3 Jurgen Moller N Based Methods 2013a PDF

8/20/2019 3 Jurgen Moller N Based Methods 2013a PDF

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[email protected]

N based methods for the

determination of protein

FOSS Laboratory Conference

September 16-17 · 2013 ·Hillerød · Denmark

1

mailto:[email protected]:[email protected]


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Synopsis

Kjeldahl method

Dumas combustion method

NIR methods Alternative methods (AAA & DBC)

Adulterations

Conclusions & Summary

2


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Nitrogen-based methods – do they

still have a future?

Jean Baptiste Dumas,

1833

Johan Kjeldahl, 1883


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Kjeldahl - Reference

Kjeldahl from 1883For Nitrogen / Protein

Dumas method 1980’s For Nitrogen / Protein

NIR/FTIR methods 1980’sFor Crude Protein


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Kjeldahl / Protein standards in OMA

920.53 (Hg)

920.70 (Hg)

920.87 (Hg)

920.109 (Cu)

920.123 (Cu)

920.155 (Hg)

920.176 (Hg)

925.31 (Hg)

928.08 (Hg)

930.01 (Hg)

930.02 (Hg)

930.25 (Hg)

930.29 (Cu)

930.33 (Cu)

932.08 (Hg)

939.02 (Hg)

940.25 (Hg)

941.06 (Cu)

945.01 (Hg)

945.18 (Hg)

945.23 (Hg)

945.48 (Cu)

950.09 (Hg)

950.10 (Hg)

950.48 (Hg)

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)

http://www.eoma.aoac.org/


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Recovery of Nitrogen

Lysine hydrochloride

10

11

12

13

14

15

16

10 20 30 40 50 60 70

digestion time (min)

% P

r o t e i n

Hg

Se

Cu

Ti

100%

90-93%


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Recovery in real samples

84

86

88

90

92

94

96

98

100

Lysine Dog food Meat Fishmeal Wheat

% recovery of protein

Se

Cu

Ti

Hg


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EGN collaborative study

Method % CP

ICC 105/2 (Kjeldahl, Cu) 12,46

ISO 5983-2 (Kjeldahl, Cu) 12,45

ISO 20483 (Kjeldahl, Cu/Ti) 12,39

ISO 16634 (Dumas/Combustion) 12,55


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Kjeltec™ from the 1970’s

1970 introduction of block

digestion by FOSS Tecator

Since 1974 introduction of

direct steam distillation and

other improvements

Decreased use of chemicals

Improved digestion efficiency

No sample transfer

Alkali added in closed system

Distillation into boric acid

receiver, reducing the distillation

times and avoiding back titration


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AOAC 2001.11

A method based on block digestion/ steam distillation /boric acid receiversolution, having a wide scope of applicability fullfilled definitely a need of theinternational laboratory society.

Method for the determination of Crude Protein in Animal Feed,

Forage, Grain, and Oilseed using Block Digestion, CopperCatalyst and Steam Distillation into Boric Acid

Study director: Nancy J. Thiex, SD State University, Brookings, US

Study report: JAOAC, 85 (2), 2002, p 309 – 317

Summary: In Focus, 26 (2), 2002, p 10 - 12


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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

—

Part 2: Block digestion/steam distillation method

ICS 65.120

EN ISO method on basis of AOAC 2001.11

http://www.cenorm.be/default.htm


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Performance of EN ISO 5983-2 standard (AOAC 2001.11)

Range: 0,3 – 70 % protein

Samples:

1: protein block

2: swine pellets

3: corn silage

4: grass hay

5: fish meal

6: dog food

7: chinchilla feed

8: albumin

9: bird seed

10: meat and bone meal

11: milk replacer

12: soybeans

13: sunflower seed

14: legume hay

15: fish feed, small floating pellets

16: fish feed, large floating pellets 17: shrimp feed, crumble

18: shrimp feed, large sinking pellets

19: shrimp feed, small sinking pellets

20: larvae feed, flake

21: wheat grain

•Validated by 24-26 international labs

•Photometric and pH end point

•Reference method also for Dumas

•Excellent repeatability and reproducibility


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Reapeatability and reproducibility

sdR and sdr vs % protein ISO 5983-2

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

0 20 40 60 80 100

sdr

sdR

Linear (sdR)

Linear (sdr)


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EN ISO 20483:2006

Cereals and pulses –

Determination of the nitrogen

content and calculation of the

crude protein content – Kjeldahl

method

Block digestion with Cu/Ti

catalyst, steam distillation into

boric acid, automatic titration

with photometric (or pH) end

point determination

2 h digestion time, 20 ml acid


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prEN ISO/DIS 20483:2006

Perf or man ce EN ISO 20483

y = 0,0129x + 0,061

y = 0,0058x + 0,0248

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

0 20 40 60 80 100

Protein %

s d %

sdr

sdR

Linear (sdR)

Linear (sdr)


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ISO 8968–3:2004 and IDF

20-3 (2004) Milk -- Determination ofnitrogen content -- Part3: Block-digestionmethod (Semi-microrapid routine method)

Joint development withIDF and AOAC

http://www.iso.ch/iso/en/ISOOnline.frontpage


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Non-protein N and protein N

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


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Dumas: EN ISO 16634 series

(2008) Food products - Determination of the total

nitrogen content by combustion according tothe Dumas principle and calculation of thecrude protein content

Part 1: Oilseeds and animal feeding stuffs

Part 2: Cereals, pulses and milled cereal

products (TS)

Using the same factors as Kjeldahl

AOAC methods:

992.15 (meat) 992.23 (cereal grains & oilseeds)


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Protein = Protein ?

Dumas determines totalNitrogen, including

inorganic fractions like

NO2/NO3.

Kjeldahl determinesorganic nitrogen plus

ammonia.

For many samples the

difference might benegligible – but you have

to check.


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Can Dumas replace Kjeldahl ?

S. Seling et al., Max Rubner Institute, DE (2005)

Wheat harvest 2000-2004:

Some 2% of ”Dumas

protein” is not determined

by Kjeldahl method

Kjeldahl protein =

0,959*Dumas + 0,258

Difference depends on

growing year, cultivar andgrowing condition


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Protein = Protein ?

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

Sample Nitrate

French Bean 8,9 / 6,9

Summer Barley 0,1 / 150

Lettuce 33,2 / 9,0

Cucumber 7,2 / 10,3

Yam (dioscorea) 4,9 / 9,6

Cabbage 7,1 / 5,2

Spinach 27,2 / 5,0

Saw-dust 0,074/ 113%


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Trade conflicts ?

Argentine supplier of soymeal claims protein content of 47,2

%

Malaysian importer of soymeal claims protein content of 44,9

%

Reason: Seller uses Dumas method, buyer applies Kjeldahl

method


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Can Dumas replace Kjeldahl ?

European Commission confirms the Kjeldahl method

as the community method for official controls

(Commission Regulation (EC) No 152/2009)


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Kjeldahl factors

Most common: 6,25 = 16% N

Established on basis of the respective amino acid profile and the composition


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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

Cysteine C3H7NO2S 121,16 11,55 8,66

Glutamic acid C5H9NO4 147,13 9,52 10,50

Glutamine 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

Tryptophane C11H12N2O2 204,23 13,71 7,29

Tyrosine C9H11NO3 181,19 7,73 12,94

Valine C5H11NO2 117,15 11,95 8,37


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Comparison of ”Protein contents”

Sample 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)

Sum of 18 reported AA: Alanine, Arginine, Aspartic Acid, Cystein. Glutamic

acid, Glycine, Histidine, Iso-Leucine, Leucine, Methionine, Phenylalanine,

Proline, Serine, Threonine, Tryptophane, Tyrosine, Valine

Significant differences. Sources:

Non-Protein Nitrogen (Ammonia, Urea…) can be excluded.

Incomplete recovery in AAA ?


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N-based methods

Most widely used for (crude) protein analysis

Simple to use, with adequate accuracy and wide applicability

Probably > 50,000 installed units

Probably > 30 Mio analyses / year

NIR calibrations based on Kjeldahl or Dumas

Susceptible to adulterations

Method

cost/analysis

(USD)

thruput

(samples/day) Accuracy Appl icabi li ty

Kjeldahl 1,50 - 2,50 100-200 +++ +++

Dumas 1 - 2 100-200 ++ +++

NIR 0,1 400-500 + +


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Dye Binding Method

Protein

Dye

A negatively charged dye

binds to the positively

charged amine groups

Epsilon N

Alpha-amino-N


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DBC calibrated against Kjeldahl

Dye Binding Capacity

Absorbance of the

Original dye –

Absorbance of dye

After filtration

DBC detects irregularities

Like ”high” protein contents

When there is no DBC


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Adulterations

Positive (allowed), e.g. urea, biuret …

Negative (prohibited), e.g. melamin …

At contamination levels (ppm, ppb) At ”intended adulteration” levels ( > 0,2-0,4 % CP)

http://upload.wikimedia.org/wikipedia/commons/2/21/Melamine-3D-balls.png


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Melamine

66% Nitrogen

”Protein” content = >400%Solubility in water: 3,2 g/l

http://upload.wikimedia.org/wikipedia/commons/2/21/Melamine-3D-balls.pnghttp://upload.wikimedia.org/wikipedia/commons/2/21/Melamine-3D-balls.pnghttp://upload.wikimedia.org/wikipedia/commons/2/21/Melamine-3D-balls.png


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Examples for N-fractionation

Nitrogen fraction in wort and beer:

Total nitrogen

“Heat coagulable protein”

HMW protein (MgSO4 precipitation)

Nitrogen in malts (ale, lager, distilling) Total Nitrogen

Soluble Nitrogen (hot water extract)

Nitrogen in animal feed / forage Crude protein/ Urea/Biuret/NH4

ADIP / ADIN and NDIP / NDIN


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.. 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)


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1000 1100 1200 1300 1400 1500 16000

0.05

0.1

wavenumber

a b s o r b a n c e d i f f e r e n c e

Ammonium sulphate dissolved in milk - FT6000

0 ppm

500 ppm

1000 ppm

2000 ppm

3000 ppm

4000 ppm

5000 ppm

6000 ppm

1000 1100 1200 1300 1400 1500 16000

0.05

0.1

wavenumber


Urea dissolved in milk - FT6000

0 ppm

250 ppm

500 ppm1000 ppm

1500 ppm

2000 ppm

2500 ppm

3000 ppm

Adulterants have different mid-IR signatures

1000 1100 1200 1300 1400 1500 16000

0.05

0.1

wavenumber


Melamine dissolved in milk - FT6000

0 ppm

250 ppm

500 ppm

1000 ppm1500 ppm

2000 ppm

2500 ppm

3000 ppm

Adulterants have different spectral

signatures, making it easy to

distinguish even related

compounds

Adulterants show different

absorption at similar

concentrations – detection limits

are therefore dependent on the

adulterant

http://en.wikipedia.org/wiki/Image:Ammonium_sulfate.pnghttp://en.wikipedia.org/wiki/Image:Urea.pnghttp://en.wikipedia.org/wiki/Image:Melamine.svg


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FTIR: Spectral integrity / Principle Component Analysis (PCA)

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4

-0.1

-0.05

0

0.05

0.1

0.15

Score PC2

S c o r e P C 5

Melamine+water

(diluted samples)

Cyanuric acid

Urea Melamine

Ammonium sulphate

• Normal milk• Adulterated milk


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NIR spectra of pure Melamine and skim milk powder

Melamine

Skim milk powder


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NIR example: PCA plot incl

adulterated samples

Cal set

Test set 1

Test set 2


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Spectral integrity

Only spectral information from ”normal samples” is needed

No reference measurements needed

Samples, deviating from ”normal samples”, can be identified

The LoD for melamine as unknown adulterant is 500 ppm


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NIR: Validation of NIR calibration

Test set 2, 144 samples, SEP 0.05

Values in % melamine

SO 2099 20 0 id li f h li i f


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EN ISO 12099:2010 Guidelines for the application of

near infrared spectrometry Focuses on the validation of calibration models with

independant test sets Defines statistics for performance measurements, i.e. SEP,

Bias, Slope, confidence limits for bias and unexplained

error

Covers also Standardization of instruments if operated in a network

Validation on local samples before use

Instrument diagnostics at regular intervals

Checking instrument stability – Control sample/chart

Running performance check of calibration during use –

Control charts

Jürgen Möller, May 8, 2012 42


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How good is NIRS? AAFCO - Protein

Jürgen Möller, May 8, 2012 43


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Conclusions

N based methods for the determination of

protein will still be in use in the foreseeable

future (at least a couple of decades)

There are simply no alternatives that couldreplace N based methods with regard to

accuracy, precision, sample throughput and cost

per analysis NIR (and FTIR) as fast and cost effective N based

methods that can screen for adulterants

Jürgen Möller, February 7, 2012 44


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Summary Kjeldahl is still the most

important reference androutine method for thedetermination of crudeprotein / protein fractions

New standards reflect theinstrumental andmethodological progress

Fractionation schemes totrace adulterations

FTIR and NIR methodshave a potential as fastscreening for adulterants

Accreditation of NIRmethods using EN ISO12099 possible

Thank you for your attention.

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