Escolar Documentos
Profissional Documentos
Cultura Documentos
determination of protein
FOSS Laboratory Conference
September 16-17 2013
Hillerd Denmark
Jurgen.moller@bredband.net
1
Synopsis
Kjeldahl method
Dumas combustion method
NIR methods
Alternative methods (AAA & DBC)
Adulterations
Conclusions & Summary
Kjeldahl - Reference
920.53 (Hg)
930.33 (Cu)
920.70 (Hg)
932.08 (Hg)
920.87 (Hg)
939.02 (Hg)
920.109 (Cu)
940.25 (Hg)
920.123 (Cu)
941.06 (Cu)
920.155 (Hg)
945.01 (Hg)
920.176 (Hg)
945.18 (Hg)
925.31 (Hg)
945.23 (Hg)
928.08 (Hg)
945.48 (Cu)
930.01 (Hg)
950.09 (Hg)
930.02 (Hg)
950.10 (Hg)
930.25 (Hg)
950.48 (Hg)
930.29 (Cu)
955.04 (Hg)
962.10 (Hg)
969.37 (Hg)
976.05 (Hg)
977.02 (Hg)
978.04 (Hg)
979.09 (Hg)
981.10 (Hg)
984.13 (Cu)
988.05 (Cu/Ti)
991.20 (Cu)
2001.11 (Cu)
Recovery of Nitrogen
Lysine hydrochloride
16
100%
15
90-93%
% Protein
14
Hg
Se
13
Cu
Ti
12
11
10
10
20
30
40
50
60
70
Se
Cu
92
Ti
90
Hg
88
86
84
Lysine
Dog food
Meat
Fishmeal
Wheat
% CP
12,46
12,45
12,39
12,55
AAFCO PTS
Cu
Cu/TiO2
%
C
P sd %
Sample
% CP
sd %
AAFCO 0826
18,2
0,35
18,2
AAFCO 0827
12,9
0,36
AAFCO 0828
40,8
AAFCO 0829
Dumas
% CP
sd %
0,29
18,4
0,32
12,8
0,46
13,0
0,46
0,77
40,0
0,68
41,0
0,43
23,6
0,42
23,0
0,58
23,5
0,33
AAFCO 0830
18,2
0,27
18,2
0,24
18,5
0,32
AAFCO 0831
27,5
0,40
27,3
0,64
27,8
0,46
23,5
0,43
23,2
0,48
23,7
0,39
Average
AOAC 2001.11
Global Proteinstandard on
basis of AOAC 2001.11
EN ISO 5983-2:2005
Animal feeding stuffs Determination of nitrogen
content and calculation of crude protein content
0,6
sdR
0,5
Linear (sdR)
0,4
Linear (sdr)
0,3
0,2
0,1
0
0
20
40
60
80
100
EN ISO 20483:2006
1,6
1,4
1,2
y = 0,0129x + 0,061
1
sd %
sdr
sdR
0,8
Linear (sdR)
Linear (sdr)
0,6
0,4
y = 0,0058x + 0,0248
0,2
0
0
20
40
60
Protein %
80
100
Milk -- Determination of
nitrogen content -- Part
3: Block-digestion
method (Semi-micro
rapid routine method)
Joint development with
IDF and AOAC
Nitrogen in milk
Total Nitrogen (AOAC 991.20 / ISO 8968-2/3 / IDF 20-2/3 )
Nonprotein Nitrogen (AOAC 991.21 / ISO 8968-4/ IDF 20-4)
Protein Nitrogen (AOAC 991.23/AOAC 991.24, ISO 8968-5, IDF 20-5 )
TCA precipitation of proteins allows a separation of non-protein Nitrogen
(Urea, Ammonia) in the filtrate from protein nitrogen (casein..) in the
filter cake
Protein = Protein ?
Protein = Protein ?
Sample
French Bean
Summer Barley
Nitrate
8,9 / 6,9
0,1 / 150
Lettuce
33,2 / 9,0
Cucumber
Yam (dioscorea)
Cabbage
Spinach
Saw-dust
7,2 / 10,3
4,9 / 9,6
7,1 / 5,2
27,2 / 5,0
0,074/ 113%
Example:
33 000 mg/kg NO3 = 7,45
g N/kg = 0,75 % Nitrogen
0,75 x 6,25 = 4,7 % Protein
Conclusion:
Dumas is a routine
method
Applicability has to be
checked vs Kjeldahl
Trade conflicts ?
Kjeldahl factors
Tryptophan
Protein factors
Amino acid
Formula
MW (g/mol)
%N
Factor
Alanine
C3H7NO2
89,09
15,71
6,37
Arginine
C6H14N4O2
174,2
32,15
3,11
Asparagine
C4H8N2O3
132,12
21,19
4,72
Aspartic acid
C4H7NO4
133,1
10,52
9,51
C3H7NO2S
121,16
11,55
8,66
C5H9NO4
147,13
9,52
10,50
C5H10N2O3
146,14
19,16
5,22
Glycine
C2H5NO2
75,07
18,65
5,36
Histidine
C6H9N3O2
155,15
27,07
3,69
Leucine
C6H13NO2
131,17
10,67
9,37
Lysine
C6H14N2O2
146,19
19,15
5,22
Methionine
C6H11NO2S
149,21
9,38
10,66
Phenylalanine
C9H11NO2
165,19
8,48
11,79
Proline
C5H9NO2
115,13
12,16
8,22
Serine
C3H7NO3
105,09
13,32
7,51
Threonine
C4H9NO3
119,12
11,75
8,51
C11H12N2O2
204,23
13,71
7,29
Tyrosine
C9H11NO3
181,19
7,73
12,94
Valine
C5H11NO2
117,15
11,95
8,37
Cysteine
Glutamic acid
Glutamine
Tryptophane
Type
Kjeldahl
Dumas
Amino Acid
AAFCO 200921
Chicken
17,29 (0,15)
17,64 (0,33)
14,22 (0,17)
AAFCO 200922
Pig starter
23,94 (0,33)
24,51 (0,39)
19,73 (1,18)
AAFCO 200923
Chow
12,3 (0,52)
12,51 (0,65)
7,16 (0,19)
N-based methods
Method
cost/analysis
(USD)
thruput
(samples/day)
Accuracy
Applicability
Kjeldahl
1,50 - 2,50
100-200
+++
+++
Dumas
1-2
100-200
++
+++
NIR
0,1
400-500
Dye
Protein
Adulterations
Melamine
66% Nitrogen
Total nitrogen
Heat coagulable protein
HMW protein (MgSO4 precipitation)
Total Nitrogen
Soluble Nitrogen (hot water extract)
.. more examples
Nitrogen in milk
TN, NPN and protein N (AOAC/ ISO / IDF )
(after TCA precipitation)
Nitrogen in eggs
Nitrogen in eggs (AOAC 925.31)
Water-soluble N and Crude Albumin (AOAC 932.08)
(pI precipation at pH 4; salting out with NaCl / EtOH)
Nitrogen in soymeal
Crude protein
Protein dispersibility index
NPN (after TCA precipitation)
0.1
absorbance difference
absorbance difference
0 ppm
250 ppm
500 ppm
1000 ppm
1500 ppm
2000 ppm
2500 ppm
3000 ppm
0.05
1000
1100
1200
1300
wavenumber
1400
1500
absorbance difference
0.1
0.05
1000
1100
1200
1300
wavenumber
1400
1500
0.1
0.05
1600
1600
0 ppm
250 ppm
500 ppm
1000 ppm
1500 ppm
2000 ppm
2500 ppm
3000 ppm
1000
1100
1200
1300
wavenumber
1400
1500
1600
0.15
Normal milk
Adulterated milk
Cyanuric acid
Score PC5
0.1
Melamine+water
(diluted samples)
0.05
Urea
Melamine
-0.05
Ammonium sulphate
-0.1
-0.5
-0.4
-0.3
-0.2
0
-0.1
Score PC2
0.1
0.2
0.3
0.4
Melamine
Skim milk powder
Test set 2
Spectral integrity
Values in % melamine
42
43
Conclusions
44
Summary