97o Nitrogen Determination Manual May - 07 Gerthard

MANUAL - NITROGEN DETERMINATION PAGE OF
APPLICATION KJELDAHL ANALYSIS 1 23
Manual - Nitrogen determination
Content
1. Introduction
1.1 Nitrogen cycle
Kjeldahl analysis
2. Principle
2.1. Aim
2.2. Digestion
2.3. Distillation
2.4. Titration
2.5. Calculation of the result
3. Error diagnosis
3.1. Possible Digestion failures

3.2. Possible Distillation failures
3.3. Possible Titration failures
4. Criteria for the selection of instruments
4.1. Selection of the Digestion Units

4.1.1. Serial Heating Units
4.1.2. Turbotherm
4.1.3. Kjeldatherm
4.1.4. Criteria for the selection of Digestion Units
4.2. Selection of the Distillation Units

4.2.1 Vapodest 20
4.2.2 Vapodest 30
4.2.3 Vapodest 40
4.2.4 Vapodest 45
4.2.5 Vapodest 50
4.2.6 Vapodest 50 carousel
4.2.7 Special Models
Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

Name: M. Kranz
1. Introduction
Ever since Justus von Liebig made his examination of the reasons for the growth of plants, it is known that
nitrogen has a big impact. Thus, it also became an important subject for the food production in general. The
total nitrogen content in soils as well as the single components as ammonia, nitrate, nitrite and organically
bound nitrogen are of special interest. The nitrogen, which can be used by plants, has to be judged
depending on the kind of nitrogen fixation. The statement:...the higher the nitrogen content the higher the
yield...dates from that time. However, by using the land continuously, a natural regeneration process is no
longer possible and thus, the nutrients have to be added artificially.
Today, we know that too much nitrogen has a negative impact on the environment and the quality of the
products. Some examples are the increase of algaes in lakes (a direct consequence when water contains
too much nitrogen) or the giant beet which has a high weight but a low sugar content. Furthermore, the
addition of nitrogen using fertilizers or natural fertilizers like manure, compost, or sewage sludge is also a
question of money. The main concern nowadays is the addition of nitrogen and other macro nutrients in a
well balanced relation of economic profit and environmental concern
The benefit of the Kjeldahl analysis is the fact that all nitrogen components can be traced as well as the
versatility of the analyzed matrices. Manure, sewage sludge, compost, soils as well as water leachate and
water can be examined for their nitrogen content. This feature has become important with the increasing
amount of environmental damages. Sewage plants as well as industrial providers for water have to be
examined continuously for the total nitrogen content. The quality of crude oil is determined according to the
nitrogen content as too high nitrogen contents lead to high nitrogen oxide emissions when they are burnt in
car engines.
The protein content is a very important quality indicator for the human and animal nutrition.
The method presented by J. Kjeldahl on March 7th 1883 for the determination of nitrogen in organic
materials was a revolution in the food analysis of those days. Even today, it is the still the standard method
for the determination of protein. The Kjeldahl analysis is still the method required for the determination of
protein in §35 of the Food Law of the Federal Republic of Germany. The protein content is of utmost
importance not only for the human food but also for the animal food. Thus, one of the factors according,
which the quality of feeds is measured, is the raw protein content.
The Kjeldahl analysis and thus, the nitrogen determination is not only limited to the environment and food
analysis but this versatile method is also found in the general or pharmaceutical industry, or where ever the
nitrogen determination is important.
Ever since this method has been published, Gerhardt has been trying to optimize it by developing various
types of instruments. In the beginning huge, iron cast racks were offered but nowadays; highly-precise
block systems and water steam distillation systems with an calculation of the result and automatic sample
feeding are on the market. The chemistry has hardly changed but Gerhardt sees clients as friends and
partners and wants to introduce the future into their labs - today.

Name: M. Kranz
1.1. Nitrogen cycle
When J. Kjeldahl published his paper about the ‘New Methods for the Determination of Nitrogen in Organic
1
Bodies’ in 1883 the nitrogen analysis was changed forever . The universal applicability of the method, the
simple way of running the analysis, yet at the same time achieving exact results, all these features helped
to make this method to be used as a reference in the eg. food and fodder industry. But the method is also
used in the soil and water analysis or where ever organically bound nitrogen has to be located.
1
Zeitschrift für Analytische Chemie, Herausgeber Dr. C.Remigius Fresenius. Zweiundzwanzigster Jahrgang. C.W. Kreidels Verlag
1883. S.366-382 J.Kjeldahl, “Neue Methode zur Bestimmung

Name: M. Kranz
2. Principle
2.1. Aim
The objective of the Kjeldahl technique is to determine quantitatively the amount of nitrogen in a sample.
The assay can be divided in three separate steps:
• Digestion with sulfuric acid and a catalyst

• Distillation with water steam
• Titration and calculation of results
2.2. Digestion
The underlying principle of all Kjeldahl digestions is the oxidative disintegration of the N-containing bonds
with concentrated sulfuric acid in order to obtain N as ammonium-ion as a result.
Digestion time ~1 - 1.5 hours + color change
2.2.1. Various Mixtures of Catalysts
To speed up the reaction

potassium or sodium sulfate and
catalysts are added. In 1973, Dr.
H. Hadorn and Ch. Obrist
published their investigation about
the function of the various
2
catalysts . In this study, the
conclusion is made that when
using mercury or vanadium
pentoxide the best digestion
results are achieved. Nowadays,
these additions are omitted due to
ecological reasons and the
digestion times are prolonged
accordingly in order to obtain the
same results. In the feeds industry, a mixture of potassium sulfate and copper sulfate in the relation 10:1 is
used widely. In other areas, as in the dairy industry a mixture of 100:1 for potassium sulfate to cooper
sulfate has been proven most successful. Thus, the catalysts that have to be used for the various
applications as recommended in the respective regulations should be chosen. The usage of a conditioned
mixture of catalysts in tablets has proven to be most handy. These are also available from C.Gerhardt in
the most popular compositions. The digestion temperature depends mainly on the chosen catalyst.
The boiling point of concentrated sulfuric acid is at 337 °C. When salt is added in the right relation then the
boiling point can be raised to about 380 °C (Van’t Hoff’sche Rule).
2
Dr.H.Hadorn, Ch.Obrist, Systematische Versuche mit verschiedenen Katalysatoren für den Kjeldahl-Aufschluß, Deutsche
Lebensmittel Rundschau, Heft 3, 1973 S. 109/114.

Name: M. Kranz
The addition of catalysts like e.g. mercury, selenium, cooper, or titanium reduces the activating energy and
speeds up the reaction.
If the temperature is raised, the oxidation and the transformation into ammonium sulfate is speeded up
(Equation of Arrhenius). The optimum relation of sulfuric acid in ml to salt in g is at the beginning of the
digestion between 1,7 and 2. Is the relation too little and at around 410°C nitrogen is lost.
When mercury and selenium are added then the transformation into ammonium ion is much faster.
Usually, additaments like cooper sulfate or titanium dioxide are taken. C.Gerhardt offers the most widely
used catalysts as tablets, e.g.:
Catalog number Composition

6123 5,0 g K2SO4 + 0,5 g CuSO4 x 5H2O
6124 5,0 g K2SO4, 0,15 g CuSO4 x 5H2O + 0,15 g TiO2
Further sizes and compositions on request
2.2.2. Sample Taking and Sample Preparation
Not only the chemical aspects are important for the accuracy of the analysis, the sample taking and
preparation are also very important. There are numerous methods available for those procedures, which
you find in official information and application books.
2.2.3. Amount of Sample
The content of nitrogen expected influences the amount of the weighted-in quantity of the sample and also
depends on the consumption of the titration solution used respectively on the precision of the titration done
afterwards. As a rule of thumb, the initial sample weight for homogenous samples should be 0.2-1g, for
samples with an average homogenity 1-2g and for samples with a bad homogenity 1.5-3g or more.
By raising the amount of sample the influence of certain particle sizes is getting less in relationship to the
final result.The expected nitrogen content of the sample also has an impact on the size of the initial sample
weight and
and also depends on the amount of standard solution used respectively on the preciseness of the following
titration. Table 1 in Chapter 1.1.2 can be used as a guideline for the selection of the standard solution.
Usually, the weighing of solid and paste-like samples are done using N-free weighing paper (Cat. No.:
6601). Liquid samples are pipetted if the result should be given in volume percentage or mg N/l. Should a
result be required in weight percentage then the weighing is done using one-way syringes. The weighted in
quantity can be precisely determined by difference weighing.
The amount of sample needed for aqueous samples depends on the nitrogen content.
In order to obtain reasonable results, the following amounts are recommended:
Amount of sample in ml Concentration range in mg/l N

500 <1
250 1 to 5
100 5 to ca.35
50 20 to ca.70
20 50 to ca. 175
10 100 to ca.350
5 300 to ca.700
2.2.4. Amount of Sulfuric Acid
Three factors determine the amount of sulfuric acid needed:

- Amount needed for the oxidation of the organic matrix to carbon dioxide and water
- Evaporation during the analysis
- Transformation of potassium sulfate into potassium hydrogen sulfate.
The later two factors are constant if the digestion conditions are the same. However, the amount of sulfuric
acid needed for the oxidation depends on the weighted-in quantity and the organic composition. For the
oxidation of fat and protein about 2 - 3 times as much sulfuric acid is needed then for carbohydrates. As an
example: for 1 g wheat about 3.5. ml sulfuric acid are needed compared to 9.5 ml for gelatin.

Name: M. Kranz
Sample material Consumption of sulfuric acid in g per g sample

Cane sugar 8,36 g
Flour 6,27
Casein 9,67
Gelatin 17,64
Stearic acid 18,26
Oleic acid 19,87
During the evaporation a maximum amount of 1,5 ml should be lost. The transformation of potassium
sulfate takes about 0.3ml per gram salt. At the end of the digestion process, there should be a surplus of
unconsumed sulfuric acid in the sample. The relation between sulfuric acid in ml to the amount of salt in
gram should not go below 0.9.
Sample calculation for the amount of sulfuric acid needed for cereal
Initial sample weight 1,000 g, digestion time ca.1 hour, 10 g potassium sulphate
Consumption oxidation: = 3,5 ml

Consumption for vaporization: = 1,5 ml
Consumption for conversion: = 3 ml
Consumption total: = 8 ml
The following calculation shows the non-consumed sulfuric acid:
Sulfuric Acid (in ml)

> 0.9 ml / g
Amount of Salt 10 g
This means, that the remaining amount of sulfuric acid should be more than 9 ml. The amount of sulfuric
acid lost and residue together are 17ml, which is the minimum that has to be added at the beginning of the
digestion.
2.2.5. Digestion Times and Temperatures
The digestion times depend largely on the kind of

sample. There is only a rule of thumb that can be given.
After the digest assumes a translucent appearance, it
has to be digested for an additional 30 minutes. The
various digestion times are around one, two or up to
four hours. Please also see the methods and
applications given in the annex. In order to bring the
catalyst mixture which is low in sulfur to a boiling, 400
°C have to be set in the Kjeldatherm block system.
Drawing:
Flow chart of the digestion unit Kjeldatherm

Name: M. Kranz
2.2.6. Digestion Gases
All gases building up during the digestion process should be gotten rid off immediately by either using a
water jet pump or a gas scrubber. Only as much as necessary yet at the same time only as little as
necessary should be scrubbed in order not to contaminate the environment but on the other hand not too
much sulfuric acid should be lost during the process. It is a good idea to have two washing bottles between
the suction device and the suction pump. The first bottle takes the condensate from the sulfuric acid and
the water from the sample whereas in the second bottle, the gas mixture that is built up during the digestion
is neutralized. For this purpose it is recommended to use e.g. 1l of a 15% NaOH (or similar), which is
sufficient for the neutralization of ca. 60 digestions. In order to be able to check on it you might want to add
a few drops of an indicator solution as e.g. bromothymol blue, which is not effected by the gas mixture.
Mixture: 0,5g bromothymol blue are diluted in 20 ml ethanol and then mixed with 1l H2O. 2ml are put into
the neutralisation bottle. Colors: neutral = green, acid = yellow, alkaline = blue).
2.3.DIstillation
2.3.1. Dilution and Neutralization
The digestion tubes containing the strongly acids sulfuric acid solution have to be cooled down to 50-60°C
and the content is liquefied with VE-water which helps the absorption of the heat formed during the solution
and neutralization process. The cooling down must not be too sudden as a salt crust might form then.
About 30 minutes would be the ideal time frame. The rule of thumb here is 30 ml water for 5 ml remaining
digestion solution. The ammonium sulfate, which is attached when it is acid, is expulsed completely as
ammonia when it is alkaline (with an pH value of about 12)
Then, a 30% caustic soda is used to neutralize it and make it alkaline.
Theoretical consumption in ml of a standard solution:
H2SO4 + 2NaOH Na2SO4 + 2 NH3- + 2 H2O
About 4ml of 30% caustic soda per ml concentrated sulfuric acid are needed for the neutralization.

Name: M. Kranz
2.3.2. Watersteam Distillation
After the neutralization, steam is blown in, to heat up the sample to the boiling point and ammonia gas is
released. Power and duration of the steam introduction depend on the instrument used. To give an
example, for the analysis of meat samples using a Vapodest 30 the distillation time is 4 min and the steam
power is 100%. A guideline is the amount of distillate, which should be around 100 - 150 ml. See annex for
details.
2.3.3. Classic Distillation
When doing a classic Kjeldahl distillation 200 ml water is put into the Kjeldahl flask. Water is used as an
entrainer for the ammonia conversion into the receiver; in general 100 ml distillate is collected.
2.3.4. Collection of the Ammonia in Boric Acid
The collection of the ammonia is done in a receiver!

The expelled ammonia is put into a boric acid solution with a known pH value respectively a known
concentration. As a standard a 2 to 4% boric acid solution has been proven successful. Depending on the
nitrogen content about 25-70 ml are used. Ammonia is complexed as boric acid. Below please find the
simplified presentation in an empirical formula, since depending on the pH different complex are made)
Whatever method is chosen, it is important that the outlet tubing of the distillation condenser is completely
immersed in the receiver. Should there be only small amounts then it is recommended to dilute with exact
amounts of distilled water. (Careful: the blind value will change).
Reaction equation:
H3BO3 + NH3 NH4BO4

Name: M. Kranz
2.3.5. Receiving Ammonia in a Defined Hydrochloric Acid or Sulfuric Acid
It is also possible to use an exactly defined amount of acid as a receiver. The amount to be pipetted
depends on the nitrogen content and has to be measured so that it is available in abundance and in a way
so that the amount needed can be determined via a back titration with NaOH as a titration solution. As a
guideline the table ‘Consumption of titration solution’ can be used.
Reaction equation:
HCl + NH3 NH4Cl

HCl + NaOH NaCl + H2O
2.3.6. Collection of Ammonia for the Photometric Content Determination
In case of very little amount of nitrogen, less than 0,1 mg N absolute per sample the precision of the
titrimetric determination of the end point recognition is not sufficient any more. The lower detection limit for
the titrimetric nitrogen determination is at around 0,1 mg N absolute per sample. In this range, special
requirements have to be taken into consideration as far as working precisely and mixing of the solutions are
concerned (also see application: Ammonia Determination in Aqueous Samples). If lower nitrogen content is
expected, the photometric detection has to be taken; here the detection limit is at about 0,01mg N absolute
per sample.
The determination of the content from the distillate has to be done photometrically e.g. using Nessler´s
Reagenz. Also see the most commonly used methods of the photometry.
2.4. Titration
The volumetric determination of the nitrogen content is now done using the end point titration to a set pH
value. The advantage being that in such a case no curve has to be determined and thus a considerable
reduction of the time needed for the dosing of the standard solution.
.
2.4.1. Manual Titration with Visual Endpoint Determination and Boric Acid as Receiver
The advantage of working with boric acid is the fact that the volume of this solution does not have to be
measured exactly. The ammonia reacts with the boric acid to give ammonium borate, which can be directly
titrated with diluted mineral acids as e.g. hydrochloric acid. The boric acid, which is then in excess, does not
have any influence on the color of the indicator used as it has such a low dissociation constant. Also see
titration curve when working with different boric acid solutions:

Name: M. Kranz
The following indicators can be used:
Indicator Type Transition Amount per sample Preparation

Merck mixing indicator Purple to clear to green Ca. 7 drops Can be bought
M5 for pH 5
Tashiro indicator Purple to green Ca. 5 drops 100ml 33% methyl red
4.0 – 6.0 solution with 15 ml 0,1%
methyl-blue solution
Congo red (for direct Blue to red Ca. 10 drops 300mg Congo red in
titration with strong acids) for pH 3.0 – 5.2 100ml hot water, let cool
off and filter
Mixing indicator Green to grey Ca. 5 drops 0,099 g Bromkresolrün +
3.8 – 5.4 0,066 g methyl red in 100
ml 95 % ethanol
2.4.2. Manual Titration with Hydrochloric Acid as a Receiver
If hydrochloric acid or sulfuric acid is used, a precisely defined volume, e.g. with a full pipette has to be
given and the content is determined using back titration with NaOH.
2.4.3. Titration of the pH value with Automatic Endpoint Detection
The detection of the endpoint using a pH electrode makes the process much more comfortable and easy to
be reproduced. With a set endpoint – usually the pH value of the boric acid in the distillate – the subjective
influence at the recognition of the color change can be eliminated. This solution allows an automatization of
the titration. Gerhardt offers the models Vapodest 45, 50, and 50 carousel. At the beginning of the
distillation the pH value is measured automatically. After the distillation, the titration goes back to that value.
The Vapodest 50 offers a very special titration: the online titration allows a titration during the distillation.
This procedure saves time during the titration. In this case, the back titration is not directly to the endpoint
but only to a defined ∆ pH endpoint.
2.4.4. Consumption of Titration Solution and Precision of Burette
The amount of titration solution used for the titration is not relevant for the precision of the titration. Thus,
the consumption of the titration solution relates to the least reading respectively to the preciseness of the
burette. The gray parts of the following table are supposed to be used as a guideline for the selection of the
titration solution in relation to the absolute nitrogen content per sample.

Name: M. Kranz
Table: Consumption of Standard Solution:
Nitrogen content Theoretical Theoretical Theoretical Theoretical

in mg N absolute consumption in consumption in consumption in consumption in
per sample ml of a standard ml of a standard ml of a standard ml of a standard
solution solution solution solution
1 mol/l 0,1 mol/l 0,01 mol/l 0,001 mol/l
0.5 0,03 0.35 3,57 35,71
1 0,07 0,71 7,14 71,42
2 0,14 1,42 14,28 142,85
3 0,21 2,14 21,42 214,28
4 0,28 2,85 28,57 285,71
5 0,35 3,57 35,71 357,14
6 0,42 4,28 42,85 428,57
7 0,5 5 50 500
8 0,57 5,71 57,14 571,42
9 0,64 6,42 64,28 642,85
10 0,71 7,14 71,42 714,28
15 1,07 10,71 107,14 1071,42
20 1,42 14,28 142,85 1428,57
30 2,14 21,42 214,28 2142,85
40 2,85 28,57 285,71 2857,14
50 3,57 35,71 357,14 3571,42
100 7,14 71,42 714,28 7142,85
200 14,28 142,85 1428,57 14285,71
300 21,42 214,28 2142,85 21428,57
1000 71,42 714,28 7142,85 71428,57
2.4.4. Blind Value
Due to the impurity of the chemicals used and the dilution of the boric acid with distillate the pH of the blank
value without N-content is raised. This fact is already taken into account by using a blank value when the
consumption of titration solution is calculated. Varying boric acid solutions and titration solution influence
the blind value. When changing the program parameter, e.g. the distillation time or the steam power or the
settings as new chemicals, a change of the blind value can result.
The determination of the blind value is done under the same circumstances as the analysis! The blind value
should have constant values!!
Should no blind value be set then the turning point of the indicator should be reached at the end of the
distillation or the pH-value at the end of the blind value distillation has to be used as the endpoint during the
analysis. Should the initial pH value of the receiver at the beginning of the distillation be taken as the
endpoint, then the consumption of the blind value has to be taken into account for the following calculation:
2.5 Calculation of the Result
The following formula is used to calculate the nitrogen content in percentage:
%N = (c x (V - VBL) x M x100 %) / E
c = Concentration of the titration solution in mol/ml

V = Consumption of titration solution in 1
VBL = Consumption titration solution blank value in 1
M = Molar mass nitrogen in g/mol
E = Initial sample weight

Name: M. Kranz
Derivation of the formula
1. Calculation of the amount
n =cxV
n = Amount of substance in mol

Example: 0,1 mol/l x 0,015 l= 0,0015 mol
2. Calculation of the amount of nitrogen
m(N) = n x M (N)
m(N) = Mass nitrogen per sample in g

n = Amount of substance in mol
M(N) = Molar mass of nitrogen in g/mol
Example: 0,0015 mol x 14 g/mol = 0,021 g
3. Calculation of the nitrogen content in percent
N% = (m / E) x 100 %
m(N) = Mass nitrogen per sample in g

Example: (0,021g/ 1,000g) x 100% = 2,1 %
4. The following final formula is obtained when using 2 and 1 for 3a. and the blank value VBl is taken
into consideration:
%N = (c x (V - VBL) x M x 100% ) / E

VBl = Consumption of titration solution blank value in 1
M(N) = Molar mass nitrogen in g/mol
Example: (0,1 mol/l x (0,015 l –0,0001 l ) x14,007 g/mol x 100 % ) / 1,000 g = 0,2 %
5. Calculation of the Nitrogen Content in g/l

For the calculation the used sample volume is taken in 4. instead of the initial sample weight, which gives
g/l N = ( c x (V - VBL) x M) / Vsample
Vsample = Sample volume used in l
Example: (0,1 mol/l ×(0,015 l –0,0001 l ) ×14 g/mol) / 0,1 l = 0,2 g N /l or 200 mg N per liter
6. Calculation of the protein content
For the calculation the result obtained in 4. has to be multiplied with the protein factor that is specific for the
product group. Proteins are made from amino acids, which contain nitrogen atoms (also see formula).
Depending on the structure of the protein, they participate in various relations at the set up of the molecules
of the proteins. The result thus is a different proportion for the mass of nitrogen to the mass of the protein

Name: M. Kranz
molecule. With the given the protein factor, conclusions are made from the nitrogen content of the
molecule to the rest. Depending on the sample type various factors result:
P = %N x F
P = Protein content in %
F = Protein content
Example: 0,2 % × 6,35 =
Table Protein Factors
Product Protein factor

Milk and dairy products 6,38
Meat, meat products 6,25
Grains and grain products with the exception of 6,25
wheat and wheat products 5,7
Feeds, egg and egg products, margarine 6,25
Reference Substances
Reference Substances Empirical formula Working range/Verification of Nitrogen

content
Acetanilide C8H9NO General/Digestion 10,36 %
Ammonium chloride NH4Cl General /Water steam distillation 26,18 %
Ammonium dihydrogen (NH4)H2PO4 General /Water steam distillation 12,15 %
phophate
Ammonium iron II Sulfat (NH4)2 Fe(SO4)2 ⋅ 6H2O General /Water steam distillation 7,15 %
Ammonium-PTSA C/H11O3SN General /Digestion 7,40 %
Ammonium sulfate (NH4)2SO4 General /Water steam distillation 21,20 %
(Distillation)
Cystine C6H12N2O4S2 General /Digestion 11,66 %
Glycine C2H5NO2 General /Digestion 37,31 %
Glycine-PTSA C9H13O5SN General /Digestion 5,66 %
Urea CH4N2O Water analysis/Digestion 46,67 %
Potassium nitrate KNO3 Nitrate determination 13,85 %
Food standards Meat, milk etc.*
Lysin-Mono-Hydrochlorid C6H15ClN2O2 Milk/Digestion 15,34 %
Niacin C6H5NO2 Milk/Digestion 11,38 %
Niacin -PTSA C13H13O5SN Milk/Digestion 4,743%
Phenylalanine C9H11NO2 Milk/Digestion 8,47%
Phenazine C12H8N2 Milk/Digestion 15,54 %
Ring tests General
Tryptophane C11H12N2O2 Milk/Digestion 13.71 %
2.6. Percentage Recovery, Reproducibility, Standard Recovery
In order to check the quality of the distillation, the ammonium sulfate should be used as a standard.
2.6.1. Standard Recovery of Ammonium Sulfate
Production of the Standard Solution:

4,717 g (NH4) 2SO4 (dried at 105°C) are weighed in, dissolved in a 1l-measuring flask and distilled water
is added to make 1l. This corresponds to a content of 1 mg N per ml. For the standard recovery a certain
amount corresponding to the measuring range is put in using a pipette.
If the measuring range is lower then this solution has to be diluted!

Name: M. Kranz
20 ml of this stock solution are put into a measuring flask using a pipette and distilled water is added to
make 1l. 50 ml of this solution are put into a sample glass and distilled water is added to make 100 ml.
This corresponds to a content of 0,02 mg N per ml. For the standard recovery a certain amount
corresponding to the measuring range is put in again using a pipette.
Analysis
Each digestion tube is rinsed with distilled water. 100ml of the sample or an aliquot part filled up to 100 ml
is pipetted into the digestion tubes.
Distillation and Programming of the Vapodest
Ammonia is distilled off the alkaline solution. It is then received in a boric acid and determined titrimetrically.
A wide neck Erlenmeyer flask, filled with 50 ml boric acid (Vap20 & 30, Vap 40 empty, none with Vap 50) is
put under the outlet tubing. It has to reach into the boric acid. The program is started, NaOH is added
automatically. The distillation takes place (Amount of distillate about 100 ml).
After the distillation is terminated, the receiver is taken out and the outlet tubing is rinsed with distilled water.
Programming
Vapodest takes over all of the above steps in various grades of automatization.
The dosing of the chemicals is done by volumetric flow rate. In 1 second 10 ml of a solution, which has the
same density as water, are transported. If the density is higher then it is less (Caustic soda 30% ca. 8ml/s).
The following table shows you the suggested programs for the various models of Vapodest. Whenever
chemicals have to be added manually, these have to be carried out with utmost care and all the dangers
involved when handling chemicals have to be observed.
Vap 20 Vap 30 Vap 40 Vap 50
Addition of water in sec manually 0 0 0

Addition of NaOH in sec 1 1 1 1
Reaction Time in sec 0 0 0 0
Distillation time in sec 240 240 240 240
Power Output in % 100 100 100 100
Sample Removal by suction in sec manually 30 30 30
Boric Acid Receiver in sec manually manually 6 6
Boric Acid Removal by suction in sec manually manually manually 30
Titration manually manually manually auto.
Calculation manually manually manually auto.
Titration
It is a titration to the end point. The mixture indicator M5 changes the color at pH 5 from green to gray.
However, other indicators can be chosen as well. The transition point should be within the pH range of the
boric acid (when working with a 2% about 4.3). In case of using an electrode for the titration, the pH -value
of the undiluted boric acid is measured, then it is back titrated to this value and the amount of titration
solution needed is noted (Vap20 - Vap40)
Sample Results:
A series of 4 blank values and 12 samples are run with the distillation program as shown above.
Position Amount used 0,1mol/l Result Comments

1 0,088 - Blank
2 0,087 - Blank
3 0,090 - Blank
4 0,086 - Blank

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Mean value 0,088

5 7,200 995,7
6 7,188 994,0
7 7,186 993,7
8 7,170 991,5
9 7,184 993,4
10 7,178 992,6
11 7,150 988,7
12 7,221 998,6
13 7,172 991,8
14 6,966* Outlier
15 7,159 989,9
Mean value 7,18 ml 993,0 mg/l
Standard deviation 0,02 ml 2,7 mg/l
Relative standard 0,28% 0,27 %
deviation
2.6.2. Calculation of the Recovery
- Initial weight: 4,7185 g

- Theoretical content acc. to package > 99,5 %
- Corrected content: 4,6949g makes a nitrogen concentration of 0,9954 g/l
- 995,4mg correspond to 100 % then the mean value corresponds to 99,75 % of the theoretical
content.
There is a recovery of > 99,7 %.
2.6.3. Repeatability
The repeatability is the value, under which the absolute difference between two single test results using the
same method and the same conditions (same lab, same instruments, same user) can be expected under a
given probability; if no other value is given, then this probability is 95%. The value for the repeatability
depends on the methods and regulations; instead of these values a confidence range of the measurements
done is set up.
2.6.4. Confidence Range for the Actual Value
If the value or the limits are determined using the student’s or t-distribution, a confidence range of the mean
value of ± 16,4 mg/l for an error probability,
of 5% is obtained.
2.6.5. Confidence Range for the Standard Deviation
If the confidence range for the standard deviation is determined, there is a error probability of 5 %. The true
value of the standard deviation is between 1,7 mg/l and 3,7 mg/l.
2.6.6. Reproducibility
The comparability is the value below which the absolute difference between two single test results, which
are obtained under different conditions (different lab technicians, different instruments, different labs, and/or
at different times) but using identical material can be expected with a given probability; if nothing else is
stated, this probability is 95%.

Name: M. Kranz
2.6.7. Quality Control
The check of the quality of the analysis has become an important part of the quality control. The
determination of standards is non-disputed. For the check of an entire analysis course, substances with
defined nitrogen contents or certified standards with test results are recommended. The participation at
public ring tests makes sure that the own analysis results can be compared and a positioning of the own
results can be done.
Recommendation:
If electrodes for the determination of the end point be used (Vap50) they have to be calibrated daily.
At least 3 times a day every day, a 10 mg/l NH4- N standard has to be measured as well and the respective
file has to be kept updated.
In order to find the cause for a failure the Kjeldahl analysis can be divided into 2 steps:
1. Quality of the Distillation with Titration
First, the constancy and value of the blanks are checked. At least 4 blanks should be consistent (also see
example given).
Then, the recovery of readily soluble ammonium salts, like e.g. ammonium sulfate should be checked. In
this case the recovery should be > 99 %, the repeatability should be better than 0,5 %.
2. Quality of the Entire Analysis
If good results are obtained for the blanks and for the ammonium standards, the quality of the digestion has
to be checked. In this case, the standards listed in the table can be used. The recovery should also be >
99 %, the repeatability should be better than 0,5 %.

Name: M. Kranz
3. Error diagnosis
3.1. Possible Digestion Failures
Result too low
Reason for failure Cause and correction of failure

Duration of digestion too short Because the clarity of the digestion solution is not
a guarantee of a complete digestion, further
heating for 30 minutes after the clear appearance
should be performed.
Digestion temperature too low Digestion temperature should be at approx. 400
°C, which means that the acid should be boiling.
Check, whether this temperature is reached and
whether the temperature measurements are
correct
Insufficient amount of catalyst Too little salt gives a too low digestion
temperature; the relation should be 20 ml acid to
10 g potassium sulfate
Too much catalyst The temperature in the digestion tube is above
390 °C ; loss of nitrogen may occur, the amount of
sulfuric acid should be increased or reduction of
the amount of catalyst, reduce heat of IR
instruments, reduce suction of the gas scrubber
so that not too much sulfuric acid is sucked off
Insufficient digestion Sample particles remaining on the walls of the
glassware are not digested, heat up more
carefully, these particles should be washed down
with the sulfuric acid condensate, the sample tube
should be clear upon complementation of the
digestion
Delay in boiling during digestion Loss of sample and thus values obtained too low,
use boiling stones for liquid samples
Excessive reaction and thus partial loss of sample Anti foam tablets might be useful or add foam
breaker, smaller sample size, temperature-time-
program, use bigger digestion tubes
Result too high

Tubes are contaminated with nitrogen Clean the tubes without detergent or Klarspülmittel
The suction is contaminated with sample residue, Clean suction
which are digested as well
The suction carries the ammonia into the sample Check the environment
tube as well
Variable end result

Un-homogeneous sample After taking the sample and the preparation, the
mixture has seperated again, check sample
preparation
Varied analysis conditions As many conditions as possible should be held
constant

Name: M. Kranz
3.2. Possible Distillation Failures
End result too low or no result at all

Distillation time too short Ammonia is not quantitatively conveyed into the
receiving solution; the amount of the distillate should
be 100ml.
Leaking instrument, ammonia is lost Broken or soiled viton cone, clean or replace
Check seals on the distribution head and replace if
needed
Check valve at the condenser, might be soiled; clean
or replace
Digestion tube is broken at the wide neck opening
Glass of the distribution head is not tight, replace
Too little NaOH is added, no ammonia can develop - Check volume transported by the NaOH pump,
about 8-9 ml should be discharged per sec
Too little boric acid in the receiver, escaping Increase amount of boric acid
ammonia can not be bound completely
Tube is not immersed entirely into the acid receiver Increase amount of acid
Formation of stable ammonia complexes, which This problem is only found when working with
cannot be destroyed with NaOH catalysts containing mercury, sodium thiosulfate
destroys these complexes
End result too high

The chemicals used are soiled with nitrogen Thoroughly check the chemicals, determination of a
combinations blind value and if necessary replace chemicals
Violent reactions in the digestion tube, NaOH is - Increase amount of water added
carried over into the receiver - Check the amount of the displacement of water
pump; about 10 ml per sec should be delivered
Part of the glass condenser is broken; drops of Replace glass condenser
NaOH can get into the receiver
Detergent is in the digestion tube Clean distillation tube with distilled water
Carry over of ammonia from the previous sample Prolong distillation time or respectively check,
whether the sample has been sufficiently alkaline

Name: M. Kranz
3.3. Possible Titration Failures
End result too low

Titration solution too concentrated There is less consumption of standard solution, thus
less calculation. Check acid strength using a primary
standard, such as potassium hydrogenphthalate
Indicator in a wrong break The break point of the indicator is too high, thus less
titration solution is used for the back titration, The
break range is between pH 4,7 and 5,0
The break point has not been reached Some drops are missing, comparing of the color
change with a stand recovery
End result too high
Reason for failure Cause and correction of Failure

Too little concentration of titration solution More standard solution is used and thus more is also
calculated; check the acid strength with a primary-
standard chemical, e.g. potassium
hydrogenphthalate
Indicator in the wrong range of color change The point of change of the indicator is too low, thus
there is more standard solution being used for the
back titration, range of change is between pH 4,7
and 5,0
Over titration Make sure that the entire standard solution gets into
the distillate and does not stay on the walls; mix or
shake during the titration
Air in the dosing pump for the titration solution Measurement of the amount used which has no
consequences on the titration; check the reservoir of
the tubing connections and the connection pipes at
the dosing pump.
Electrode reacts too slow, determination of the end The calibration time for the electrode should be
point is too late, thus too much acid is added about 5 -15 seconds, otherwise it is faulty and should
be exchanged.
The life expectancy of an electrode is ca. 1- 1,5
years, exchange it.
Variable end result
Reason for failure Cause and correction of the failure

Titration solution has not been mixed well The titration solution is pumped into the receiver
vessel with varying concentrations

Name: M. Kranz
4. Criteria for the selection of instruments
4.1. Selection of the digestion Units
GERHARDT offers various digestion units for digestions acc. to Kjeldahl. The descriptions given below
should help to select the unit most suitable for the customers’ needs.
4.1.1. Serial Heating Units
Serial heating units whose hotplates can be controlled individually, flexible as far as the number and size of
digestion tubes is concerned, at an affordable price, robust, simple control, for low sample through put.
4.1.2. Turbotherm
The Turbotherm is a versatile infrared digestion system, user friendly control with the opion of programming
time and heating level, extremly short heating up, for various sample types due to a vast selection of tubes,
excellent price performance ratio, for labs with strongly variing applications and a medium range sample
through put.
4.1.3. Kjeldatherm
This block digestion unit is temperture controlled with an external electronic controller. The digestion
conditions are reproducable, high sample throughput taking only a minimum of bench space, ergonomic
working conditions, extrnal temperature control, automatisation possible thanks to lift system and
temperature-time-programmer VARIOSTAT, for high and homogenous sample through put.
4.1.4. Criteria for the selection of Digestion Units
1. Have you done this determintation beforet?
2. Orientieren Sie sich an externem Erfahrungsaustausch?
3. Do you have to follow instructions /- Normen gebunden ?
4. If so which ones?
5. How many samples do you ?
6. During which time frame they have to be done?
7. Are the samples coming in on a continuous or discontinuous basis?
8. Type of sample (various types or just one)?
9. Amount of sample (Initial sample weight or volume)
10. How is the sample treated ?
11. Which chemicals are being used? Amount used?
12. Which connections (water/electricity/compressed air) are available in the lab?
13. Is there enough time for the entire presentation, installation and initial traing (min. 2-3 h, ½ day)
14. Transport: Is there a lift or is help available ?
Please be kind enough to write down the answers in the following sheet:

Name: M. Kranz
Aim of the digestion: e.g. Kjeldahl digestion, trace metal digestion , CSB, etc.?
__________________________________________________________________________________
__________________________________________________________________________________
According to which method are you working? e.g. DIN, ISO, AOAC etc.?
__________________________________________________________________________________
Type of sample Amount of sample Content or recovery
Sample preparation:
__________________________________________________________________________________
__________________________________________________________________________________
__________________________________________________________________________________
Details of instrument:
Type (Block digestion, Turbotherm, heating unit, flask heater) :______________________________
Number of positions and size of flasks: :______________________________
Fume removal (Water jet pump, gas scrubber etc.) :______________________________
Type of controller: :______________________________
Location (e.g. in fume cabinet, on the open bench): :______________________________
Chemicals used for digestion:
Acid (type/amount) :_______________________________________________________
Catalyst (type/amount) :_______________________________________________________
Conditions for digestion
Open/closed/air condenser: :_______________________________________________________
Temperature/time profile
Time Power / Temperature Remark e.g. draining, foaming, decoloration
Remarks:
__________________________________________________________________________________
__________________________________________________________________________________

Name: M. Kranz
4.2. Selection of the Distillation Systems
C.Gerhardt offers water steam distillation systems for various demands. Depending on the requirements
one of 6 models can be choosen. The various features are listed in the table below.
Standard configuration Vap 20 Vap 30 Vap 40 Vap 45 Vap 50 Vap 50c

Automatic addition H3BO3 o o o o
Automatic addition H2O o o o o o
Automatic addition NaOH o o o o o o
Programmable reaction time o o o o o o
Destillationszeit programmierbar o o o o o o
Automatic steam generator o o o o o o
Programmable steam o o o o o o
power 40 - 100 %
Automatic suction of sample waste o o o o o
Automatic suction of receiver o o
Main programs 1 1 10 10 20 20
Selection of language o o o o
Automatic check of chemical o o o o o o
reservoir
Stand-by-function o o o o o o
Optical and acousitcal error o o o o o o
messages
Kjeldatherm digestion tube o o o o o o
Kjeldahl-flasks can be used o o o o o o(1)
External titrator can be used o
2 x RS 485 interface o o o o o o
2 x parallel interface (Centronix) o o
1 x serial interface (RS 232) o o
1 x VGA interface o o
2 x PCMCIA-socket o o
Card PC o o
Automatic Titration o o
Printer o o
Display of analysis results o o
Microdosing pump o o
Calibration data can be stored and o o
recalled
Sample results can be stored and o o
recalled
Result print out o o
Autosampler (carousel) o
Set of storage tanks o
4.2.1. Vapodest 20
Basic model for low sample through put. Especially good for determinations without prior digestion, e.g.
ammonium distillation, alcohhol distillation, water steam volatile components etc. Good price value relation.
Can be used e.g. together with a serial flask heater with Kjeldahl flasks with wide neck opening.

Name: M. Kranz
4.2.2. Vapodest 30
Basic model for the Kjeldahl analysis. All dosing for the digestion tube are done automatically according to
the programming. For labs with an average amount of samples and similar analysis conditions. Can be
used e.g. together with Kjeldatherm Type KB8 or Turbotherm Type TT6. Very good price-value relation
ship.
4.2.3. Vapodest 40
The deluxe edition with the option of defining several programs. All dosing steps for the Kjeldahl analysis
can be programmied. For labs with an average to high sample through put and changing analysis
conditions. Can be used e.g. together with Type KB20 or Turbotherm Type TT12.
4.2.4. Vapodest 45
Same functions as Vapodest 40 but with the optional use of working with two different external iitrators.
When connected to the small titrator model type TL96 it offers only the display of the acid volume used as
well as a titration to the end point or equivalent point. The results are shown on the display of the Vap45.
The bigger model Alpha offers the possibility of various titrations, as well as a connection to a printer. This
set up is ideal for users who need the titrator for other titrations as well.
4.2.5. Vapodest 50
The high light of the product line with a maximum of user friendlyness with an interactive menu and built in
titrator. This model has been developed especially for labs with a high sample throughput. Customer needs
were taken into account when developing the calculation of the results, protocl of the analysis data and the
transfer of data. It is best used together with Kjeldatherm type KBL20 with Variostat and Turbosog. The
Vapodest50 OT gives the option to add an autosampler should the need arise.
4.2.6. Vapodest 50 Carousel
Identical to the Vapodest 50 plus the autosampler. The user has the option to work with carousel racks for
20 x 250ml glasses, 16 x 400ml or 12 x 800ml glasses which are then fed into the Vapodest 50
automatically. Ideal for a even higher sample throughput, e.g. when working with 2 digestion systems
Kjeldatherm type KB20.
4.2.6. Special Models
When working with digestion tubes from other manufacturers, Gerhardt
offers adapters. Furthermore, there are acid resistant models available for applications that require working
with acids. Last but not least, there are various distribution heads the user can choose from.

Name: M. Kranz

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MANUAL - NITROGEN DETERMINATION PAGE OF

APPLICATION KJELDAHL ANALYSIS 1 23

Manual - Nitrogen determination

3.1. Possible Digestion failures

4. Criteria for the selection of instruments

4.1. Selection of the Digestion Units

4.2. Selection of the Distillation Units

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 2 23

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 3 23

1.1. Nitrogen cycle

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 4 23

The assay can be divided in three separate steps:

• Digestion with sulfuric acid and a catalyst

Digestion time ~1 - 1.5 hours + color change

2.2.1. Various Mixtures of Catalysts

To speed up the reaction

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 5 23

Catalog number Composition

2.2.2. Sample Taking and Sample Preparation

2.2.3. Amount of Sample

Amount of sample in ml Concentration range in mg/l N

2.2.4. Amount of Sulfuric Acid

Three factors determine the amount of sulfuric acid needed:

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 6 23

Sample material Consumption of sulfuric acid in g per g sample

Consumption oxidation: = 3,5 ml

The following calculation shows the non-consumed sulfuric acid:

Sulfuric Acid (in ml)

2.2.5. Digestion Times and Temperatures

The digestion times depend largely on the kind of

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 7 23

2.2.6. Digestion Gases

Then, a 30% caustic soda is used to neutralize it and make it alkaline.

Theoretical consumption in ml of a standard solution:

H2SO4 + 2NaOH Na2SO4 + 2 NH3- + 2 H2O

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 8 23

2.3.2. Watersteam Distillation

2.3.3. Classic Distillation

2.3.4. Collection of the Ammonia in Boric Acid

The collection of the ammonia is done in a receiver!

H3BO3 + NH3 NH4BO4

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 9 23

2.3.5. Receiving Ammonia in a Defined Hydrochloric Acid or Sulfuric Acid

HCl + NH3 NH4Cl

2.3.6. Collection of Ammonia for the Photometric Content Determination

Date: May 2007 Revision: A File: Nitrogen determination manual May_07.doc

APPLICATION KJELDAHL ANALYSIS 10 23

The following indicators can be used:

Indicator Type Transition Amount per sample Preparation

2.4.2. Manual Titration with Hydrochloric Acid as a Receiver