PROTE.

03-1

NITROGEN / PROTEIN by COMBUSTION

PRINCIPLE

The sample is combusted in the presence of oxygen at high temperature, whereby

nitrogen-containing material is converted to molecular nitrogen and nitrogen
oxides. Water vapor and other gaseous oxidation products are removed through a
purification train. The oxides of nitrogen are catalytically reduced to molecular
nitrogen. The total nitrogen obtained is detected in a thermal conductivity cell and
quantified by integration relative to standard reference material of known nitrogen
content. A measure of the protein content can be obtained by multiplying the
nitrogen concentration by an appropriate factor, e.g. 6.25 for corn products.

SCOPE

This method is applicable to the determination of nitrogen in corn starch, co-

products, starch slurry, and any corn product with a protein value ≥ 0.2% dry solids
basis.

SPECIAL APPARATUS

1. Nitrogen Combustion Analyzer and supporting equipment as per manufacturer’s

instructions (Note 1).

2. Analytical Balance accurate to 0.0001 grams.

REAGENTS

1. Oxygen: 99.99% purity zero grade in gas cylinder equipped with a regulator
capable of delivering 40 psi followed by an in-line gas purifier (Note 2).

2. Helium: 99.99% purity zero grade in gas cylinder equipped with a regulator
capable of delivering 40 psi followed by an in-line gas purifier.

STANDARDS

Nitrogen Calibration Standards: High purity EDTA (9.57% nitrogen), glycine

(18.658% nitrogen), or other appropriate nitrogen standard. Solutions of these

Standard Analytical Method of the Member Companies of the

Corn Refiners Association, Inc.
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standards at lower concentrations can be prepared (Note 3).

Optional Standards:

High Protein (Gluten Meal) Standard: A large sample of gluten meal is ground
through a Wiley mill or equivalent using the 1 mm screen (to pass U.S.
Standard Sieve No. 20) and blended well to insure homogeneity. Test portions
are analyzed repeatedly (3 times minimum, within tolerance) for protein by the
Kjeldahl method. The bulk of the ground sample is then packaged in 8 oz.
screw cap bottles which are stored in a freezer. Use as the standard for meal,
feed, germ, steepwater, and other high protein process samples.

Starch Standard: A large sample of unmodified corn starch is blended well and
test portions are analyzed repeatedly for protein by the Kjeldahl method. The
bulk of the sample is packaged in 8 oz. screw cap bottles which are stored in a
freezer. Use as standard for unmodified dry starch samples only.

Modified Starch Standard: A large sample of modified corn starch is blended

well and test portions are analyzed repeatedly for protein by the Kjeldahl
method. The bulk of the sample is packaged in 8 oz. screw cap bottles which
are stored in a freezer. Use as standard for a specific type of modified dry
starch samples only. Each starch modified with a nitrogen-containing reagent
will have its own standard.

CRA Check Sample Standards may be used.

PROCEDURE

Proceed according to manufacturer’s instructions.

General Operating Guidelines:

A sample run is composed of a series of blanks, calibrants run as samples, calibrants,

sample blanks, and samples.

General Blank Procedure:

The first several blanks (3-5) serve to flush and equilibrate the analyzer. These
should not be used to evaluate the performance of the instrument. The next several
blanks (3-5 more) should become consistently low in nitrogen. At this point, the
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blank factor can be adjusted according to manufacturer’s instructions. If the samples

to be analyzed are high in nitrogen, it is not necessary to adjust the blank factor at all.
This adjustment, however, becomes essential for samples that are low in nitrogen.

General Calibration Procedure:

Select an appropriate nitrogen calibration standard to match the sample matrix and
expected nitrogen level. For aqueous samples, it is better to use a liquid calibrant.
For starch slurries, best results will be obtained if a starch slurry calibrant is used.
This rule-of-thumb for the selection of the calibrant is especially important for
samples with low levels of nitrogen.

After the blanks, a set (2-3) of calibrants run as samples should be analyzed. These
serve to condition the analyzer for the testing of the calibrant. A calibrant run as a
sample can also be used to check the accuracy of a daily factor.

The next several spots (4-7) in the run should be calibrants. These establish the daily
factor for the determination of nitrogen in the samples. It is advisable to analyze
inter-sample calibrants throughout a run to assure a robust daily factor.

General Sample Preparation:

After the calibrants, the next couple of spots (1-2) in the run should be sample
blanks. Samples blanks condition the analyzer for the testing of samples. These
sample blanks are especially important if there is a considerable difference in
nitrogen content between the calibrant and the sample or if there are major matrix
differences.

Use a sample preparation procedure appropriate to the matrix being analyzed to

ensure a homogenous sample. Weigh sample according to table below. Ensure
instrument response is within the response for the calibration standards (Note 4).

Sample Weight Range, g Check Sample Expected Nitrogen

Level, %, dsb
Corn 0.6000-1.0000 High Protein 1.28 – 2.00
Dry Starch 1.0000-1.6000 Starch 0.015 – 0.130
Cationic Starch 0.5000-0.8000 Cationic Starch 0.100 – 0.500
Corn Gluten Meal 0.3000-0.7000 High Protein 5.5 – 10.5
Corn Gluten Feed 0.6000-1.0000 High Protein 2.5 – 4.0
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NITROGEN / PROTEIN by COMBUSTION –continued

Heavy Steepwater 0.4000-0.6000 High protein 0.5 – 1.5

Dorrclone Starch 1.8000-2.5000 Starch 0.015 – 0.130
Gluten Slurry 0.3000-0.7000 High Protein 5.5 – 10.5
Corn Germ Meal 0.6000-1.0000 High Protein 2.5 – 6.0

The next spots in the run should be samples. It is advisable to include a calibrant
after a fixed interval of samples, for example, after every five or ten samples. For
better accuracy, run samples in duplicate if possible.

Finally, some blanks (1-2) are included at the very end of the run to clean out the
instrument.

CALCULATIONS

The instrument will provide a printed table of results as either % nitrogen or %

protein. For manual calculations, use the following formula.

Percent Crude Protein “as is” = % N x 6.25 (for corn products).

NOTES AND SAFETY PRECAUTIONS

1. Follow manufacturer’s recommendations for safe operation of the instrument. Be

cautious of hot surfaces and electrical hazards during routine maintenance.

Nitrogen Analyzer: Leco Model FP-528 or FP-2000 Nitrogen Determinator

equipped with automatic sampler or autoloader, and printer
http://www.leco.com/products/organic/fp_528/fp_528.htm; or Elementar Vario
MAX CN – Carbon/Nitrogen Analyzer; or equivalent. Elementar Americas help
line is 856-787-0022 or http://www.elementar-inc.com.

2. Many materials are very flammable in the presence of oxygen. Keep away from
flame or sparks.

3. Many materials are very flammable in the presence of oxygen. Keep away from
flame or sparks.

4. Glycine Solution Standard: Dissolve glycine (aminoacetic acid, H2NCH2COOH)

in water to obtain a nitrogen concentration just above that of the samples to be
analyzed. Calculate amount of glycine to be used on the basis of a theoretical
nitrogen contents of 18.658%. Weigh the calculated amount of glycine powder in
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a dry, tared 125 mL Erlenmeyer flask with a rubber stopper. Add 100 mL of
purified water and reweigh. Seal the flask and mix to dissolve. Recalculate the
exact concentration taking into account the actual weights measured. The
solution(s) may be stored in a refrigerator and discarded after 2 days, or may be
kept frozen indefinitely.

5. Care must be taken in weighing samples. Samples which are high in carbohydrates
may not combust completely if the sample weights are too high. Liquid slurry
samples should be shaken immediately before being weighed to insure
homogeneity. For samples which are high in nitrogen, sample weights should be
low but not so low as to introduce balance errors into the analysis (minimum
weight should be 0.1g).

REFERENCES

1. AOAC Official Method 990.03, Protein (Crude) in Animal Feed, Combustion

Method. First Action 1990. AOAC Official Methods of Analysis (Current
Edition).

2. Comparison of Kjeldahl Method for Determination of Crude Protein in Cereal

Grains and Oilseeds with Generic Combustion Method: Collaborative Study.
Bicsak, R. C., Journal of AOAC International, Vol. 76, No. 4, (780- 786) 1993.

3. Performance of an Automated High Temperature Combustion-Thermal

Conductivity Method for Measurement of Protein Content of Food Products.
King-Brink, M, & Sebranek, J. G. Paper presented March 1993 Pittsburgh
Conference and latter published in The Analyzer, Vol. IV, Leco Corporation.

4. Protein-Nitrogen Combustion Study Results, Brown. J. S, INFORM, Vol. 5 No.

5, May 1994.

5. Rose A. Sweeney, Generic Combustion Method for Determination of Crude

Protein in Feeds: Collaborative Study, J. Assoc. Off. Anal. Chem. 72, no. 5,
770-774 (1989).

6. Robert W. Sachen and Nancy J. Thiex, Effect of Sample Introduction and

Atmospheric Blank on Determination of Nitrogen (Crude Protein) by
Combustion, J. of AOAC Int’l 80, no. 1, 14-19 (1997).
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NITROGEN / PROTEIN by COMBUSTION –continued

METHOD HISTORY

Combined the Nitrogen / Protein by Combustion methods for Corn Starch

(Unmodified) (B-66), Corn Starch (Modified) (C66) and Feedstuffs (G-66) on 4-
15-2010.

Corn Starch (Unmodified), Nitrogen / Protein by Combustion (B-66) Date of

Acceptance 5-31-2007.

Corn Starch (Modified), Nitrogen / Protein by Combustion (C-66) Date of

Acceptance 5-31-2007.

Feedstuffs, Nitrogen / Protein by Combustion (G-66) Date of Acceptance 5-31-

2007.