Application Note No. 197/2015 Nitrogen and protein determination in dairy products Nitrogen determination in sodium nitrate KjelDigester K-449, KjelMaster K-375 with KjelSampler K-376: Accelerated Nitrogen and Protein Determination in Dairy Products According to the Kjeldahl Method by Digestion with Kjeldahl Tablets and Hydrogen Peroxide Followed by Colorimetric Titration
1. Introduction A reliable method for the determination of total nitrogen and protein in dairy products using hydrogen peroxide for digestion acceleration, according to ISO 8968-3 and AOAC 991.20 is presented [1,2]. The samples are digested by the KjelDigester K-449. The distillation and boric acid titration are performed using the KjelMaster system K-375 / K-376. Equivalent to the international normative procedures [1,2], the measuring method of the boric acid titration is colorimetric. Therefore, a mixed indicator accoding to Sher is added to the boric acid solution and the KjelMaster K-375 is equipped with a colorimetric sensor. The combination of the accelerated digestion method, using the Kjeldahl Tablet Titanium in combination with hydrogen peroxide, the KjelMaster system K-375 / K-376 increases the sample throughput to up to 120 samples per workday (9 h).
2. Equipment ⋅ ⋅ ⋅ ⋅ ⋅
KjelDigester K-449 (the parameters used are also valid for K-446) Scrubber K-415 TripleScrub ECO KjelMaster K-375 with KjelSampler K-376 Mixer, Retsch Grindomix GM200 Analytical balance (accuracy ± 0.1 mg) Safety accessories: ⋅ User protection shield, BUCHI (11057889) ⋅ Hirschmann bottle top dispenser ceramus® 5-30 mL, VWR (613-3243) with ceramus® discharge tube, spiral-shaped, VWR (612-0917)
Figure 1: Safety accessories for the digestion with hydrogen peroxide and Kjeldahl Tablet Titanium
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3. Chemicals and Materials Chemicals: ⋅ ⋅ ⋅ ⋅ ⋅
⋅ ⋅
⋅
Sulfuric acid, conc. 98 %, analytical reagent, Beijing Chemical Works Titanium, BUCHI Kjeldahl Tablets (11057980) Hydrogen peroxide, 30 %, analytical reagent, Beijing Chemical Works Sodium hydroxide 32 %, analytical reagent, Sinopharm chemical reagent Boric acid 4 %, 200 g boric acid (analytical reagent, Tianjin Guangfu Fine Chemical Research Institute) diluted to 5 L with deionized water, 12.5 mL of mixed Sher indicator BUCHI (003512) are added to the solution, adjust pH to 4.65 Sulfuric acid 0.05 mol/L, Aladdin (S112273-1L) Neutralization solution for the Scrubber: 600 g sodium carbonate (analytical reagent, Sinopharm chemical reagent) about 2 mL ethanol and a spatula tip of bromthymol blue (analytical reagent, Tianjin Kemiou chemical reagent) diluted to 3 L with distilled water L(+) Glutamic acid, assay 99.5 %, Aladdin (G103979-100g)
For a safe handling please pay attention to all corresponding MSDS. Samples: Table 1: The labelled protein and fat contents of the samples
Sample
Protein
Fat
Skimmed milk
3.4 g/100 mL
0 g/100 mL
Whipping cream
2.2 g/100 g
36.0 g/100 g
Shake milk drink
1.5 g/100 mL
1.7 g/100 mL
Parmesan cheese
33.0 g/100 g
38.4 g/100 g
The samples were purchased at a local supermarket.
4. Procedure The determination of nitrogen and protein in dairy products includes the following steps: ⋅ ⋅ ⋅
Homogenization of the Skimmed milk, Whipping cream and Shake milk drink by shaking and the Parmesan cheese by grinding Digestion of the sample, using the K-449 (K-446 respectively) Distillation and colorimetric titration of the sample, using KjelMaster system K-375 / K-376
4.1. Digestion method – glutamic acid (verification of the method) Start the KjelDigester K-449 according to the parameters listed in Table 2 1. 2. 3. 4. 5. 6. 7. 8.
9.
Preheat the KjelDigester K-449 to 330 °C Place 0.12 g glutamic acid in a 300 mL sample tube Add 1 Titanium tablet and 10 mL of sulfuric acid (conc. 98 %) to each tube Prepare additional blanks, chemicals without sample Connect the Scrubber K-415 to the K-449 for absorbing the acid fumes created during digestion Place the rack under the fume hood and attach the protection shield Dropwise add 8 mL hygrogen peroxide (30 %) with the dispenser down the glass wall of the sample tube, wait until the fuming stops and the reaction subsides Insert the rack containing the protection shield and the samples into the cooling position of the KjelDigester and immediately start the digestion according to the parameters listed in Table 2 Let the samples cool down when the digestion is completed.
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Table 2: Temperature profile for digestion with the K-449
Step
Temperature [°C]
Time [min]
1
330
0
2
420
60
Cooling
–
25
4.2. Digestion method – samples 1. Start the KjelDigester K-449 according to the parameters listed in Table 2 2. Place each sample in a 300 mL sample tube as described in Table 3 Table 3: Weight for each sample
Sample
Weight [g]
Skimmed milk
2.0
Whipping cream
2.0
Shake milk drink
2.5
Parmesan cheese
0.25
3. Add one Titanium tablet and 10 mL of sulfuric acid (conc. 98 %) to each tube 4. Prepare additional blanks, i.e. add 10 mL sulfuric acid and one Titanium tablet without sample 5. Connect the Scrubber K-415 to the K-449 for absorbing acid fumes created during digestion 6. Place the rack under the fume hood and attch the protection shield 7. Dropwise add 8 mL hygrogen peroxide (30 %) with the dispenser down the glass wall of the sample tube, wait until the fuming stops and the reaction subsides. 8. Insert the rack containing the protection shield and the samples into the cooling position of the KjelDigester and immediately start the digestion according to the parameters listed in Table 2. 9. Let the samples cool down when the digestion is completed. NOTE: If the liquid inside the sample tube is not clear and blue-green, digest for additional 15 min at 420 °C. The samples should be clear-green immediately after the digestion. A darkening of the clear liquid samples during the cooling down process is possible but does not affect the results. NOTE: Due to the use of hydrogen peroxide, the indicator bromthymol blue in the neutralisation solution of the K-415 could be decomposed faster compared to a classical Kjeldahl digestion, hence, a fading of the blue color is possible.
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4.3. Distillation and titration For colorimetric titration it is necessary to determine the setpoint of the boric acid solution in advance to the blank and sample determinations. It is necessary to determine the setpoint every day before starting sample determinations, and when the method is changed or fresh chemicals are used to adjust the device to the current conditions. The detailed procedure, including the preparation of the sensor, is described in the Technical Note 179/2015 “Colorimetric titration procedure using Sher indicator” [3]. The setpoint was measured three times. ⋅ ⋅ ⋅
1st setpoint 2nd setpoint 3rd setpoint
preheating 1st measurement 2nd measurement, confirms the 1st measurement
The last setpoint measurement is used as endpoint for all following determinations including priming, blanks and samples. 1. Determine the setpoint and check it’s range and deviation: Select all parameters for the setpoint determination according to Table 4. Table 4: Parameters for setpoint determination
Parameter
Setting
Preheating before setpoint
Yes
Setpoint runs
3
Setpoint cycle
Via sampler
Boric acid
4%
Indicator
Sher
Method
Select the same method as for sample determination
NOTE: The selected method, boric acid and indicator for setpoint determination must be identical to the method used for sample determination. 2. Check the setpoint range and deviation ⋅ The determined setpoints should be in a range of 700 – 900 mV ⋅ The deviation between the two last measured setpoints should be ≤ 20 mV 3. Perform a priming to remove all residues 4. Determine blanks according to the parameters listed in Table 6 5. Determine samples according to the parameters listed in Table 6 Table 5: Setpoint measurements and deviation
Setpoint 2nd
843.2 mV
Setpoint 3rd
846.1 mV
Deviation
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Table 6: Distillation and titration with the KjelMaster system K-375 / K-376
H2O volume
50 mL
Titration solution
H2SO4 0.05 mol/L
NaOH volume
45 mL
Sensor type
Colorimetric
Reaction time
5s
Titration mode
Online
Distillation mode
Fixed time
Titration start time
90 s
Distillation time
180 s
Measuring mode
Setpoint
Stirrer speed distillation
5
Stirrer speed titration 10
Steam output
100 %
Titration start volume 0 mL
Titration type
Boric acid
Titration algorithm
Receiving solution vol.
60 mL
Optimal
NOTE: The sample throughput for this application was increased by using the “Online” titration mode: By applying the “Online” titration the time for the distillation and titration process is reduced to about 5 minutes per analysis because titration starts during the distillation is still in progress. 4.4. Calculation The results are calculated as a 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. For the reference substance, the purity of the glutamic acid is considered in equation (4).
wN =
(VSample - VBlank ) ⋅ z ⋅ c ⋅ f ⋅ MN
(1)
m Sample ⋅ 1000
%N = wN · 100 %
(2)
%P = wN · PF · 100 %
(3)
%N Glu =
wN VSample VBlank z c f MN mSample 1000 %N %NGlu %P P PF
%N • 100 P
(4)
: weight fraction of nitrogen : amount of titrant for the sample [mL] : mean amount of titrant for the blank [mL] : molar valence factor (1 for HCl, 2 for H2SO4) : titrant concentration [mol/L] : titrant factor (for commercial solutions normally 1.000) : molecular weight of nitrogen (14.007 g/mol) : sample weight [g] : conversion factor [mL/L] : percentage of weight of nitrogen : percentage of weight of nitrogen corrected for the purity of reference substance : percentage of weight of protein : purity of the reference substance glutamic acid [%] : sample-specific protein factor (6.38 for dairy products)
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5. Results
5.1. Recovery of glutamic acid The results of nitrogen determination and recovery for glutamic acid analysis (assay ≥ 99.5%) are presented in Table 7. The nominal value of glutamic acid is 9.47 % nitrogen. The recoveries are within the specification between the 98 % and 100 % [1-3]. Table 7: Results of the recovery of nitrogen in glutamic acid
Glutamic acid
mSample [g]
VSample [mL]
%NGlu
Recovery [%]
Sample 1
0.1182
8.663
9.497
100.3
Sample 2
0.1213
8.902
9.530
100.6
Sample 3
0.1175
8.680
9.574
101.1
Sample 4
0.1208
8.758
9.403
99.3
Average [%]
–
–
9.501
100.3
Rsd [%]
–
–
0.76
0.76
The mean blank volume (VBlank) was 0.649 mL (n = 4). 5.2. Protein determination in dairy products The results of the determination of nitrogen and protein contents in dairy products are presented in Tables 8-11. Table 8: Results of the determination of nitrogen and protein in skimmed milk (declared protein content 3.4 g/100 mL)
Skimmed milk mSample [g]
VSample [mL] %N
%P [g/100g]
%P [g/100mL]
Sample 1
2.0269
8.880
0.569
3.629
3.516
Sample 2
2.1194
9.178
0.564
3.596
3.484
Sample 3
1.9377
8.404
0.561
3.577
3.466
Average [%]
–
–
0.565
3.600
33.489
Rsd [%]
–
–
–
0.73
0.73
The mean blank volume (VBlank) was 0.649 mL (n = 4). The experimental protein content [%] was re-calculated taking the density (1.032 g/mL) into account in order to obtain the protein content as g/100 mL for milk. Table 9: Results of the determination of nitrogen and protein in whipping cream (declared protein content 2.2 g/100 g)
Whipping cream
mSample [g]
VSample [mL]
%N
%P [g/100g]
Sample 1
1.8244
4.883
0.332
2.077
Sample 2
1.7929
4.769
0.329
2.058
Sample 3
1.8164
4.829
0.330
2.060
Average [%]
–
–
0.330
2.065
Rsd [%]
–
–
0.51
0.51
The mean blank volume (VBlank) was 0.554 mL (n = 3). Table 10: Results of the determination of nitrogen and protein in shake milk drink (declared protein cont. 1.5 g/100 mL)
Shake milk drink mSample [g]
VSample [mL]
%N
%P [g/100g]
%P [g/100mL]
Sample 1
2.2986
5.114
0.272
1.736
1.682
Sample 2
2.2965
5.131
0.273
1.744
1.690
Sample 3
2.6041
5.706
0.272
1.735
1.681
Average [%]
–
–
0.272
1.738
1.684
Rsd [%]
–
–
–
0.28
0.28
The mean blank volume (VBlank) was 0.649 mL (n = 4). Application Note 197/2015
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Table 11: Results of the nitrogen and protein determination in parmesan cheese (declared protein content 33 g/100 g)
Parmesan cheese mSample [g]
VSample [mL]
%N
%P [g/100 g]
Sample 1
0.2789
10.796
5.116
32.640
Sample 2
0.2484
9.746
5.152
32.871
Sample 3
0.2498
9.529
5.002
31.910
Average [%]
–
–
5.090
32.474
Rsd [%]
–
–
1.54
1.54
The mean blank volume (VBlank) was 0.609 mL (n = 3).
6. Comparison to Standard Methods This application note is based on the standard method ISO 8968-3 and AOAC 991.20 with minor differences. These differences are shown in Table 11. Table 11: Differences to ISO 8968-3 and AOAC 991.20
Application note
ISO 8968-3
AOAC 991.20
Notes / Impact
Sample tube
300 mL
250 mL
250 mL
No impact
Sample weight
2.5 g
2g
5 mL
No impact, homogeneous sample
Catalyst
1 × 3.7 g Tablets Composition 94.4 % K2SO4 2.8 % TiO2 2.8% CuSO4*5H2O
1 × 3.7 g Tablets Composition 94.4 % K2SO4 2.8 % TiO2 2.8% CuSO4*5H2O
12 g K2SO4 + 1 mL CuSO4 Solution 5g CuSO4 x 5 H2O in 100 mL water
The choice of catalyst does not influence the result. Digestion time is reduced using Titanium Tablets, see Application Note 078/2012.
Sulfuric acid
10 mL
10 mL
20 mL
No impact, same ration of sulfuric acid/catalyst.
hydrogen peroxide
8 mL
5 mL
No
No impact, hydrogen peroxide can reduce the digestion time and foaming.
Sodium hydroxide
45 mL (Conc. 32 %)
40 mL (Conc. 40 %)
65 mL (Conc. 40 %)
No impact, same ratio of sodium hydroxide/sulfuric acid.
Titration solution
H2SO4 0.1N
HCl 0.1N
HCl 0.1N
No impact consumption of the titration solution should be between 3-17 mL.
Titration
colorimetric
Colorimetric or potentiometric
colorimetric
No impact, the results are equal
1.75 – 3 h
1.75 - 2.5 h
No impact
Digestion time 60 min Indicator
Sher mixed indicator Methyl red / Methyl red / No impact. With the Sher bromocresol green bromocresol green mixed indicator the color 1:5 1:5 changes more sharply at pH 4.65 than methyl red / bromocresol green indicator.
7. Conclusion The determination of nitrogen and protein in dairy products using the KjelDigester K-449 and KjelMaster system K-375 / K-376 by colorimetric titration provides reliable and well reproducible results. By applying accelerated digestion using hydrogen peroxide, the process is intensified. These results correspond well to the labelled values of the dairy products. In addition, the protein content determined is in accordance the tolerance level as controlled by the Swiss Cantonal Chemists [4].
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With the KjelDigester K-449 the digestion process (including preheating, digestion and cooling) is very fast and is fully automated. The combination with the fully-automatic KjelMaster system K-375 / K-376, allows unattended operation and highest sample throughput of about 120 samples per 9 hours working day.
8. References [1] ISO 8968-3:2007 Milk-Determination of nitrogen content Part 3:Block-digestion method (Semi-micro rapid routine method) [2] AOAC 991.20 Nitrogen (Total) in Milk [3] Technical Note No.179/2015 Colorimetric titration procedure using Sher indicator [4] FIAL, Verband der Kantonschemiker der Schweiz; Empfehlung: Genauigkeit der Angaben bei der Nährwertkennzeichnung, 3. Ausgabe 2008.
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