054/2010

SpeedDigester K-436, K-439

Kjeldahl Sampler System K-370/K-371
Nitrogen and Protein Determination in Milk by
Digestion with Hydrogen Peroxide and Sulfuric Acid
 SHORT NOTE
054/2010
SpeedDigester K-436 / K-439
Kjeldahl Sampler System K-370/K-371

Nitrogen and Protein Determination in Milk by Digestion with

Hydrogen Peroxide and Sulfuric Acid
A simple and very fast procedure for protein determination in milk is introduced below. The sample is digested with hydrogen
peroxide and sulfuric acid using the SpeedDigester K-436 or K-439, followed by distillation and titration with the Kjeldahl
Sampler System K-370/K-371. The determined protein contents correspond to the results obtained with the Kjeldahl method.

Table 2: Parameters for distillation and titration using the Kjeldahl Sampler
Introduction System K-370/K-371
Distillation Titration
The digestion with hydrogen peroxide 30% instead of
Kjeldahl tablets for nitrogen and protein determination [1] is Water 50 ml Boric acid 4% 50 ml
a very fast (30 min instead of 85 min (K-439) or 100 min Sodium 90 ml Titration solution H2SO4
(K-436)) and environmentally friendly (free of any heavy hydroxide 0.1 mol/l
metal) alternative to the classical Kjeldahl methods. Reaction time 5s Min. titration time 5s
Distillation time 240 s Max. titration vol. 40 ml
Experimental
Steam power 100% Titration method Standard
Instrumentation: SpeedDigester K-436, K-439, with H2O2 Sample tube 500 ml Type Endpoint
suction module, Kjeldahl Sampler System K-370/K-371
Stirrer sp. dist. 5 pH 4.65
Samples: Whole milk UHT, partially skimmed milk UHT, Stirrer sp. titr. 7 Algorithm 1
and chocolate milk beverage: labeled protein contents
3 g/100ml (whole milk) and 3.5 g/100ml (partially skimmed
and chocolate milk). Protein determinations according to Results
the classical Kjeldahl method [2] measure 3.15% for the The tryptophan recoveries (n = 4) were 99.0%, rsd 0.04%
whole milk, 3.19% for the partially skimmed milk, and (K-439) and 98.9%, rsd 0.13% (K-436).
3.30% for the chocolate milk beverage.
The determined protein contents are presented in Table 3.
Determination: Approx. 5 g of the homogenized sample
Table 3: Determined protein contents in milk samples (relative standard
were weighed directly into a sample tube. A portion of
deviation in brackets, n=4)
20 ml of sulfuric acid was added, and the digestion was
started using the parameters specified in Table 1. Initially, Protein content Protein content
15 ml of hydrogen peroxide 30% was added to the capillary K-439 [g/100g] K-436 [g/100g]
funnel 2 minutes after the start of the digestion. Then, Whole milk 3.13 (0.20%) 3.13 (0.20%)
another 15 ml of hydrogen peroxide 30% was added to the Part. skimmed milk 3.18 (0.10%) 3.18 (0.09%)
capillary funnel 10 minutes after the start of the digestion.
Chocolate milk 3.28 (0.25%) 3.29 (0.22%)
The method was verified by using 0.18 g tryptophan as the
reference substance. Conclusion
Table 1: Temperature profile for digestion with the K-439, K-436
The determination of the protein contents in milk according
K-439 K-436 to the hydrogen peroxide method using SpeedDigester
Step Temp Time [min] Level Time K-436, K-439 and Kjeldahl Sampler System K-370/K-371 is
[°C] [min] very fast and gives reliable and reproducible results that
correspond to the results obtained with the standard
Preheat 450 - 7¾ 10 Kjeldahl method using catalysts with low relative standard
1 450 20 7¾ 20 deviations.
2 480 10 8½ 10
Cooling - 30 - 30 References
[1] Rossi et al., Comparison between the Kjeldahl and the
After the digestion, the ammonia of the sample was distilled Hach methods, J. Argentine Chemical Society, Vol. 92, No
into a boric acid solution by steam distillation and then 4/6, 99-108 (2004)
titrated with sulfuric acid using the parameters specified in [2] AN 020/2010 “Nitrogen and Protein Determination in
Table 2. Milk according to the Kjeldahl Method”
Operation manual SpeedDigester K-425 / K-436
Operation manual SpeedDigester K-439
Operation manual Kjeldahl Sampler System K-370/K-371

For more detailed information please refer to Application

Note 054/2010

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 1 Introduction
An easy, fast, and reliable method for the determination of nitrogen and protein in milk
samples by digestion with hydrogen peroxide and sulfuric acid is introduced below.
In literature both methods were compared and had comparable results [1].
The main differences to the standard methods [2] and [3] are as follows:
- Considerably shorter digestion time (30 min instead of 85 min / 100 min) [4]
- More environmentally friendly (H2O2 is used instead of heavy metal catalysts)
The samples are digested using the SpeedDigester K-436 and K-439. The distillation
and boric acid titration are performed with the Kjeldahl Sampler System K-370/K-371.

2 Equipment
− SpeedDigester K-436, K-439 (the parameters used for K-436 are also valid for
SpeedDigester K-425)
− Suction module for H2O2 digestion, Büchi (11055853)
− Stand with drip tray, Büchi (11055216)
− Scrubber B-414 with condenser
− Kjeldahl Sampler System K-370/K-371 (or any other Büchi Kjeldahl Distillation
Unit)
®
− Hirschmann bottle top dispenser ceramus 5-30 ml, VWR (613-3243) with
® 1
ceramus discharge tube, spiral-shaped, VWR (612-0917)
− Analytical balance (accuracy ± 0.1 mg)

3 Chemicals and Materials

− Sulfuric acid conc 98%, Fluka (84727)
− Hydrogen peroxide 30%, Fluka (95302); always store the hydrogen peroxide in a
refrigerator with a temperature range of 4 – 8°C to prevent degradation
− Sodium hydroxide 32%, Brenntag (81980-452)
− Boric acid 4%, 200 g boric acid, Brenntag (80948-155), diluted to 5 l with
deionized water, pH adjusted to 4.65
− Sulfuric acid 0.1 mol/l, Riedel de Haën (35357), standard solution
− Neutralization solution without indicator (H2O2 vapor will destroy the indicator) for
the Scrubber: 600 g sodium carbonate, calcined, technical, Synopharm
(0179420), diluted to 3 l with distilled water: a couple drops of Silicone Antifoam,
Fluka (85390), were added to prevent foaming of the scrubber solution
− Tryptophan, VWR (28821.131; assay: > 99%)

4 Samples
− Whole milk UHT (labeled protein content: 3 g/100ml; determined according to
the standard Kjeldahl method [4] 3.15%)
− Partially skimmed milk UHT (labeled protein content: 3.5 g/100ml; determined
according to the standard Kjeldahl method [4] 3.19%)
− Chocolate milk beverage (labeled protein content: 3.5 g/100ml; determined
according to the standard Kjeldahl method [4] 3.30%)

The samples were purchased at a local supermarket.

The samples were homogenized by shaking at ambient temperature.

1
A bottle top dispenser makes dosing of hydrogen peroxide much easier than using a
graduated cylinder but the dispenser has to be approved for hydrogen peroxide 30%.
Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 3/11
 5 Procedure
The determination of nitrogen and protein in milk includes the following steps:

− Homogenization of the sample

− Digestion of the sample using SpeedDigester K-436, K-439
− Distillation and titration of the sample using
Kjeldahl Sampler System K-370/K-371

5.1 Digestion method - tryptophan (verification of the method)

− Place approx. 0.18 g tryptophan in a 300 ml sample tube
− Add a portion of 20 ml of sulfuric acid (98%)
− Prepare additional blanks, chemicals without sample
− Carefully suspend the sample by gently swirling the tube
− Connect the Scrubber B-414 to the SpeedDigester K-436 or K-439 for absorbing
the acid fumes created during digestion
− Insert the rack containing the samples into the preheated unit
− Digest the samples according to the parameters listed in Table 1
− The capillary funnel must be mounted correctly (see Figure 1); ensure that the
capillary funnel is free of fat or dust, as fat/dust can block the capillary
− Add 15 ml of hydrogen peroxide 30% to the capillary funnel 2 minutes after the
start of the digestion
− Then, add another 15 ml of hydrogen peroxide 30% to the capillary funnel
10 minutes after the start of the digestion

Figure 1: Correct mounting of the capillary funnel: the hydrogen peroxide drops have to travel down the glass wall. If the
drops drip directly into the digestion solution, boiling retardation may occur.

5.2 Digestion method - samples

− Place approx. 5 g of the sample in a 300 ml sample tube
− Add a portion of 20 ml of sulfuric acid (98%)
− Prepare additional blanks, chemicals without sample
− Carefully suspend the sample by gently swirling the tube
− Connect the Scrubber B-414 to the SpeedDigester K-436 or K-439 for absorbing
the acid fumes created during digestion
− Insert the rack containing the samples into the preheated unit
− Digest the samples according to the parameters listed in Table 1
− The capillary funnel must be mounted correctly (see Figure 1); ensure that the
capillary funnel is free of fat or dust, as fat/dust can block the capillary

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 4/11

 − Add 15 ml of hydrogen peroxide 30% to the capillary funnel 2 minutes after the
start of the digestion
− Then, add another 15 ml of hydrogen peroxide 30% to the capillary funnel
10 minutes after the start of the digestion

Table 1: Temperature profile for digestion with the K-439, K-436

K-439 K-436
Step Temperature [°C] Time [min] Heating level Time [min]

Preheating 450 - 7¾ 10
1 450 20 7¾ 20
2 480 10 8½ 10
Cooling 30 30

− If the liquid inside the sample tube is not clear and colorless after Step 1, add
another 15 ml of hydrogen peroxide 30% into the capillary funnel and digest for
an additional 10 min as described in step 1
− Let the samples cool down to ambient temperature

NOTE: When the samples are placed in the cooling position it takes approx. 30 min to
cool them down: when they are left in the heating chamber it takes at least 60 min.
NOTE: If the preheat temperature or the temperature at step 1 is too high, H2O2
evaporates too fast => insufficient digestion.
If not all positions are used, the suction circuit has to be sealed by placing rubber
plugs (Büchi No. 11056016) on top of the suction module, and glass caps (Büchi No.
040049) have to be used instead of sample tubes. Install insulation caps (Büchi No.
11056024) in unused positions of the insulation plate to ensure even heat distribution
inside the heating chamber.
Unused positions should always be located at the rear end of the rack.
For easy and safe handling and storage of the suction module and it’s funnels, the use
of the stand with drip tray (Büchi No. 11055216) is recommended.

5.3 Distillation and titration

Distill the samples according to the parameters listed in Table 2.
Table 2: Distillation and titration with the Kjeldahl Sampler System K-370/K-371

Distillation Titration

Water 50 ml Boric acid 4% 50 ml

Sodium hydroxide 90 ml Titration solution H2SO4 0.1 mol/l
Reaction time 5s Min. titration time 5s
Distillation time 240 s Max. titration volume 40 ml
Steam power 100% Titration method Standard
Sample tube 500 ml Type Endpoint
Stirrer speed dist. 5 pH 4.65
Stirrer speed titr. 7 Algorithm 1

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 5/11

 5.4 Calculation
The results are calculated as percentage of nitrogen. In order to calculate the protein
content of the sample, the nitrogen content is multiplied with a sample-specific protein
factor. The following equations (1), (2), and (3) are used to calculate the results.

(VSample - VBlank ) ⋅ z ⋅ c ⋅ f ⋅ MN
wN = (1)
m Sample ⋅ 1000

%N = wN · 100% (2)

%P = wN · PF · 100% (3)

wN : weight fraction of nitrogen

VSample : amount of titrant for the sample [ml]
VBlank : mean amount of titrant for the blank [ml]
z : molar valence factor (1 for HCl, 2 for H2SO4)
c : titrant concentration [mol/l]
f : titrant factor (for commercial solutions normally 1.000)
MN : molecular weight of nitrogen (14.007 g/mol)
mSample : sample weight [g]
1000 : conversion factor [ml/l]
%N : percentage of weight of nitrogen
%P : percentage of weight of protein
PF : sample-specific protein factor (6.38 for dairy products)

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 6/11

 6 Results

6.1 Digestion with SpeedDigester K-439

6.1.1 Recovery of tryptophan

The results of the nitrogen determination and recovery in tryptophan are presented in
Table 3. The nominal value of tryptophan is 13.72% nitrogen. The recoveries are
within the specification of > 98% [2], [3].

Table 3: Results for the recovery of nitrogen in tryptophan with K-439

Tryptophan mSample [g] VSample [ml] %N Recovery [%]

Sample 1 0.2037 10.136 13.58 99.0

Sample 2 0.1865 9.298 13.57 99.0
Sample 3 0.2137 10.624 13.58 99.0
Sample 4 0.1979 9.860 13.59 99.1
Average - - 13.58 99.0
Rsd [%] - - 0.04 0.04
The mean blank volume for this sample was 0.261 ml (n = 2).

6.1.2 Protein determination in milk samples

The results of the determination of nitrogen and protein in milk samples are presented
in Tables 4 to 6.

Table 4: Results for the determination of nitrogen in whole milk UHT with K-439

Whole milk UHT mSample [g] VSample [ml] %N %P

Sample 1 5.1635 9.306 0.491 3.13

Sample 2 5.1584 9.325 0.493 3.14
Sample 3 5.1494 9.266 0.490 3.13
Sample 4 5.1485 9.289 0.492 3.14
Average - - 0.491 3.13
Rsd [%] - - 0.20 0.20
The mean blank volume for this sample was 0.255 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.15%P
(rsd 0.14%).

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 7/11

 Table 5: Results for the determination of nitrogen in partially skimmed milk UHT with K-439

Partially skimmed milk mSample [g] VSample [ml] %N %P

UHT

Sample 1 5.1553 9.425 0.498 3.18

Sample 2 5.1584 9.429 0.498 3.18
Sample 3 5.1494 9.390 0.498 3.18
Sample 4 5.1485 9.410 0.499 3.18
Average - - 0.498 3.18
Rsd [%] - - 0.10 0.10
The mean blank volume for this sample was 0.253 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.19%P
(rsd 0.23%).

Table 6: Results for the determination of nitrogen in chocolate milk beverage UHT with K-439

Chocolate milk beverage mSample [g] VSample [ml] %N %P

Sample 1 5.2087 9.792 0.513 3.27

Sample 2 5.2225 9.833 0.514 3.28
Sample 3 5.2527 9.894 0.514 3.28
Sample 4 5.1874 9.810 0.516 3.29
Average - - 0.514 3.28
Rsd [%] - - 0.25 0.25
The mean blank volume for this sample was 0.253 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.30%P
(rsd 0.17%).

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 8/11

 6.2 Digestion with SpeedDigester K-436

6.2.1 Recovery of tryptophan

The results of the nitrogen determination and recovery in tryptophan are presented in
Table 7. The nominal value of tryptophan is 13.72% nitrogen. The recoveries are
within the specification of > 98% [2], [3].

Table 7: Results for the recovery of nitrogen in tryptophan with K-436

Tryptophan mSample [g] VSample [ml] %N Recovery [%]

Sample 1 0.1998 9.923 13.56 98.8

Sample 2 0.1895 9.440 13.58 99.0
Sample 3 0.1865 9.296 13.58 99.0
Sample 4 0.1926 9.566 13.54 98.7
Average - - 13.57 98.9
Rsd [%] - - 0.13 0.13
The mean blank volume for this sample was 0.255 ml (n = 2).

6.2.2 Protein determination in milk samples

The results of the determination of nitrogen and protein in milk samples are presented
in Tables 8 to 10.

Table 8: Results for the determination of nitrogen in whole milk UHT with K-436

Whole milk UHT mSample [g] VSample [ml] %N %P

Sample 1 5.1382 9.250 0.491 3.13

Sample 2 5.1750 9.306 0.491 3.13
Sample 3 5.1569 9.264 0.490 3.13
Sample 4 5.1588 9.288 0.491 3.13
Average - - 0.491 3.13
Rsd [%] - - 0.10 0.10
The mean blank volume for this sample was 0.243 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.15%P
(rsd 0.14%)

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 9/11

 Table 9: Results for the determination of nitrogen in partial skimmed milk UHT with K-436

Partial skimmed milk UHT mSample [g] VSample [ml] %N %P

Sample 1 5.1555 9.416 0.498 3.18

Sample 2 5.1354 9.387 0.498 3.18
Sample 3 5.1251 9.349 0.497 3.17
Sample 4 5.1225 9.356 0.498 3.18
Average - - 0.498 3.18
Rsd [%] - - 0.09 0.09
The mean blank volume for this sample was 0.254 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.19%P
(rsd 0.23%)

Table 10: Results for the determination of nitrogen in chocolate milk beverage UHT with K-436

Chocolate milk beverage mSample [g] VSample [ml] %N %P

Sample 1 5.2330 9.873 0.515 3.29

Sample 2 5.2035 9.820 0.515 3.29
Sample 3 5.2058 9.801 0.514 3.28
Sample 4 5.2072 9.855 0.516 3.30
Average - - 0.515 3.29
Rsd [%] - - 0.22 0.22
The mean blank volume for this sample was 0.255 ml (n = 2).
Determined protein content according to the classical Kjeldahl method [4]: 3.30%P
(rsd 0.17%)

Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 10/11

 7 Conclusion
The determination of nitrogen and protein in milk samples according to the hydrogen
peroxide method using the SpeedDigester K-436, K-439 and Kjeldahl Sampler System
K-370/K-371 provides reliable and reproducible results that correspond to the results
obtained with the standard Kjeldahl method using catalysts with low relative standard
deviations. The digestion time is very fast: 30 min for both the K-439 and the K-436.
The recoveries with tryptophan were 99.0%, rsd 0.04% (K-439), and 98.9%, rsd
0.13% (K-436), respectively and are within the specification of > 98% [2], [3].

8 References
[1] Rossi et al., Comparison between the Kjeldahl and the Hach methods, J. Argentine
Chemical Society, Vol. 92, No 4/6, 99-108 (2004)
[2] LFGB § 35 L01.00-10/1
[3] AOAC 991.20
[4] AN 020/2010 “Nitrogen and Protein Determination in Milk according to the Kjeldahl
Method”

Operation manual of SpeedDigester K-425 / K-436

Operation manual of SpeedDigester K-439
Operation manual of Scrubber B-414
Operation manual of Kjeldahl Sampler System K-370/K-371

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T +41 71 394 63 63
F +41 71 394 65 65
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Application Note 054/2010 Version 1, Copyright © 2010 Büchi Labortechnik AG 11/11