Amino acid analysis methods review: Post column detection method compared to Kjeldahl and Dumas methods

Three methods have been widely used for the acquisition of protein content in food products. In this article, all methods are briefly explained and the advantages and disadvantages of each explored. In addition, the methodology that is currently utilised by AltaBioscience is reviewed and comparisons drawn.

1. Post Column Detection Method

This is the method used by AltaBiosience for amino acid analysis.

This method passes the unknown sample through an ion-exchange column to separate the protein content into corresponding amino acids. These post-column amino acids are then derivatised with a ninhydrin dye and their absorbance measured at 440 and 570nm.

Advantages

• Individual amino acids in the protein are separated BEFORE detection therefore any other nitrogen-containing compound will not distort the results thus giving greater accuracy.
• Both amino acids bound in proteins and those that are free in solution can be measured by performing hydrolysis prior to analysis.
• The sample does not have to be pure to produce accurate measurements.
• Mixed samples including food, drink, nutraceutical supplements, grains as well as raw materials can be tested accurately.
• No harsh chemicals are required.

Disadvantages

• Requires specialist equipment and experienced technical analysis.

2. Kjeldahl Method

The Kjeldahl method utilises concentrated sulfuric acid to digest the nutritional sample liberating ammonium sulfate from any nitrogen-containing compounds that are present. Its advantages and disadvantages are as follows:

Advantages

• Used in a wide variety of samples for many years so is considered a measuring stick for other methods.

Disadvantages

• Measures non-protein nitrogen as well as proteinogenic nitrogen giving a false high reading.
• Harsh conditions required of a high temperature of ~ 400°C, concentrated sulfuric acid and long reaction times.
• Correction factors must be calculated and applied for different proteins dependent on amino acid sequence.
• Pure samples are required for accurate measurements.

These disadvantages were exacerbated during the 2007 pet food incident and the 2008 milk powder scandal where melamine was intentionally added to vegetable proteins used for human and animal consumption. Cyanuric acid, a further compound, was found to cause kidney failure when used in conjunction with melamine. This combination has been reported as widely used overseas.

3. Dumas Method

The Dumas method utilises a combustion technique by heating the sample to ~ 900°C. This releases carbon dioxide, water and nitrogen as gases, which are then passed through a column to absorb the carbon dioxide and water leaving only the nitrogen. This nitrogen content is then measured. The advantages and disadvantages of this method are as follows:

Advantages

• Fully automatable.
• Run times of only a few minutes per sample.
• No toxic chemicals or catalysts.

Disadvantages

• High setup cost.
• Registers non-protein nitrogen, which leads to false positives regarding the protein content of the sample.
• Pure samples required for accurate measurements

In conclusion, the methodology of amino acid analysis using post-column detection utilised by AltaBioscience can produce a more accurate measurement of protein content than other methods discussed in this article. This is displayed by its usage on a wider variety of sample types such as food, drink, cultured media etc. where other types of compounds such as fats and carbohydrates may be present. Only amino acids associated with proteins or as free amino acids are detected.

For more information about the amino acid analysis service we provide, please contact us.

E: info@altabioscience.com T: +44 (0)1527 584495