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best@buchi No. 58
Nitrogen Determination with Kjeldahl
How to Achieve Low Detection and Quantification Limits
for the Nitrogen Determination with Kjeldahl
Authors: Dr. Claudia Blum-Fretz, Stephan Buschor, Jürgen Müller
Key words: nitrogen determination, Kjeldahl, detection limit, and quantification limit
Abstract
Kjeldahl is one of the most commonly used techniques to determine the protein content in food and
feed samples. The detection and quantification limits are important characteristics of analytical
methods. The impact of the concentration of boric acid, the addition of potassium chloride, and the
concentration of the titration solution on the detection and quantification limits were investigated. The
best results were obtained by using 2 % boric acid with 3 g potassium chloride per liter. A titration
solution of 0.005 M HCl worked best. With these parameters, detection limits for distillation of standard
n solutions as low as 0.008 mg nitrogen and quantification limits of 0.02 mg nitrogen can be achieved.
0 e How to Achieve Low Detection and Quantification Limits for the Nitrogen
1
0 Introduction Often the potential of the reference electrode E , which should be constant, shows a small variability
/ 2 Determination with Kjeldahl ref
8 which can lead to measurement variations. The variability of the potential is largest when the solution
i 5
h is stirred. To demonstrate the stirring effect, a detailed view of the pH sensor is provided below.
c Kjeldahl
u
b
@
t For almost 130 years, the determination of nitrogen using the method developed by the Danish
s
e Authors: Dr. Claudia Blum-Fretz, Theoretical background of pH
b chemist Johan Kjeldahl (1849–1900) has been an internationally accepted standard. The method,
Stephan Buschor, Jürgen Müller measurements and boric acid
which is named after its inventor, has since found widespread application in life science and chemistry
titration
and has extended its scope to the determination of nitrogen and proteins in dairy products, meat
products, beer, cereals, and other food materials [1].
Abstract The pH value is the negative logarithm
The Kjeldahl procedure involves three major steps:
Kjeldahl is one of the most commonly of the hydronium ion activity and is
used techniques to determine the protein In the digestion step, the organically bonded nitrogen is converted into ammonium ions by oxidation
measured with an electro-chemical
with concentrated sulfuric acid.
content in food and feed samples. The sensor. In practice this is a measurement
In the distillation step, the sample is alkalinized to convert the ammonium ions to ammonia. The latter
detection and quantification limits are of a potential difference between a
is then distilled into a boric acid solution (via steam distillation).
important characteristics of analytical reference electrode E and the mea-
In the final titration step, the ammonia is titrated and the nitrogen content can be calculated.
ref
methods. The impact of the concentra- suring electrode E. The measured volt-
Theoretical background of pH measurements and boric acid titration
tion of boric acid, the addition of age U is the potential difference of E and
potassium chloride, and the concentra- E . The calculation of pH is performed
The pH value is the negative logarithm of the hydronium ion activity and is measured with an electro-
ref
chemical sensor. In practice this is a measurement of a potential difference between a reference
tion of the titration solution on the according to the following equations
electrode and the measuring electrode. The measured voltage U is the potential difference of E and
detection and quantification limits were (1 - 2), which are derived from the Nernst
E . The calculation of pH is performed according to the following equations (1 - 2), which are derived
ref
investigated. The best results were equation [2 - 4].
obtained by using 2 % boric acid with from the Nernst equation [2 - 4].
3 g potassium chloride per liter. A
titration solution of 0.005 M HCl E−Eref (1)
pH = pH0 − slope (1)
worked best. With these parameters,
detection limits for distillation of
The quotient in equation (2) represents the slope of the pH function and shows that the slope is a
standard solutions as low as 0.008 mg The quotient in equation (2) represents
function of the temperature.
Neue Formeln
Neue Formeln
nitrogen and quantification limits of the slope of the pH function and
0.02 mg nitrogen can be achieved. Figure 1: Schematic description of the pH Electrode:
shows that the slope is a function of
f *R*T Figure 1: Schematic representation of
ln−log
the temperature.
slope = (2)
the pH electrode
Introduction z*F
1 measuring electrode (e.g., Ag/AgCl)
2 internal reference solution
pH negative logarithm of the hydronium ion activity
f •R*T
f •R*T
ln−log
Kjeldahl ln−log (2) 1 measuring electrode (e.g., Ag/AgCl)
3 pH sensitive glass membrane
slope = (2)
slope = (2)
pH pH at zero point of pH sensor (i.e., the pH when the sensor signal is 0 mV)
0 z•F
z•F 4 sample solution (e.g., boric acid as receiving solution)
For almost 130 years, the determination 2 internal reference solution
E potential at measuring electrode
5 liquid junction (e.g., ceramic diaphragm)
E potential of the reference electrode (should be constant)
of nitrogen using the method developed ref 3 pH sensitive glass membrane
6 reference electrolyte (e.g., 3 M KCl)
f conversion factor for the change of the natural (ln) to the common logarithm (2.303)
ln-log
by the Danish chemist Johan Kjeldahl pH negative logarithm of the 4 sample solution
7 reference electrode (e.g., Ag/AgCl)
R universal gas constant (8.3145 J/(K*mol))
(1849–1900) has been an internationally hydronium ion activity (e.g., boric acid as receiving solution)
x = x •k (5)
x = x •k (5)
(LOQ) (LOD) U voltage measurement
(LOQ) (LOD)
T absolute temperature [K]
accepted standard. The method, which pH pH at zero point of pH sensor 5 liquid junction
0
z number of electrons transferred (for pH: 1)
is named after its inventor, has since (the pH when the sensor 4 The variability of the potential is produced at the liquid junction (zeta potential, different mobility of
(e.g., ceramic diaphragm)
F Faraday constant (9.6485*10 C/mol)-
found widespread application in life sci- borate and hydronium ion, etc.). In diluted solutions, the variability is higher than in concentrated
signal is 0 mV) 6 reference electrolyte (e.g., 3 M KCl)
ence and chemistry and has extended solutions. If the solution is not stirred, a cloud of potassium and chloride ions (black dots in Figures 2
E potential at measuring electrode 7 reference electrode (e.g., Ag/AgCl)
and 3) is created at the exterior of the liquid junction and reduces the surface potential. If the solution
its scope to the determination of nitrogen E potential of the reference U voltage measurement
ref Zusätzlicher Satz auf Seite 4:
Zusätzlicher Satz auf Seite 4:
and proteins in dairy products, meat electrode (should be constant) is stirred, the cloud of potassium and chloride ions is removed from the surface so that the potential
increases and the measured pH value decreases.
products, beer, cereals, and other food f conversion factor for the change The variability of the potential is
ln-log In this study, the LOD and LOQ were always calculated according to the direct and
materials [1]. In this study, the LOD and LOQ were always calculated according to the direct and
of the natural (ln) to the common produced at the liquid junction (zeta
the indirect method to be able to compare the findings. For Kjeldahl, the direct
The Kjeldahl procedure involves three the indirect method to be able to compare the findings. For Kjeldahl, the direct
logarithm (2.303) potential, different mobility of borate
method is well suited, because the matrix is completely destroyed by the digestion
major steps: method is well suited, because the matrix is completely destroyed by the digestion
R universal gas constant and hydronium ion, etc.). In diluted
with sulfuric acid.
with sulfuric acid.
In the digestion step, the organically (8.3145 J/(K•mol)) solutions, the variability is higher
bonded nitrogen is converted into
T absolute temperature [K] than in concentrated solutions. If the
ammonium ions by oxidation with z number of electrons transferred solution is not stirred, a cloud of
concentrated sulfuric acid. (for pH: 1) potassium and chloride ions (black
Zusätzlicher Satz auf Seite 5:
Zusätzlicher Satz auf Seite 5:
In the distillation step, the sample is F Faraday constant dots in Figures 2 and 3) is created at
alkalinized to convert the ammonium • 4 C/mol) the exterior of the liquid junction and
(9.6485 10
The experiment was set up in the following way: 1) optimization of the boric acid
The experiment was set up in the following way: 1) optimization of the boric acid
ions to ammonia. The latter is then reduces the surface potential. If the
concentration 2) optimization of the KCl addition and finally 3) optimization of the
concentration 2) optimization of the KCl addition and finally 3) optimization of the
distilled into a boric acid solution Often the potential of the reference solution is stirred, the cloud of potas-
titrant concentration.
titrant concentration.
(via steam distillation). electrode E , which should be constant, sium and chloride ions is removed
ref
In the final titration step, the ammonia shows a small variability which can from the surface so that the potential
is titrated and the nitrogen content can lead to measurement variations. The increases and the measured pH value
be calculated. variability of the potential is largest when decreases.
the solution is stirred. To demonstrate the
stirring effect, a detailed view of the pH
sensor is shown in Fig. 1.
2
n
0 e
1
0
/ 2
8
i 5
h
c
u
b
@
t
without stirring s
1. The pH increase due to dilution of the receiving solution by distillate is less e
important in low concentrated boric acid. The variability of the amount of distilled b
water has less impact on the pH value and will therefore lead to less variability of
the blank values. The pH change related to dilution is shown in Figure 4.
pH change by dilution of boric acid
5.9
5.7
5.5
pH5.3
5.1
4.9
4.7
Figure 2: Liquid junction without stirring 4.5
Figure 2: Liquid junction without stirring Figure 3: Liquid junction with stirring
0 20 40 60 80 100 120 140
Added H O [ml]
2 4 % 2 % 1 %
The stirring effect can be minimized by adding potassium chloride to low concentrated (< 4%) boric
acid to ensure that a sufficient amount of potassium chloride is always at the surface of the liquid
junction. Figure 4: pH change when diluting 60 ml receiving solution at different
concentrations of boric acid
with stirring
The use of diluted boric acid is beneficial for the determination of low nitrogen amounts for the
following three main reasons: 2. The blank values in less concentrated boric acid are smaller for the same
1. The pH increase due to dilution of the receiving solution by distillate is less important in low
reason as above. This is particularly important because usually low concentrated
concentrated boric acid. The variability of the amount of distilled water has less impact on the pH value
titration solutions are used for the determination of low nitrogen amounts. For
and will therefore lead to less variability of the blank values. The pH change related to dilution is
shown in Figure 4. the determination of low nitrogen contents it is advantageous to have smaller blank
values, because the difference in titration volumes between the blanks and the
samples gets larger.
pH change by dilution of boric acid
3. The pH change caused by the distilled nitrogen is more important the lower
5.9 the concentration of the receiving solution is. Small amounts of nitrogen cause
a considerable increase in pH, thus making the titration more accurate.
5.7
5.5 Detection limit and quantification limit
The so-called detection limit (limit of detection LOD) and quantification limit (limit of
5.3 quantification LOQ) are important characteristics of analytical methods. They
H have to be determined for each method, analyte, and matrix.
p
Figure 3: Liquid junction with stirring
5.1
Figure 2: Liquid junction without stirring Figure 3: Liquid junction with stirring The DIN 32 645 standard defines the two terms and describes the procedure used to
calculate these values based on analytical results [5]. In this best@buchi, the definitions
The stirring effect can be minimized by adding potassium chloride to low concentrated (< 4%) boric
4.9 of the aforementioned standard are used (the terminology used in other standards may
acid to ensure that a sufficient amount of potassium chloride is always at the surface of the liquid
junction. The stirring effect can be minimized be slightly different).
4.7
by adding potassium chloride to low
concentrated (< 4%) boric acid to ensure Definitions
The use of diluted boric acid is beneficial for the determination of low nitrogen amounts for the
4.5
following three main reasons: that a sufficient amount of potassium Detection limit: The smallest content of the analyte that is significantly different
0 20 40 60 80 100 120 140
chloride is always at the surface of the from the blank value.
1. The pH increase due to dilution of the receiving solution by distillate is less important in low O [mL]
liquid junction. Added H2 4% 2% 1%
concentrated boric acid. The variability of the amount of distilled water has less impact on the pH value
and will therefore lead to less variability of the blank values. The pH change related to dilution is
Quantification limit: The smallest content of the analyte that can be determined
shown in Figure 4. Figure 4: pH change when diluting 60 ml receiving solution with different concentrations of boric acid
The use of diluted boric acid is benefi- quantitatively.
cial for the determination of low nitrogen
2. The blank values in less concentrated boric acid are smaller for the same reason as above. This is
amounts for the following three main In general, the quantification limit is three times higher than the detection limit [5].
particularly important because usually low concentrated titration solutions are used for the
pH change by dilution of boric acid
reasons: There are two ways to calculate these limits. The results achieved from these two
determination of low nitrogen amounts. For the determination of low nitrogen contents it is
methods are not equal but are equivalent:
5.9 advantageous to have smaller blank values, because the difference in titration volumes between the
blanks and the samples is more important.
5.7 3
5.5
H5.3
p5.1
4.9
4.7
4.5
02040 60 80 100 120 140
Added H2O [mL] 4% 2% 1%
Figure 4: pH change when diluting 60 ml receiving solution with different concentrations of boric acid
2. The blank values in less concentrated boric acid are smaller for the same reason as above. This is
particularly important because usually low concentrated titration solutions are used for the
determination of low nitrogen amounts. For the determination of low nitrogen contents it is
advantageous to have smaller blank values, because the difference in titration volumes between the
blanks and the samples is more important.
n
0 e
1 3. The pH change caused by the distilled nitrogen is more important the lower the concentration of the
0
/ 2receiving solution is. Small amounts of nitrogen cause a considerable increase in pH, thus making the
8
i 5titration more accurate.
h
c
u
b
@
t
s Detection limit and quantification limit
e
b
The so-called detection limit (LOD) and quantification limit (LOQ) are important characteristics of
analytical methods. They have to be determined for each method, analyte, and matrix.
Direct method (“Blank method”) k factor used to calculate the Experimental
With the determination of a large number x based on x ;
(LOQ) (LOD)
The DIN 32 645 [5] standard defines the two terms and describes the procedure used to calculate
of blanks (n ≥ 10), the detection and the factor is usually k=3 [5]. Equipment
these values based on analytical results. In this paper, the definitions of the aforementioned standard
quantification limit can be calculated AutoKjeldahl Unit K-370 with Kjeldahl
are used (the terminology used in other standards may be slightly different).
based on the standard deviations of the Indirect method (“Calibration line Sampler K-371; Schott Titronic Uni-
blank measurements and the slope of the method”) versal, dosage instrument (Buchi P/N
Definitions:
calibration line. For Kjeldahl the slope A calibration line (in the range of the limit 043596); Analytical balance, reading
would be the linearity between the nitro- of quantification) is established (range 0 precision +/- 0.1 mg; Statist24cp, Ver-
Detection limit: The smallest content of the analyte that is significantly different from the blank value.
gen content and the consumption of the to 10 times x LOD). Based on the slope sion 2.0., statistical program for method
titration solution. The calibration line of of this line, the detection and quanti- validation for analytical laboratories,
Quantification limit: The smallest content of the analyte that can be determined quantitatively.
the entire working range is used. fication limit can be calculated. In this ©2000-2005, Georg Schmitt, Michael
This method can only be used if a suit- case, the uncertainty of the blank is Herbold, Arvecon GmbH, Walldorf,
In general, the quantification limit is three times higher than the detection limit [1]. There are two ways
able blank is available. A blank should estimated by extrapolation of the Germany.
to calculate these limits. The results achieved from these two methods are not equal but are
have identical properties to those of the calibration data. This method is more
equivalent:
actual sample, but without any analyte. laborious and needs more statistical Chemicals
This is rarely the case, as most analyses know-how than the direct method, but Ammonium dihydrogen phosphate
Direct method (“Blank method”)
are done in complex matrices such as is often necessary due to the reasons 99.99 % (Merck, 1.01440), dried; boric
With the determination of a large number of blanks (n ≥ 10), the limit of detection and quantification
food or environmental samples, which mentioned above. acid (Brenntag, 80948-155); potassium
can be calculated based on the standard deviations of the blank measurements and the slope of the
cannot be imitated easily. The calculations are explained in detail chloride (Merck, 104936); 0.05 M
calibration line. For Kjeldahl the slope would be the linearity between the nitrogen content and the
The calculations of the detection and in the DIN 32 645 standard. Several hydrochloric acid (Riedel de Haën,
consumption of the titration solution. The calibration line of the entire working range is used.
quantification limits are performed statistical programs can be used to 35320), the titration solutions were
This method can only be used if a suitable blank is available. A blank should have identical properties
according to equations (3 - 5). calculate the detection and quantification prepared by diluting this standard
to those of the actual sample, but without any analyte. This is rarely the case, as most analyses are
Neue Formeln limit using the indirect method solution.
done in complex matrices such as food or environmental samples, which cannot be imitated easily.
The calculations of the detection and quantification limits are performed according to equations (3 - 5).
according to DIN 32 645.
x =Φ •sL (3) In this study, the LOD and LOQ were Samples
(LOD) n;α b (3) always calculated according to the direct Solutions of ammonium dihydrogen
fln−log • R*T and the indirect method to be able to phosphate were diluted to obtain an
slope = (2)
z•F compare the findings. For Kjeldahl, the absolute nitrogen amount per sample
1 1 (4)
Φn;α =tf;α • + (4) direct method is well suited, because the between 0.005 mg and 0.5 mg. Each
m n matrix is completely destroyed by the sample was determined in triplicate.
digestion with sulfuric acid. The solution was dosed into the
(5)
(5)
x = x •k (5)
x(LOQ) = x(LOD) *k
(LOQ) (LOD) Kjeldahl flasks using the Titronic
Universal dosage instrument.
detection limit
x(LOD)
detection limit The determination was carried out
x
x quantification limit
(LOD)
(LOQ)
x quantification limit with the AutoKjeldahl unit K-370
Φ factor, depending on number of blank measurements (n), sample replicates (m), and
(LOQ)
n,α
Zusätzlicher Satz auf Seite 4:
Φ factor, depending on number with Kjeldahl Sampler K-371 using
n,α significance level (α)
of blank measurements (n), the parameters given in Table 1.
s standard deviation of blank measurements
L
b slope of the calibration line; for Kjeldahl the relation between titration solution consumption
sample replicates (m), and
In this study, the LOD and LOQ were always calculated according to the direct and
and the nitrogen content (example: 14.28 ml of 0.005 M HCl corresponds to 1 mg Nitrogen,
significance level (α)
the indirect method to be able to compare the findings. For Kjeldahl, the direct
s b= 14.28). Table 1: Parameters for the Kjeldahl sampler system K-370/K-371
L standard deviation of blank
method is well suited, because the matrix is completely destroyed by the digestion
t quantile of the Student t-distribution, depending on degree of freedom f (f = n-1) and
measurements
f;α
with sulfuric acid.
significance level α
b slope of the calibration line; for Distillation Titration
n number of blank measurements
Kjeldahl the relation between
m number of sample replicates
titration solution consumption Water 50 ml Type Boric acid
k factor used to calculate the x based on x ; the factor is usually k=3 [1].
Zusätzlicher Satz auf Seite 5: (LOQ) (LOD)
and the nitrogen content
NaOH 90 ml Titration solution HCl 0.005 M
(example: 14.28 ml of 0.005 M
Indirect method (“Calibration line method”)
HCl corresponds to 1 mg
The experiment was set up in the following way: 1) optimization of the boric acid
A calibration line (in the range of the limit of quantification) is established (range 0 to 10 times x LOD).
nitrogen, b= 14.28). Reaction time 5 s Volume receiving sol. 60 ml
concentration 2) optimization of the KCl addition and finally 3) optimization of the
Based on the slope of this line, the limit of detection and quantification can be calculated. In this case,
t quantile of the Student
f;α
titrant concentration.
the uncertainty of the blank is estimated by extrapolation of the calibration data. This method is more
t-distribution, depending on Distillation time 240 s Titration mode Standard
degree of freedom f (f = n-1) Steam power 100 % End-point pH 4.65
α
and significance level
n number of blank measurements Algorithm 1
m number of sample replicates
4
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