4. Acknowledgment
The author expresses his gratitude to Dr. Matthias Wüst, University of Applied
Sciences Western Switzerland, for his conscientious examination and complementation of the references to the scientific literature and the careful reviewing of
the manuscript. I owe thanks to my colleagues at work with BÜCHI Labortechnik Flawil and I would like to convey my appreciation to Dr. Claudia Blum, Mr.
Thomas Ziolko, Ms. Michaela Buffler and Mr. Jürgen Müller for their invaluable
proof-reading. Last but not least Mr. Darvin Martin and Dr. Susanna Shaw from
BUCHI UK deserve citation for their linguistic checking of the English text.
Dr. Huldrych Egli
Foreword
In the past 200 years the techniques supporting chemical analysis have made
tremendous progress from the invention of the Bunsen-burner and its use for
flame tests to the atomic force microscope sent to Mars for the exploration of
martian soil. At the time when Johan Kjeldahl published his method for the determination of nitrogen in 1883 the electric lamp was just patented and the technical age in its childhood. Seldom in human history has an invention remained
basically unchanged for such a long time as Kjeldahl’s method for nitrogen determination. As in 1883 a Kjeldahl nitrogen determination starts with sample preparation, proceeds to the mineralization followed by separation using distillation
and subsequent volumetric determination of the amount of ammonia formed in
the process. Kjeldahl’s visionary idea of providing a simple method for nitrogen
and protein determinations, which also can be carried out by non academic lab
personnel, has been put into practice by BUCHI’s Kjeldahl systems since 1961.
The BUCHI Kjeldahl Guide you have in your hands is addressed to laboratory
personnel, laboratory supervisors, students and teachers. It is our intention to
revive the basic knowledge needed to understand the chemical and physical
background associated with nitrogen determinations according to Kjeldahl and
provide clear instructions in a wide area of Kjeldahl applications. The mayor theoretical part of the Kjeldahl Guide contains basic knowledge followed by an extensive list of actual BUCHI Application Notes describing successful nitrogen
determinations.
With this Kjeldahl Guide BUCHI would like to live up to its company slogan
«Quality in your hands»
and support you in your daily work by not only providing high quality instrumentation but also offering comprehensible theoretical background information and showcase applications.
Dr. Huldrych Egli
8. Introduction
1
7
Introduction
This introduction gives an overview of the history of the Kjeldahl Method and of
its evolution to the current state of the art.
1.1
History of the Kjeldahl Method
For almost 130 years the determination of nitrogen by means of the method
developed by the Danish chemist Johan Gustav Christoffer Thorsager Kjeldahl
(1849–1900) has been an internationally accepted standard. The method was introduced in 1883 at a meeting of the Danish Chemical Society by Johan Kjeldahl
as a means to determine nitrogen in barley and yeast [1]. The method named after its inventor has since found wide-spread application in life science and chemistry and has extended its scope to the determination of nitrogen and proteins in
dairy products, meat products, beer, cereals and other food materials.
Kjeldahl was a member of the innovative laboratory team at the Carlsberg
brewery in Copenhagen, also famous in microbiology for isolating the famous
beer yeast Saccharomyces Carlsbergensis which is still used today. As the head
of the chemistry department at the Carlsberg brewery he was involved in a very
modern problem: quality management and optimization of productivity. Kjeldahl
intended to determine the protein content of grain in order to find out how the
protein content influences quality and quantity of the brewed beer.
Figure 1:
Johan Kjeldahl in his laboratory at Carlsberg Brewery
in Copenhagen in the year
1880 (image by courtesy of
Carlsberg Archives, Copenhagen).
9. 8
Introduction
Table 1:
Citation from Kjeldahl’s
original publication of 1883.
Original citation
Translation
«Le principe de la nouvelle méthode
consiste donc à chauffer pendant quelque
temps la matière à analyser avec une forte
proportion d’acide sulfurique concentré
jusqu’à une température voisine du point
d’ébullition de l’acide, et à oxyder la
dissolution ainsi obtenue avec un excès
d’hyperpermanganate de potasse sec en
poudre. Dans ces conditions, l’azote des
substances organiques, … , se transforme
complètement en sulfate d’ammoniaque,
qui, l’oxydation une fois terminée et après
saturation avec la soude, peut être distillé
et dosé par les méthodes ordinaires.»
«The principle of the new method is to
heat the test material for some time with
a large quantity of concentrated sulfuric
acid at a temperature close to the acid’s
boiling point and to oxidize the solution
thus obtained with an excess of dry potassium per-manganate powder. Under these
conditions the nitrogen of the organic substances, … , is completely transformed
into ammonium sulfate which, once the
oxidation is completed and after saturation with caustic soda can be distilled and
determined by ordinary methods.»
Although individual chemicals used in the Kjeldahl method have changed over
the years it is possible to give a concise general definition:
The Kjeldahl Method consists in a procedure of catalytically supported mineralization of organic material in a boiling mixture of sulfuric acid and sulfate salts
at digestion temperatures between 340 and 370 °C. In the digestion process
the organically bonded nitrogen is converted into ammonium sulfate. Alkalizing the solution liberates ammonia which is quantitatively steam-distilled and
determined by titration.
1.2
Product Classes Amenable to the Kjeldahl Method
Proteins are of indispensable nutritional value for humans and animals and are
contained in juices, dairy products, food and feed [2]. The Kjeldahl method allows the calculation of protein contents in food samples based on the determined nitrogen which is a general constituent of all proteins. Organically bonded
nitrogen, amenable to the Kjeldahl method, is found in beer, yeast, barley, wine,
wheat, corn, rice, vegetables, beans, soy and nuts, milk and related dairy products, eggs, meat and sausages, fish and seafood.
The scope of Kjeldahl nitrogen determinations today also includes applications in the fields of environmental analysis, research and development, pharmaceutical, chemical and cosmetics industries and is also used in governmental
and regulatory laboratories.
10. Introduction
9
Food
Table 2:
Protein contents of some
foodstuffs.
Protein [%]
Apple
0.3
Peach
0.8
Carrot
1.0
Raspberry
1.3
Potatoes
2.0
Elderberry
2.5
Spinach
2.7
Horse-radish
2.8
Rose hip
3.6
Milk
3.2
Pea sprouts
5.1
Corn
9.2
Flour
11.0
Oats
12.6
Chicken
19.9
Halibut
20.1
Red beans
21.2
Beef
22.0
Lentils (dry)
22.9
Cheese (eg. Cheddar)
24.7
Peanuts
24.7
Sunflower seed
26.5
Ostrich
35.3
Soybeans
37.6
For packed and processed food nutrition fact labels are in use almost everywhere in the world, examples are given in Figure 2. The protein content is one of
the important parameters declared on nutrition fact labels.
Nutrition Facts
Nährwerte/valeurs nutritives/
valori nutritivi
Serving Size 67 g
Amount Per Serving
Calories 33
Calories from Fat 4
% Daily Value*
Total Fat 0g
1%
Saturated Fat 0g
0%
Trans Fat
Cholesterol 0mg
0%
Sodium 29mg
1%
Total Carbohydrate 7g
2%
Dietary Fiber 1g
5%
Sugars
Protein 2g
*Percent Dialy Values are based on a 2,000
calorie diet. Your daily values may be higher
or lower depending on your calorie needs.
100 g
enthalten/
contiennent/
contengono:
1 Stängel (17 g)
enthält/
bâton contient/
bastoncino contrine:
Energiewert/
valeur énergétique/ 1870 KJ
calore energetico (447 kcal)
320 KJ
(77 kcal)
Eiweiss/
protéines/
proteine
15 g
Kohlenhydrate/
glucides/carboidrati
56 g
davon/dont/di cui
· Zucker/sucres/zuccheri 30 g
Fett/lipides/grassi
davon/dont/di cui
· gesättigte Fettsäuren/
acides gras saturés/
acidi grassi saturi
· Cholesterin/
cholestérol
colesterolo
Ballaststoffe/
fibres alimentaires/
fibres alimentari
2.5 g
10 g
5g
18 g
3g
6g
1g
0 mg
0 mg
6g
1g
Natrium/sodium sodio 0.15 g
0.02 g
Figure 2:
Typical Nutrition Facts
Labels as used in North
America and Europe.
11. 10
Introduction
1.3
Procedures for Kjeldahl Nitrogen Determinations
The Kjeldahl procedure involves three major steps
Figure 3:
The three major steps in
Kjeldahl nitrogen determinations.
1.3.1
Digestion
In the digestion step the organically bonded nitrogen is converted into ammonium ions. Organic carbon and hydrogen form carbon dioxide and water, very
much reminiscent to an incineration process. In this process the organic material carbonizes which can be visualized by the transformation of the sample into
black foam. During the digestion the foam decomposes and finally a clear liquid
indicates the completion of the chemical reaction. The generalized non-stoichiometric chemical equation (1) shows how a general nitrogen containing starting
material (CHNO) is mineralized to dissolved ammonium ions.
(CHNO) + H2SO4
CO2 + SO2 + H2O + NH4+
(1)
In the original procedure published by Kjeldahl the mineralization was carried
out in boiling sulfuric acid. The oxidation was supported by the addition of the
strong oxidizing agent potassium permanganate. After its introduction by Kjeldahl,
the digestion reaction was further improved and optimized. Examples were the
addition of salts and the use of catalysts which allowed for shorter digestion time.
The most common salt used historically was potassium sulfate and the catalysts
were selenium and metal salts, particularly of mercury, copper or titanium.
Two types of heating units are used to heat up the sample together with the
reagents to boiling temperatures of 340 to 370 °C. One type are IR-digesters and
the other are block digesters (see «2.2.11 IR-Digestion versus Block-Digestion»,
p. 26).
Figure 4:
SpeedDigester K-425/439
(left), Block-Digestion Unit
K-437 (right).
12. Introduction
11
Figure 5:
Scrubber B-414 connected
to a Digestion Unit K-438.
After the digestion has lead to a clear liquid, an additional digestion time of
e.g. 30 minutes is usually added, in order to allow complete mineralization [3].
For the digestion working in a fume hood is highly recommended and the use
of the Scrubber B-414 provides additional safety to laboratory personnel and
environment as well as offering protection of the equipment against corrosion.
1.3.2
Distillation
After digestion the sample is allowed to cool to room temperature and the glass
sample tube is transferred to a distillation unit.
Neutralization of sulfuric acid
Prior to the distillation the acidic sample is neutralized by means of concentrated
sodium hydroxide solution (NaOH) as shown in equation (2).
H2SO4 + 2 NaOH
2 Na+ + SO42- + 2 H2O
(2)
Distillation in glass sample tube
In the distillation step the ammonium ions are converted into ammonia which is
transferred into the receiver vessel by means of steam distillation.
In a chemical equilibrium (see equation (3)) the solvated ammonium ions (NH4+)
produce ammonia gas (NH3) by reacting with hydroxyl ions (OH-) of excess sodium hydroxide. By the steam distillation ammonia is separated from the glass
sample tube and condensed together with water in the receiving vessel.
13. 12
Introduction
Figure 6:
Distillation units:
1. stand alone distillation
(K-355),
2. with external titrator
(K-360),
3. with built-in titrator
(K-370).
o
NH4+ + OH-
NH3(gas) + H2O
(3)
Condensate collection in receiving vessel
A common procedure to collect the ammonia in the receiver involves the presence of boric acid B(OH)3 dissolved in water which forms ions with ammonia
according to equation (4). The ammonia is quantitatively captured by the boric
acid solution forming solvated ammonium ions. See also «1.4 Blanks», p. 13.
B(OH)3 + NH3 + H2O
1.3.3
-
NH4+ + B(OH)4
(4)
Titration
The concentration of the captured ammonium ions in the boric acid are determined by means of an acid base titration commonly using standard solutions
of sulfuric or hydrochloric acid. Depending on the amount of ammonium ions
present, concentrations in the range of 0.01 N to 0.5 N are used. Titrations may
be carried out by means of a burette using an appropriate pH-indicator such as
Sher mixed indicator [4] (BUCHI 003512) to indicate the end point of pH = 4.65
(see «2.4.1 Boric Acid Titration», p. 31). For the preparation see «5.1 Two-Stage
Mixed Indicator according to Sher for Boric Acid Titration» (p. 44). A second
option is to attach a titration stand to the distillation unit and read the volume
of consumed acid from the display of the titrator. The most sophisticated procedure is the use of a Kjeldahl distillation unit with a built-in titrator and have the
calculation done by the software of the instrument. Whatever the choice of the
determination method, the chemical reaction is described by equation (5) showing the reaction of the tetrahydroxyborate anion B(OH)4- with a generalized strong
acid HX (X = Cl- etc.).
B(OH)4- + HX
X- + B(OH)3 + H2O
(5)
14. Introduction
1.4
13
Blanks
Blanks, containing all reagents apart from the test material, are necessary if a
Kjeldahl distillation is carried out. In the following outline this is discussed for the
case of boric-acid titrations (see chapter «2.4 Titration», p. 31):
During distillation of a blank an increase of the pH-value in the receiving vessel
is observed. This change in pH is due to dilution. The effect on the pH-value by
diluting boric acid can be explained by equation (10) for the pH-value of a weak
acid. In Table 3 the effect is demonstrated by means of experimental pH readings
as a function of the volume of added water to 60 mL of 4%, 2% and 1% boric
acid. In addition to the increase of pH due to dilution, also effects of traces of
volatile bases, inevitably present in reagents and equipment, are taken into account by blank determinations.
Vol H2O
4%
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
pH exp
2%
1%
4.65
4.85
5.02
5.14
5.25
5.35
5.42
5.48
5.54
5.59
5.64
5.67
5.71
5.75
5.78
4.64
4.74
4.84
4.91
4.96
5.00
5.05
5.08
5.11
5.13
5.15
5.17
5.18
5.19
5.20
4.65
4.72
4.80
4.83
4.85
4.88
4.92
4.93
4.95
4.97
4.99
5.01
5.03
5.04
5.05
In a distillation the extent of dilution depends on the distillation time and is
identical for blank and sample determinations. The determination of the samples
includes the pH increase due to dilution. This is taken into account in the calculations of the nitrogen contents in which the blank volumes are subtracted from
the volumes found for the samples (see chapter «2.5.1 Calculation for Boric Acid
Titration», p. 34)
As can be seen in Figure 7 the increase of the pH-value depends on the concentration of the boric acid. At lower concentrations the increase in pH due to the
distillation is less than for higher concentrations. This also leads to lower blank
consumptions if less boric acid is used.
A typical blank volume in a boric-acid titration, determined using a distillation
time of 4 minutes, is in the range of 0.1–0.2 mL if 0.25 mol/L H2SO4 is used as
a titrant.
For a better understanding of the chemistry involved in the boric-acid titration
and the associated pH-increase effected by dilution during the distillation, a view
at the chemical reaction and the chemical equilibria is given below:
Table 3:
Measured pH-values as a
function of the volume of
added water to 60 mL of
4%, 2% and 1% boric acid.
15. 14
Introduction
Figure 7:
Representation of experimental pH-values as a function of the volume of water
distillate added to 4%, 2%
and 1% boric acid.
Boric acid acts as a Lewis acid, an electron pair acceptor, in the chemical
equilibrium described by equation (6):
B(OH)3 + 2 H2O
B(OH)4- + H3O+
(6)
The chemical equilibrium is described by the law of mass action expressed
by equation (7).
K=
c(B(OH)4–) · c(H3O+)
c(B(OH)3) · c2(H2O)
(7)
The derivation of equation (10) is based on equations (8) and (9). The pKa-value
for boric acid can be found in the literature [5].
Ka =
c(B(OH)4–) · c(H3O+)
= 5.8 · 10–10 mol/L
c(B(OH)3)
pKa = 9.27
pH = pKa + log
(8)
(9)
c(B(OH)4–)
c(B(OH)3)
(10)
16. Determination of Nitrogen by the Kjeldahl Method
2
15
Determination of Nitrogen by the Kjeldahl Method
Nitrogen in the oxidized form of nitrates and nitrites and often nitrogen in aromatic heterocycles do not add quantitatively to the finally determined Kjeldahl
reaction product ammonia. In Table 4 some of the most important refractory
compounds for Kjeldahl nitrogen determination are summarized and in Table 8,
p. 17, a classification of organic nitrogen containing compounds is given.
Compound
Recovery [%]
Nitrate, nitrite (organic and inorganic)
<1
Aromatic N-heterocycles:
Pyridine, pyrimidine, thiazole, imidazole,
pyrazole
1–<80
Azo compounds
30–85
Hydrazines
<50
Table 4:
Refractory compounds
which show poor contribution to Kjeldahl nitrogen.
In the context of studies on the oxidation of organic nitrogen containing substances, a general chemical equation was published by Jurecek et al. [6] [7]
[8] in order to describe the oxidation. Kjeldahl digestions of nitrogen containing
chemical compounds/substances can either lead to ammonia, nitrate or elemental nitrogen. A theoretical consideration on chemical reactions in the Kjeldahl
digestion was published by Morita [9]. A short form of the chemical equations
described in [8] and [9] is given in equation (11) and the indices a, b, c are explained to the right.
CHONa+b+c + O
a NH3 + b HNO3 + c/2 N2
(11)
In Table 5 organic functional groups are listed and correlated to the respective
nitrogen degradation products a, b, and c as described above in equation (11)
[9] [10].
The chemical pathways of degradation processes include simultaneous reduction, dehydration, hydrolysis, substitution and other reactions. For (-NH2,
=NH, N, [R4N]x) the first step is protonation by sulfuric acid. The more alkaline
an amine the easier it is protonated aiding the cleavage of the C-N bond. Primary
amines are the easiest amines to be digested. In Table 6 the ease of reaction for
a series of amines is expressed by relative recovery rates referenced to methylamine.
a = groups producing NH3
b = groups producing HNO3
c = groups producing N2
17. 16
Table 5:
Degradation products
depending on functional
groups.
Determination of Nitrogen by the Kjeldahl Method
Amide
Amino
Heterogenic Nitrogen
Imino
IsocyanideIsooxocyanate
Isothiocyanate
Oxocyanate
Peptide
Nitrile group
-CONH2
-NH2
=N=NH
-NC
-NCO
-NCS
-OCN
-CONH-CN
-NHOH
-NOOH
-NO2
=NOH
NitrosoAzoAzino
DiazoniumHydrazone
Hydrazine-Group
1 100% is easiest degradation,
results based on nitrogen
recoveries of Kjeldahl reactions
without catalyst.
Functional Group
HydroxyamineIsonitro
NitroOxim-Group
Table 6:
Ease of degradation depending on type of amine.
Name
-NO
-N=N=N-N=
-N N+
-N-NH-R
-NHNH2
Type of Amine
Primary Amines
Secondary Amines
Tertiary Amines
Quarternary Ammonium
Methylamine
Aniline
2-Nitroaniline
Degradation
Product
Symbol
NH3
a
HNO3
b
N2
c
Ease of reaction1 [%]
90
80
76
54
100
85
50
A comparison of recovery rates is given in Table 7 for a selection of typical
amino acids. In amino acids neighboring carboxyl groups weaken the C-N bond
and the conversion into ammonia is facilitated compared to primary amines.
More difficult is the dissolution of a C-N bond if two amino groups are attached
as in lysine shown in Figure 9 because a stable piperidine carboxylic acid is generated [11]. In this case only approx. 50% of the nitrogen is recovered. Amino
acids containing aromatic heterocycles show a reduced nitrogen recovery, for
tryptophan for instance a recovery of 67% is reported [10]. With modern optimized digestions e.g. by using selenium and mercury free Kjeldahl tablets, higher
recoveries can be obtained for tryptophan but the problem of difficult digestions
remains. In studies dealing with protein determinations an average nitrogen recovery of 98% can be reached [10], and protein contents are derived from experimentally found nitrogen contents by means of a protein factor (see chapter
«2.5.3 Protein Content» p. 35)
18. Determination of Nitrogen by the Kjeldahl Method
17
Ease of reaction1 [%]
Type of amino acid
100
98
98
67
66
50
Aspartic acid
Proline
Arginine
Tryptophan
Histidine
Lysine
Table 7:
Ease of degradation depending on type of amino
acid.
1 100% is easiest degradation,
results based on nitrogen
recoveries of Kjeldahl reactions
without catalyst.
Figure 8:
Structure of the amino acid
lysine which contains two
amino-groups and formation of a stable piperidine
carboxylic acid.
Depending on the ease of Kjeldahl degradations the duration of a particular digestion needs to be adjusted for the highest possible recovery rate (see «2.2.10
Optimization of the Digestion», p. 25). Ammonia salts do not need digestion.
In Table 8 the ease of degradation is given as a function of the nitrogen containing chemical groups [10]. As can be seen, especially nitrogen containing heterocycles exhibiting a strong stabilization by resonance of the aromatic system
are not easy or even impossible to be digested. Organic nitrates and nitrites are
not accessible to Kjeldahl because of the high oxidation state which does not
allow the formation of ammonia.
Group
Azides (MeN3)
Azo compounds (-N=N-)
Carbamine group
Heterocycles
Hydrazine (NH2-NH2)
Imides, oximes
Nitrates
Nitrides
Nitrites (Me-NO2)
Nitro (R-NO2)
Purines
(uric acid, guanine, caffeine)
Acid amides
2.1
N recovery by Kjeldahl digestion
Recovery approx. 20%2
only partly3
very good
The higher the resonance stability the worse
30–54%
up to 100%
1%
10%
0%
50%
100%
100%
Sample Preparation
Two critical points involved in sample preparation are the amount of sample and
its homogeneity which will be discussed below in chapters «2.1.1 Amount of
Sample», p. 18, and «2.1.2 Mincing and Homogenization», p. 19. A further aspect is the expected titrant consumption which, for reasons of accuracy, should
be in a range of 5 to 20 mL for titrant concentrations of 0.5 to 0.01 mol/L if a
20 mL burette is used. An immanent problem associated to Kjeldahl digestions
is foam formation (see «1.3.1 Digestion», p. 10) and, especially if large sample
volumes are present, the risk of foaming over into the suction module. In such
cases the use of anti-foam agents can be of help. A common substance used as
Table 8:
Nitrogen containing groups
and typical recovery rates
for nitrogen by means of
Kjeldahl digestion.
2 Produces explosive HN3
3 Produces nitrogen N2
19. 18
Determination of Nitrogen by the Kjeldahl Method
anti-foam agent in Kjeldahl digestions is stearic acid of which a tip of a spatula is
added to the sample. Digestions of liquid samples, e.g. in TKN determinations,
may be affected by bumping caused by boiling retardation. In older descriptions
of such applications boiling stones or similar boiling aids were recommended.
With modern distillation units the distillation process may be completed by an
aspiration of the sample in which case the presence of boiling aids leads to
blockages of the hoses. For this reason digestion rods (BUCHI order number
043087 for a set of 10 pieces) are recommended. The digestion rods remain in
the glass sample tube during the distillation. Digestion rods also do not impede
the transfer of samples from the Kjeldahl Sampler K-371 to the Auto Kjeldahl Unit
K-370.
Amount of Sample
Depending on the expected nitrogen content, optimal amounts of sample are in
the range of 0.01 – 5 g as presented in Table 9. Most samples from food and feed
containing between 6 –30% proteins require sample amounts of 0.5 –1 g. Such
amounts of organic material can be digested using two Kjeldahl tablets of 5 g (see
table 15) and 20 mL of 98% sulfuric acid. With samples consisting predominantly
of pure proteins like ovalbumine, lactalbumine or other globular proteins, amounts
of 1–2 mg can still be digested in 300 mL sample tubes. One Kjeldahl tablet of
5 g in combination with 10 mL of 98% sulfuric acid is sufficient to break up the
organic matrix. Dealing with low nitrogen contents on the other hand requires
large sample amounts in order to produce a detectable amount of ammonia.
Maximum sample amounts depend on composition and the size of the digestion
glass tube. With milk samples a typical size amounts to 5 mL in 300 mL tubes and
one Kjeldahl tablet together with 13 mL 98% sulfuric acid are added. Low nitrogen
wax samples however may require 10 g. The limit of sample amounts normally
needs to be found experimentally and would be around 10 g in 500 mL glass
tubes. For water samples a maximum volume of 200 mL in 300 mL sample tubes
and a maximum of 330 mL in 500 mL sample tubes should be used (see chapter
«2.6 Limit of Detection and Limit of Quantification», p. 36)
For the weighing of samples analytical balances accurate to 0.1 mg should
be used. The actual weight of a sample required for analysis also depends on its
2.1.1
Table 9:
Correlation of nitrogen
contents (N[mg], N[%]) with
sample weight, number
of Kjeldahl tablets (KT),
volume of sulfuric acid
(Acid [mL]), concentration
of titrant (c(HX) [mol/L] and
titrant consumptions (Titrant
consumption sample [mL]
in 4% boric acid and Titrant
consumption blank [mL] in
4% boric acid).
Sample weight [g]
c(HX) [mol/L]
5
KT
Acid [mL]
2
1
0.5
0.125
4
40
3
30
2
20
1
10
1
10
N [mg]
0.5
2
2.5
7
10
50
100
Nitrogen N [%]
0.01%
0.04%
0.05%
0.14%
0.20%
1.00%
2.00%
0.03%
0.10%
0.13%
0.35%
0.50%
2.50%
5.00%
0.05% 0.10% 0.40%
0.20% 0.40% 1.60%
0.25% 0.50% 2.00%
0.70% 1.40% 5.60%
1.00% 2.00% 8.00%
5.00% 10.00% 40.00%
10.00% 20.00% 80.00%
0.01
0.05
0.1
c(HX) [mol/L]
0.5
Titrant consumption
sample [mL]
3.6
14.3
2.9
3.6
10.0
14.3
0.01
1.4
7.1
14.3
0.1
0.5
Titrant consumption
blank [mL]
<6
<6
1.8
5.0
7.1
0.05
<1.2
<1.2
<1.2
<1.2
<0.6
<0.6
<0.6
<0.2
<0.2
<0.2
For more refined optimization of Kjeldahl parameters the BUCHI Kjeldahl Calculator (Excel template) is
available on request.
20. Determination of Nitrogen by the Kjeldahl Method
19
homogeneity (see «2.1.2 Mincing and Homogenization», p. 19). More sample is
needed to achieve reproducible results for less homogeneous material.
As described above optimal sample amounts depend on the expected nitrogen contents but also affect the choice of titrant concentration. In Table 9 recommended sample weights are correlated with the amounts of catalyst, the volume
of 98% sulfuric acid and suggested titrant concentrations.
The effect of the Kjeldahl tablets added to 98% sulfuric acid is an increase of
the boiling temperature (see chapter 2.2.2, p. 21).
Depending on the nitrogen content per sample the terminology macro, semimicro- and micro Kjeldahl is used [10] [13]:
Kjeldahl Type
Tube Size
Macro Kjeldahl
Semimicro Kjeldahl
Micro Kjeldahl
2.1.2
Sample Amount
10–30 mgN/sample
0.1–3 mgN/sample
1–15 μgN/sample
300, 500 mL
300 mL
100 mL
Table 10:
Terminology macro, semi
micro and micro Kjeldahl.
Mincing and Homogenization
As a general rule of thumb particle sizes should not be larger than 1 mm. Inhomogeneous samples would lead to increased standard deviations in repeated
determinations. A useful measure for inhomogeneity is the relative standard deviation (rsd) expressed in % of the mean value. Protein contents of 6–30% of
homogeneous samples show rsd values < 1% and in turn rsd values > 1% are
strong evidence for insufficient homogeneity. It is essential to homogenize solid
samples.
Figure 9:
BUCHI Mixer B-400.
For inhomogeneous samples high precision of the measurements can not be
obtained using small sample sizes. It is recommended to homogenize samples in
order to be able to carry out the analysis with sample amounts as low as possible
since homogeneous samples are less critical in this respect.
21. 20
Determination of Nitrogen by the Kjeldahl Method
2.1.3
Drying of Samples
Drying of samples may be necessary if results referring to dry matter are required.
Alternatively the water content of a sample may be determined by classical water
analyses like Karl-Fischer titration or by gravimetric methods.
2.1.4
Aqueous Samples
In waste water applications, involving the determination of nitrogen by means of
the Kjeldahl digestion method, liquid samples are used and in a first step the water is evaporated. Together with organic nitrogen, waste water samples may also
contain ammonia and ammonium ions which will add to the result of determined
nitrogen. This type of Kjeldahl determination is referred to as a determination of
Total Kjeldahl Nitrogen (TKN). Depending on the expected nitrogen contents
suitable sample volumes have to be chosen (see Table 11).
Table 11:
Sample volumes for TKN
determinations.
Nitrogen concentration [mgN/L]
< 20
20–50
50–100
2.2
Volume of Sample [mL]
100
50
25
Digestion
Two different types of digestion units are used to heat up samples for Kjeldahl
digestions. One is referred to as an IR-digestion unit and the other one is called a
block digestion unit. IR digesters are equipped with a heating rod similar to those
used in an oven. The sample is directly irradiated in a heating zone of approximately one third of the tube’s height. With block digesters the sample tubes are
embedded in a boring of 3.5 cm if 300 mL tubes are used or of 8 cm for 500 mL
tubes respectively. The heat is transferred from the aluminum block through the
glass wall to the sample. IR- and block digesters are suitable for all applications
provided correct parameters are chosen. Important parameters are the speed
of heating up samples, the digestion temperature and the duration of the digestion. A major technical difference between IR- and block digestion consists
in the temperatures generated in the oven and in the block respectively. In IR
digestion a temperature of 600 °C is reached in the oven and in block digesters
the aluminium block is heated up to 420–450 °C. In both types of digestion units
the heat transfer to the sample ensures that in the sample mixture the required
temperatures of 350–380 °C are maintained during the Kjeldahl digestion (see
also «2.2.11 IR-Digestion versus Block-Digestion», p. 26).
IR digesters exhibit a heating zone of 8 cm. By means of an additional aluminium
block (BUCHI order number 040088) the heating zone can be increased to 8
cm in a regular block digestor also. A higher heating zone has a positive effect
in foaming samples by allowing rising gas bubbles to burst and reducing the
level of foam in the digestion tube. An additional aluminium block improves equal
evaporation of all positions of water samples e.g. in TKN determinations.
A further technical aspect associated with IR- and block digestion is the time
needed to heat up the respective digestion unit to the operation temperature.
IR digesters need 15 minutes and an aluminium block with 20 sample positions
approximately 60 minutes. If an additional heating block is used the heat-up time
increases to 80 minutes.
22. Determination of Nitrogen by the Kjeldahl Method
2.2.1
21
Acid Consumption per Sample
The volume of consumed concentrated sulfuric acid in a Kjeldahl digestion depends on the sample [10] [13] [14]. In Table 12 the consumption of 98% sulfuric
acid for fats, proteins and carbohydrates is shown.
Sample Component
Fat
Protein
Carbohydrate
A
[mL H2SO4/g]
B
[g H2SO4/g]
9.7
4.9
4.0
18–19
8.7–9.7
8.4
Table 12:
Consumption of volumes
(A) and weights (B) of 98%
H2SO4 for different components in samples.
Based on column A in Table 12 the consumption of H2SO4 for typical food
samples can be calculated by adding the individual acid consumptions per constituent. An example is given below for a wheat sample of 1 g:
Constituent
Calculation
Fat (assume 3.5%)
Protein (assume 12.5%)
Carbohydrate (assume 66.5%)
3.5% x 1.0 x 9.7 = 0.34 mL
12.5% x 1.0 x 4.9 = 0.61 mL
66.5% x 1.0 x 4.0 = 2.66 mL
Acid consumption
3.61 mL
Bradstreet published a recommended amount of 6.27 g H2SO4 for corn meal
which can be compared to the calculated 3.61 mL shown in Table 13 [15]. An
excess of sulfuric acid is needed because it ensures the formation of ammonium
ions and nitrogen loss by evaporation of ammonia is avoided.
2.2.2
Ideal Ratio of Salt to Sulfuric Acid
Pure sulfuric acid boils at approx. 290 °C and upon boiling it releases a surplus
of SO3 together with H2O until a 98.3% sulfuric acid is obtained finally boiling at
338 °C [12]. Commercially available sulfuric acid of 98% for this reason shows
a boiling temperature of 338 °C. With the addition of Kjeldahl tablets the boiling
temperature of the mixture in the sample tube is increased to a desired 350 to
370 °C. With time and duration of the digestion, the concentration of the sulfuric
acid in the sample tube decreases, leading to even higher temperatures. The
ratio of sulfuric acid to sulfate-salts is crucial for the boiling temperature actually
found. A good ratio is 1 g of Kjeldahl catalyst mixture to 2 mL of 98% sulfuric
acid. Typically for 1 g sample two Kjeldahl tablets of 5 g are used together with
20 mL of 98% sulfuric acid and digestion times of 90 minutes are applied.
The ideal ratio of sulfuric acid to sulfate salts by the end of a digestion should
be such that the sample temperature does not exceed 380 °C. Above 390 °C the
formation of elemental nitrogen gas (N2) becomes a possibility, eventually leading to too low nitrogen results. In Table 14 the ideal ratio of acid to salt is given.
Samples which exhibit final ratios close to the limits are prone to crystallization
after cooling. This is not desirable especially when the Kjeldahl Sampler is used
because then the suction tube would not be able to reach the bottom of the
glass and sample transfer would be hampered leading to an error message on
the distillation unit K-370.
Table 13:
Calculation of the consumption of concentrated sulfuric
acid by 1.0 g of sample.
23. 22
Determination of Nitrogen by the Kjeldahl Method
2.2.3
Acid Mixtures
In general food and feed applications 98% sulfuric acid is used for digestions.
Special applications may however call for modifications in the concentration of
sulfuric acid or mixtures of acids could be envisaged. As an example protein
determinations of milk and cream are often carried out using a 69% sulfuric acid
in order to reduce the risk of foaming (see chapter «2.2.6 Digestion by Hydrogen
Peroxide», p. 24). In modified Kjeldahl digestions mixtures of sulfuric and chromic
acid [6] [7] are described. Oxidative destruction of organic matrices however
is not limited to the nitrogen determination by the Kjeldahl method. Digestions
aiming at the analysis of phosphates in waste water are performed in a mixture
of sulfuric and nitric acid [31] and for the analysis of heavy metals hydrochloric
and nitric acid (acqua regia) are described in the literature [16]. Mixtures of sulfuric and nitric acids bear a potential to nitrify organic materials for instance in
electrophilic aromatic substitutions or if hydroxyl-groups are present. This could
lead to the production of very hazardous explosives. It is very important to follow
the published methods carefully and always assess the explosion potential when
nitric acid is involved.
2.2.4
Catalyst Used for Digestion
The composition of Kjeldahl tablets consists of more than 97% of a salt which
increases the boiling temperature of the sulfuric acid and of 1–3% of one or
more types of catalysts. The ideal digestion temperature for the Kjeldahl reaction is 370 °C. Usually potassium or sodium sulfate is used for the purpose of
increasing the boiling temperature. Typical catalysts are selenium or metal salts
of mercury, copper or titanium.
The ratio of sulfuric acid to salt and catalyst ideally is 20 mL H2SO4 to 10 g of
salt and catalyst e.g. in the form of 2 Kjeldahl tablets of 5 g.
Table 14:
Effect of ratio H2SO4 [mL]
to K2SO4 [g] on optimal
digestion temperature [°C].
H2SO4
[mL]
20
20
20
20
20
K2SO4
[g]
Boiling
temperature [°C]
Remarks
–
–
5
10
15
290
338
350
370
390
pure H2SO4
98% H2SO4
optimal digestion temp.
start of nitrogen loss
In Table 15 some of the commonly commercially available Kjeldahl tablets are
given.
Table 15:
Names, composition, use
and weight of the new
BUCHI Kjeldahl Tablets.
See www.buchi.com.
Name / Order
no.
Composition
Use
Weight
Titanium
# 11057980
3.5 g K2SO4
0.105 g CuSO4 5 H2O
0.105 g TiO2
Optimal compromise
between environmental and
performance priorities
3.71 g
Titanium Micro
# 11057981
1.5 g K2SO4
0.045 g CuSO4 5 H2O
0.045 g TiO2
Same as Titanium
(11057980) but for semimicro-Kjeldahl & micro
Kjeldahl applications.
1.59 g
4.98 g K2SO4
0.02 g CuSO4 5 H2O
The digestion with the
Missouri catalyst is more
environmentally friendly.
5g
Missouri
# 11057982
24. Determination of Nitrogen by the Kjeldahl Method
23
ECO
# 11057983
3.998 g K2SO4
0.002 g CuSO4
Our most environmentally
friendly catalyst, due to the
very low copper content.
4g
Antifoam
# 11057984
0.97 g Na2SO4
0.03 g Silicone Antifoam
Used as a general purpose
foam suppressant. This
catalyst has to be combined
with Titanium Micro or
Copper Micro.
1g
1.5 g K2SO4
0.15 g CuSO4 5 H2O
Combo tablets for antifoam
or for micro Kjeldahl
applications.
1.65 g
Copper Micro
# 11057985
Mercury- and selenium-free catalyst
Mercury-containing catalysts are hardly ever used at all today, due to their toxicity. The mixture of titanium dioxide and copper sulfate [17], however, also results
in a considerable reduction in the reaction time.
Missouri catalyst
The main feature of this catalyst is its environmental compatibility, due to the low
content of copper. This does, however, mean that the digestion takes longer.
2.2.5
Selecting the correct catalyst
The decisive factors for selecting a particular catalyst are the ecological and
toxicity aspects or practical facts like reaction time or foaming and sputtering.
While a toxic, selenium-containing catalyst reacts fastest, a copper-containing
catalyst is considerably safer for both humans and the environment. An ideal
compromise in this regard is the mixed catalyst consisting of copper- and titanium sulfate.
In water containing samples, e.g. Total Kjeldahl Nitrogen (TKN) determinations, strong foam formation and sputtering often is caused by Kjeldahl tablets.
In such a situation a catalyst mixture in powder form and the use of boiling rods
is appropriate.
Alternatively to selenium and metal-salt catalysts peroxides like hydrogen
peroxide or sodium persulfate together with sulfuric acid are used with very good
results (see chapter «2.2.7 The Chemical Process of the Kjeldahl Digestion Reaction», p. 24).
Figure 10:
Suction module for
digestion with H2O2.
25. 24
Determination of Nitrogen by the Kjeldahl Method
2.2.6
Digestion by Hydrogen Peroxide
Digestion supported by the oxidative power of hydrogen peroxide often leads to a
substantial reductions in digestion time and foaming. The digestion of milk using
Kjeldahl tablets for example needs 120 minutes digestion time compared to 30
minutes if hydrogen peroxide is the oxidant. A typical digestion of milk with H2O2
would use 5 g sample, 20 mL of 98% sulfuric acid and 30 mL of aqueous 30%
H2O2. The procedure is described in the BUCHI Application Note ‘AN 054/2010’
and available on request. A comparison between Kjeldahl digestions and digestions using H2O2 was presented in [19] for various food samples. Mechanistic
studies of the Kjeldahl microwave digestion of amino acids under the influence
of H2O2 were reported in [20]. Method descriptions of nitrogen determinations in
salami and milk [21] [22] [23] [24] can be found in chapter «7 Application Notes»,
p. 47 ff.) added to this publication.
2.2.7
The Chemical Process of the Kjeldahl Digestion Reaction
The oxidation process of organic material with sulfuric acid involves a first step
of carbonization in which the formation of water can be observed when it condenses at cool parts of the glass ware. Carbonization starts at room temperature
and is enhanced by increased temperatures. At higher temperatures the decomposition of carbonized material to carbon dioxide commences and is reflected
by foam formation expanding black sample material up in the Kjeldahl digestion
tube. It is important to keep foam formation under control in order to avoid poor
reflux of carbonized sample material attached to the glass wall. The choice of the
right parameters as described in «2.2.8 Parameters for Digestion», p. 24, is a key
for a successful digestion.
2.2.8
Parameters for Digestion
The significant parameters for digestion of a given sample are the amounts of
reagents and the temperature setting of the digestion unit. The volume of sulfuric
acid used is a function of the expected consumption of sulfuric acid in the redox
reaction converting sulfuric acid to sulfur dioxide. The digestion time depends on
the chemical structure of the sample, the temperature, the amounts of sulfate
salt and the catalyst. A practical recommendation distinguishes a first period of
time until the sample becomes translucent and a second period for the completion of the digestion. The second period of time is meant for the completion of
the conversion of the nitrogen degradation products to ammonia [3] [14]. As a
rule of thumb, after the observation of a clear translucent solution, 30 minutes
are sufficient to complete the reaction [3]. By the end of the digestion a surplus
of acid has to be present in a sufficient amount in order to keep the non-volatile ammonium ions in solution and prevent the loss of volatile ammonia. If the
amount of sulfuric acid is too low in the boiling sulfuric acid the organic material
may produce carbon smoke leaving the Kjeldahl glass tube. The excess sulfuric
acid ideally is at a concentration which is high enough to keep the sulfate salt in
solution such that after cooling no crystallization occurs. The added sulfate salt
increases the boiling temperature of the sulfuric acid solution. The ratio of sulfate
salt to sulfuric acid should be high enough to allow a starting boiling temperature
of 350 °C and must never lead to an increase of the sample temperature higher
than 380 °C during the entire digestion. If the temperature is below 350 °C the
reaction is slowed down and at temperatures above 390 °C nitrogen loss will
26. Determination of Nitrogen by the Kjeldahl Method
25
be encountered. In this temperature range of 350–380 °C the Kjeldahl reaction
takes place and the speed of reaction is further enhanced by the added catalysts
(see «2.2.4 Catalyst Used for Digestion», p. 22).
2.2.9
Temperature
The digestion temperature is controlled by the boiling temperature of the mixture
of sulfate salts and sulfuric acid (see «2.2.2 Ideal Ratio of Salt to Sulfuric Acid»,
p. 21). As discussed above in «2.2.7 The Chemical Process of the Kjeldahl Digestion Reaction», p. 24, foam formation is a function of the temperature. Monitoring
the temperature along the digestion process is a way to control foam formation.
In practice the sample temperature is increased stepwise parallel to the chemical
degradation process under way. This can be done manually but also is an instrumental feature with semi-automated digestion units. A typical temperature ramp,
as it can be programmed on a Digest Automat K-438 or by means of a Control
Unit B-436, could look as given in Table 16.
Step
Temperature
[°C]
Time
[min]
1
2
3
4
cool
100
150
250
420
Process
5
10
25
90
Evaporation of water
Beginning of carbonization
Foam control
Kjeldahl reaction
Ready for distillation
Table 16:
Temperature program used
on the Digest Automat
K-438.
Typical digestion parameters are given in Table 17:
Parameter
Sulfuric acid
5 g Kjeldahl tablets
Warm up time
Digestion time
<0.5 g sample
1 g sample
<5 g sample
10 mL
1
30 min
90 min
20 mL
2
30 min
90 min
30 mL
3
30 min
90–120 min
The ratio of sulfuric acid [mL] to sulfate salt [g] for a general sample is 2 and
for samples containing fat 2–3. The digestion of fat consumes more sulfuric acid
than a non fat containing sample [14].
2.2.10
Optimization of the Digestion
Digestion times depend on the type of sample, the volume of sulfuric acid, the
ratio of acid to salt and the type of catalyst (see «2.2.8 Parameters for Digestion»,
p. 24).
Dependence on Sample
Aromatic substances take longer for complete digestion. In Table 18 digestions
for urea, glycine and tryptophan clearing times and times used for total degradation are correlated with experimental recovery rates. The aromatic tryptophan
shows the typical dependence of the digestion time on resonance stabilized
nitrogen.
Table 17:
Digestion parameters for
typical sample weights.
27. 26
Determination of Nitrogen by the Kjeldahl Method
Table 18:
Correlation of times when
samples become translucent (clearing) and total
digestion time with recovery
rates using selenium and
mercury free Kjeldahl tablets
(see Table 15).
Compound
Clearing
[min]
Digestion time
[min]
Recovery
[%]
Urea
Glycine
Tryptophan
Tryptophan
Tryptophan
11
15
16
16
16
90
90
90
200
300
99 –101
99.5
97.8
98.8
99.5
Dependence on Volume of Sulfuric Acid
Samples consisting of or containing fat, oil or carbohydrates tend to carbonize under strong foam formation. By increasing the amount of sulfuric acid the degradation of the carbonized material is facilitated and the formation of carbon smoke
reduced. Samples with low nitrogen contents require increased sample amounts
in order to bring determinable amounts of nitrogen into the sample tube. Large
samples require more sulfuric acid in order to achieve complete digestion.
Dependence on Ratio of Sulfuric Acid to Sulfate Salt
With samples requiring larger amounts of sulfuric acid the ratio of sulfuric acid
to sulfate salts has to be kept constant in order to maintain an optimal boiling
temperature.
Dependence on Type of Catalyst
As described in «2.2.4 Catalyst Used for Digestion», p. 22, different types of
catalysts are commercially available for specific types of samples. Fat, oil and
heterocyclic aromatic compounds are more easily digested if the catalyst contains selenium.
Further optimizations of the conditions of digestions can be achieved by the
measures shown in Table 19. Large liquid samples for the determination of total
Kjeldahl nitrogen (TKN) can be handled more easily if boiling rods are used. Boiling chips are not recommended because they can cause problems to the distillation units by congesting and blocking hoses and they can lead to damage of
membranes in valves and pumps. Antifoam agents generally reduce the surface
tension and viscosity of samples which facilitates the release of gases from the
foam. A commonly appropriate antifoam agent is stearic acid of which a tip of
a spatula usually is sufficient to suppress foaming. The amount of foam formed
is proportional to the sample amount and thus foaming can be controlled by
minimizing this amount. Consequently also larger glass sample tubes of 500 mL
instead of 300 mL are a solution to the problems. Critical samples may also be
pretreated or predigested at room temperature with hydrogen peroxide.
Table 19:
Further measures for improved conditions in digestions, of boiling and foaming
samples.
2.2.11
Sample
TKN: large volume
Foaming
Boiling Rods
Antifoam Agent
Temperature Ramp
yes
no
no
yes
no
yes
IR-Digestion versus Block-Digestion
IR and block digestions are both suitable for all applications but optimal digestion parameters usually differ slightly. IR digestors usually show a reduced risk
for foaming because the higher heating zone apparently breaks the bubbles of
28. Determination of Nitrogen by the Kjeldahl Method
27
Figure 11:
Heating systems in IR and
block digesters.
rising foam such that the carbonizing material flows back. In addition carbon
deposits at the glass walls are refluxed more efficiently in IR digestion units. The
preheating period for an IR digestor is in the range of ten minutes compared to
60 minutes for a block digester.
Block digesters however fit into the scheme for automatization of a subsequent distillation and titration, especially if a sampler is used.
2.2.12
Limits of the Kjeldahl Method
In some applications for nitrogen determinations the digestion is not necessary
or does not lead to the desired conversion to ammonia. A sample already containing ammonia or ammonium-ions can be distilled directly without digestion.
A classical Kjeldahl digestion would not be successful if a sample contains inorganic nitrates or nitrites. In this case a reductive method however can convert the
nitrogen entity into ammonia. In the following reference is made to such methods
outside the limits of the Kjeldahl Method.
Ammonia and Ammonium Ions
If ammonium ions or ammonia have to be determined the digestion step is not
needed and the method of choice would be to raise the pH to about 9–10 e.g. by
means of MgO or phosphate buffer followed by a steam distillation [25].
Reduction of Nitrates and Nitrites
The Kjeldahl digestion method can be applied to the determination of organically bonded nitrogen but would not yield results in samples containing nitrates
and nitrites. For the determination of nitrogen in nitrates and nitrites the use of
Devarda alloy powder reduces the nitrogen compound to ammonium ions. The
Devarda reagent consists of aluminium (44–46%), copper (49–51%) and zinc
(4–6%). The reduction reaction described in equation (12) is carried out in the
distillation unit and no digestion is done.
3 NO3- + 8 Al + 5 OH- + 18 H2O
3 NH3(g) + 8 [Al(OH)4]-
(12)
Besides the Devarda reagent other reductive distillations are reported using Fe(II)
or salicylic acid as reductive agents [26].
2.2.13
Suction Manifold
For both types of digestion units, the IR and the block digesters, specifically
designed suction units are used to conduct the reaction gases into an absorbing media be it by means of a water jet pump or a scrubber. The intention of the
29. 28
Determination of Nitrogen by the Kjeldahl Method
Figure 12:
BUCHI Scrubber B-414.
suction manifold is to avoid a health hazard to laboratory personnel. The use of
a water jet pump would at least serve this purpose but is not environmentally
sound. A better solution consists in the use of a scrubber filled with a neutralizing
solution. BUCHI offers the Scrubber B-414 which is specially designed for Kjeldahl applications. A final remark to the strength of suction may be appropriate
at this point. The suction must only be weak, approximately 200 mbar below air
pressure. This is sufficient to transport the toxic fumes but does not enhance the
evaporation of sulfuric acid.
2.3
Table 20: Typical distillation
parameters for a boric acid
titration on the Auto Kjeldahl
K-370.
4 Setting for K-370
Distillation
Typical distillation parameters for a boric acid titration are given in Table 20. The
titration types «boric acid titration» and «back titration» are explained in «2.4
Titration», p. 31.
Water
NaOH (32%)
Reaction time
Distillation time
Steam power
Stirrer speed
50 mL
90 mL
5s
240 s
100%
54
Typical distillation parameters for a back titration (see «2.4.2 Back Titration»,
p. 32) are given in Table 21. The slower stirrer speed during distillation reduces
the risk of losing sulfuric acid due to splashes to the glass wall.
Table 21: Typical distillation
parameters for a back titration on the Auto Kjeldahl
K-370.
5 Setting for K-370
Water
NaOH (32%)
Reaction time
Distillation time
Steam power
Stirrer speed
50 mL
90 mL
5s
240 s
100%
45
30. Determination of Nitrogen by the Kjeldahl Method
29
Figure 13:
Autokjeldahlsystem
K-370/371.
2.3.1
Diluting the Digestion Solution
Before the sulfuric acid is neutralized by adding concentrated sodium hydroxide
solution the sample is diluted with distilled water. This is done to avoid splashing
of the sample due to boiling induced by the heat of reaction dissipated when the
concentrated acid and base are mixed.
If digested samples cannot be processed directly after cooling, when standing for some minutes to hours, crystallization of the samples can be observed.
In manual distillations this may lead to too low results for nitrogen if the solids
are not dissolved. If the Kjeldahl Sampler K-371 is used solid samples would
hamper sample transfer resulting in an error message and the determination
to be skipped. If samples are diluted with 10–20 mL of water just after cooling,
crystallization can be avoided.
2.3.2
Neutralizing the Digestion Solution
The strong acid keeps the ammonium ions dissolved in the sample tube. Neutralizing the acid by means of concentrated sodium hydroxide solution drives
the chemical equilibrium between ammonium ions and ammonia towards the
production of ammonia. In a basic environment ammonia can be driven out of
the sample by means of steam distillation. The chemical reactions for the neutralization and the distillation are described by equation (2) p. 11.
2.3.3
Steam Distillation
After the neutralization of the acid a waiting period of some seconds, called
«reaction time» is set by the distillation unit in order to avoid splashes due to
overheating when the hot steam enters the mixture already heated by the neutralization reaction. A distillation should last long enough such that more than
99.5% of the ammonia is recovered in the receiver vessel. A typical distillation
time is 4 minutes at a steam power setting of 100%.
The starting volume for a distillation in 300 mL glass sample tubes should not
be larger than 200 mL and in 500 mL tubes a volume of 300 mL should not be
exceeded. If the starting volume is too large a risk of carry over of sample solution into the cooler and receiver is possible.
31. 30
Determination of Nitrogen by the Kjeldahl Method
2.3.4
Receiving Vessel of Distillate
The receiving vessel for the distillate is filled with an absorbing solution in order to
capture the dissolved ammonia gas. Common solutions are aqueous boric acid
of 2–4% concentration if a boric acid titration is performed. In back titrations the
Figure 14:
Titration unit with receiving
vessel.
absorbing solution is a precisely dosed volume of H2SO4 standard solution. In
the first case, the ammonia forms solvated ammonium cations and tetrahydroxyborate (B(OH)4-) anions (see equation (4)) and in the second case the ammonia
reacts with sulfuric acid to ammonium ions. In the boric acid type of titration the
tetrahydroxyborate is titrated with a standard solution of a strong acid. In the
case of back titration sodium hydroxide standard solution is used in order to
determine the amount of sulfuric acid which reacted with ammonium ions.
It is important that the inlet from the condenser is immersed in the absorption
solution contained in the receiver vessel.
2.3.5
Distillation Time
The actual distillation time needed for a quantitative distillation can be evaluated
and optimized by running verifications on the distillation unit under consideration.
A reference material of choice is ammonium di-hydrogen phosphate. A recovery
rate indicating a complete transfer of ammonia into the receiving vessel should
be in the range of 98.5–101.5%. If dry ammonium di-hydrogen phosphate is
used and provided the determination is carried out with appropriate care, recoveries of > 99.5% can be reached (see «2.7.1 Verification», p. 38).
2.3.6
Steam Power
Depending on the steam generator 130–160 mL of condensate are distilled into
the receiver vessel in five minutes time if set to 100% steam power. The distillation units K-355, K-360 and K-370 allow settings of 30–100% steam power. This
allows distillations at a lower pace which can be of help if the speed of distillation
is prescribed by legal regulations or if optimizing recovered substance with a
minimum of distilled volume is sought.
32. Determination of Nitrogen by the Kjeldahl Method
2.4
31
Titration
After the completed steam distillation the determination is concluded by a quantification of ammonia. This is most commonly done by means of titration. With
the BUCHI distillation units K-350 and K-355 titrations have to be carried out
separately either manually using a burette or by means of titrator using either a
pH-electrode or a colorimetric electrode. With the BUCHI distillation unit K-360 it
is possible to connect a titration unit and initiate titration by a TTL signal. Finally
the high end distillation unit K-370 is equipped with a built-in titrator and the
software for the respective data evaluation.
Two types of titrations are commonly used, the boric acid titration being the
method of choice because it allows automatization without further equipment.
The alternative type of titration is called back titration. The two types of titrations
are described in the following.
It is necessary to verify that there are no air bubbles in the tubing of the titrant.
Air bubbles will cause a false titrant consumption reading, yielding high results. Incorrect results may also be caused by wrong titrant, calculation errors or a defective or badly calibrated electrode (see also chapter «3 Troubleshooting» p. 41).
Figure 15:
Semiautomatic distillation
unit with external titrator
(BUCHI KjelFlex K-360,
Metrohm DMP 785).
2.4.1
Boric Acid Titration
As shown in equation (4) ammonia and boric acid form ammonium- and tetrahydroxyborate ions. According to equation (10) the pH rises upon addition of NH3
during the distillation when the NH3 is captured by the transformation into NH4+ions. In the titration with sulphuric acid standard solution the tetrahydroxyborate
anions react to ammonium sulfate and boric acid. The reaction scheme is given
in equation (13).
2 NH4+B(OH)4- + H2SO4
(NH4+)2SO42- + 2 B(OH)3 + 2 H2O
(13)
By the addition of acid titrant the ratio of tetrahydryoxborate and boric acid
becomes smaller and in agreement with the equation (10) the pH decreases.
As can be seen from Figure 16 the point of inflection of the titration curve is at
pH = 4.65 (see «5.1 Two-Stage Mixed Indicator, p. 44). This is the reason for
33. 32
Determination of Nitrogen by the Kjeldahl Method
Figure 16:
Titration curve for ammonium tetrahydroxyborate
with acid.
preferably adjust the pH of the boric acid to 4.65 before distillation and use an
end-point pH = 4.65 for the titration.
The detection of the end point for a manual titration can be carried out by
means of Sher mixed indicator. If a titrator is used the end point is set to pH =
4.65, the volume of acid titrant is determined and the nitrogen content calculated. Alternatively instead of the pH-electrode a colorimetric electrode can be
used to detect the color change of the Sher indicator at pH = 4.65.
Typical parameters for a boric acid type of titration are given in Table 22.
Table 22:
Typical titration parameters
for a boric acid titration on
the Auto Kjeldahl K-370.
2.4.2
Type
Boric acid
Titration solvent
Volume receiving sol.
Min. titration time
Max. titration volume
Titration mode
Titr. pH meas. Type
End-point pH
Stirrer speed
HCl or H2SO4 standard solution
60 mL
5s
40 mL
Standard
Endpoint
4.65
7
Back Titration
The back titration is an indirect determination of the amount of distilled ammonia.
As shown in equation (15) an excess of sulfuric acid standard solution reacts with
the NH3 in the distillate. The residual sulfuric acid is titrated with sodium hydroxide standard solution and by difference the amount of ammonia is calculated.
Reaction scheme for back titration
During distillation:
H2SO4 (total) + 2 NH3
SO42- + 2 NH4+
(14)
34. Determination of Nitrogen by the Kjeldahl Method
33
During titration:
H2SO4 (residual) + 2 OH-
SO42- + 2 H2O
(15)
In equation (14) the chemical reaction during the distillation is described and
in (15) the chemical reaction during the titration with sodium hydroxide standard
solution is given. The difference of the initial amount of sulfuric acid (H2SO4 (total))
and the amount of sulfuric acid present after the distillation (H2SO4 (residual)) corresponds to the distilled ammonia.
The titration of a strong acid with a strong base requires an indicator exhibiting
a pKa close to 7 if a manual titration is performed. If a titration stand is used an
end point of pH = 4.5–9.5 is appropriate. The amount of ammonia is calculated
according to equations (21) to (25) (see chapter «2.5.1 Calculation for Boric Acid
Titration», p. 34). The blank titration is carried out with an empty sample tube and
basically corresponds to the initial amount of H2SO4. Small amounts of ammonia
however, stemming from contaminations of the system, are accounted for by
including blank determinations into the procedure.
Typical parameters for a titration of the type «back titration» are shown in
Table 23.
Type
Titration solvent
Receiving solvent
Volume receiving sol.
Min. titration time
Max. titration volume
Titration mode
Titr. pH meas. Type
End-point pH
Stirrer speed
2.5
Back titration
NaOH standard solution
H2SO4 0.1 M
60 mL
1s
40 mL
Standard
Endpoint
4.8
4
Table 23:
Typical titration parameters
for a back titration on the
Auto Kjeldahl K-370.
Calculations
Calculations can aim at results expressed in absolute amounts of nitrogen mgN
per sample or in terms of concentrations either %N, mgN/kg, mgN/L etc.
The parameters needed for the calculations are:
Known parameters
cacid
True concentration of standard acid solution6
z
Valency of reaction
cNaOH Nominal concentration of standard
base solution7
M(N) Atomic mass of nitrogen
0.005 mol/L–0.5 mol/L
14.0067 g/mol
Experimentally determined parameters
Vblank Titrant consumption for blank
msample Sample weight
Volsample Sample volume
Vsample Titrant consumption for sample
fNaOH
Titer of NaOH titrant
[mL]
[g]
[mL]
[mL]
dimensionless
0.005 mol/L–0.5 mol/L
1 for HCl, 2 for H2SO4
6 It is assumed that a commercially available standard acid
solution is used.
7 Unless a fresh commercially
available standard NaOH solution is used, the titer has to be
determined.
35. 34
Determination of Nitrogen by the Kjeldahl Method
Calculated results
W(N) Weight of nitrogen in sample
w(N) Weight fraction of nitrogen
c(N)
Volume fraction of nitrogen
%(N)wt Weight percent of nitrogen
%(N)vol Volume percent of nitrogen
2.5.1
[g]
[gN/g sample]
[gN/mL sample]
[g/100 g sample]
[g/100 mL sample]
Calculation for Boric Acid Titration
Nitrogen Content
Absolute amount of nitrogen [g]
W(N) =
(Vsample – Vblank) · M(N) · cacid · z
1000
(16)
Weight fraction of nitrogen [gN/g sample]
(Vsample – Vblank) · M(N) · cacid · z
W(N)
=
msample · 1000
msample
w(N) =
(17)
Volume fraction of nitrogen [gN/mL sample]
c(N) =
(Vsample – Vblank) · M(N) · cacid · z
W(N)
=
Volsample · 1000
Volsample
(18)
Weight percentage of nitrogen
%(N)wt =
(Vsample – Vblank) · M(N) · cacid · z
= 100 · w(N)
msample · 10
(19)
Volume percentage of nitrogen
%(N)vol =
2.5.2
(Vsample – Vblank) · M(N) · cacid · z
= 100 · c(N)
Volsample · 10
(20)
Calculation for Back Titration
Nitrogen Content
Absolute amount of nitrogen [g]
W(N) =
(Vblank – Vsample) · M(N) · cNaOH · fNaOH
1000
(21)
Weight fraction of nitrogen [gN/g sample]
w(N) =
(Vblank – Vsample) · M(N) · cNaOH · fNaOH W(N)
=
msample · 1000
msample
(22)
Volume fraction of nitrogen [gN/mL sample]
c(N) =
(Vblank – Vsample) · M(N) · cNaOH · fNaOH
W(N)
=
Volsample · 1000
Volsample
(23)
36. Determination of Nitrogen by the Kjeldahl Method
35
Weight percentage of nitrogen
%(N)wt =
(Vblank – Vsample) · M(N) · cNaOH · fNaOH
= 100 · w(N)
msample · 10
(24)
Volume percentage of nitrogen
%(N)Vol =
2.5.3
(Vblank – Vsample) · M(N) · cNaOH · fNaOH
= 100 · c(N)
Volsample · 10
(25)
Protein Content
Especially in food and feed applications the determination of Kjeldahl nitrogen is
carried out with the intention to calculate the protein content. Over the time from
1900 to 1982 the concept of multiplying the experimentally determined nitrogen
content by an empirical protein factor fprotein was widely used [10]. For a «general protein», the concept assumes an average nitrogen content of 16%N which
leads to a «general protein factor» of 6.25 as described by equation (26).
%P =
%(N) · 100%
= %(N) · 6.25 = %(N) · fprotein
16%
(26)
Empirical protein factors for individual protein containing foodstuffs were determined based on their average nitrogen contents. In Table 24 specific protein
factors are listed [10] [27]. Already in 1982 the concept of protein factors was
heavily disputed because with improved analytical tools available for the analysis of amino acids and proteins more precise protein determinations became
feasible [10]. As a consequence adjusted protein factors were derived and are
supported by official bodies [27] [28] [29].
%(N) Weight percent of
nitrogen.
fprotein Empirical protein
factor.
Figure 17:
Typical food sample.
37. 36
Determination of Nitrogen by the Kjeldahl Method
Table 24:
Empirical protein factors
for the Kjeldahl method.
Food
General
6.25
Animal origin
Eggs and egg products
Gelatine
Meat and meat products
Fish, sea animals
Milk, milk products, cheese, whey
6.25
5.55
6.25
6.25
6.38
Grains & cereals
Barley, oats, rye
Corn
Rize
Wheat
Barley, oats, rye
Full grain products
Bran
5.83
6.25
5.95
5.70
5.83
5.83
6.31
Fruits
Fruits and fruit products
6.25
Legumes
Vegetables and products made of vegetables (except of soy)
Beans
Soy and soy products
6.25
6.25
5.71
Nuts
Nuts (treenut, coconut, chestnut etc.
except of peanuts and almonds)
Peanuts
Almonds
5.30
5.46
5.18
Seeds
Oilseed (except of peanuts)
2.6
Factor
5.30
Limit of Detection and Limit of Quantification
For the evaluation of the limit of detection and the limit of quantification in Kjeldahl
nitrogen determinations established rules are given by the German and European Norm DIN EN 32 645 [30]. In a rough approximation the limit of detection
(xLOD) can be estimated according to equation (27) and the limit of quantification
(xLOQ) is expressed by equation (28).
xLOD =
sB Standard deviation of
blank determination.
b Slope of calibration
curve.
3 · sB
b
xLOQ = 3 · xLOD
(27)
(28)
Ten blank determinations and a total of 10 data points of equidistant calibration steps in the range of the xLOD and xLOQ are required as a basis for the evaluation according to equations (27) and (28). sB is determined from the 10 blank determinations and the calibration curve consists of a plot of the known quantities
in standard solutions against the experimental results. After the determination of
the limits of the analytical procedure results are reported according to a scheme
given in Table 25.
38. Determination of Nitrogen by the Kjeldahl Method
37
Result
Expression
Additional Information
x > = xLOQ
xLOD < x < xLOQ
x < xLOD
determined content
detected
not detected
standard deviation
can not be determined, xLOQ
not higher than 2*xLOD
Table 25:
Reporting of analytical
results [DIN EN 32645).
By means of volumetric titrations nitrogen amounts per sample of as little as
0.05 mg can be detected however associated with relative standard deviations
of >1%. Based on evaluations following DIN EN 32 645 for the BUCHI distillation
units values of xLOD between 0.1 mg and 0.2 mgN per sample can be found in
BUCHI brochures. Using equation (28), it can be estimated that nitrogen quantities of 0.3–0.6 mgN per sample are accessible for quantification.
A short discussion of the limit of quantification and its practical effect on optimization of sample amounts seems appropriate in this place: Food and feed
samples like milk, meat, cereals, beans contain high amounts of proteins and the
determined nitrogen content are significantly higher than XLOQ. Fats and carbohydrates are more critical samples in this respect. This class of samples tends to
foam and requires a large amount of sulfuric acid due to its chemical structure.
In addition such materials are usually low in nitrogen thus requiring large sample
sizes in order to perform determinations above xLOD. For low N samples an optimum has to be found with regard to the limitation of sample size due to the size
of the sample tube, the threat of foaming and the demand on sulfuric acid, salt
and catalyst. Generally 2 g of sample is the maximum in 300 mL glass sample
tubes and 4 g in 500 mL tubes can be handled by Kjeldahl. Larger amounts can
be envisaged but need optimized procedures resorting to the use of anti-foam
agents, pre digestion by hydrogen peroxide and temperature ramps.
In total Kjeldahl nitrogen (TKN) determinations aqueous samples are involved
of which the major portion is evaporated in the digestion process. In TKN determinations xLOD and xLOQ often are referred to in terms of concentrations [mg/L]. In
TKN analyses sample volumes used in the BUCHI digestion- and distillation units
are at a maximum 200 mL if 300 mL glass sample tubes are used and can reach
330 mL in 500 mL glass tubes. Consequently the LOQ’s of nitrogen expressed
in [mg/L], sometimes simply referred to as [ppm], is between 0.5 and 1 ppm if
300 mL glass tubes are used and 0.3 and 0.6 ppm in 500 mL sample tubes.
Figure 18:
IQ/OQ documentation.
39. 38
Determination of Nitrogen by the Kjeldahl Method
2.7
2.7.1
Verification
In laboratories working under GLP or similar regulations it is mandatory to prove
the correct function of the instrumentation used to perform an analysis. Instrument qualifications (IQ), operation qualification (OQ) and performance qualification (PQ) also require verification of the equipment.
Distillation units are verified by a determination of a stoichiometric, pure and
dry ammonium salt of which the nitrogen content can be calculated. The ratio of
the determined to the expected nitrogen content is expressed as recovery in %.
Recovery rates in a range of 98.5–101.5% are proof for a well performing distillation unit. A well suited substance is ammonium dihydrogen phosphate.
The digestion unit is verified by running a digestion of a stoichiometric, pure
and dry nitrogen containing substance which undergoes an unproblematic Kjeldahl reaction. A well suited substance is glycine.
Verification of Distillation Unit
Reagents / Chemicals
– Sulfuric acid c = 0.25 mol/L
– Sulfuric acid conc. 98%
– Boric acid 4% (adj. to pH 4.65)
– Sodium hydroxide 32% / 33%
– Ammonium dihydrogen phosphate > 99.5%
– Ammonium dihydrogen phosphate Suprapur (99.99%)
The theoretical total nitrogen content of ammonium dihydrogen phosphate is
N% = 12.18%.
Procedure
If the ammonium dihydrogen phosphate contains moisture drying may be required. This extra step is recommended if the resulting recovery rate of a preliminary trial did not yield results in a range of 98.5–101.5%. If drying of the reference
substance is needed, use portions of 5–10 g of ammonium dihydrogenphosphate salt and dry in the oven at a temperature of 105 °C for 8 hours. Let the salt
cool down to room temperature in a desiccator filled with activated silica gel or
other desiccant. After cooling transfer dry ammonium dihydrogen phosphate to
a tight glass container.
Three empty Kjeldahl glass tubes are used as blank samples and three samples of ammonium dihydrogen phosphate are prepared. For each sample weigh
in 0.8 g of dried ammonium dihydrogen phosphate with an accuracy of ± 0.1 mg
on nitrogen free paper. Transfer the reference including the paper into a Kjeldahl
glass sample tube. If nitrogen free paper is not available perform a weight by
difference.
40. Determination of Nitrogen by the Kjeldahl Method
39
Distillation and boric acid titration
Distillation
Titration
Water
80 mL
Type
NaOH (32%)
Reaction time
Distillation time
Steam power
90 mL
5s
300 s
100%
Asp. sample
Asp. rec. soln.
Stirrer speed
yes
yes
5
Titration solvent
Volume receiving sol.
Min. titration time
Max. titration volume
Titration mode
Titr. pH meas. Type
End-point pH
Algorithm
Stirrer speed
4% boric acid
pH = 4.65
H2SO4 0.25 M
60 mL
1s
40 mL
Standard
Endpoint
4.65
1
7
Table 26:
Distillation parameters
for boric acid titration of
ammonium dihydrogen
phosphate.
Calculation
Based on equation (19) and taking the purity of the reference substance into
account in equation (29) for %N(det) the results are calculated as percentage of
nitrogen and the recovery is calculated as given in equation (30).
%(N)det =
R=
%(N) · P
100
%(N)det · 100%
%(N)theor
(29)
(30)
Results
The relative standard deviation (rsd) of the blanks should be lower than 5%. If
the rsd is higher, the blank determination should be repeated until it meets the
condition for three consecutive blanks. Recoveries should be between 98.5 and
101.5% and the rsd for the %(N)det should be lower than 1%.
2.7.2
Verification of Digestion Unit
Reagents / Chemicals
– Sulfuric acid c = 0.25 mol/L
– Sulfuric acid conc. 98%
– Boric acid 4% (adj. to pH 4.65)
– Sodium hydroxide 32% / 33%
– Kjeldahl tablets Hg/ Se-free
– Glycine of purity >= 99.7%
The theoretical total nitrogen content of glycine is 18.66%.
Procedure
If the glycine contains moisture drying may be needed. The same procedure is
applicable as it was described for ammonium dihydrogen phosphate in chapter
«2.7.1 Verification of Distillation Unit», p. 38.
Prepare three blanks as follows: Add two Kjeldahl tablets and 20 mL of sulfuric acid (98%) to the three Kjeldahl glass sample tubes. Prepare three reference
samples of 0.5 g with an accuracy of ± 0.1 mg. Add 2 Kjeldahl tablets (10 g) and
carefully add 20 mL of sulfuric acid (98%).
%(N)det Nitrogen percent
corrected for the
purity of the sample.
%(N) Not corrected nitrogen percent.
P
Purity [%].
R
Recovery rate [%].
41. 40
Determination of Nitrogen by the Kjeldahl Method
Digestion
Preheat the digestion system to the maximum heat prescribed in the operation
manual. Subsequently, digest blanks and reference samples for 90 min at the
same temperature. Let the samples cool down to room temperature prior to
distillation.
Distillation and boric acid titration
The digested glycine samples are distilled and titrated using the conditions described for the distillation and titration of ammonium dihydrogen phosphate.
Calculation
The same equations are used for the calculation as for ammonium dihydrogen
phosphate.
Results
The same criteria as described for ammonium dihydrogen phosphate apply for
glycine.
42. Troubleshooting
3
41
Troubleshooting
In Table 27 reasons for failures or incorrect results are given and correlated to
corrective measures.
Result
Reason
Correction
Too high
carry over in distillation
air bubbles in titration
wrong titrant
error in calculation
use less volume
refill burette
use right concentration
e.g. check molar reaction
factor and factor of titrant
calibrate, replace
defective pH electrode
incomplete digestion
insufficient H2SO4
incorrect ratio Kjeldahl
tablets to sulphuric acid
too high nitrogen
too low NaOH
leaks
wrong titrant
error in calculation
defective pH electrode
sampler leak
dip tube loose, too short
empty reagent containers
repeat corrected
repeat corrected
air bubble in system
aspiration failure
incorrect pH calibration
sample homogeneity
weighing problems
incomplete digestion
venting suction module
digestion time too short
fix tubings
check for leaks and fix
repeat with fresh buffer
homogenize
e.g. use weighing paper
improve procedure
repeat
fix pump, or scrubber
check color of samples
Poor accuracy
Inhomogeneous sample
improper distillation
homogenize sample
verification of distillation
Foaming digestion
too high sample amount
not enough sulfuric acid
no anti-foam agent
high fat/sugar content
correct
correct
add stearic acid
optimize digestion conditions
Run dry digestion
bad sealings
not enough acid
wrong acid/salt ratio
too long digestion
replace sealings
correct
correct
use more acid/salt
Crystallization
bad end conditions
dilute cooled sample
Too low
Poor reproducibility
repeat corrected
use less sample
repeat until sample is blue
seal
check and replace
check and correct
calibrate, replace
repair
repair, pull down
refill
Table 27:
Reasons for failures and
corrective measures.
43. 42
Official Norms and Regulations
4
Official Norms and Regulations
In Table 28 official methods according to DIN / ISO regulations for the determination of nitrogen in various types of samples are listed. In Table 29 official methods
according to AOAC and EPA methods are given.
Table 28:
Kjeldahl nitrogen determinations according to DIN/ISO
regulations.
Nitrogen Content of
DIN/ISO Method
Agricultural food products
ISO 1871
Ammonium sulfate for industrial use
ISO 3332
Milk
DIN 8968-1
Non-protein content (NPN)
ISO 8968-4
Meat and meat products
ISO 937
Starches & derived products
ISO 3188
Water quality, Kjeldahl nitrogen
ISO 5663
DIN 38409H11
Water quality, ammonium
ISO 5664
Water quality, ammonium, potentiometric method
ISO 6778
Ammonia-nitrogen in water, waste water and sludge
DIN 38406-E5-2
Animal feeding stuffs – Determination of nitrogen
content and calculation of crude protein content
ISO 5983-2
Rubbers
Rubber, raw natural and rubber latex,
natural – Determination of nitrogen content
ISO 1656 (1996 E)
44. Official Norms and Regulations
43
Nitrogen content of
AOAC / EPA method
Animal feed and pet food
953.01
Animal feed
976.06
Beer
920.53
Brewing sugars
945.23
Milk powder
930.29
Milk
991.20
Whole milk
991.22
991.23
Eggs
925.31
Fertilizers
955.04
Food dressings
935.58
Fruit products
920.152
Grains
979.09
Ice cream, frozen desserts
930.33
Laboratory malt
950.09
Laboratory wort
950.10
Liquid eggs
932.08
Meat
928.08
Milk chocolate
939.02
Nuts and nuts products
950.48
Plants
978.04
Prepared mustard
920.173
Sugars and syrups
920.176
Tobacco
959.04
Water
973.48
Total Kjeldahl nitrogen (TKN)
EPA 351.3
Ammonia (as nitrogen)
EPA 350.2
Table 29:
Kjeldahl nitrogen determinations according to
AOAC/EPA regulations.
45. 44
Attachments
5
5.1
Attachments
Two-Stage Mixed Indicator according to Sher for Boric Acid
Titration
Reagents
Bromo cresol green, e.g. Merck 8121
p-Nitrophenol, e.g. Merck 6798
Ponceau 4 R, e.g. Siegfried 143130
0.1 mol/L Sodium hydroxide, e.g. Titrisol Merck 9959
Ethanol 96%
Distilled water
Preparation
Dissolve 0.35 g of bromocresol green in a 250 mL measuring flask in 10 mL of
ethanol. Add 10 mL 0.1 N sodium hydroxide, 200 mL distilled water, 22 mL of
1% aqueous Ponceau 4 R solution and 0.75 g of p-nitro phenol dissolved in
5 mL ethanol. Fill up to the mark with distilled water.
5.2
Mixing Indicator according to Mortimer for Back-Titration
Method and Colorimetric Titration
Reagents
Dissolve 20 mg methyl-red
100 mg bromocresol-green in 100 mL ethanol
Color-change
From red to green (= pH 4.5–4.6)
5.3
Recommended Monographs on Kjeldahl
R.B. Bradstreet, The Kjeldahl Method for Organic Nitrogen, Academic Press
1965, Library of Congress Catalog Card Number: 64-24653
46. References
6
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
J. Kjeldahl, Sur une nouvelle méthode de dosage de l’azote dans les substances organiques, Résumé du compte-rendu des travaux du Laboratoire
de Carlsberg, Copenhague, en commission chez H. Hagerup, Imprimerie
de Thiele 1983
Souci Fachmann Kraut, Food Composition and Nutrition Tables, 6th revised
and completed edition (2000), compiled by Heimo Scherz und Friedrich
Senser, medpharm Scientific Publishers, ISBN 3-88763-076-9
H. Hadorn, R. Jungkunz und K.W. Biefer, Über die Stickstoffbestimmung
in Lebensmitteln nach Kjeldahl und den Einfluss des Katalysators im
Besondern, (1953) Mitt. Gebiete Lebensm. Hygiene 44:14
Irving H. Sher, Two-Step Mixed Indicator for Kjledahl Nitrogen Titration, Analytical Chemistry, 831 (1955)
Handbook for Chemistry and Physics, D.R. Lide, Editor-in-chief, 88th Edition
2007–2008, p. 8–40.
M. Jurecek, Einige analytische Aspekte der Oxydation organischer Stickstoffverbindungen mit Chromsäure, Mikrochim. Acta 926–938, 5 (1962)
V. Novak, P. Kozak, P. Matousek und M. Jurecek, Analytische Aspekte der
Oxydation organischer Stickstoffverbindungen mit Chromsäure. Bestimmung der Nitro- und Nitrosogruppen, Mikrochim. Acta 1101–1107, 6
(1962)
P. Kozak, Vl. Novak, Zd. Bohackova und M. Jurecek, Analytische Aspekte
der Oxydation organischer Stickstoffverbindungen mit Chromsäure, Oxydation aromatischer Azoverbindungen und Bestimmung der Azogruppen,
Mikrochim. Acta 643–654, 4 (1963)
Y. Morita, A Theoretical Consideration on Chemical Reactions in the Kjeldahl
Digestion, Bulletin of the Chemical Society of Japan, Vol. 41, 2029 (1968),
A theoretical consideration on chemical reactions in the Kjeldahl digestion.
Die Stickstoffbestimmung nach Kjeldahl, Die Umrechnung von Stickstoff zu
Protein, Literaturstudie und Erfahrungsbericht, M. Ugrinovits, BÜCHI Laboratoriumstechnik GmbH, Göppingen
S.P.L. Sörensen und A.C. Andersen (1905). Lässt sich der Stickstoffgehalt
in Lysin und ähnlichen Verbindungen nach Kjeldahl bestimmen?, HoppeSeylers Zeitschrift für physiologische Chemie 44(5/6): 429-447
Römpps Chemie-Lexikon, Frankhsche Verlagshandlung Stuttgart, ISBN
3-440-04510-2 (1987)
R. Lange, R. Friebe und F. Linow (1979). Zur Anwendung der Methodenkombination Kjeldahl-Aufschluss/Bertholet-Reaktion bei der Stickstoffbestimmung in biologischen Materialien, 1. Mitt. Stand der Kenntnisse – Teil I,
Die Nahrung 23, 3, 1979, 255-261 referring to Exley, D., Biochem. J. 63,
496 (1956)
Handbook for Kjeldahl digestion, Jan-Ake Persson, ISBN 91-630-3471-9
(1995)
R.B. Bradstreet, Acid Requirements of the Kjeldahl Digestion, Analytical
Chemistry, 29, 6, 945–946 (1957)
RoHS: Sample Preparation (Digestion with Aqua Regia) for the Determination of the Heavy Metals Cadmium and Lead in Electronic Components,
BUCHI Application Note No. K-435-B-436-001 V.1.1. (2007)
45
47. 46
References
[17] P.R. Rexroad and R.D. Cathey, Pollution-Reduced Kjeldahl Method for
Crude Protein, Journal of the AOAC 59, 6, 1213–1217 (1976)
[18] F.M. Wieninger, Wochenzeitschrift für Brauerei 53, 251 (1936)
[19] A.M. Rossi, M. Villareal, M.D. Juárez, N.C. Sammán, Nitrogen Contents In
Food: A Comparison Between The Kjeldahl And Hach Methods, J. Argent.
Chem. Soc. 92, 4–6 (2004)
[20] C.L. Suard, R-M. Mourel, D. Didenot and M.H. Feinberg, Mechanisms Involved in Kjeldahl Microwave Digestion of Amino Acids, J. Agric. Food
Chem. 45, 1202–1208 (1997)
[21] Fast and Environmental friendly Hydrogen Peroxide-Sulfuric Acid Digestion
Method for the Total Nitrogen Determination in Meat, BUCHI Application
Note No. K-424-K-370-014 V.1.0 (2007)
[22] Fast and Environmental friendly Hydrogen Peroxide-Sulfuric Acid Digestion
Method for the Total Nitrogen Determination in Dried Meat, BUCHI Application Note No. K-424-K-370-015 V.1.0 (2008)
[23] Fast and Environmental friendly Hydrogen Peroxide-Sulfuric Acid Digestion Method for the Total Nitrogen Determination in Salami, Application No.
K-424-K-370-010 V1.0 (2007)
[24] Protein Determination in Skimmed Milk by Digestion with Hydrogen Peroxide and Sulfuric Acid, BUCHI Application Note No. K-435-K-370-371-001
V.1.0
[25] Determination of ammonia in water and waste water, BUCHI Application
Note No. K370-006 Version B (2007)
[26] Nitrogen-Information No. 7, Determination of nitrogen content in fertilizers
containing nitrate: Iron reduction method, Devarda method, Salicylic acid
method, BUCHI Application Report (1994)
[27] USDA Agric. Handbook No. 8 by Watt&Merril referring to Lee I. Chiba, Animal Nutrition Handbook, online: http://www.ag.auburn.edu/~chibale/an07protein.pdf
[28] Schweizerisches Lebensmittelbuch, SLMB, online: http://www.slmb.bag.
admin.ch/slmb/index.html
[29] Association of Analytical Communities (AOAC), online: http://eoma.aoac.
org check online for methods given in chapter «4 Official Norms and Regulations», p. 3432
[30] German industry norm and European norm DIN EN 32 645, Beuth Verlag
GmbH, Berlin
[31] Standard Methods for the examination of water and waste water, 19th edition 1995, 4–146, Inorganic Nonmetals (4000), 4500-P Phosphorus
48. Application Notes
7
7.1
47
Application Notes
List of available Application Notes
Order
Segment
Field
Sample
Analyte
Code
Method
Processing
Analysis
C01
Cosmetics
Hair
Hair dye
Ammonia
Distillation
Titration
C02
Cosmetics
Hair
Hair dye
Ammonia
Distillation
Back titration
C03
Cosmetics
Cremes
Lotion / face
cream / repair
ointment
Nitrogen /
Urea
Kjeldahl
Kjeldahl
E01
Environment
Sewage
Water /
waste water
Ammonia
Distillation
(Kjeldahl)
Distillation
(Kjeldahl)
E02
Environment
Sewage
Water /
waste water
Nitrogen
TKN
TKN
E03
Environment
Sewage
Water /
waste water
Nitrogen
TKN
TKN
E04
Environment
Sewage
Water /
waste water
Nitrogen
TKN
TKN
E05
Environment
Sewage
Water /
waste water
Nitrogen
TKN
TKN
E06
Environment
Sewage
Water /
waste water
Nitrogen
TKN
TKN
E07
Environment
Sewage
Water /
waste water
COD
COD digestion
Back titration
E08
Environment
Sewage
Water /
waste water
Cyanide
Distillation
Spectrometry
E09
Environment
Sewage
Water /
waste water
Phosphate
Digestion
Photometry
E10
Environment
Sewage
Standard /
beer / sludge
Phenol
Distillation
Spectrometry
E11
Environment
Sewage
Sludge
Volatile
acid
Distillation
Titration
E12
Environment
Material
control
Standard
Formaldehyde
Distillation
Spectrometry
E13
Environment
Electronic
Electronic
scrab
Heavy
metal
Aqua regia
digestion
ICP-MS
E14
Environment
Agriculture
Fertilizer
(nitrate
containing)
Nitrogen
Pre-treatment
with salicylic
acid, Digestion
Kjeldahl
E15
Environment
Agriculture
Fertilizer
(without
nitrate)
Nitrogen
Kjeldahl
Kjeldahl
E16
Environment
Agriculture
Fertilizer
(nitrate
containing)
Nitrogen
Pre-treatment
with salicylic
acid, Digestion
Kjeldahl
E17
Environment
Agriculture
Fertilizer
(without
nitrate)
Nitrogen
Kjeldahl
Kjeldahl
E18
Environment
Power Plant
Fly ash
Ammonia
Direct
distillation
