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The Kjeldahl Process
From sample preparation to result calculation
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The Kjeldahl Process
Introduction
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 digestion 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 can also be carried out by non-academic lab personnel, has been put into
practice by BUCHI’s Kjeldahl systems since 1961.
With this short guide BUCHI would like to support you in your daily work by not only providing high quality instrumentation but also offering theoretical background information
and useful tables for your daily routine. The laminated tables can be taken out of the guide
to be placed at the locations in the lab where they are needed.
For more detailed information please refer to the BUCHI Kjeldahl Guide.
www.buchi.com/kjeldahl/en/applications/literature/
In addition to this short guide you could find detailed information including our application
database at: www.buchi.com/kjeldahl/applications
And to download BUCHI’s practical tablet guide or Kjeldahl calculator for iOS, Android and
Windows Phone 7: www.buchi.com/kjeldahl/applications
2
The Kjeldahl Process
From sample preparation to result calculation
∙∙
∙∙
∙∙
Sample
preparation ∙∙
Grinding
Sample tube size
Weighing table
Kjeldahl Tablets
Samples must be homogeneous. The actual weight of a
sample depends on the nitrogen content as well as on the
inhomogeneity of the sample.
∙∙ Digestion:
(CHNO) + H2SO4 → CO2 + SO2 + H2O + NH4+
Digestion
∙∙ Digestion parameters
∙∙ Neutralization/Alkalinization:
H2SO4 + 2 NaOH → 2 Na+ + SO4 2- + 2 H2O
Distillation
∙∙ Distillation:
NH4+ + OH ⇌ NH3 (gas) + H2O
∙∙ Receiver:
B(OH)3+ NH3+ H2O ⇌ NH4+ + B(OH)4–
Titration
Result
∙∙ Titration:
–
B(OH)4– + HX → X + B(OH)3 + H2O
∙∙ Calculation
∙∙ LIMS
∙∙ KjelLink
Organic matter is destroyed by
boiling in concentrated sulfuric
acid. Kjeldahl Tablets raise the
boiling point and accelerate the
process.
The digestion mixture is alkalized with NaOH prior to distillation to free up the ammonia.
The ammonia is steam distilled
into an acidic receiver solution.
The pH in the acidic receiver
solution rises upon addition of
ammonia. The nitrogen and
protein content is then determined by titration of the borate
complex.
The nitrogen content is then
calculated. To calculate the
protein content the nitrogen is
multiplied by a sample specific
proteinfactor.
3
Sample preparation
Sample tube selection and weight
Micro
Weight
< 0.2 g
Sample volume
2 – 3 mL
H2SO4
2 – 5 mL
Kjeldahl Tablets
1 (Micro)
Recommendation Homogeneous samples high in nitrogen/protein
Benefit
Reduction of chemicals of ca. 80 % compared to 300 mL
tubes
300 mL
Weight
0.1 – 5 g
Sample volume
< 200 mL
H2SO4
5 – 30 mL
Kjeldahl Tablets
2
Recommendation Standard tube for most applications
Benefit
All round sample tube
500 mL
4
Weight
>4g
Sample volume
< 400 mL
H2SO4
> 10 mL
Kjeldahl Tablets
2
Recommendation
Especially for high sample volumes or strongly foaming
samples
Benefit
Problem-free digestion of strongly foaming samples
Sample preparation
Weighing
The actual weight depends on
Sample
preparation
∙∙ The protein or rather the nitrogen content of the sample
▶ the higher the N-content, the lower the weight can be
∙∙ The Homogeneity of the sample
▶ By increasing the sample amount the impact of the grain size is reduced
▶ Example: homogeneous samples < 1 g, inhomogeneous samples > 2 g
∙∙ The concentration of the titrant
▶ The consumption of the titrant should be in a range of 3 and 17 mL
(optimal accuracy of measurement when using a 20 mL burette)
5
Sample preparation
Usage of the weighing table
Example for the usage of the weighing table
1
Expected %N of the sample must be selected (here 2 %)
2
Selection of the titrant concentration used (e.g. 0.05 mol/L)
3
Determination of the expected titrant consumption in mL ▶ here 3.6 and
14.3 mL
4
Result: For samples containing 2% N and with titrant concentration of
0.05 mol/L, the expected consumption should be in a range of 3 – 17
mL. Therefore the weight must be between 0.125 and 0.5 g.
Sample: weight [g]
5
2
1
4
0.5
0.125
Titrant conc.: [mol/L]
0.01
N [mg]
per glas
N [%]
0.5
0.01
0.03
0.05
0.10
0.40
3.6
2.0
0.04
0.10
0.20
0.40
1.60
14.3
2.5
0.05
0.13
0.25
0.50
2.00
7.0
0.14
0.35
0.70
1.40
5.60
10.0
0.20
0.50
1.00
2.00
8.00
50.0
1.00
2.50
5.00 10.00 40.00
100.0
2.00
5.00 10.00 20.00 80.00
0.05
0.1
0.5
Titrant consumption for sample
[mL]
1
1
2
2.9
3.6
10.0
1
14.3
3
3
X
1.8
5.0
7.1
1.4
7.1
14.3
3
The limit of determination is 0.02 mg N per sample tube.
However, optimal would be nitrogen content of 1 - 200 mg per sample tube.
6
Sample preparation
Addition of chemicals and Kjeldahl Tablets
Sample
preparation
Addition of chemicals, rule-of-the-thumb:
∙∙ sulfuric acid: 2 mL H2SO4 per 1 g catalyst
∙∙ Kjeldahl Tablets: frequent practice 2 pieces per sample tube
The aim of the Kjeldahl Tablets is the acceleration of the digestion process by
means of:
∙∙ catalysis by metal salts
∙∙ raising the boiling point of the H2SO4 by sulfate salts (K 2SO4)
The selection of Kjeldahl Tablet depends on:
For problem-free samples:
1.
2.
3.
4.
Ideal digestion conditions are
∙∙ Boiling point at 370 °C
∙∙ No nitrogen losses
∙∙ Minimal time needs
Safety aspects
Digestion time
Ecological aspects
Foam formation of the sample
www.buchi.com/kjeldahl/application
These are achieved with
▶ 2 mL H2SO4 to 1 g catalyst
For more demanding samples or samples that are high in fat or carbohydrate see
page 10.
7
Sample preparation
Weighing table for solid and liquid samples
Solid samples
Sample: weight [g]
5
2
1
Titrant concentration [mol/L]
0.5
0.125
N [mg] N [%]
0.01
0.05
0.1
0.5
Titrant consumption sample [mL]
0.5
0.01
0.03
0.05
0.10
0.40
3.6
2.0
0.04
0.10
0.20
0.40
1.60
14.3
2.5
0.05
0.13
0.25
0.50
2.00
3.6
1.8
7.0
0.14
0.35
0.70
1.40
5.60
10.0
5.0
10.0
0.20
0.50
1.00
2.00
8.00
14.3
7.1
50.0
1.00
2.50
5.00 10.00 40.00
7.1
100.0
2.00
5.00 10.00 20.00 80.00
14.3
2.9
1.4
Procedure:
A: Select N % of sample
B: select titrant concentration
C: Choose weight in order that the titrant consumption can be expected
between 3 and 17 mL
Liquid samples
Sample
[mL]
Titrant
N [%]
N mg/L
Titrant [N]
4
0.1 – 0.6
0.10
6
0.06 – 0.4
0.10
10
100 – 200
0.01
25
50 – 100
0.01
50
20 – 50
0.01
100
10 – 20
0.01
250
5 – 10
0.01
400
<5
0.01
8
Sample preparation
Kjeldahl Tablets overview
Article
Composition
Titanium
# 11057980
3.5 g K2SO4 / 0.105 g CuSO4 • 5 H2O
0.105 g TiO2
Benefit:
Recommendation:
Time saving
Optimal compromise between environmental and performance
priorities.
Titanium Micro 1.5 g K2SO4 / 0.045 g CuSO4 • 5 H2O
0.045 g TiO2
# 11057981
3.71 g
1.59 g
Benefit:
Recommendation:
Reduced chemical amount
Same as Titanium (11057980) but for semi-micro & microKjeldahl applications.
Missouri
# 11057982
4.98 g K2SO4
0.02 g CuSO4 • 5 H2O
Benefit:
Recommendation:
Easy to use and universally applicable
The digestion with Missouri is more eco-friendly
ECO
# 11057983
3.998 g K2SO4
0.002 g CuSO4
Benefit:
Recommendation:
Eco-friendly
Most environmentally friendly catalyst, due to the very low
copper content
Antifoam
# 11057984
0.97 g Na2SO4
0.03 g Silikon Schaumhemmer
Benefit:
Recommendation:
Maximum foam reduction
Used as general purpose foam suppressant. This tablet has to
be combined with Titanium Micro (11057981) or Copper Micro
(11057985).
Copper Micro
# 11057985
1.5 g K2SO4
0.15 g CuSO4 • 5 H2O
Benefit:
Recommendation:
9
Weight
5g
4g
1g
1.65 g
Reduced chemical amount
Combo tablets for Antifoam or micro Kjeldahl applications.
Sample preparation
Amount of sulfuric acid
The amount of H2SO4 is given by:
1. Conversion of K2SO4 to KHSO4 (K2SO4 is a component of Kjeldahl Tablets)
ca. 2 – 3 mL
2. Consumption by organic matter
Organic matter
H2SO4/ g
[mL]
Example:
Salami
e.g. for 1.5 g weight
(weight ∙ org. matter):
Fat
9.7
27.3 %
1.5 ∙ 9.7 ∙ 27.3 = 3.97 mL
100
Protein
4.9
20.6 %
1.5 ∙ 4.9 ∙ 20.6 = 1.51 mL
100
Carbohydrates
4.0
0.0 %
1.5 ∙ 4.0 ∙ 0.0 = 0.0 mL
100
3. Losses due to evaporation
ca. 1 mL/h
4. Remaining volume
> 10 mL
H2SO4 volume =
conversion + (total consumption by org. matter) + evaporation + remaining volume
3 mL + (3.97 + 1.51 + 0.00) mL + 1 mL + 10 mL = 18.48 mL ~18 mL
10
The digestion
Hints
Inlet for ambient air
Safety zone (≈ 5 cm)
Condensation zone
Digestion
Boiling /digesting sample
∙∙ Optimal conditions are achieved when the condensation zone remains 5 cm below the constriction of the sample tube.
∙∙ For foaming samples one Kjeldahl Tablet «Antifoam» or stearic.
∙∙ To reduce the digestion time H2O2 can be added.
∙∙ When samples crystalize despite optimal H2SO4/catalyst ratio ▶ the suction power of the
Scrubber should be reduced.
∙∙ The Scrubber must only be placed on the left of the digester.
∙∙ Minimal use of chemicals due to micro-Kjeldahl (only SpeedDigester).
∙∙ For liquid samples boiling rods prevent boiling delays.
∙∙ Boiling rods can in contrast to boiling stones, also be used for the following automated analysis
via the KjelSampler.
∙∙ Slowly increasing the digestion temp. helps to reduce foam formation of problematic samples.
11
Digestion
Digestionparameter: SpeedDigester (IR)
TKN using 500 mL sample tube
Temp. [°C]
Digestion
First H2O has to
evaporate before
the temperature
can increase
█ T nominal
█ T sample
1)
Step
Temp. [°C] Level Time [min] Vol. [mL]
Preheating
380
7
Digestion
520
9
Time [min]
Cooling
1)
The sample is heated to boil as fast
as possible.
Temp. [°C]
The sample is kept
constantly boiling.
█ T nominal
█ T sample
45
10
50
25
60
50
80
100
135
250
185
400
30
The digestion time depends on the sample volume
Standard Kjeldahl using 300 mL sample tube
Step
Temp. [°C] Level Time [min]
Preheating
480
8.5
Digestion
480
550
490
8.5
9.5
8.5
Cooling
10
10
65
30
Temp. [°C]
Time [min]
The sample is heated to boil as fast
as possible.
Due to the low volume used
for micro-Kjeldahl lower
temperatures are required
(480 compared to 490°C) to
reach the boiling temperature and the digestion time
can significantly reduced.
█ T nominal
█ T sample
Time [min]
12
Micro Kjeldahl using 100 mL sample tubes
Step
Temp. [°C] Level Time [min]
Preheating
480
8.5
Digestion
480
500
480
8.5
9.5
8.5
Cooling
10
5
45
15
Digestion
Digestion parameter: Block digester
TKN using 500 mL sample tubes
Step
Temp. [°C]
Preheating
300 – 420
Digestion
420
1)
Time [min]
Cooling
1)
Vol. [mL]
45
10
50
25
60
50
90
100
165
250
45
The digestion time depends on the sample volume.
Standard Kjeldahl using 300 mL sample tubes
Step
Temp. [°C]
Preheating
320
Digestion
420
Cooling
Time [min]
120
45
13
Digestion
Highest Sample throughput
Reduce heat-up / cool-down periods
█ Block: tablets
█ IR: tablets
Reduce digestion period
█ Block: tablets + H2O2
█IR: H2O2 (operator’s presence required)
60 min
1
Time saving: 65 min
Time saving: 135 min
Use optimized method using Solution 1 to get the highest sample throughput
(batch size: 20 samples)
3. Distillation
Per batch: 100 min
t [min]
14
batch #4
batch #6/7 of the day before batch #1
with K-377
Start 8:00 a.m.
batch #6
batch #2
batch #7
cooling
cooling
batch #5
cooling
batch #2
batch #3
cooling
2. Digestion (K-438 #2)
Per batch: 140 min
batch #1
cooling
2. Digestion (K-438 #1)
Per batch: 140 min
cooling
batch batch batch batch batch batch batch
#1
#2
#3
#4
#5
#6
#7
cooling
1. Sample preparation
Per batch: 40 min
batch #3
#4/5 with
K-377
(unattended)
Finish 05:00 p.m.
Distillation
Overview
General overview
1. Dilution of the acidic digestion mixture ▶
2. Alkalinization to convert NH4+ in NH3 ▶
3. Steam distillation to drive out the NH3 ▶
4. NH3 collection in acidic receiver ▶
H 2O
NaOH
H2O steam
acid (H3BO3 or H2SO4)
Distillation
Step 4 can be varied as required for
∙∙ Boric acid titration
∙∙ Back titration
15
Distillation
Parameter for boric acid titration
Step
Why
How much
Rule-of-the-thumb
Step 1: Dilution
H2O dist.
Dilution of the strongly acidic 25 – 90 mL
solution, prevents violent
reactions
4 mL per mL
used H2SO4
Step 2: Alkalinization
NaOH 32 %
Conversion of NH4+ in
NH3 (gaseous)
15 – 90 mL
4.5 mL per mL
used H2SO4
40 – 70 mL
2 % H3BO3 with KCl for
low N contents
0.02 − 6.75 mg/L
Step 3: Preparation of the receiver
H3BO3
(pH 4.65)
To collect the distilled NH3.
NH3 is bound as borate
complex (NH4BO4).
4 % H3BO3 for medium
and high N content
6.75 – 125 mg/L
Step 4: Distillation
Water steam
(100 %)
Separation of NH3 by boiling
of the sample
180 – 300 s
Distillation time:
180 s with KjelMaster
240 s with others
Step 5: Collection
NH3
In boric acid receiver of
pH 4.65
Condenser outlet tube
and electrode must be
completely immersed
For the following analysis potentiometric as well as colorimetric titration can be chosen.
16
Distillation
Parameter for back titration
Step
Why
How much
Rule-of-the-thumb
Dilution of the strongly
acidic solution, prevents
from violent reactions
25 – 90 mL
4 mL per mL
used H2SO4
15 – 90 mL
4.5 mL er mL
used H2SO4
Step 1: Dilution
H2O dist.
Step 2: Alkalinization
NaOH 32 %
Conversion of NH4+ in
NH3 (gaseous)
Step 3: Preparation of the receiver
H2SO4
(0.25 mol/L)
To collect the distilled surplus 10 – 20 mL
of NH3. Is titrated with H2SO4
Volume must be exactly
dosed (usually 20 mL)
Separation of NH3 by boiling
of the sample
Distillation time:
180 s with KjelMaster
240 s with others
Step 4: Distillation
Water steam
(100 %)
180 – 300 s
Step 5: Collection
NH3
In sulfuric acid receiver
Condenser outlet hose and
electrode must be immersed
completely.
For the following analysis only potentiometric titration can be done.
17
Distillation
Hints
∙∙ After digestion the samples must cool-down to 50 – 100 °C before they can be further
processed.
∙∙ Optimal NaOH concentration = 32 %
∙∙ For very low N-contents 2% H3BO3 with KCI (3 g/L) should be chosen as receiving solution,
to achieve lower detection limits.
∙∙ With KjelSampler samples can be processed overnight.
∙∙ With the «IntelliDist» function of the KjelMaster preheating of the instrument can be avoided.
∙∙ Distillation and titration can be synchronized by means of the «Online-Titration».
∙∙ Waste from the sample tube and the receiver can be collected separately with the
KjelMaster.
18
Titration
Overview
Boric acid titration
Potentiometric titration
Colorimetric titration
1. Standard
a) endpoint titration / b) startpoint titration
2. Online
a) endpoint titration / b) startpoint titration
1. Standard
a) endpoint titration / b) startpoint titration
Start volume before titration can be used for all titration variants.
Back titration
Potentiometric Titration
1. Standard
Advantage
a) endpoint titration
Disadvantage
Potentiometric: direct pH measurement
Lower detection limit / online titration possible /
calibration required / relatively short lifetime
shorter analysis time / „IntelliDist“ possible
of electrode
Colorimetric: detection of the color change
longer analysis time / „IntelliDist“ impossible
No calibration necessary / longer lifetime of the
probe / end point visible
/ indicator required / more boric acid necessary
(compared to pot.)
Titration
Standard: the titration starts after the distillation is finished
Standardized process: Distillation → titration / easier Longer analysis time as for Online-Titration
monitoring
Online: the titration takes place whilst the distillation is still in progress
Time saving: Synchronizes distillation and titration
Useful for higher titration volume (> 9 mL)
Fixed and known pH
Adjustment of pH not needed
Huge time saving, higher sample throughput
Endpoint
pH of boric acid must be adjusted to 4.65
Startpoint
More boric acid required (compared to end
point titration), to detect the startpoint
Start volume
Only for well-known titration volume
19
Titration
Endpoint and Startpoint titration (potentiometric)
Endpoint titration
Parameter
What
Setting
Rule-of-the-thumb
1. Algorithm
Dosage steps
for titration
Optimal
Optimal for Titrant < 0.5 N
Slow for Titrant ≥ 0.5 N
2. Start volume
Titrant
HCI / H2SO4
Only for well-known samples /
N-content and high titration volume
3. Titration
Titrant
Consumption should be between 3 and
17 mL
4. Endpoint
pH-value
pH 4.65
pH-value of the used H3BO3 must previously be adjusted to 4.65
Startpoint titration (only step 4 must be changed)
Parameter
What
Setting
Rule-of-the-thumb
4. Startpoint
pH is
detected
pH-value before
NaOH addition
Adjustment of the pH not necessary
Back titration (potentiometric)
Endpoint titration
20
Parameter
What
Setting
Rule-of-the-thumb
1. Algorithm
Dosage steps
for titration
Optimal
„Optimal“ for Titrant < 0.5 N,
„Slow“ for Titrant ≥ 0.5 N
2. Start volume
Titrant NaOH
Only for well-known samples /
N-content and high titration volume
3. Titration
Titrant
Consumption should be between
3 and 17 mL
4. Endpoint
Titrant NaOH
pH-value
Back titration
pH 7.00
Endpoint neutral pH 7.00
Titration
Endpoint titration (Colorimetric)
Endpoint titration
Parameter
What
Setting
Rule-of-the-thumb
1. Algorithm
Dosage steps
for titration
Optimal
Only „Optimal“, not „Slow“
2. Start volume
Titrant
HCI / H2SO4
Only for well-known samples /
N-content and high titration volume
3. Titration
Titrant
Consumption should be between 3 and
17 mL
4. Endpoint
Color change
Sher Indicator in H3BO3
Startpoint titration (only step 4 must be changed)
Parameter
What
Setting
Rule-of-the-thumb
4. Startpoint
pH is
detected
pH before
NaOH addition
Adjustment of the pH not necessary
21
Titration
Hints
Endpoint Titration Boric Acid
B(OH)3 + 2 H2O ⇌ B(OH)4– + H3O+ pKa = 9.24
B(OH)3 + 2 H2O
Sher Indicator
Endpoint pH 4.65
B(OH)4– + H3O+
Acid consumption [mL]
∙∙ Depending on the regulation or method, either potentiometric or colorimetric titration can be
chosen.
∙∙ For endpoint and colorimetric titration the pH of the boric acid must be adjusted to 4.65.
∙∙ Back titration can be used when boric acid should be avoided.
∙∙ For well-known samples a start volume for titration might be used to accelerate the titration
step. (only useful for high titration volumes)
∙∙ The distillation and titration process can be synchronized, by means of online titration.
22
Result calculation
Hints
∙∙ The KjelMaster K-375 performs the calculation automatically.
∙∙ With the PC software KjelLink results/methods can be transferred to a PC for printing, storage or
recalculation.
∙∙ To determine the optimal method parameters, the BUCHI Kjeldahl-calculator can be used as a
helpful tool.
[V(1) - V(Bl)] ∙ F ∙ c ∙ f ∙ M(N)
w(N) =————————————————
m ∙ 1000
% N = w(N) ∙ 100 %
% P = w(N) ∙ PF ∙ 100 %
Example (Titrant is 0.25 M H2SO4,f=1.000):
(7.5mL - 1mL) ∙ 2 ∙ 0.25 ∙ 1 ∙ 14
—————————————————— = 0.0607 g N
0.750 g ∙ 1000
0.0607 g N ∙ 100 %
= 6.07 % N
0.0607 g N ∙ 6.25 ∙ 100 % = 37.94 % P
weight fraction of N
consumption of titrant, sample, [mL]
average consumption of titrant, blank, [mL]
molar reaction factor (1 = HCI, 2 = H2SO4)
concentration of titrant [mol /L]
factor of titrant
molecular weight of (14,007)
sample weight [g]
conversion factor (mL in L)
Protein factor
% of weight of N
% of weight of protein
Result
w(N):
V(1):
V(Bl):
F:
c:
f:
M(N):
m:
1000:
PF:
% N:
% P:
23
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