CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF
CHEMICAL AND BIOENGINEERING
TECHNOLOGY.

CFB 40503
INSTRUMENTAL FOOD ANALYSIS

LABORATORY
MANUAL
SEMESTER JANUARY 2020

FOOD ENGINEERING TECHNOLOGY SECTION

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

CONTENTS

Introduction 3

Safety Declaration From 4

Experiment 1 :
Measurement of Colour of Food Product 5

Experiment 2 :
Measurement of Viscosity of Food Product 7

Experiment 3 :
Application of Food Using UV-Vis Spectroscopy 10

Experiment 4 :
Determination of Copper in Tea By AAS 14

Experiment 5 :
Sugar Analysis with High Performance Liquid Chromatography (HPLC) 16

Experiment 6 :
Determination of Fatty Acid Methyl Esters (Fame) in Oil by Gas Chromatography 19

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

INTRODUCTION

This manual provides the laboratory guidelines, safety declaration form, lab report format and
experimental procedures for subject Instrumental Food Analysis (CFB40503). The manual also
contains specific instructions for completing the experiments and general information covering
laboratory conducts and safety rules.

Students are compulsory to read and understand all the laboratory guidelines provided in the
manual. The safety declaration form should be submitted to the lecturer/instructor before starting
the experiment works.

In the laboratory report format, there are report writing guidelines and requirements that should
be followed to produce a good quality of lab report.

Laboratory Jotter Notes are required for each of the experiment. In the jotter note, the basic
information of the experiment, the procedure and all primary data and observations during the
experiment should be clearly written. The jotter note must be prepared BEFORE coming to the
lab and need to be verified by the lecturer/instructor BEFORE leaving the lab on the day of
experiment. Then the jotter note of each group member MUST be attached to the lab report during
submission.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

SAFETY DECLARATION FORM

The Dean/Head of Campus

Universiti Kuala Lumpur
Malaysian Institute of Chemical and Bioengineering Technology
Lot 1988, Vendor City Industrial Area
Taboh Naning, 78000 Alor Gajah
Melaka.

Dear Sir,

SAFETY DECLARATION (Subject: INSTRUMENTAL FOOD ANALYSIS CFB40503)

I……………………………….………………………………………….......................................
ID No ………………………......... declare that I have read and understood the safety rules and
regulations in UniKL MICET. I hereby agree to abide by all the rules and regulations stated in
the safety guidelines.
2. I hereby understood the contents and disciplinary action will be taken against me, if I do not
abide by the stated rules.
3. I am fully responsible for all my actions during laboratory sessions.

Thank you.

Yours faithfully,
……………………………….
Name:
Matrix No:
Date:

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 1: MEASUREMENT OF COLOUR OF FOOD PRODUCT

OBJECTIVE
1. To determine colour properties of food products using Lovibond tintometer and
Chromameter.

INTRODUCTION
Colour is one of the three principal quality attributes that influence the food acceptance and
judgement. Customer examines the food product and expecting the colour must be ‘right’.
Instrumental colour measurements are extensively applied due to subjective assessment through
human’s visual. The colour measurement instrumentals provide stability, easy to use and most
importantly achieve the desired objective colour measurement for both research and industrial
applications.

List of Manuals needed

1. Lovibond tintometer
2. Chromameter

SAMPLES
Two types of samples needed
1. Liquid samples – drinks with some coloring
2. Solid samples – fruits, labels etc

METHODOLOGY

Sample preparation
1. Liquid samples – degas the carbonated drinks. Separate drinks into three parts – original
conditions and dilute it into 50% and 25% of original conditions. You will need a
minimum of 25 ml from each part.
2. Solid samples – to use as it is.

Lovibond tintometer
1. This system needs to be calibrated.
2. Ensure that you have selected the Transmission or X, Y, Z mode for the determination.
You can do using the Mode selection button.
3. Prepare samples into cuvet, you will need about 15 ml liquid. Press READ button and
Press PRINT button after sometimes.
4. Remove existing sample for the next determination.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

5. Rinse cuvette with next sample and fill it with sample to be determined. Repeat step
number 2.

Chromameter
1. Calibrate the chromameter
2. Prepare sample into cuvette of the system. You will need about 10 ml liquid sample.
3. Press button on the system to determine the reflextance value. You may need to reposition
the sample for another 2 times during data acquisitions.
4. Remove existing sample for the next determination.
5. Rinse cuvette with next sample and fill it with sample to be determined. Repeat step
number 2.

DATA COLLECTION
1. Please ensure that you have read and understand the operation manuals for both Lovibond
and Chromameter. Brief operation for both systems is included in this Manual.
2. For Lovibond tintometer, adjust the system to get data for X, Y and Z and data for
Transmission from 410 to 700 nm. This data will be used in your report.
3. For Chromameter, adjust the system to get a list of data for the X, Y, and Z; L ab and L
a*b*.
4. Use data from tintometer to calculate the x and y value utilizing excel sheet as provided
in the e-learning system.

RESULTS AND DISCUSSION

1. Prepare data obtained from the experiments to calculate the X, Y and Z; x and y and to
identify the point for each liquid portion in the x, y chromaticity diagram.
2. Differentiate result between both tintometer and chromaticity system and discuss the
method that was used by both systems that give results as obtained in your experiment.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 2: MEASUREMENT OF VISCOSITY OF FOOD PRODUCT

INTRODUCTION
Whether working in product development, quality control, or process design and scale-up,
rheology plays an integral role in the manufacture of the best products. Rheology is a science
based on the fundamental physical relationships concerned with how all materials respond to
applied forces or deformations.
Determination and control of the flow properties of fluid foods is critical for optimizing
processing conditions and obtaining the desired beneficial effects for the consumer.
Transportation of fluids (pumping) from one location to another requires pumps, piping, and
fittings such as valves, elbows, and tees. Proper sizing of this equipment depends on a number of
elements but primarily on the flow properties of the product. For example, the equipment used to
pump a dough mixture would be very different from that used for milk. Additionally, rheological
properties are fundamental to many aspects of food safety. During continuous thermal processing
of fluid foods, the amount of time the food is in the system (known as the residence time or RT),
and therefore the amount of heating or “thermal dose” received, relates directly to its flow
properties. The rheological properties of a fluid are a function of composition, temperature, and
other processing conditions. Identifying how these parameters influence the flow properties may
be performed using a variety of rheometers. In this laboratory, we will measure the viscosity of
three liquid foods using Brookfield rotational viscometers – common rheological instruments
widely used throughout the food industry.

EQUIPMENT
1. Brookfield rotational viscometer model LV and spindle #3
2. Refrigerator

MATERIALS
Prepare 1%, 2%, 5%, 8% 10% and 12% starch solution by adding with hot water to 400ml for
each preparation. Divide each preparation into 200 ml each, one of preparation will be placed in
refrigerator for 1 hour and another one is kept at room temperature.
Each group to test 2 samples from above preparation, including the refrigerated samples, eg 1%
and 5% samples.

METHODOLOGY
1. Prior to evaluating the samples, make sure the viscometer is level. Use the leveling ball and
circle on the viscometer.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

2. Fill a beaker with 200 ml sample A and the two remaining beakers with 200 ml Sample B.
Place one of the beakers of salad dressing in a refrigerator 1 hr prior to analysis. The remaining
beakers shall be allowed to equilibrate to room temperature.
3. Because rheological properties are strongly dependent on temperature, measure and record
fluid temperatures prior to each measurement.
4. On the data sheet provided, record the viscometer model number and spindle size, product
information (type and brand, etc.), and the sample temperature.
5. Immerse the spindle into the test fluid (i.e., honey, salad dressing) up to the notch cut in the
shaft; the viscometer motor should be off.
6. Zero the digital viscometers if necessary.
7. Set the motor at the lowest speed revolutions per minute (rpm) setting. Once the digital display
shows a stable value, record the percentage of full-scale torque reading. Increase the rpm
setting to the next speed and again record the percentage of full-scale torque reading. Repeat
this procedure until the maximum rpm setting has been reached or100% (but not higher) of
the full-scale torque reading is obtained.
8. Stop the motor and slowly raise the spindle from the sample. Remove the spindle and clean
with soap and water, then dry.
9. A factor exists for each spindle-speed combination:

Factors for Brookfield Model LV (Spindle #3)

Speed (rpm) Factor
0.3 4000
0.6 2000
1.5 800
3 400
6 200
12 100
30 40
60 20

For every dial reading (percentage full-scale torque), multiply the display value by the
corresponding factor to calculate the viscosity with units of mPa-s.
Example:
A French salad dressing was tested with a Brookfield LV viscometer equipped with spindle #3.
At a speed of 6 rpm, the display read 40.6%. For these conditions, the viscosity is calculated:
 =40.6 x 200 =8120mPa-s =8.12 Pa-s
10. Repeat Steps 3–9 to test all samples.
11. Once all the data have been collected for the product samples, remove the sample kept in the
refrigerator and run the same procedure. Be sure to record the sample temperature.
12. You may choose to run the samples in duplicate or perhaps triplicate. Data from samples
collected under identical conditions may be pooled to generate an average reading.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

DATA
Date:
Product information:
Viscometer make and model:
Spindle size:
Spindle speed (rpm) % Reading Factor Viscosity (mPa-s)

CALCULATIONS
1. Sketch and describe (label) the experimental apparatus.
2. Calculate the viscosity of the test fluids at each rpm.
3. Plot viscosity versus rpm for each fluid on a single graph.
4. Label the plots with the type of fluid based on the response of viscosity to speed (rpm). Keep in mind,
the speed is proportional to the shear rate. In other words, as the speed is doubled, the shear rate is
doubled.

QUESTIONS
1. What is viscosity?
2. What is a Newtonian fluid? What is a non-Newtonian fluid? Which of your materials responded as a
Newtonian fluid?
3. What effect does temperature have on the viscosity of fluid foods?
4. How may food composition impact the viscosity? What ingredient in the salad dressing may impart
deviations from Newtonian behavior?
5. Describe the importance of viscosity in food processing, quality control, and consumer satisfaction.
6. For samples at similar temperatures and identical speeds, was the viscosity of honey ever less than the
viscosity of salad dressing? Is this behavior representative of the sample rheology at all speeds?
7. Why is it important to test samples at more than 1 speed?

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 3: APPLICATION OF FOOD USING UV-VIS SPECTROSCOPY

OBJECTIVE
1. To determine  max for carmoisine or Potassium Dichromate sample (wavelength scan)
2. To determine ɛ the molar absorptivity of carmoisine or Potassium Dichromate
3. To prepare a serial dilution and generate a standard calibration graph for sample quantitation
(photometric scan)

INTRODUCTION
Absorption of light by solution is one of the oldest and still one of the more useful instrumental
methods. The wavelength of light that a compound will absorb is characteristic of its chemical
structure. Specific regions of the electromagnetic spectrum are absorbed by exciting specific
types of molecular and atomic motion to higher energy levels. Absorption of microwave radiation
is generally due to excitation of molecular rotational motion. Infrared absorption is associated
with vibrational motions of molecules. Absorption of visible and ultraviolet (UV) radiation is
associated with excitation of electrons, in both atoms and molecules, to higher energy states. All
molecules will undergo electronic excitation following absorption of light, but for most molecules
very high energy radiation (in the vacuum ultraviolet, <200 nm) is required. For molecules
containing conjugated electron systems however, light in the UV-visible region is adequate (e.g.,
benzene absorbs in the 260 nm region). As the degree of conjugation increases, the spectrum
shifts to lower energy.
Electromagnetic radiation in the UV-VIS portion of the spectrum ranges in wavelength from
approximately 200 to 700 nm. The UV range is colorless to the human eye, while different
wavelengths in the visible range each have the characteristic color, ranging from violet at the
short wavelength end of the spectrum to red at the long wavelength end of the spectrum. Because
absorption spectra are characteristic of molecular structure, they can be used to qualitatively
identify atomic and molecular species.
The amount of light, I, transmitted through a solution of an absorbing chemical in a transparent
solvent can be related to its concentration by Beers Law:
-Log I/Io = A=bc

The standard equation for absorbance is A = ɛbc, where A is the amount of light absorbed by the
sample for a given wavelength (), ɛ is the molar absorptivity, b is the distance that the light
travels through the solution, and c is the concentration of the absorbing species per unit volume.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

Absorbance can also be calculated using the ratio between the intensity of a reference sample and
the unknown sample. It is given by the equation A = log10(Io/I).

Intensity is obtained using a spectrophotometer. The absorbance of a solution will change based
on the wavelength that is passed through the solution. Some wavelengths will be absorbed more
than others depending upon the makeup of the solution. Remember to state which wavelength is
being used for your calculation.

Molar absorptivity, also known as the molar extinction coefficient, is a measure of how well a
chemical species absorbs a given wavelength of light. It allows you to make comparisons between
compounds without taking into account differences in concentration or solution length during
measurements. The standard units for molar absorptivity are liters per mole centimeter (L mol -1
cm-1).

REAGENTS
• 100ppm Carmoisine stock (1000ml).
• 100ppm K2CrO7 stock (1000ml) - Potassium Dichromate
• Unknown concentration of Carmoisine (2 samples) and Potassium Dichromate (2
samples)
• Distilled water

Choose either one of sample only – carmoisine or Potassium Dichromate

APPARATUS
• Sample cuvettes, path length 1 cm
• Volumetric flasks 50mL (five)
• Pipette 5 ml, 10 ml, 25 ml (one each)
• Rubber bulb (three)
• Beaker 100 ml (one)
• Graduated cylinder 50 ml (one)
• Dropper (one)
• Labeling sticker
• Tissue paper

METHODOLOGY
1. Prepare serial dilutions (5ppm, 15ppm, 25ppm, 35 ppm, 45ppm) from the 100ppm stock.
Calculate into units for concentration in molar or moles/liter. Given Formula carmoisine
is C20H12N2Na2O7S2, Mw 502.43 g/mol and Mw K2CrO7 is 242.18 g/mol.

2. Calculate volume needed, V1 from the 100ppm stock for all dilutions.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

3. In order to prepare a dilution, an exact volume of V1 was drawn up from the carmoisine
or Potassium Dichromate stock and was poured into a 50ml volumetric flask. Add distilled
water up to the mark level of the volumetric flask. Shake the volumetric flask properly.
4. The procedure previous was repeated for all dilutions. The formula used is :

M1V1= M2V2 to find the V1

Where M1= concentration of stock

V1= volume of stock to be drawn
M2= concentration of diluted
V2= volume of diluted

5. Your instructor will brief on the standard operating procedure of Perkin Elmer UV-VIS
Spectrophotometer Lambda EZ210.
6. Fill a cuvette with 45ppm dilution and another cuvette with blank solution. Insert both
cuvettes in the sample compartment and blank compartment. Wipe clean the clean sides
of the cuvettes and not touched. Conduct wavelength scan was to obtain the λmax. Record
data obtained from this scan.
7. For the photometric scan, the cuvette was filled as step 6 but the serial dilution prepared
was used and scanned one by one. Record all absorbance readings and prepare the
standard calibration graph.
8. Calculate the molar absorptivity of carmoisine or Potassium Dichromate at the maximum
wavelength for each concentration. Rearrange the Beer-Lambert equation to solve for
molar absorptivity. Using the equation, we can divide absorbance by the length and the
concentration to get molar absorptivity on one side of the equation: ɛ = A/lc. Present result
into table form.
9. Determine the concentrations of two unknown solutions using calibration curve of
absorbance (Y) vs concentration (X).
10. Clean your workstation before leaving the laboratory.

RESULT
Plot a calibration curve for the standard solutions to demonstrate that the absorbance follows
Beer's Law. Perform a linear regression to determine the slope of the line and its standard
deviation. Determine the amount of carmoisine or Potassium Dichromate in the unknown sample
in mg/L based on the absorbance of both sample solutions.

DATA
Date:
Product information:

Absorbance of standards and samples:

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

Absorbance

Standards
(ppm stock) Msmt 1 Msmt 2 Average
0 (blank)
5
15
25
35
45

Samples
Rep 1
Rep 2

DISCUSSION & CONCLUSION

Calculation of total carmoisine or Potassium Dichromate in sample:

1. Plot the standard curve and determine the content of carmoisine or Potassium Dichromate in
the dissolved solution.
2. Calculate the carmoisine or Potassium Dichromate (µg /g, ppm and ppb) in the sample.
3. Report the maximum wavelength of carmoisine or Potassium Dichromate

Using the calibration curve find the molar absorptivity of carmoisine or Potassium Dichromate
and compare this result with result obtained as above.

Example: The absorbance at a .2 molar concentration is 0.27 and at 0.3 molar is 0.41. The
absorbance values are Y-values, while concentrations are X-values. Using the equation for a line
(Y2 - Y1)/(X2 - X1) = (0.41-0.27)/(0.3-0.2) = 0.14/0.1 = 1.4 is the slope of the line. If 1.4 is the
slope of the line and the path length is 0.5 cm, then the molar absorptivity is 1.4/0.5 = 2.8 L mol-
1
cm-1.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 4: DETERMINATION OF COPPER IN TEA BY AAS

OBJECTIVE
1. To determine the mineral content in sample and to prepare the standard concentrations from
given standard stock solution.

INTRODUCTION
Atomic Absorption Spectroscopy (AAS) played a major role in measuring trace amount of
mineral elements in biological samples. AAS quantifies the absorption of electromagnetic
radiation by well separated neutral atoms in the gaseous state. Atomic spectroscopy is particularly
well suited for analytical measurements because atomic spectra consist of discrete lines and every
element has unique spectrum.

REAGENTS
Equipment and Supplies:
Perkin Elmer Atomic Absorption Spectrometer model 100 Analyst
Copper hollow cathode lamp
Eight 100 ml volumetric flasks
Beakers
Pipette (5 ml, 10 ml and 25 ml)
Copper stock solution: 1000 ml
8 M nitric acid
Tea leaves
Furnace
Steam bath

Blank Preparation (1% Nitric acid)

a. Pipette 1 ml (v/v) of concentrated nitric acid into 100 ml volumetric flask.
b. Mark up with deionized water and label as blank.

Copper Standard Preparation (External standard solutions)

a. Pipette 10 ml of 1000 ppm copper standard stock solution and dispense into 100
ml volumetric flask. Label it concentration in pp using the following equation:
M1V1 = M2V2

Where
M1 = Initial concentration in ppm
M2 = Final concentration in ppm
V1 = Initial volume in ml (request solution volume)

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

V2 = Final volume in ml (volume of volumetric flask used)

b. Using the 1000 ppm copper standard solution, prepare 0.5 ppm, 1.0 ppm, 1.5 ppm,
2.0 ppm, 2.5 ppm and 4.0 ppm copper standard solution in 100 ml volumetric
flask. Add 1% (v/v) of concentrated nitric acid to each of the serial standard
solution and mark up to the volume.
c. The standard solution prepared was ready to analyze.

Sample Preparations
• Dry ashing of black tea leaves: duplicate sample
a. Ash 5 g of the dry food at 500 - 5500C for 1 hour.
b. After cooling, moisten the ash with 10 ml of 8M nitric acid and evaporate on a
steam bath to near dryness.
c. Then, transfer the moist render to 50 ml volumetric flask and dilute to the mark
with distilled water. Add another 1% concentrated nitric acid into one of the
sample.
d. Filter the sample for analysis using AAS.

• Tea infusion; duplicate sample

a. Prepare a tea infusion by adding 200 ml of boiling distilled water to 2.5 g of tea
leaves, and let it stand for 5 minutes.
b. Make sure to use the same sample and brand as for the dry ashing determination.
c. Cool, dilute to 250 ml volumetric flask and filter for analysis by AAS.
d. Add 1% of nitric acid to one of the sample.

PRECAUTIONS
Use fume hood when dealing with concentrated acid. Be careful when diluting acid with water.

Page 15 of 21
CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 5: SUGAR ANALYSIS WITH HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)

OBJECTIVE
1. The objectives of our study were to develop an uncomplicated and rapid HPLC method for
quantitating individual sugars, particularly in various fresh and fermented products.
2. To determine sugar content of several fruit and food sources.
3. To prepare a serial dilution and generate a standard calibration graph for sample quantitation

INTRODUCTION
The mandate of nutrition labeling by Food Act 1983 and Food Regulation 1985 has made sugar
analysis (mono and disaccharides) a necessity for all of the food industry. Previously, sugar
content in various food products were based on the total sugar or sucrose, with little emphasis on
the other individual sugars. Colorimetric and iodometric methods (Somogyi 1952, Ting 1956)
were unable to quantitate individual sugars. Gas-liquid chromatography (GLC) has been
successful in determining individual sugars, but it requires a derivatization procedure (Li et al
1985). Recently, high-performance liquid chromatography (HPLC) has become the preferred
method for quantitating simple sugars in a variety of food products.
Sample preparation varies depending on the food product in question. High-fat samples, such as
chocolates, require defatting before analysis. High-fat samples have been pretreated by various
methods, including boiling water (AACC 1983), a Carrez reagent (Bugner and Feinberg 1992),
or a petroleum ether wash (AOAC 1990). Extraction methods for other food products include
boiling in 80% ethanol (Picha 1985), 50% alcohol in an 80-850C water bath (Zygmunt 1982), or
a water extraction (AACC 1983). Li et al (1985) extracted samples using hexane followed by
water or 80% ethanol. The objectives of our study were to develop an uncomplicated and rapid
HPLC method for quantitating individual sugars, particularly in various fermented products.

MATERIALS
1. Materials:
Sample used in this experiment are:
• Fresh fruit and fermented samples
Standards used:
• Glucose, acetic acid, levulinic acid

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

• Acid sulfuric, deionized water

METHODOLOGY
1. Weigh accurately 100 to 200 mg samples into 25 ml glass beaker. Add 1.5 ml cold 72%
sulfuric acid into beaker while ensuring all material parts are thoroughly wetted with glass
rod. Material was thoroughly mixed for 60 minutes in temperature-controlled bath at 30oC.
Accurately 10 ml water was then added into beaker while stirring and mixing to ensure all
materials were dissolved in water and subsequently transferred into 150 ml glass bottle with
tight cap. Rinsed beaker with small volume of water and add into glass bottle. Total volume
of water of 56 ml was required to make final acid solution 4%. Hydrolysis was done in an
autoclave at 120oC for 60 minutes followed by immersing glass bottle in cold iced water to
stop hydrolysis reaction. Final volume of water was recorded to ensure each hydrolysis was
done at the same acid concentration within batch and during other hydrolysis batch.
2. Following the hydrolysis of polysaccharides in materials, hydrolysate containing solid
residues was filtered and solution obtained (hydrolysate) was prepared for glucose
determination with HPLC as prescribed in the glucose determination procedure with HPLC.
3. Sample that was ready for glucose determination was either already centrifuges at 5000 rpm
for 10 minutes or filtered through minimum 0.25 or 0.45 m nylon filter size and already
placed in respective HPLC vials with label.
4. Hydrolysate from sample will be diluted with mobile phase or deionised water to be in the
range of standard if necessary.
5. Typical standard chromatogram (RID and DAD 210 nm) are represented as follows:

Figure 1: Standard chromatogram obtained for glucose, formic acid, acetic acid and levulinic
acid for (a) RID and (b) DAD 210 nm, mobile phase 0.5 ml/min.

6. Standard (glucose, acetic acid, levulinic acid and formic acid) were prepared from stock
solution containing 1000 ppm each component. Standard were diluted to 20, 100, 300 and
500 ppm in respective vials.
7. Mobile phase was dilute sulfuric acid (5 mM) in deionised water at a flow rate of 0.5 ml/min.
10 µl sample and standards of different concentration are injected separately into HPLC
system.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

8. Representative of standard chromatograms for glucose, formic acid, acetic acid and levulinic
acid were presented in Figure 1.
9. Selected glucose degradation products as described above are acetic acid (AA), levulinic acid
(LA) and formic acid (FA). Standards were prepared by diluting glucose, acetic acid, formic
acid and levulinic acid in deionized water [density and purity of each chemical were used to
calculate actual amount of standard in solution]. Concentration of each component was in the
range 10 ppm to 1200 ppm.
10. Determine the concentrations the content of Sugar, Acetic acid and levulinic acid in samples.
11. Clean your workstation before leaving the laboratory.

RESULT
Plot a calibration curve for the standard solutions to demonstrate that the absorbance follows
Beer's Law. Perform a linear regression to determine the slope of the line and its standard
deviation.

DISCUSSION
Discuss your result using relevant tables and graphs obtained from experiments.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

MALAYSIAN INSTITUTE OF CHEMICAL AND

BIOENGINEERING TECHNOLOGY.

INSTRUMENTAL FOOD ANALYSIS (CFB 40503)

UNIKL MICET
DOC. NO. : JAN 2020
LAB MANUAL (REV. NO. 1)

EXPERIMENT 6: DETERMINATION OF FATTY ACID METHYL ESTERS (FAME)

IN OIL BY GAS CHROMATOGRAPHY

OBJECTIVE
1. To determine fatty acid methyl ester content of several vegetable oil source
2. To prepare a serial dilution and generate a standard calibration graph for sample quantitation

INTRODUCTION
Gas chromatography uses high temperatures to volatilize compounds that are separated as they
pass through the stationary phase of a column and are detected for quantitation.
Information about fatty acid profile on food is important for nutrition labeling, which involves
the measurement of not only total fat but also saturated, unsaturated, and monounsaturated fat.
Gas chromatography is an ideal instrument to determine (qualitatively and quantitatively) fatty
acid profile or fatty acid composition of a food product. This usually involves extracting the lipids
and analyzing them using capillary gas chromatography. Before such analysis, triacylglycerols
and phospholipids are saponified and the fatty acids liberated are esterified to form fatty acid
methyl esters (FAMEs) so that the volatility is increased.

MATERIALS

Materials:
a. Sample: oil (palm, olive, sunflower)
b. Chemicals: Fatty acid Standard, hexane, sodium methoxide (dissolve sodium oxide with
methanol), sodium sulphate
c. 2 ml vial, 5µL micro-syringe

Equipment:
a. Perkin Elmer Gas Chromatography with silica capillary column; Supelco SP2340 (60m X
0.25mm id X 0.2µm).
b. Vortex mixture

Preparation of Fatty Acid Methyl Ester (FAME)

a. Dissolve 0.05g of the oil with 0.95ml hexane and 0.5ml sodium methoxide into 2ml vial.
b. Shake the mixture in a vortex mixture vigorously. See the separation.
c. Take out the clear, separate methyl ester layer and dry with anhydrous sodium sulphate.

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

d. Take out 10 µl of the extract of FAME and diluted with hexane in 1ml volumetric flask.
e. Then inject 1µl of sample into the gas chromatography for analysis.

METHODOLOGY
1. Determine the concentrations the content of FAME in vegetable oil samples.
2. Clean your workstation before leaving the laboratory.

RESULT, DISCUSSION & CONCLUSION

Data and Calculations

1. Report retention times and relative peak areas for the peaks in the chromatogram from the
FAME reference standard mixture. Use this information to identify up to 14 peaks in the
chromatogram.

Peak Retention time Peak area Identity of peak

1
2
3
4
5
6
7
8
9
10
11
12
13
14

2. Using the retention times for peaks in the chromatogram from the FAME reference standard
mixture, and your knowledge of the profile of the oil, identify the peaks in the chromatograms
for each type of oil analyzed. [Cite your source(s) of information on the fatty acid profile of
oil] Report results for samples from of derivatization method.
3. For the one oil analyzed by your group, prepare a table (with appropriate units) comparing
your experimentally determined fatty acid profile to that found in your cited literature source.

Quantity determined
Quantity in literature Sodium methoxide method
C4:0
C6:0
C8:0
C10:0
C12:0
C14:0

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CFB 40503 INSTRUMENTAL FOOD ANALYSIS LAB MANUAL

C14:1
C16:0
C16:1
C18:0
C18:1
C18:2
C18:3
C20:0

QUESTIONS
1. Why is the fatty acid profiling method used in lab inadequate to quantify the fatty acids?
2. What are the characteristics required of a suitable internal standard for FAME quantification
by GC and how does this overcome the problem(s) identified in (a)?
3. Would the internal standard be added to the reference standard mixture and the sample, or
only to one of these?
4. When would the internal standard be added?

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