substance in the body.
lyzed precipitation reaction can occur. Brown-colored
Animals make antibodies against many different
“dots” indicate the presence of gliadin-specific anti-
“antigens,” defined as foreign substance that will elicit
body and hence gliadin antigen. The color intensity
a specific immune response in the host. These include
indicates the amount of antigen present in the food
foreign proteins, peptides, carbohydrates, nucleic
sample. The stronger the color, the higher amount of
acids, lipids, and many other naturally occurring or
the antigen (gliadin) in the food sample extract.
synthetic compounds.
Immunoassays are tests that take advantage of the
25.1.5 Chemicals
remarkably specific and strong binding of antibodies
to antigens. Immunoassays can be used to determine
the presence and quantity of either antibody or anti- CAS no. Hazards
gen. Antibodies that identify a specific protein (anti- Bovine serum albumin 9048-46-8
gen) can be developed by injection of a laboratory (BSA)
animal with this protein, much as humans are vacci- Chicken egg albumin (CEA) 9006-59-1
nated against a disease. These antigen-specific anti- 3,3’-Diaminobenzidine 7411-49-6
bodies can be used to identify the antigen in a food tetrahydrochloride (DAB)
(e.g., detection of gliadin in food products) through Gliadin standard protein 9007-90-3
the appropriate use of a label, such as an enzyme or Hydrogen peroxide, 30 % 7722-84-1 Oxidizing,
fluorescent molecules linked covalently to either the (H2O2) corrosive
antibody or a reference antigen. This type of immuno- Rabbit anti-gliadin
assay concept also can be used to determine the pres- immunoglobulin
conjugated to
ence of specific antibodies in blood. For example, by
horseradish peroxidase
analyzing for the presence of gliadin-specific antibod- (RAGIg-HRP, Sigma,
ies in an individual’s blood, one can determine if the A1052)
individual has celiac disease. Sodium chloride (NaCl) 7647-14-5 Irritant
Sodium dodecyl sulfate 151-21-3 Harmful
25.1.2 Reading Assignment (SDS)
Sodium phosphate, 7558-80-7 Irritant
Hsieh, Y-H.P. and Rao, Q. 2017. Immunoassays. Ch.27, monobasic (NaH2PO4 ·
in Food Analysis, 5th ed. S.S. Nielsen (Ed.), Springer, H2O)
New York.
www.dbooks.org
Chapter 25 • Gliadin Detection by Immunoassay 209
• Disposable tips, for pipettors

CAS no. Hazards
• Filter paper, Whatman #1
Tris(hydroxymethyl) 77-86-1 Irritant • Food samples (e.g., flour, crackers, cookies, starch,
aminomethane (TRIS) pharmaceuticals, etc.)
Tween-20 detergent 9005-64-5 • Funnels, tapered glass
• Mechanical, adjustable volume pipettors, for 2 μL,
25.1.6 Reagents 100 μL, and 1000 μL ranges, with plastic tips (glass
capillary pipettes can be substituted for 2 μL
pipettors)
• Microcentrifuge tubes, 1.5 mL, two per sample
processed
• Blocking solution ** • Nitrocellulose paper (Bio-Rad 162–0145) cut into
3 % (g/mL) BSA in PBST; 5–10 mL per student 1.7 × 2.3 cm rectangular strips (NC strips)
• DAB substrate** • Petri dishes, 3.5 cm
60 mg DAB dissolved in 100 mL 50 mM TRIS • Test tubes, 13 × 100 mm, six per student
(pH 7.6) and then filtered through filter paper • Test tube rack, one per student
Whatman #1. (Note: DAB may not completely • Tissue paper
dissolve in this buffer if it is the free base form • Tweezers, one set per student
instead of the acid form. Just filter out the undis- • Wash bottles, one per two students for PBST
solved DAB and it will still work well.) Just • Wash bottles, one per two students for distilled
5 min prior to use, add 100 μL 30 % H2O2, water
5–10 mL per student.
• Gliadin antibody probe **
25.1.9 Equipment
1:500 (mL/mL) diluted RAGIg-HRP using 0.5 %
(g/mL) BSA in PBST; 5–10 mL per student • Mechanical platform shaker
• Gliadin extraction detergent** • Microcentrifuge
1 % (g/mL) SDS in water; 10 mL per student • pH meter
• Gliadin standard protein, 4000 μg/mL, in 1 % SDS** • Vortex mixer
One vial of 150 μL per student
• Negative control sample**
3 % CEA (or other non-gliadin protein) in PBST; 25.2 PROCEDURE
100 μL per student
• Phosphate-buffered saline (PBS)** 25.2.1 Sample Preparation
0.05 M sodium phosphate, 0.9 % (g/mL) NaCl, (Note: Sample preparation by the students may take place
pH 7.2 on a separate day prior to the immunoassay. In this initial
• Phosphate-buffered saline + Tween 20 (PBST) ** sample preparation lab, the principles of differential cen-
0.05 M sodium phosphate, 0.9 % (g/mL) NaCl, trifugation, with respect to the Osborne protein classifi-
0.05 % (mL/mL) Tween 20, pH 7.2, 250 mL per cation system, may be studied. Sample preparation and
student immunoassay may not be reasonable to achieve in one
day. If only one day can be allocated for this lab, the sam-
25.1.7 Hazards, Precautions, and Waste ples can be prepared ahead of time for the students by
Disposal the technical assistant, and this lab will demonstrate con-
Adhere to normal laboratory safety procedures. Wear cept and techniques of a simple immunoassay.)
gloves and safety glasses at all times. Handle the DAB 1. Weigh accurately (record the mass) about 0.1 g
substrate with care. Wipe up spills and wash hands of flour, starch, or a ground processed food and
thoroughly. The DAB, SDS, and hydrogen peroxide add to a 1.5 mL microcentrifuge tube. Add
wastes should be disposed of as hazardous wastes. 1.0 mL distilled water and vortex for 2 min.
Other wastes likely may be put down the drain using Place in the microcentrifuge with other samples
a water rinse, but follow good laboratory practices and centrifuge at 800 × g for 5 min. Discard the
outlined by environmental health and safety protocols supernatant (albumins). Repeat.
at your institution. 2. Add 1.0 mL of 1.5 M NaCl to the pellet from
Step 1 and resuspend it by vortexing for 2 min.
25.1.8 Supplies If the pellet is not resuspending, dislodge it
(Used by students) with a spatula. Centrifuge at 800 × g for 5 min.
Discard the supernatant (globulins). Repeat.
• 1–10 μL, 10–100 μL, and 200–1000 μL positive dis- 3. Add 1.0 mL of 1 % SDS detergent to the pellet
placement pipettors from Step 2 and resuspend it to extract the glia-
dins. Vortex for 2 min. Centrifuge at 800 × g for
210 Y.H.P. Hsieh and Q. Rao
5 min. Carefully pipette off most of the A1 2 3 B1 2 3

supernatant and transfer to a clean microcentri-
fuge tube. Discard the pellet.
4 5 6 4 5 6
25.2.2 Standard Gliadin
The standard pure gliadin is dissolved at a concentra-
tion of 4000 μg/mL in 1 % SDS. To provide for a series
A series (food sample): B series (food sample):
of standards to compare unknown samples, dilute the
standard serially by a factor of 10 in 13 × 100 mm test
tubes to make 400 μg/mL, 40 μg/mL, and 4 μg/mL A series (food sample): B series (gliadin
standards in 1 % SDS. Use 100 μL of the highest stan- standards):
dard transferred to 900 μL of 1 % SDS detergent for the 1 = food sample 1 1 = gliadin standard
first tenfold dilution. Repeat this procedure serially to @ 1× dilution @ 4000 μg/mL
produce the last two standards. As each standard is
made, mix it well on a vortex mixer. 2 = food sample 2 2 = gliadin standard
@ 1× dilution @ 400 μg/mL
25.2.3 Nitrocellulose Dot Blot Immunoassay 3 = food sample 3 3 = gliadin standard
@ 1× dilution @ 40 μg/mL
[Note: The nitrocellulose (NC) strips should only be
handled with tweezers to prevent binding of proteins 4 = food sample 4 4 = gliadin standard
and other compounds from your fingers. Hold the @ 1× dilution @ 4 μg/mL
strips with the tips of the tweezers on the corners of 5 = 1 % SDS control 5 = 1 % SDS control
the nitrocellulose to avoid damaging or interfering
with the spotted surface.] 6 = 3 % protein negative 6 = 3 % protein negative
control (CEA) control (CEA)
1. Mark two NC strips with a pencil into six equal
boxes each (see drawing below).
2. Pipette 2 μL each of sample, standard or nega- 6. Wash the NC strips with PBST using a wash
tive controls onto the NC paper. Lay the NC bottle and then incubate them for 5 min in a
strips flat onto some tissue. clean petri dish half full with PBST. Rinse again
(a) On NC strip A, pipette 2 μL of four different well with PBST, and rinse one last time with dis-
SDS food sample extracts. tilled water.
(b) On NC strip B, pipette 2 μL of four different 7. Add NC strips to a petri dish containing the
gliadin standards. 5 mL of DAB/H2O2 substrate, and watch for the
(c) On the remaining two squares (5 and 6) on development of a brown stain. (Note: Handle
strips A and B, add 2 μL of 1 % SDS and 2 μL the substrate with care. Wipe up spills, wash
of the 3 % CEA protein negative control, hands thoroughly, and wear gloves. Although
respectively. there is no specific evidence that DAB is a carci-
(d) Let the spots air-dry on the tissue. nogenic compound, it should be treated as if it
were.) Stop the reaction in 10–15 min, or when
the background nitrocellulose color is becom-
Below are diagrams of the NC strips marked off
ing noticeably brown, by rinsing each strip in
in boxes and numbered by pencil. The circles are
distilled water.
not penciled in, but rather they represent where
8. Let the NC strips air-dry on tissue paper.
the 2 μL sample or standard spots will be applied.
3. Place both NC strips into a petri dish containing
5 mL of blocking solution (3 % BSA in PBST) 25.3 DATA AND CALCULATIONS
and let incubate for 20 min on the mechanical
shaker so that the NC strips are moving around Make any observations you feel are pertinent to this
slightly in the solution. laboratory. Attach the developed NC strips to your lab
4. Rinse the strips well with PBST using a wash report with a transparent tape.
bottle over a sink, holding the strips by the tips Describe the results based on observations of the
of their corners with tweezers. degree of brown-colored stain in standards and samples
5. Place the NC strips into a petri dish containing relative to negative controls. You can use a crude quanti-
about 5 mL of 1:500 diluted RAGIg-HRP tative rating system like +++, ++, +, ±, and − to describe
conjugate, and incubate for 60 min on the and report the relative intensities of the dot reactions
mechanical shaker. (note: the brown dot images will fade in several days).
www.dbooks.org
Chapter 25 • Gliadin Detection by Immunoassay 211
Make very crude approximations of the quantity 2. Why should you block unbound sites on nitro-
of gliadin in each substance relative to (more or less cellulose with 3 % BSA in a special blocking step
than) the standard gliadin dots. Comment on this after applying samples to the membrane?
crude estimate relative to the food product’s gluten 3. Why is a spot of 1 % SDS (gliadin extraction
status [i.e., gluten free or not; Codex Alimentarius detergent) used in the dot blot?
(http://www.codexalimentarius.net/) defines less 4. Why is a protein negative control spot (3 %
than 20 mg of gluten/kg in total, based on the food as CEA) used in the dot blot?
sold or distributed to the consumer to be classified as 5. Describe the basic role of horseradish peroxidase
“gluten-free”]. enzyme (i.e., why is it attached to the rabbit anti-
Tabulate your results in a manner that is easy to body), and what roles do DAB and H2O2 play in
interpret. the development of the colored dot reaction in
To make the gluten status estimation, you must this immunoassay? Do not describe the actual
know the values of both the concentration of the food chemical reaction mechanisms, but rather explain
sample (g food/mL extraction solution) extracted and why a color reaction can ultimately infer a gliadin
the concentration of the gliadin standards (mg glia- antigen that is present on the nitrocellulose paper.
din/mL extraction solution) to which you are making
a comparison. Acknowledgment This laboratory exercise was initially
developed for the first edition of the laboratory manual by
Example calculation: Mr Gordon Grant and Dr Peter Sporns, Department of
Agricultural, Food, and Nutritional Science, University of
Alberta, Edmonton, Alberta, Canada. Dr. Hsieh, who has
If the food sample has a concentration of 100 mg/
now updated the laboratory for two editions, gratefully
mL and reacts equivalently to a 4 μg/mL gliadin stan- acknowledges the original contribution.
dard, it can be estimated that 4 μg gliadin is in 100 mg
food sample, because both are applied at equal vol-
umes so the two concentrations can be related frac- RESOURCE MATERIALS
tionally (i.e., 4 μg gliadin/100 mg or 40 mg gliadin/kg
food sample). The gliadin content of gluten is gener- Hsieh Y-HP (2017) Immunoassays. Ch.27. In: Nielsen SS (ed)
ally taken as 50 %. Since this sample has a gliadin con- Food analysis, 5th edn. Springer, New York
centration higher than the limit set by Codex Miletic ID, Miletic VD, Sattely-Miller EA, Schiffman SS (1994)
Alimentarius (10 mg gliadin/kg or 20 mg gluten/kg Identification of gliadin presence in pharmaceutical prod-
food), the food cannot be considered as “gluten-free.” ucts. J Pediatr Gastroenterol Nutr 19:27–33
Rubio-Tapia, A.; Ludvigsson, J. F.; Brantner, T. L.; Murray,
J. A.; Everhart, J. (2012) The prevalence of celiac disease in
the United States. Gastroenterology 142: S181-S182
Sdepanian VL, Scaletsky ICA, Fagundes-Neto U, deMorais
25.4 QUESTIONS MB (2001) Assessment of gliadin in supposedly gluten-free
foods prepared and purchased by celiac patients. J Pediatr
Gastroenterol Nutr 32:65–70
1. Draw a set of symbolic pictures representing the
Skerritt JH, Hill AS (1991) Enzyme immunoassay for deter-
stages of the dot blot assay used in this labora-
mination of gluten in foods: collaborative study. J Assoc
tory, including the major active molecular sub- Off Anal Chem 74:257–264
stances being employed (i.e., nitrocellulose solid WHO and FAO (2008) CODEX STAN 118-1981-Standard for
matrix, antigen, BSA blocking reagent, antibody- Foods for Special Dietary Use for Persons Intolerant to
enzyme conjugate, substrate, product). Gluten
26
chapter
Viscosity Measurements
of Fluid Food Products
Helen S. Joyner
School of Food Science, University of Idaho,
Moscow, ID, USA
26.1 Introduction 26.3 Data

26.1.1 Background 26.3.1 Brookfield Viscometer
26.1.2 Reading Assignment 26.3.2 Zahn Cup
26.1.3 Objectives 26.3.3 Bostwick Consistometer
26.1.4 Supplies 26.4 Calculations
26.1.5 Equipment 26.4.1 Brookfield Viscometer
26.2 Procedure 26.4.2 Zahn Cup
26.2.1 Brookfield Viscometer 26.4.3 Bostwick Consistometer
Measurements 26.5 Questions
26.2.2 Zahn Cup Measurements
26.2.3 Bostwick Consistometer
Measurements
www.dbooks.org
214 H.S. Joyner
26.1 INTRODUCTION 26.1.4 Supplies

• 6 beakers, 250 mL
26.1.1 Background • 3 beakers, 600 mL
Whether working in product development, quality • French salad dressing
control, or process design and scale-up, rheology plays • Honey (make sure there are no additional ingredi-
an integral role in the manufacturing of high-quality ents aside from honey)
food products. Rheology is a science based on funda- • Thermometer or thermocouple with digital reader
mental physical relationships concerned with how all • Stopwatch
materials respond to applied forces or deformations.
Flow behavior is one such response to force or 26.1.5 Equipment
deformation.
• Brookfield rotational viscometer model LV and
Determination and control of the flow properties of
spindle #3
fluid foods are critical for optimizing processing condi-
• Brookfield Zahn cup #5 (hole diameter of at least 0.2
tions and obtaining the desired sensory qualities for the
in)
consumer. Transportation of fluids (pumping) from one
• Bostwick consistometer
location to another requires pumps, piping, and fittings
• Refrigerator
such as valves, elbows, and tees. Proper sizing of this
equipment depends on a number of elements but pri-
marily on the flow properties of the product. For exam-
26.2 PROCEDURE
ple, the equipment used to pump a dough mixture
would be very different from that used for milk. 26.2.1 Brookfield Viscometer Measurements
Additionally, rheological properties are fundamental to
many aspects of food safety. During continuous thermal 1. Fill a 250 mL beaker with 200 mL honey and the
processing of fluid foods, the amount of time the food is two remaining 250 mL beakers with 200 mL
in the system (known as the residence time or RT), and salad dressing each. Label the beakers appro-
therefore the amount of heating or “thermal dose” priately. Place one of the beakers of salad dress-
received, is directly related to its flow properties. ing in a refrigerator at least 1 h prior to analysis.
The rheological properties of a fluid are a function The remaining beakers shall be allowed to
of composition, temperature, and other processing equilibrate to room temperature.
conditions, such as the speed the fluid travels through 2. Prior to evaluating the samples, make sure the
a pipe. Identifying how these parameters influence viscometer is level. Use the leveling ball and
flow properties may be accomplished by measuring circle on the viscometer to check.
viscosity with a viscometer or rheometer. In this labo- 3. On the data sheet provided, record the viscom-
ratory, we will measure the viscosity of two fluid foods eter model number and spindle size, product
using common rheological instruments widely used information (type, brand, etc.), and the sample
throughout the food industry. These instruments temperature. Because rheological properties are
include a Brookfield rotational viscometer, a Zahn cup, strongly dependent on temperature, sample
and a Bostwick consistometer. temperatures must be measured and recorded
prior to performing measurements.
4. Immerse the spindle into the test fluid (i.e.,
honey, salad dressing) up to the notch cut in the
Joyner, H.S., and Daubert, C.R. 2017. Rheological prin- shaft. The viscometer motor should be off.
ciples for food analysis. Ch. 29, in Food Analysis, 5th ed. 5. Zero the digital viscometers before turning on
S.S. Nielsen (Ed.), Springer, New York. the motor.
Singh, R.P., and Heldman, D.R. 2001. Introduction 6. Set the motor at the lowest speed revolutions
to Food Engineering, 3rd ed., pp. 69–78, 144–157. per minute (rpm) setting. Once the digital dis-
Academic Press, San Diego, CA. play shows a stable value, record the percent-
age of full-scale torque reading. Increase the
26.1.3 Objectives rpm setting to the next speed and again record
the percentage of full-scale torque reading.
1. Explain the basic principles of fluid rheology. Repeat this procedure until the maximum rpm
2. Gain experience measuring fluid viscosity setting has been reached or 100 % (but not
using different equipment. higher) of the full-scale torque reading is
3. Describe the effects of temperature and (shear) obtained. Do not increase the speed once 100 % of
speed on viscosity. the full-scale torque has been reached.
Chapter 26 • Viscosity Measurements of Fluid Food Products 215
7. Stop the motor, then slowly raise the spindle analysis. The remaining beakers shall be
from the sample. Remove the spindle and clean allowed to equilibrate to room temperature.
with soap and water, then dry. Be sure not to 2. On the data sheet provided, record the Zahn
use any abrasive scrubbers or soaps on the spin- cup hole diameter and volume, product infor-
dle, as these can scratch the spindle and result mation (type, brand, etc.), and the sample tem-
in measurement errors. perature. Because rheological properties are
8. Calculate the viscosity using the spindle factors. strongly dependent on temperature, sample
A factor exists for each spindle-speed combina- temperatures must be measured and recorded
tion (Table 26.1): prior to performing measurements.
For every dial reading (percentage full-scale 3. Completely immerse the Zahn cup into the test
torque), multiply the display value by the cor- fluid (i.e., honey, salad dressing) so that the top of
responding factor to calculate the viscosity with the cup is below the surface of the fluid. Allow
units of mPa-s. the cup to fill with fluid. The cup must remain in
the fluid for at least 5 min before measurements
Example: begin to equilibrate it to the sample temperature.
A Ranch salad dressing was tested with a 4. Put a finger through the ring at the top of the
Brookfield LV viscometer equipped with spin- handle on the cup and lift it straight up out of
dle #3. At a speed of 6 rpm, the display read the beaker. Start the stopwatch as soon as the
40.6 %. For these conditions, the viscosity is: top of the cup breaks the surface of the fluid.
5. Allow the cup to drain into the beaker with the
40.6 200 8120 mPa s 8012 Pa s bottom of the cup no more than six inches above
the surface of the fluid.
9. Repeat Steps 2–8 to test all samples. 6. Stop the stopwatch as soon as there is a clear
10. Once all the data have been collected for the break in the stream of fluid flowing out of the
salad dressing and honey at room temperature, cup. Record the time at break, also called the
remove the salad dressing sample from the Zahn time.
refrigerator, and repeat Steps 2–8. Be sure to 7. Clean the cup with soap and water, then dry. Be
record the sample temperature before perform- sure not to use any abrasive scrubbers or soaps
ing any measurements. on the cup, as these can scratch the cup and
11. You may choose to run the samples in duplicate result in measurement errors.
or triplicate. Data from samples collected under 8. Calculate the viscosity using the time conver-
identical conditions may be pooled to generate sion factors and the density of the test fluid.
an average reading. There are two conversion factors, K and C, to
convert the Zahn time to viscosity in centistokes
26.2.2 Zahn Cup Measurements (cS) using the following equation:
1. Fill a 600 mL beaker with 450 mL honey and the n K t C
two remaining 600 mL beakers with 450 mL
salad dressing each. The beakers must be tall where:
enough so that the Zahn cup can be completely ν = kinematic viscosity (cS)
immersed in the sample. Label the beakers t = Zahn time (s)
appropriately. Place one of the beakers of salad
dressing in a refrigerator at least 2 h prior to For a Brookfield Zahn Cup #5:
K = 23
26.1 C = zero
table Factors for Brookfield model LV (spindle #3) The kinematic viscosity can be converted to vis-
cosity with units on mPa-s by multiplying by
Speed (rpm) Factor the specific gravity of the fluid. The specific
0.3 4000 gravities of honey and French dressing are
0.6 2000 approximately 1.42 and 1.10, respectively.
1.5 800
3 400 Example:
6 200 A Ranch salad dressing was tested with a #5
12 100
Brookfield Zahn cup. The stream broke at 80 s.
30 40
Given that the specific gravity of the dressing is
60 20
about 1.2, the viscosity is:
www.dbooks.org
216 H.S. Joyner
n SG K t C SG record the sample temperature before perform-

ing any measurements.
23 80 0 1.2 2208 mPa s 2.208 Pa s 10. You may choose to run the samples in duplicate
or triplicate. Data from samples collected under
9. Repeat Steps 2–8 to test all samples. identical conditions may be pooled to generate
10. Once all the data have been collected for the an average reading.
salad dressing and honey at room temperature,
remove the salad dressing sample from the
refrigerator, and repeat Steps 2–8. Be sure to 26.3 DATA
record the sample temperature before perform-
ing any measurements. 26.3.1 Brookfield Viscometer
11. You may choose to run the samples in duplicate
or triplicate. Data from samples collected under Date:
identical conditions may be pooled to generate Product information:
an average reading. Viscometer make and model:
Spindle size:
Sample Spindle Vicosity
Measurements
Sample temp (°C) speed (rpm) % Reading Factor (mPa-s)
1. Fill a 250 mL beaker with 200 mL honey and the
two remaining 250 mL beakers with 200 mL
salad dressing each. Label the beakers appro-
priately. Place one of the beakers of salad dress- 26.3.2 Zahn Cup
ing in a refrigerator at least 1 h prior to analysis.
The remaining beakers shall be allowed to Date:
equilibrate to room temperature. Product information:
2. Prior to evaluating the samples, make sure the Zahn cup hole diameter:
consistometer is at the correct angle. Level the Zahn cup volume:
consistometer by adjusting the leveling screws
under the consistometer until the leveling bub- Fluid
ble is in the center of the black circle. Sample Zahn Vicosity density Vicosity
3. On the data sheet provided, record the consis- Sample temp (°C) time (s) (cS) (kg/m3) (mPa-s)
tometer make and model, product information
(type, brand, etc.), and the sample temperature.
Because rheological properties are strongly
dependent on temperature, sample tempera-
tures must be measured and recorded prior to
performing measurements. Date:
4. Close the product gate and hold it down while Product information:
pulling the lever arm as far up as it can go. Consistometer make and model:
5. Pour the test fluid (i.e., honey, salad dressing) in
Sample Sample temp (°C) Consistency (cm)
the product reservoir until it reaches the top of
the product gate.
6. Push the lever arm down to raise the product
gate; start the stopwatch as you push the lever
arm down. Record the consistency or the dis-
26.4 CALCULATIONS
tance the fluid traveled 30 s after the gate is
raised. The numbers on the slope are distances
in cm.
26.4.1 Brookfield Viscometer
7. Clean the consistometer with soap and water, 1. Sketch the experimental apparatus and label
then dry. Be sure the consistometer is com- the major parts.
pletely dry before using it again. 2. Calculate the viscosity of the test fluids at each
8. Repeat Steps 2–7 to test all samples. rpm.
9. Once all the data have been collected for the 3. Plot viscosity versus rpm for each fluid on a
salad dressing and honey at room temperature, single graph.
remove the salad dressing sample from the 4. Label the plots with the type of fluid (e.g.,
refrigerator, and repeat Steps 2–7. Be sure to Newtonian, pseudoplastic, Herschel-Bulkley)
Chapter 26 • Viscosity Measurements of Fluid Food Products 217
based on the response of viscosity to speed 6. Compare the viscosities determined from the
(rpm). Keep in mind that speed is proportional to three different viscosity measurement methods.
shear rate. In other words, as the speed is doubled, Are they similar? Do they give you the same
the shear rate is doubled. information about the fluids? If there are differ-
ences in the viscosity results, what might be
26.4.2 Zahn Cup responsible for the differences?
7. Which viscosity measurement method gives
1. Sketch the experimental apparatus and label
you the most information about the test fluids?
the major parts.
How could you adjust the other two measure-
2. Calculate the viscosity of the test fluids.
ment methods to give you more information
3. Compare the viscosity of the test fluids with the
about the test fluid?
Zahn cup to the viscosities measured with the
8. Consistency is not the same as viscosity.
Brookfield viscometer. What speed in rpm
Considering that different fluids have different
would the Brookfield need to be run at for each
flow behaviors, do you think it would be possi-
fluid to match the viscosity measured using the
ble to develop an equation to convert consis-
Zahn cup?
tency to viscosity? Why or why not?
9. A fluid food product was designed to have a
certain viscosity profile. When the product was
1. Sketch the experimental apparatus and label being developed, the viscosity was measured at
the major parts. several different speeds on a Brookfield viscom-
2. Compare the consistency of the test fluids with eter. Now the product is in full-scale production
the consistometer to the viscosities measured in two locations. The quality control team in
with the Brookfield viscometer. Do the data Location 1 uses a Zahn cup to check the viscos-
match? What rpm gives you the best match? ity of each batch of product. The quality control
team at Location 2 uses a Bostwick consistometer
to check the viscosity of each batch of product.
26.5 QUESTIONS What issues might occur if there is a problem
with product viscosity at one of the production
1. What is viscosity? facilities or if the two production facilities want
2. What is a Newtonian fluid? What is a non- to compare product viscosity data?
Newtonian fluid? Were your materials Newtonian
or non-Newtonian? Explain your choice.
Acknowledgment This laboratory was adapted from a labo-
3. Describe the importance of viscosity and flow
ratory created by:
properties in food processing, quality con- Dr. Christopher R. Daubert, Department of Food
trol, and consumer satisfaction. How might Bioprocessing & Nutritional Sciences, North Carolina State
food composition impact its viscosity or flow University, Raleigh, NC, USA
properties? What ingredient(s) in the salad Dr. Brian E. Farkas, Department of Food Science, Purdue
dressing may have caused deviations from University, West Lafayette, IN, USA
Newtonian behavior? What effect does temper-
ature have on the viscosity of fluid foods?
4. For samples at similar temperatures and identi- RESOURCE MATERIALS
cal speeds, was the viscosity of honey ever less
Joyner, HS, Daubert CR (2017) Rheological principles for
than the viscosity of salad dressing? Is this
food analysis. Ch 29. In: Nielsen SS (ed) Food analysis, 5th
behavior representative of the sample rheology
at all speeds? Singh RP, Heldman DR (2001) Introduction to food engineer-
5. Why is it important to test samples at more than ing, 3rd edn. Academic Press, San Diego, CA, pp 69–78,
1 speed? 144–157
www.dbooks.org
27
chapter
CIE Color
Specifications
Calculated
from Reflectance
or Transmittance
Spectra
M. Monica Giusti (*)
Department of Food Science and Technology, The Ohio State University,
Columbus, OH, USA
Ronald E. Wrolstad • Daniel E. Smith
Department of Food Science and Technology, Oregon State University,
Corvallis, OR, USA
27.1 Introduction 27.2 Procedure
27.1.1 Background 27.2.1 Weighted Ordinate Method
27.1.2 Reading Assignment 27.2.2 Expression in Other Color
27.1.3 Objectives Specification Systems
27.1.4 Materials 27.3 Questions
27.1.5 Optional
www.dbooks.org
Chapter 27 • CIE Color Specifications Calculated from Reflectance or Transmittance Spectra 221
27.1 INTRODUCTION 2 7. 1 % Transmittancea and reflectanceb data for

table Maraschino cherry
27.1.1 Background
Food color is arguably one of the most important deter- %T
Maraschino cherry %R
minants of acceptability and is, therefore, an important
λnm syrup Maraschino cherries
specification for many food products. The develop-
ment of compact and easy to use colorimeters and 400 1.00 0.34
spectrometers has made the quantitative measurement 410 2.00 0.34
of color a routine part of product development and 420 2.70 1.08
quality assurance. 430 3.40 0.89
There are several widely employed systems of 440 3.80 1.14
450 3.50 1.06
color specification: notably Munsell, Commission
460 2.40 0.85
Internationale de l’Eclairage (CIE) tristimulus, and the 470 1.30 0.83
more recent CIEL*a*b* system. The Munsell system 480 0.60 0.7
relies on matching with standard color chips. Value, 490 0.30 0.77
hue, and chroma are employed to express lightness, 500 0.30 0.75
“color,” and saturation, respectively. The CIE tristimu- 510 0.30 0.8
lus system uses mathematical coordinates (X, Y, and Z) 520 0.30 0.85
to represent the amount of red, green, and blue prima- 530 0.30 0.77
ries required by a “standard observer” to give a color 540 0.40 0.86
match. These coordinates can be combined to yield a 550 1.30 0.82
560 6.60 0.99
two-dimensional representation (chromaticity coordi-
570 7.60 1.42
nates x and y) of color. The CIEL*a*b* system employs 580 13.6 2.19
L* (lightness), a* (red-green axis), and b* (yellow-blue 590 22.4 4.29
axis) to provide a visually linear color specification. 600 33.9 7.47
Available software, often incorporated into modern 610 46.8 11.2
instruments, enables the investigator to report data in 620 59.0 15.0
any of the above notations. Understanding the different 630 68.6 17.8
color specification systems, and the means of intercon- 640 74.9 20.2
version, aids the food scientist in selecting an appropriate 650 78.8 21.8
means of reporting and comparing color measurements. 660 81.1 23.2
670 82.7 25.1
680 84.2 26.3
690 84.8 27.8
Wrolstad, R.E., and Smith, D.E. 2017. Color analysis. 700 85.7 28.4
Ch. 31, in Food Analysis, 5th ed. S.S. Nielsen (Ed.), a
1 cm pathlength; Shimadzu Model UV160A Spectrophoto
Springer, New York. meter
b
Hunter ColorQuest 45/0 Colorimeter, illuminant D65, reflec-
27.1.3 Objectives tance mode, specular included, 10° observer angle
1. Learn how to calculate the following CIE color

2. % reflectance spectrum (%R) (A spectrum of
specifications from reflectance and transmission
Maraschino cherries colored with radish extract
spectra:
is provided, Table 27.1.)
(a) Tristimulus values X, Y, and Z
3. CIE chromaticity diagram (Fig. 27.1), Munsell
(b) Chromaticity coordinates x and y and
conversion charts, or appropriate interconver-
luminosity, Y
sion software
(c) Dominant wavelength (λd) and % purity
(using the chromaticity diagram)
Examples:
2. Using readily available software, interconvert
between the CIE Y and chromaticity coordi-
nates and other color specification systems (a) An online applet http://www.colorpro.com/
including Munsell and CIEL*a*b*. info/tools/labcalc.htm is a graphical tool that
permits the user to adjust tristimulus values by
means of slider bars. Corresponding values of
27.1.4 Materials
CIE L*a*b* and Lch equivalents and a visual rep-
1. % transmittance (%T) spectrum (A spectrum of resentation of the associated color are displayed.
syrup from Maraschino cherries colored with (b) A second application, http://www.colorpro.
radish extract is provided, Table 27.1.) com/info/tools/rgbcalc.htm, provides the same
222 M.M. Giusti et al.
1964
2 7. 1 Chromaticity
figure
diagram (10
supplemental
standard
observer)
slider adjustment of RGB values with conver- 27.2 PROCEDURE

sion to equivalent values in other systems.
(c) Convert L*, a*, and b* values to other notations: 27.2.1 Weighted Ordinate Method
http://www.colorpro.com/info/tools/con-
vert.htm#TOP. 1. Determine percent transmittance (%T) or
(d) A free evaluation copy of software that permits percent reflectance (%R) at the specified
entry of numeric values for any of tristimulus, wavelengths (e.g., every 10 nm between 400
Munsell, CIE L*a*b*, and chromaticity (x, y) and 700 nm). [Note: Example data for trans-
coordinates, with conversion to the other sys- mittance and reflectance (Table 27.1) are
tems can be obtained from http://www.xrite. provided and can be used for these
com/. An annual license for this program calculations.]
(CMC) is available for purchase from http:// 2. Multiply %T (or %R) by Ex , Ey , and Ez (see
Tables 27.2 or 27.3, respectively, for %T or %R).
wallkillcolor.com.
These factors incorporate both the CIE spectral
distribution for illuminant D65 and the 1964 CIE
27.1.5 Optional standard supplemental observer curves for x, y,
and z.
Spectra of other samples can be acquired using the fol-
lowing instruments: 3. Sum the values %T or % R Ex , %T or % R Ey ,
and %T or % R Ez to give X, Y, and Z, respec-
1. Visible spectrophotometer (transmittance
spectrum) tively (Table 27.4). The sums of each are divided by
2. Spectrophotometers for color analyses (com- the sum of Ey (760.7). (By doing this, the three
monly called colorimeter) operated in transmit- values are normalized to Y = 100, which is
tance or reflectance mode “perfect” white; objects are specified relative to
www.dbooks.org
Chapter 27 • CIE Color Specifications Calculated from Reflectance or Transmittance Spectra 223
2 7. 2 2 7. 3
table Calculation of CIE specifications by the table Calculation of CIE specifications by the
weighted ordinate method: % transmittance weighted ordinate method: % reflectance
λnm %T Ex Ex %T Ey Ey %T Ez Ez %T λnm %R Ex Ex % R Ey Ey % R Ez Ez % R

400 0.60 0.10 2.50 400 0.60 0.10 2.50
410 3.20 0.30 14.90 410 3.20 0.30 14.90
420 8.80 0.90 41.80 420 8.80 0.90 41.80
430 13.00 1.60 64.20 430 13.00 1.60 64.20
440 19.10 3.10 98.00 440 19.10 3.10 98.00
450 20.40 4.90 109.70 450 20.40 4.90 109.70
460 16.50 7.00 95.00 460 16.50 7.00 95.00
470 10.20 9.70 68.90 470 10.20 9.70 68.90
480 4.20 13.30 40.60 480 4.20 13.30 40.60
490 0.80 16.90 20.70 490 0.80 16.90 20.70
500 0.20 24.10 11.40 500 0.20 24.10 11.40
510 2.10 33.80 6.20 510 2.10 33.80 6.20
520 7.00 45.00 3.60 520 7.00 45.00 3.60
530 15.70 58.10 2.00 530 15.70 58.10 2.00
540 26.10 66.60 0.90 540 26.10 66.60 0.90
550 38.10 71.40 0.30 550 38.10 71.40 0.30
560 48.70 68.90 0.00 560 48.70 68.90 0.00
570 57.50 62.60 0.00 570 57.50 62.60 0.00
580 67.30 57.70 0.00 580 67.30 57.70 0.00
590 73.50 51.10 0.00 590 73.50 51.10 0.00
600 79.90 46.80 0.00 600 79.90 46.80 0.00
610 76.30 39.10 0.00 610 76.30 39.10 0.00
620 63.50 29.50 0.00 620 63.50 29.50 0.00
630 46.00 20.10 0.00 630 46.00 20.10 0.00
640 30.20 12.60 0.00 640 30.20 12.60 0.00
650 18.30 7.30 0.00 650 18.30 7.30 0.00
660 10.70 4.20 0.00 660 10.70 4.20 0.00
670 5.70 2.20 0.00 670 5.70 2.20 0.00
680 2.70 1.10 0.00 680 2.70 1.10 0.00
690 1.20 0.50 0.00 690 1.20 0.50 0.00
700 0.6 0.2 0.00 700 0.6 0.2 0.00
SUM 760.7 SUM 760.7
1964 CIE color matching functions for 10° standard supple- 1964 CIE color matching functions for 10° standard supple-
mental observer, illuminant D65: mental observer, illuminant D65:
X Ex %T / Ey X Ex % R / Ey
Y Ey %T / E y Y Ey % R / Ey
Z Ez %T / E y Z Ez % R / Ey
224 M.M. Giusti et al.
2 7. 4 Coordinates for illuminant D65: x = 0.314

table
y = 0.331
CIE color specifications worksheet
for Maraschino cherry sample
% purity = ratio of distance (a) from the illumi-
%T %R nant to the sample over the distance (a + b) from
the illuminant to the spectrum locus. Analogous
X
to chroma.
Y (luminosity)
Z
X+Y+Z Determine Munsell value, hue, and chroma with chro-
x maticity coordinates x and y. Also convert these data
y to their L*a*b* equivalents. Calculate chroma and hue
λd as indicated.
% Purity
Munsell notation Chroma = (a*2 + b*2)1/2
CIE L* Hue angle = arctanb*/a*
a*
b*
Hue angle, arctanb/a
27.3 QUESTIONS
Chroma, (a*2 + b*2)1/2
Assume D65 illuminant and 10° supplemental stan-
luminosity of perfect white rather than the abso- dard observer for all measurements in the questions
lute level of light.) below.
4. Determine chromaticity coordinates x and y as 1. What is the analogous term in the Munsell sys-
follows: tem to luminosity in the CIE system?
x = (X)/(X + Y + Z) y = (Y)/(X + Y + Z) 2. What are the dominant wavelength and %
purity of a food with chromaticity coordinates
5. Luminosity is the value of Y following the nor- x = 0.450 and y = 0.350?
malization described above. 3. A lemon is found to have values of L* = 75.34,
a* = 4.11, and b* = 68.54. Convert to correspond-
27.2.2 Expression in Other Color ing chromaticity coordinates x and y and plot
Specification Systems on the 1964 chromaticity diagram.
Plot the x and y coordinates on the CIE chromaticity 4. Which has the greater hue angle, an apple with
diagram (Fig. 27.1) and determine dominant wave- coordinates L* = 44.31, a* = 47.63, and b* = 14.12
length and % purity: or L* = 47.34, a* = 44.5, and b* = 15.16? Which
apple has the greater value of chroma?
Dominant wavelength = λd = wavelength of spec-
trally pure light that if mixed with white light
will match a color; analogous to hue. RESOURCE MATERIALS
Berns RS (2000) Billmeyer and Saltzman’s principles of color

On the CIE chromaticity diagram (Fig. 27.1), draw a technology, 3rd edn. Wiley, New York
straight line from illuminant D65, extending through Judd DB, Wyszecki G (1975) Color in business, science and
the sample point to the perimeter of the diagram. The industry, 3rd edn. Wiley, New York
point on the perimeter will be the dominant Wrolstad RE, Smith DE (2017) Color analysis. Ch. 31. In:
wavelength. Nielsen SS (ed) Food analysis, 5th edn. Springer, New York
www.dbooks.org
28
Chapter
Extraneous Matter
Examination
S. Suzanne Nielsen
28.1 Introduction 28.3 Extraneous Matter in Jam

28.1.1 Background 28.3.1 Chemicals
28.1.2 Reading Assignment 28.3.2 Hazards, Precautions,
28.1.3 Notes and Waste Disposal
28.1.5 Principle of Methods 28.3.4 Equipment
28.2 Extraneous Matter in Soft Cheese 28.3.5 Procedure
28.2.1 Chemicals 28.4 Extraneous Matter in Infant Food
28.2.2 Reagents 28.4.1 Chemicals
28.2.3 Hazards, Precautions, 28.4.2 Hazards, Precautions,
and Waste Disposal and Waste Disposal
28.2.4 Supplies 28.4.3 Supplies
28.2.5 Equipment 28.4.4 Equipment
28.2.6 Procedure 28.4.5 Procedure
28.5 Extraneous Matter in Potato Chips 28.6 Extraneous Matter in Citrus Juice
28.5.2 Reagents 28.6.2 Equipment
28.5.3 Hazards, Precautions, and Waste 28.6.3 Procedure
Disposal 28.7 Questions
28.5.4 Supplies
28.5.5 Equipment
28.5.6 Procedure
www.dbooks.org
Chapter 28 • Extraneous Matter Examination 227
28.1 INTRODUCTION this experiment. To do this, drill a hole through a rub-

ber stopper of a size just too large for a 500-mL
28.1.1 Background Erlenmeyer flask. Thread a heavy string through the
hole in the stopper, and knot both ends of the string.
Extraneous materials are any foreign substances in Coat the sides of the rubber stopper with glycerin and
foods that are associated with objectionable conditions carefully force it (with larger end of stopper pointed
or practices in production, storage, or distribution of up) through the top of the flask. Note that the string
foods. Extraneous materials include (a) filth or objec- could be a trap for contaminants such as rodent hair
tionable matter contributed by animal contamination and insect fragments.
(rodent, insect, or bird matter) or unsanitary condi- For the parts of this laboratory exercise that require
tions, (b) decomposed material or decayed tissues due filter paper, S&S #8 (Schleicher & Schuell, Inc., Keene,
to parasitic or nonparasitic causes, and (c) miscella- NH) is recommended. It meets the specifications set
neous matter (sand, soil, glass, rust, or other foreign forth in the AOAC Method 945.75 Extraneous Materials
substances). Bacterial contamination is excluded from (Foreign Matter) in Products, Isolation Techniques Part
these substances. B(i), which suggests using “smooth, high wet strength,
Filth is classified according to its extractability. rapid acting filter paper ruled with oil-, alcohol-, and
Light filth is oleophilic and lighter than water (sepa- water-proof lines 5 mm apart.” The S&S #8 ruled filter
rated from product by floating it in an oil-aqueous paper is 9 cm in diameter and fits well into the top of
mixture). Insect fragments, rodent hairs, and feather standard 9-cm plastic petri dishes. The bottom of the
barbules are examples of light filth. Heavy filth is plastic petri dish can be used as a protective cover over
heavier than water and separated from the product by the sample filter paper in the top of the petri dish. The
sedimentation based on different densities of filth, top of the plastic petri dish provides a 9 cm flat surface
food particles, and immersion liquids (CHCl3, CCl4, (as opposed to glass petri dishes) for examining the fil-
etc.). Examples of heavy filth are sand, soil, and nut-
ter paper, making it easier to view the plate without
shell fragments. Sieved filth involves particles sepa-
having to continuously refocus the microscope. The
rated from the product by the use of selected mesh
5-mm ruled lines provide a guide for systematically
sizes. Whole insects, stones, sticks, and bolts are exam-
examining and enumerating contaminants on the filter
ples of sieved filth.
paper at 30× magnification. To obtain a moist surface
Various methods of isolation of extraneous mat-
on which contaminants can be manipulated and
ter from various food commodities can be found in
observed, apply a small amount of glycerin: 60 % alco-
the Official Methods of Analysis of the AOAC
hol (1:1) solution to the top of the petri dish before
International and in the Approved Methods of Analysis
transferring the filter paper from the Buchner funnel.
of the AACC International. Presented here are a few
Using both overhead and substage lighting with the
procedures for some food commodities, with descrip-
microscope will assist in identifying contaminants.
tions based on AOAC methods, but the quantities
reduced to half.
28.1.4 Objective
28.1.2 Reading Assignment The objective of this laboratory is to utilize techniques
to isolate the extraneous matter from various foods:
Dogan, H., and Subramanyam, B. 2017. Extraneous cottage cheese, jam, infant food, potato chips, and cit-
matter. Ch 34, in Food Analysis, 5th ed. S.S. Nielsen rus juice.
28.1.5 Principle of Methods
28.1.3 Notes
Extraneous materials can be separated from food
Regulatory examination of samples by the Food and products by particle size, sedimentation, and affinity
Drug Administration (FDA) would be based on repli- for oleophilic solutions. Once isolated, extraneous
cate samples using official methods, including the materials can be examined microscopically.
specified sample size. However, for instructional pur-
poses, the costs associated with adequate commercial
1-L Wildman trap flasks, reagents, and food samples 28.2 EXTRANEOUS MATTER IN SOFT
specified in official methods may be prohibitive. CHEESE
Procedures given below are based on AOAC methods,
but all quantities are reduced to half, and a 500-mL 28.2.1 Chemicals
Wildman trap flask (vs. 1-L trap flask) is specified in
most procedures. Commercially available 1-L trap CAS no. Hazards
flasks with the standard stopper rod would ideally be
used (with all quantities in the procedures doubled). Phosphoric acid 7664-38-2 Corrosive
However, 500-mL trap flasks can be made for use in (H3PO4)
228 S.S. Nielsen
28.2.2 Reagents the paper clears. [May also use dilute (1–5 %)
alkali or hot alcohol to aid in filtration.] Resume
• Phosphoric acid solution, 400–500 mL
addition of sample and water until sample is
Combine 1 part phosphoric acid with 40 parts
filtered.
deionized distilled (dd) water (vol/vol).
3. Examine filter paper microscopically.
Disposal
28.3 EXTRANEOUS MATTER IN JAM
safety glasses at all times. Waste likely may be put 28.3.1 Chemicals
down the drain using a water rinse, but follow good
laboratory practices outlined by environmental health CAS no. Hazards
and safety protocols at your institution. Heptane (12.5 mL) 142-82-5 Harmful, highly
flammable,
28.2.4 Supplies dangerous for the
environment
• Beaker, 1 L (for phosphoric acid solution)
• Beaker, 600 mL (to boil water) concentrated
• Buchner funnel (HCl) (5 mL)
• Cottage cheese, 115 g
• Filter paper
• Heavy gloves
Disposal
• Pipette, 10 mL (to prepare phosphoric acid
solution) Heptane is an extremely flammable liquid; avoid open
• Pipette bulb or pump flames, breathing vapors, and contact with skin.
• Spoon Otherwise, adhere to normal laboratory safety proce-
• Sidearm flask, 500 mL or 1 L dures. Wear safety glasses at all times. Dispose of hep-
• Stirring rod tane waste as hazardous waste. Other waste may be
• Tap water, ca. 500 mL (boiling) put down the drain using a water rinse.
• Tweezers
• Volumetric flask, 500 mL (to prepare phosphoric 28.3.3 Supplies
acid solution)
• Weighing boat • 2 Beakers, 250 mL (for weighing jam and heating
water)
28.2.5 Equipment • Buchner funnel
• Filter paper
• Hot plate • Glass stirring rod
• Microscope • Graduated cylinder, 100 mL
• Top loading balance • Ice water bath (to cool mixture to room
• Water aspirator system temperature)
• Jam, 50 g
28.2.6 Procedure • Graduated pipette, 10 mL (for heptane)
(Based on AOAC Method 960.49, Filth in Dairy • Pipette bulb or pump
Products) • Sidearm flask, 500 mL or 1 L
• Spoon
1. Weigh out 115-g cottage cheese and add it to • Thermometer
400–500-mL boiling phosphoric acid solution • Tweezers
(1 + 40 mixture) in a 1-L beaker, stirring with a • Volumetric pipette, 5 mL (for conc. HCl)
glass stirring rod continuously to disperse the • Waste jar (for heptane)
cottage cheese. • Water, dd, 100 mL (heated to 50 °C)
2. Filter the mixture through filter paper in a • Wildman trap flask, 500 mL
Buchner funnel, using a vacuum created by a
water aspirator. Do not let the mixture accumu- 28.3.4 Equipment
late on the paper, and continually wash filter
with a stream of hot water to prevent clogging. • Hot plate
Make sure the cheese mixture is hot as it is fil- • Microscope
tered. When filtration is impeded, add hot water • Top loading balance
or phosphoric acid solution (1 + 40 mixture) until • Water aspirator system
www.dbooks.org
Chapter 28 • Extraneous Matter Examination 229
28.3.5 Procedure 28.4.5 Procedure

(Based on AOAC Method 950.89, Filth in Jam and (Based on AOAC Method 970.73, Filth in Pureed Infant
Jelly) Food, A. Light Filth)
1. Empty contents of jam jar into beaker and mix 1. Transfer 113 g (one jar) of baby food to a 500-mL
thoroughly with glass stirring rod. trap flask.
2. Weigh 50 g of jam into a beaker, add ca. 80 mL 2. Add 10 mL of light mineral oil and mix
dd water at 50 °C, transfer to a 500-mL trap thoroughly.
flask (use the other ca. 20 mL dd water to help 3. Fill the trap flask with deaerated water (can use
make transfer), add 5 mL conc. HCl, and boil for dd water) at room temperature.
5 min. 4. Let stand 30 min, stirring four to six times dur-
3. Cool to room temperature (with an ice water ing this period.
bath). 5. Trap off mineral oil in a layer above the rubber
4. Add 12.5 mL heptane and stir thoroughly. stopper and then filter the mineral oil through
5. Add dd water to a level so heptane is just above filter paper in a Buchner funnel using vacuum
rubber stopper when in the “trap” position. created by a water aspirator.
6. Trap off the heptane, and filter the heptane 6. Examine filter paper microscopically.
through filter paper in a Buchner funnel using
vacuum created by a water aspirator.
7. Examine filter paper microscopically. 28.5 EXTRANEOUS MATTER IN POTATO
CHIPS
28.4 EXTRANEOUS MATTER IN INFANT 28.5.1 Chemicals

FOOD
CAS no. Hazards
28.4.1 Chemicals Ethanol, 95 % 64-17-5 Highly flammable
Heptane (9 mL) 142-82-5 Harmful, highly
CAS no. Hazards flammable, dangerous
to environment
Light mineral oil (10 mL) 8012-95-1
Petroleum ether 8032-32-4 Harmful, highly
(200 mL) flammable, dangerous
to environment
Disposal
28.5.2 Reagents
safety glasses at all times. Waste may be put down the • Ethanol, 60 %, 1 L
drain using water rinse. Use 95 % ethanol to prepare 1 L of 60 % ethanol;
dilute 632 mL of 95 % ethanol with water to 1 L.
28.4.3 Supplies
• Baby food, ~113 g (one jar) Disposal
• Buchner funnel
• Filter paper Petroleum ether, heptane, and ethanol are fire hazards;
• Glass stirring rod avoid open flames, breathing vapors, and contact with
• Graduated cylinder, 10 or 25 mL skin. Otherwise, adhere to normal laboratory safety
• Pipette bulb or pump procedures. Wear safety glasses at all times. Heptane
• Sidearm flask, 500 mL or 1 L and petroleum ether wastes must be disposed of as
• Spoon hazardous wastes. Other waste may be put down the
• Tweezers drain using a water rinse.
• Water, deaerated, 500 mL 28.5.4 Supplies
• Wildman trap flask, 500 mL • Beaker, 400 mL
• Buchner funnel
28.4.4 Equipment • Filter paper
• Glass stirring rod
• Microscope
• Graduated cylinder, 1 L (to measure 95 % ethanol)
• Water aspirator system
• Ice water bath
230 S.S. Nielsen
• Potato chips, 25 g 28.6 EXTRANEOUS MATTER IN CITRUS

• Sidearm flask, 500 mL or 1 L JUICE
• Spatula
• Wildman trap flask, 500 mL 28.6.1 Supplies
• Tweezers
• Volumetric flask, 1 L (to prepare 60 % ethanol) • Beaker, 250 mL
• Waste jars (for heptane and petroleum ether) • Buchner funnel
• Cheesecloth
• Citrus juice, 125 mL
28.5.5 Equipment
• Graduated cylinder, 500 mL or 1 L
• Hot plate • Sidearm flask, 250 mL
• Microscope • Tweezers
• Top loading balance
• Water aspirator system 28.6.2 Equipment
• Microscope
28.5.6 Procedure
• Water aspirator system
(Based on AOAC Method 955.44, Filth in Potato Chips)
1. Weigh 25 g of potato chips into a 400-mL beaker. 28.6.3 Procedure
2. With a spatula or glass stirring rod, crush chips [Based on AOAC Method 970.72, Filth in Citrus and
into small pieces. Pineapple Juice (Canned), Method A. Fly Eggs and
3. In a hood, add petroleum ether to cover the Maggots]
chips. Let stand 5 min. Decant petroleum ether
from the chips through filter paper. Again add 1. Filter 125 mL of juice through a Buchner funnel
petroleum ether to the chips, let stand 5 min, fitted with a double layer of cheesecloth. Filter
and decant through filter paper. Let petroleum with a vacuum created by a water aspirator.
ether evaporate from chips in hood. Pour the juice slowly to avoid accumulation of
4. Transfer chips to a 500-mL trap flask, add excess pulp on the cheesecloth.
125 mL 60 % ethanol, and boil for 30 min. Mark 2. Examine material on cheesecloth microscopi-
initial level of ethanol on flask. During boiling cally for fly eggs and maggots.
and at the end of boiling, replace ethanol lost by
evaporation as a result of boiling.
5. Cool in ice water bath.
28.7 QUESTIONS
6. Add 9 mL heptane, mix, and let stand for 5 min.
1. Summarize the results for each type of food
7. Add enough 60 % ethanol to the flask so that
analyzed for extraneous materials.
only the heptane layer is above the rubber
2. Why are contaminants such as insect fragments
stopper. Let stand to allow heptane layer to
found in food, when the Pure Food and Drug
form at the top, trap off the heptane layer, and
Act prohibits adulteration?
filter it through filter paper in a Buchner
funnel.
8. Add 9 mL more heptane to solution. Mix and
RESOURCE MATERIALS
then let stand until heptane layer rises to the
top. Trap off the heptane layer, and filter it AOAC International (2016) Official methods of analysis, 20th
through filter paper (i.e., new piece of filter edn. (On-line). AOAC International, Rockville, MD
paper, not piece used in Parts 3 and 4) in a Dogan H, Subramanyam B. (2017) Extraneous matter. Ch 34.
Buchner funnel. In: Nielsen SS (ed) Food analysis, 5th edn. Springer,
9. Examine the filter paper microscopically. New York
www.dbooks.org
3
part
Answers to Practice
Problems
29
chapter
Answers to Practice
Problems in Chap. 2,
Preparation of
Reagents and Buffers
Catrin Tyl ( ) • Baraem P. Ismail
*

St. Paul, MN, USA
www.dbooks.org
234 C. Tyl and B. Ismail
1. (a) This problem can be solved by using mL of sulfuric acid N of sulfuric acid
Eq. (2.5): The molecular weight of NaH2PO4 mL of NaOH
N of NaOH
is 120 g/mol. Make sure to use the same
units throughout. Molecular weights are 200 4
mL of NaOH 80 mL
stated in mol/g; the unit of concentration is 10
in mol/L; thus, the 500 mL should also be 5. For HCl, normality and molarity are equal,
converted into L: because 1 H+ is released per molecule HCl. The
problem can be solved like Example A3 by
mol g
mg M v L MW (2.5) using Eq. (2.13) to calculate M of concentrated
L mol HCl, followed by Eq. (2.15):
mol g g
m g 0.1 0.5 L 120 mol 6g d 1000
L mol L % wt
M (2.13)
L MW g wt
(b) Just like for Example A2, the only change in mol
the calculation is the use of 156 instead of
g
120, thus: 1.22 1000
mol
M L 0.37 12.16 mol
mol g L g L
m g 0.1 0.5 L 156 7.8 g 36.5
L mol mol
2. According to the definition of % wt/vol, as M1 v1 M2 v2 (2.15)

found in Table 2.1: v of concentrated HCl [ L ] =
w weight solute g 100  mol 
% vol of diluted HCl [ L ] × M of diluted HCl 
 L 

v total volume mL
 mol 
wt 1 M of concentrated HCl 
weight of solute = % × × volume  L 
v 100
 mol 
 g  1 0.25 [ L ] × 2 
= 10   × 100 × 150 [ mL ] = 155 g  L 
 mL  v of concentrated HCl [ L ] =
 mol 
12.16 
3. Determine NaOH’s molarity in a 40 % wt/vol  L 
solution: Due to NaOH’s equivalence of 1, = 0.041 L or 41 mL
molarity equals normality. Use the definition of
wt % to obtain the mass of NaOH in 1 L and 6. Just like for Example A3 and A5, determine the
Eq. (2.9) to calculate normality: molarity of the concentrated acetic acid with
Eq. (2.13) (ignore %wt/wt):
w weight solute g 100
%
v total volume mL 1.05 1000 g mol mol
M acetic acid 17.5
1
60.06 g L L
weight of solute [ g ] = %
w
× × volume
v 100 The desired amount is 0.04 mol; thus, take the
 g  1
= 40  × ×1000 [ mL ] amount calculated below with Eq. (2.16) and
 mL  100 dilute to 1 L:
= 400 [ g ] n mol L 0.04
v L 0.0023 L
mg 400 M mol 17.5
n mol 10 mol in 1 L
g 40 7. The weight of acetic acid in 1 L of solution can
MW
mol be found analogously to Problem 2:
w weight solute g 100
Thus, the molarity and the normality of this %
v total volume mL
solution are 10.
wt 1
4. The number of equivalents for H2SO4 is 2, weight of acetic acid = % × × volume
v 100
because it can donate 2 H+, and so the normality
 g  1
is two times the molarity. The mL of NaOH can = 1 × × 1000 [ mL ] = 10g
be found through inserting into Eq. (2.19):  mL  100
The corresponding number of moles of 10 g can 10. The molecular weight of KHP is 204.22 g/mol,
be found using Eq. (2.9): and as it only contains one unionized carboxyl
group, its number of equivalents is 1 and its
mg 10
n mol 0.167 mol in 1 L molarity equals its normality. According to Eq.
g 60.02 (2.5), 100 mL would contain:
MW
mol
mol g
mg M v L MW (2.5)
This already answers the question: A 1 % acetic L mol
acid solution contains 0.167 mol per L, not 0.1
m g 0.1 0.1 204.22 2.0422 g
mol per L.
11. The first step is to find the desired amount of Ca
8. This problem can be solved analogously to in the 1000 mL. As listed in Table 2.1, ppm cor-
Problem 14: responds to:
w weight solute g 100 mg solute

% ppm =
v total volume mL kg solution
1000 mg Ca
wt 1 Thus, 1000 ppm =
weight of sodium hydroxide = % × × volume kg standard solution
v 100
For our example, the solution’s density can be
 g  1
= 10   × × 1000 [ mL ] assumed to be 1, and thus there would be
 mL  100 1000 mg Ca/L or 1 g Ca/L. If 110.98 g of CaCl2
= 100 g contain 40.078 g Ca, then 1 g Ca is supplied by:
The corresponding number of moles of 100 g 1 110.98

m g of CaCl 2 2.7691 g
can be found using Eq. (2.9): 40.078
mg 10
12. First, the correct ratio of sodium acetate/acetic
n mol 0.25 mol in 1 L acid needs to be determined; similar to Example
g 40 problem C3, [A−] is expressed through [AH] to
MW
mol have only one unknown quantity in the equa-
This already answers the question: A 10 % tion and then inserted into Eq. (2.2):
sodium hydroxide solution contains 0.25 mol
per L, not 1 mol per L. A 0.1 AH
0.1 AH
5.5 4.76 log
9. The normality of K2Cr2O7 is 6 times the molar- AH
ity. Use (Eqs. 2.1 and 2.9) to calculate the molar-
0.1 AH
10
ity and then multiply with the number of 5.5 4.76

equivalents to obtain normality: AH
mol 5.5 AH 0.1 AH
Molarity M
L 0.1
number of moles n mol
AH 0.0154 A 0.0846
(2.1) 5. 5 1
vol v L
Preparing the buffer by approach 1 (Chap 2,
m g mol
n mol (2.9) Sect, 2.3) (1 L of 0.1 M solutions of sodium ace-
MW g tate and acetic acid):
m of sodium acetate g MW M 82 0.1 8.2 g
0.2 g
g MW M 60.02 0.1
294.187
mol
M mol 0.0068 mol v of acetic acid mL
d

1.05
5 .7 mL
L 0.1 L L
Mix them so that the resulting concentrations
correspond to 0.0846 M of sodium acetate and
equivalents
N M number of equivalents 0.0154 M of acetic acid using Eq. (2.15):
L
equivalents M1 v1 M2 v2 (2.15)
0.0068 6 0.04
L
www.dbooks.org
v of sodium acetate solution mL v of acetic acid [ mL ]

M of A in buffer v of buffer  mol   g 
v of buffer [ L ] × M of buffer   × MW 
M of stock =  L   mol 
0.0846 0.25  g 
211.5 mL  mL 
0.1
v of acetic acid [ mL ]
v of acetic acid solution mL
 mol   g 
M of AH in buffer v of buffer 0.25[ L ] × 0.1  × 60.02 
 L   mol  = 1.43 mL
M of stock = [ ]
 g 
1.05 

0.0154 0.25
38.5 mL  mL 
0.1
Preparing the buffer via approach 2 (Chap 2, 13. The molarities of Na2EDTA and MgSO4 are
Sect, 2.3) (directly dissolve appropriate amounts found by rearranging Eq. (2.5):
of sodium acetate and acetic acid in 250 mL):
mol g
mg M v L MW (2.5)
L mol
m of sodium acetate [ g ]
 mol  mol mg
= M of  A −  in buffer   M
 L 
L v L MW g
mol
 g 
× v of buffer [ L ] × MW   (2.5)
 mol  1.179 g
mol
m of sodium acetate [ g ] M of Na 2 EDTA
L g
0.25 L 372.24
 mol   g  mol
= 0.25[ L ] × 0.0846   × 82  mol  = 1.73[ g ]
 L    mol
0.0127
L
vol of acetic acid mL
mol 0.78 g
vol of buffer L M of AH in M of MgSO 4
L g
g 0.25 L 246.47
buffer
mol
MW mol
L mol mol
g 0.0127
mL L
To calculate the buffer pH, determine molarity of

v of acetic acid [ mL ] =
NH4Cl using Eqs. (2.1 and 2.3) and NH3 with
 mol   g  Eq. (2.27):
0.25[ L ] × 0.0154   × 60.02 
 L   mol  = 2.2 mL
[ ]  mol 

1.05 
g  M of NH 4Cl in buffer  =
 L 
 mL 
m [g ] 16.9  mol 
= = 1.26  
 g  53.49 × 0.25  L 
These would be dissolved in, e.g., 200 mL, and MW   × v [ L]
then the pH adjusted and the volume brought  mol 
up to 250 mL.
g
1000
mol mL
Preparing the buffer via approach 3 (Chap 2, M of conc. NH 3 % wt
Sect, 2.3) (dissolve the appropriate amount of L g
MW
acetic acid to yield 250 mL of a 0.1 M solution mol
in < 250 mL, and then adjust the pH with NaOH 0.88 1000 mol
0.28 14.5
of a high molarity, e.g., 6 or 10 M to pH 5.5): 17 L
 mol  0.1 − [ AH ]
M in buffer   −0.51 = log
 L  [ AH ]
 mol 
M of stock solutions  × v of stock solutions [ L ]
 L 
0.1 − [ AH ]
= 0.309 =
v of buffer [ L ] [ AH ]
(2.27)
[ AH ] × ( 0.309 + 1) = 0.1
 mol 
[ AH ] 
M of NH 3 in buffer  mol  0.1  mol   −   mol 
  = = 0.0764   A  
 L   L  1.219  L    L 
 mol   mol 
M of conc. NH 3  × v of conc. NH 3 [ L ] = 1 − 0.0764 = 0.0236 
=  L   L 

v of buffer [ L ]
The molarities of the stock solutions are 0.2
mol . To find the volumes to mix, use
 mol  L
M of NH 3 in buffer  
 L 
Eq. (2.27):
 mol 
14.5  × 0.143[ L ]
 L   mol  M1 v1 M2 v2 (2.27)
= = 8.229  
0.25[ L ]  L 
v of NaH 2PO 4 stock solution L
M of NaH 2PO 4 in buffer v of buffer
The normal form of the Henderson-Hasselbalch M of stock solution
equation may be used after calculating the pKa
of NH4+. NH4Cl acts as the acid, NH4OH is the 0.0764 × 0.2
v of NaH 2 PO 4 stock solution [ L ] =
base (NH4OH is just another way of writing 0.2
NH3 in water). Note: The actual pKa and pKb = 0.0764 [ L ] or 76 mL
may be slightly different because of the added
salts affecting the ionic strength. v of Na 2HPO 4 stock solution L
pK a of NH 4 14 pK b 14 4.74 9.26 M of Na 2HPO 4 in buffer v of buffer
M of stock solution
8.29
pH 9.26 log 9.26 0.82 10.08
1.26 0.0236 × 0.2
v of NaH 2 PO 4 stock solution [ L ] =
14. (a) Use Eq. (2.5) to obtain the masses: 0.2
= 0.024 [ L ] or 24 mL
mol g
mg M v L MW (2.5)
L mol (c) This problem is similar to Example C3. 1 mL
of 6 M NaOH supplies Eq. (2.2):
m g of NaH 2PO 4 .H 2 O 0.2 0.5 138 13.8 g
n of NaOH mol M v 6 0.001 0.006 mol
m g of Na 2HPO 4 .7 H 2 O 0.2 0.5 268 26.8 g
The amounts of NaH2PO4 and Na2HPO4 are
(b) Use Eq. (2.38) to express [A−] through [AH] found through Eq. (2.2) (use either the buffer
to substitute into Eq. (2.25); find the pKa in molarity or values from the stock solutions cal-
Table 2.2: culated for Problem12b):
0.1 A AH n of NaH 2 PO 4 [ mol] = M × v = 0.2 × 0.076

A 0.1 AH = 0.015[ mol]
0.1 − AH n of NaH 2 PO 4 [ mol] = M × v = 0.2 × 0.024

6.2 = 6.71 + log
AH = 0.0048 [ mol]
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Addition of NaOH changes the ratio by increas- 0.01 [A ] AH

ing the amount of Na2HPO4 and decreasing
NaH2PO4:
[A ] 0.01 AH
n of NaH 2 PO 4 after HCl [ mol] = 0.015 − 0.006
= 0.009 [ mol] 0.01 AH
3.5 3.75 log
AH
n of Na 2 HPO 4 after HCl [ mol] = 0.0048 + 0.006
0.01 AH
= 0.0108 [ mol] 0.25 log
AH
Substitute these values into Eq. (2.25) to find the
new pH. (Note: You may insert molar ratios, 0.01 AH
0.562
and you do not need to convert to concentra- AH
tions, because the ratio would stay the same):
0.01
0.0108 [AH] 0.0064 [A ] 0.0036
pH = 6.71 + log = 6.77 1.562
0.009
15. To find the pH at 25°C, the acid/base ratio M v MW
needs to be substituted into Eq. (2.25): v mL (2.52)
d
4
pH 8.06 log v of formic acid mL
1
M v of buffer MW

pH 8.06 0.6 7.46 d
Calculate the pKa at 60 °C with Eq. (2.61), and mol g
0.0064 46 1 L
then insert into Eq. (2.25): L mol
0.24 mL
g
pK a 8.06 0.023 60 25 7.26 1.22
mL
pH 7.26 0.6 6.65
16. This problem is analogous to Example C3. Find m of ammonium formate [ g ]
the ratio of acid to base through Eqs. (2.25 and
= M × v × MW of buffer
2.38), and then use Eq. (2.52) to find the volume
 mol   g 
 × 1[ L ] = 0.227 [ g ]
of formic acid and Eq. (2.5) to find the mass of = 0.0036  × 63.06 

ammonium formate:  L   mol 
30
chapter
Answers to Practice
Dilutions and
Concentrations
Andrew P. Neilson (*) • Sean F. O’Keefe
Blacksburg, VA, USA
www.dbooks.org
1. A diagram of this scheme is shown here.
total volume total volume

250 mL 5 mL
total volume
10.3 g
~150 mL
extract 3X dilute to dissolve to

w/ 50mL volume w/ 25 mL volume w/
ethyl acetate ethyal acetate evaporated methanol
GC
analysis
ethyl acetate dried methanol

applesauce extract residue solution
Step 1 Step 2
Ci=? pg/g methoxyfenozide Vi=25 mL
mi=10.3 g Vf=5 mL
Vf=250 mL Cf=0.00334 µg/mL methoxyfenozide
The calculations are performed as follows:
Ci C f
0.00334 g methoxy fenozide 250 mL ethylacettate extract 5 mL methanol solution

Ci
mL methanol solution 10.3 g applesauce 25 mL ethylactetate extract
0.0162 g methoxy fenozide

g applesauce
0.0162 g methoxy fenzoide

X Cm 113 g applesauce 1.83 g methoxy fenozide
g applesauce
The concentration of methoxyfenozide in the

applesauce is 0.0162 μg/g, and the total amount
of methoxyfenozide in the entire applesauce
cup is 1.83 μg.
2. A diagram of this scheme is shown below (note

that the first dilution is “dilute to,” while the
last two dilutions are “dilute with”).
total volume
10 mL

5.5 mL 12 mL
dilute 0.5 mL dilute 1.5 mL dilute 3 mL

to volume + 4 mL + 9 mL
stock solution solution A solution B solution C
Step 1 Step 2 Step 3

Ci=0.94 mg/mL (–)-epicatechin Vi=1.5 mL Vi=3 mL
Vi=0.5 mL Vf=5.5 mL Vf=12 mL
Vf=10 mL Ci=? mg/mL (–)-epicatechin
The calculations are performed as follows:
C f Ci
0.94 mg EC 0.5 mL stock solution 1.5 mL solution A 3 mL solution B 0.00320 mg EC

Cf
mL stock solution 10 mL solution A 5.5 mL solution B 12 mL solution C mL solution C
0.00320 mg EC 1 g EC 1 mol EC 1, 000 , 000 mol EC 1000 mL 11.0 mol EC

Cf 11.0 M EC
mL solution C 1000 mg EC 290.26 g EC mol EC 1L L
0.00320 mg EC
Cf mL
=
DF£ = = 0.00341
Ci 0.94 mg EC
mL
1 1
dilution " fold "or " =
X" = = 293
DF 0.00341
The concentration of solution C is 0.00320 mg

(−)-epicatechin/mL [or 11.0 μM (−)-epicate-
chin]. The dilution factor is 0.00341, a 293-fold
(293×) dilution of the stock solution.
3. A diagram of this scheme is shown below (note

that all of these are “dilute with,” the standards
are prepared in parallel, and 100 μL = 0.1 mL).
dilute 2 mL dilute 0.1 mL

+ 25 mL + 0.5 mL
Vi=0.1 mL
Vf=0.6 mL
solution
A
riboflavin stock
solution solution
dilute 0.1 mL
+ 0.75 mL
Ci=1.45 mg/mL riboflavin

Vi=2 mL
Vi=0.1 mL
Vf=27 mL
Cf=? mg/mL riboflavin Vf=0.85 mL
dilute 0.1 mL
+ 1 mL solution
B
Vi=0.1 mL
dilute 0.1 mL dilute 0.1 mL
Vf=1.1 mL
+ 5 mL + 2 mL
solution
C
Vi=0.1 mL Vi=0.1 mL
Vf=5.1 mL Vf=2.1 mL
solution solution
E D
www.dbooks.org
The calculations are performed as shown below. For Vi 1

the stock solution: =
DF =
Vf 320
V
1.45 mg riboflavin 2 mL Therefore, we need to find a ratio of volumes
C f Ci i

Vf mL 27 mL equal to 1/320 to do this dilution. You do not

have a 320 mL volumetric flask to do a simple
0.107 mg riiboflavin
1 mL into 320 mL total dilution. However, you
mL could use a 250 mL volumetric flask for the final
For the standard solutions: volume and calculate the starting volume of the
stock solution needed:
V 0.107 mg riboflavin 0.1 mL
A C f Ci i
Vf mL 0.6 mL
Vi
=
1

Vf 320
0.0179mg riboflavin

mL Vf 250 mL
=
Vi = = 0.781 mL
V 0.107 mg riboflavin 0.1 mL 320 320
B C f Ci i Therefore, the 0.5 mL standard is made by dilut-
Vf mL 0.85 mL
ing 0.781 mL (using a 1 mL adjustable pipettor)
0.0126mg riboflavin to 250 mL final volume. You could use a variety

mL of different dilutions to get the same final con-
centrations, as long as you don’t use less than
V 0.107 mg riboflavin 0.1 mL 0.2 mL as your starting volume (e.g., you could
C C f Ci i
Vf mL 1.1 mL
also dilute 0.313 mL to 100 mL and get the same

concentration). From the 0.5 mg/mL standard,
0.00976mg riboflavin
the other standards (0–0.3 mg/mL) can easily
mL be made in 2 mL volumes by combining vari-
able volumes of the 0.5 mg/mL standard with
V 0.107 mg riboflavin 0.1 mL water to achieve a total volume of 2 mL. These
D C f Ci i
Vf mL 2.1 mL volumes are calculated as follows:

0.00511 mg riboflavin C f Vf
CiVi C f Vf Vi
mL Ci
V 0.107 mg riboflavin 0.1 mL C fVf

E C f Ci i For 0 mg / mL Vi =
Vf mL 5.1 mL
Ci
0.00211 mg riboflavin ( 0 mg / mL )( 2 mL ) = 0 mL
=
mL 0.5 mg / mL
The riboflavin concentration in the stock solu- C fVf

For 0.1mg / mL Vi =
tion and the five standards is 0.107, 0.0179, Ci
0.0126, 0.00976, 0.00511, and 0.00211 mg/mL,
 mg 
respectively.  0.1  ( 2 mL )
=  mL 
= 0.4 mL
mg
4. First, 160 g/L = 160 mg/mL. Therefore, large 0.5
dilutions are needed to get from 160 mg/mL to mL
0–0.5 mg/mL. From this point, there are several
and so forth for each solution. The volume of
approaches that could be used. One approach
water is the amount needed to bring the start-
would be to dilute the stock solution down to
ing volume up to 2 mL:
the most concentrated standard (0.5 mg/mL)
and then do further dilutions from that concen- For 0 mg / mL: Vwater 2 mL 0 mL 2 mL
tration. To do this, we calculate the DF:
For 0.1 mg / mL: Vwater 2 mL 0.4 mL
C f 0.5 mg / mL 1
= =
DF = = 0.003125 1.6 mL
Ci 160 mg / mL 320
From this DF, we can calculate the ratio of vol- and so forth. The dilutions from the diluted
umes needed. Recall that: 0.5 mg/mL stock solution are shown in Table 30.1:
30.1
table Dilution example for a standard curve
Anthocyanins Diluted stock Water Total volume

(mg/mL) (mL) (mL) (mL)
0.5 2 0 2.0
0.3 1.2 0.8 2.0
0.2 0.8 1.2 2.0
0.1 0.4 1.6 2.0
0 0 2 2.0
A diagram of this scheme is shown here:
dilute undiluted
0.781 mL (2 mL)
to 250 mL
final
volume
0.5 mg/mL
stock diluted
solution stock
dilute 1.2 mL
(160 mg/mL) solution + 0.8 mL
(0.05 mg/mL)
dilute 0.8 mL
+ 1.2 mL 0.3 mg/mL
0 mL +
2 mL water
dilute 0.4 mL
0.2 mg/mL
+ 1.6 mL
0 mg/mL 0.1 mg/mL
www.dbooks.org
5. We know that the anthocyanin concentration of Assume 0.75 mg / mL:

the juice is probably somewhere between 750
and 3000 μg/mL (0.75–3 mg/mL). However, it Vi
C fVf

0.25 mg / mL 10 mL 3.33 mL
could literally be anywhere within this range. Ci 0.75 mg / mL
Samples with 0.75 and 3 mg/mL anthocyanins
would require very different dilutions to get Assume 1.125 mg / mL:
near the center of the standard curve (~0.25 mg/
Vi
C fVf

0.25 mg / mL 10 mL 2.22 mL
mL)—how do we handle this? The solution is to Ci 1.125 mg / mL
design a dilution scheme with various dilutions
of the same sample, which will likely yield at Assume 3 mg / mL:
least 1 dilution in the acceptable range that can
be used for quantification. To do this, we assume Vi
C fVf

0.25 mg / mL 10 mL 0.833 mL
that the anthocyanin concentration in the juice Ci 3 mg / mL
could be on the extremes (~0.75 and ~3 mg/ Therefore, we would make three dilutions using
mL) and right in the middle of those extremes 3.33 mL, 2.22 mL, and 0.833 mL of the juice and
(~1.125 mg/mL). Then, we can calculate 3 dif- dilute each to a final volume of 10 mL. We would
ferent dilutions (assuming the 3 different antho- then analyze the standard solutions and the 3 dilu-
cyanin levels and diluting to the middle of the tions and use the diluted sample (and correspond-
curve) so that we will have at least 1 useable ing dilution factor) with an analytical response
sample regardless of the actual sample concen- within the range of the standard curve to calculate
tration. Let’s assume that we want a final sam- the anthocyanin concentration in the juice.
ple volume of 10 mL (we could pick any volume
that corresponds to a volumetric flask we have 6. This problem is set up as shown below. Note
available, but it wouldn’t make sense to make that all of the minerals from 2.8 mL milk are
50–1000 mL of diluted sample if we only need diluted to a total volume of 50 mL, so these
2 mL for the analysis): steps can be simplified somewhat in the calcula-
tion as shown in the figure.

~12 mL 50 mL
total volume
20 mL
dilute all solution

dry ash dissolve ash in dilute 7 mL
to 50 mL
2.8 mL milk 12 mL HC1 + 13 mL HC1
with 1 N HC1
analyze by AAS
(28.2 ppm Ca)
milk minerals only solution A solution B solution C
Step 1 Step 2
Ci=? ppm Ca Ci=? ppm Ca
mi=2.8 g Vi=7 mL
Vf=50 mL Vf=20 mL
Cf=? ppm Ca Cf=28.2 ppm Ca
The problem can then be set up and solved as a mul-

tistep dilution as follows:
Ci C f
50 mL 20 mL
Ci 28.2 ppm Ca 1440 ppm Ca
2.8 mL 7 mL
7. First, you need to get the sample and standards

into the same units. Convert the sample caffeine
concentration to mM:
170 mg caffeine 1000 mL 1 g 1 molcaffeine 1 106 molcaffeine 2190 molcaffeine

2190 M caffeine
400 mL 1 L 1000 mg 194.2 caffeine 1 molcaffeine L
Then, determine the starting volume needed to Therefore, if 5.17 mL of the energy drink is
dilute to 250 mL total volume and obtain a con- diluted to 250 mL final volume, the caffeine
centration at the center of the standard curve concentration will be ~50 μM, which is right in
(50 μM caffeine): the middle of your standard curve.
CiVi = C f V f →
Vi =
C fVf
=
( 50 m M caffeine )( 250 mL )
Ci ( 2190 m M caffeine )
= 5.71mL
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31
chapter
Answers to Practice
Use of Statistics
in Food Analysis
Andrew P. Neilson ( ) • Sean F. O’Keefe *

Blacksburg, VA, USA
1. What is the mean of the observations (mg/cup)? The lower limit of the CI is:
SD mg mg
x t 327.85 10.7983
x
xix x2 xn 1 xn 1967.1 mg / cup
1 2
, df n 1 n cup cup
n n 6 317.052 mg / cup
327.85 mg / cup, round to 328mg / cup
Use a t-test to determine if the sample mean provides
What is the standard deviation of the observations strong enough evidence that the population is “out of
(mg/cup)? Since we have < 30 observations, the cor- spec” (i.e., the actual population mean ≠ 343 mg cup).
rect formula for sample standard deviation is: Since we have mean a “target” (μ = 343 mg/cup) that
we want to compare a sample against, we use a one
2 2
SD n 1
x i x

x i 327.85 mg / cup sample t-test:
n 1 6 1 x
2 2 tobs
460.215 mg / cup SD
9.593904 mg / cup
5 n
Plugging in the mean, SD, n, and μ values:
Calculate the 96 % confidence interval for the true pop-
ulation mean (use t-score, not Z score, because n is x 327.85 mg / cup 343 mg / cup
small). We know that the formula for a CI is: tobs
SD 9.593904 mg / cup
SD n 6
CI : x t 15.15mg / cup
2
, df n 1 n 3.868
3.91669 mg / cup
We know the mean, SD, and n already, so we just need
the t-score. First, calculate df: Based on tobs, is there sufficient evidence that the popu-
df n 1 6 1 5 lation is “out of spec” (99 % confidence)?
The decision rule is that we need to compare tdf, α/2
Next calculate C and α/2: vs. tobs. Find tdf, α/2:
1 C 1 0.96
for 96% CI, C 0.96 0.02 for 99% confidence,
2 2 2
∝ 1 − C 1 − 0.99
C = 0.99 → = = = 0.005
Then, go to the t-table and find the t-score that corre- 2 2 2
sponds to df = 5, α/2 = 0.02: df = n − 1 = 6 − 1 = 5
t t0.02 , 5 2.757 From the t-score table shown, t5, 0.005 = 4.032.
, df n 1
2 Next, compare tdf, α/2 vs. tobs:
Putting it all together: if tobs tcritical there IS strong evidence that
SD the true pop mean
CI : x t
2
, df n 1 n
if tobs tcritical there IS NOT strong evidence that
9.593904 mg / cup the true pop mean
327.85 mg / cup 2.757
6
327.85 mg / cup 10.7983 mg / cup In this case, since tobs (3.868) < tcritical (4.032), there IS
NOT sufficient evidence that the population is “out of
What are the upper and lower limits of the 96 % confi- spec” with 99 % confidence.
dence interval for the population mean?
The upper limit of the CI is:
2. You should use a two-sample t-test to determine if
SD mg mg the means are statistically different. We need the
x + tα × → 327.85 +10.7983
, df = n −1 n cup cup mean and the SDn-1 for each population. Using your
2
calculator or Excel, we get:
= 338.648 mg / cup
www.dbooks.org
Chapter 31 • Use of Statistics in Food Analysis 249
Line 1: n =5, x–=86.98, and SD=0.81670068

tobs t strong evidencethat means
Line B: n = 5, x–=89.02, and SD=0.195 0.192353841 , df n1 n2 2
2 aresignificantllydifferent
2
Next, calculate the pooled variance (sp ) from the
two sample standard deviations:
tobs t insufficient evidence that means
2 2 , df n1 n2 2
pooled variance sp2
n1 1 SD n2 1 SD
1 2
2 aresignifficantlydifferent
n1 n2 2
2 2
5 1 0.81670068 5 1 0.192353841 1 C 1 0.95
0.352 95% confidence C 0.95,
552 2 2 2
Now, calculate tobs: 0.05
0.025
2
x1 x2 86.98 89.02
tobs df n1 n2 2 5 5 2 8
1 1 1 1
s 2p 0.352
n1 n2 5 5 Then, go to the t-table and find:
2.04
5.43662
0.1408 tcritical t : t0.025, 8 2.306
, df n1 n2 2
2
Based on the t-test, is there strong evidence that the
sample means are statistically different? For a two- Since tobs (5.44) > tcritical (2.31), there is strong evi-
sample t-test, the decision rule is: dence that the means differ (95 % confidence).
Correction to: Food Analysis
Laboratory Manual
S. Suzanne Nielsen
CORRECTION TO:
S.S. Nielsen, Food Analysis Laboratory Manual, Food Science Text Series,
https://doi.org/10.1007/978-3-319-44127-6
An error in the production process unfortunately led to publication of the book before incorporating the
below corrections. This has now been corrected and approved by the Editor.
p. 35 Col 1, 3.4.2, line 6 M = mass” changed to “m = mass”

p. 67 Col 2, 5.2.1, step 5, line 4 8” changed to “2/3”
p. 67 Col 2, 5.2.1, step 5, line 5 oz” changed to “cup“
p. 67 Col 2, 5.2.1, step 5, line 7 227” changed to “170 g”
p. 69 Col 1, step 7, 2nd equation Change denominator from “227” changed to “170”
p. 69 Col 2, step 7, 3rd equation 286” changed to “382”
p. 69 Col 2, step 8, line 4 286” changed to “382”
p. 69 Col 2, step 8, line 6 286” changed to “382”
p. 69 Col 2, step 8, line 16 data” changed to “% wt/wt for Dairy Calcium”
p. 139 Col 2, step 7, line 2 Part 5” changed to “Part 6”
p. 140 Col 2, 3 lines above step 2 99.14 ug/mL” changed to “99140 μg/mL”
p. 144 Col 1, line 4 Deleted entire sentence “The US Food and Drug Administration
requires the vitamin C content to be listed on the nutrition label of
foods.”
p. 146 Col 2, top line of Titer eq 5 mL” changed to “50 mL”
p. 168 Col 1, line 3 300-600 ppm” changed to “30-600 ppm”
p. 168 Col 1, line 4 Deleted “30-600 mg/L”
The updated online version of this book is available at

https://doi.org/10.1007/978-3-319-44127-6
S. S. Nielsen, Food Analysis Laboratory Manual, Food Science Text Series, C1

https://doi.org/10.1007/978-3-319-44127-6_32, © Springer International Publishing 2019
www.dbooks.org
C2 S. S. Nielsen
p. 188 Col 2, question 2, line 2 “name” changed to “nature”

p. 190 Col 1, line 7 W in denominator from “W” changed to “W x 1000”
p. 191 Col 1, sect. 22.4.9, %FFA eq. W in denominator from “W” changed to “W x 1000”
p. 191 Col 2, sect. 22.5.4, last 4 lines Change 0.2 N to 0.1 N
Change 50 g to 25, add “pentahydrate” after “sodium thiosulfate”,
and change “l” to “L”, to read “Dissolve ca. 25 g sodium thiosulfate
pentahydrate in 1 L dd water

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What is the purpose of the editorial board?

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What corrections were made to the book?

Food Science Text Series

For other titles published in this series, go to

ISSN 1572-0330 ISSN 2214-7799 (electronic)

Library of Congress Control Number: 2017942968

© Springer International Publishing 2017, corrected publication 2019

Printed on acid-free paper

This Springer imprint is published by Springer Nature

The original version of this book was revised.

9 Mass Spectrometry with High-Performance 16 Water Hardness Testing by Complexometric

15 Vitamin C Determination by Indophenol 22 Fat Characterization 185

23 Proteins: Extraction, Quantitation, 27 CIE Color Specifications Calculated

1.1 INTRODUCTION cal relationship between the analyte concentration and

balances are usually sensitive to 0.001–0.00001 g,

1.4.1 Operation 10. With plunger fully depressed, withdraw

1. 4 Accuracy and precision of PIPETMAN 1. 5 Recommended volume ranges for mechani-

Reproducibilitya Maximum volume Lowest recommended volume

volumetric measurements can result in significant

dilutions and concentrations whenever possible to 1. 6 Volume tolerances of Class A glassware

2.1 Preparation of Reagents of Specified 2.4 Notes on Buffers

2.1 PREPARATION OF REAGENTS

Unit Symbol Definition Relationship

2.2 USE OF TITRATION TO DETERMINE

Using equivalent weights can facilitate calculations,

Example B1 100 mL of vinegar is titrated with a

A buffer is an aqueous solution containing compara-

æ mol ö n of Na 2HPO 4 = 0.072 ´ 0.1 = 0.0072 ( mol ) (2.32)

æ mol ö n of Na 2HPO 4 after HCl addition ( mol ) :

v of sodium acetate stock solution ( L ) =

There are three ways to prepare such a buffer: 0.0635 ´ 0.25

REFERENCES 3. Harakany AA, Abdel Halim FM, Barakat AO (1984)

3.1 Introduction 3.5 Special Cases

3.1 INTRODUCTION (c) Post-extraction evaporation of solvent (see

4. For converting an analytical result obtained

Recall that the concentration of an analyte in a sample  1.12 mg thiamine 

C  concentration Note that we are only concerned with the

Example D2 If a pesticide is present at 2.8 parts

Once we know the concentration of a sample or CiVi V 

V  1.12 mg thiamine  1 mL  Example D4 Suppose that soybean oil contains

A special case of concentration is when a solution 3.4.5 Multiple-Step Dilutions

Then, a general formula for multistep forward calcula-

Step 2 : 25 mL V3  diluted to 125 mL Step 3 : 75 mL V5 , solution B  concentratedto 50 mL

In reality, only a fraction (generally unknown) of

It is very common for analytical procedures to require

Example F2 Suppose the 1 M stock solution of

Preparing standards in parallel is typically more 3.6.2 Designing Dilution Schemes

error in solution 2    1.01 1.01 X 3.1

error in solution 3    1.01 1.01 1.01 X Ci Dilute To final Cf (%

either with “dilute to” or “dilute with” procedures.

3.7 UNIT CONVERSIONS

C f Vf  750 ppm 1 mL   Vf 

 Vf  0.0725 ng riboflavin  1000 L 

 Vi  Ldilutedsample  mL  1.2 mL milk +

 250 mLdilutedsample  1, 260 ng riboflavin 88.8 mL water

By keeping track of the units, you easily catch an

2. Lack of needed unit conversion or incorrect unit dilute to

takes or catch them if they have been made:

Recall that the concentration of an analyte in a sample 1.12 mg thiamine

Once we know the concentration of a sample or CiVi V

V 1.12 mg thiamine 1 mL Example D4 Suppose that soybean oil contains

Step 2 : 25 mL V3 diluted to 125 mL Step 3 : 75 mL V5 , solution B concentratedto 50 mL

error in solution 2 1.01 1.01 X 3.1

error in solution 3 1.01 1.01 1.01 X Ci Dilute To final Cf (%

C f Vf 750 ppm 1 mL Vf

Vf 0.0725 ng riboflavin 1000 L

Vi Ldilutedsample mL 1.2 mL milk +

250 mLdilutedsample 1, 260 ng riboflavin 88.8 mL water

0 x 3.8 2.783 1.017

x 3.5 2.783 0.717

x 2.9 2.783 0.117 P Z1 Z Z2 P Z Z2 P Z Z1

Population x standard deviation x (4.9) sx

1C (4.16) Usually you do not know the actual population