Chromatography amino acid analysis

However, the revision of many naturally occurring amino acids is under progress. For limitation of other amino acids various methods such as ion chromatography, amino acid analysis, HPEC, and CZE are available and have to be tested case by case. In addition, tests for other non-amino acid related substances have to be developed. Possibly substances of different origin (production by synthesis, by fermentation, or by protein hydrolysis) may need different methods. In addition, a test for enantiomeric purity has to be included. 

Gel-filtration column chromatography, amino acid analysis and carbohydrate determination... 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

Heinrikson, R. L. and Meredith, S. C., Amino acid analysis by reverse-phase high-performance liquid chromatography precolumn derivatization with phenylisothiocyanate, Anal. Biochem., 136, 65, 1984. 

Houpert, Y., Tarallo, P., and Siest, G., Amino acid analysis by ion-exchange chromatography using a lithium elution gradient. Influence of methanol concentration and sample pH, /. Chromatogr., 115, 33, 1975. 

The most popular current techniques for amino acid analysis rely on liquid chromatography and there are two basic analytical methods. The first is based on ion-exchange chromatography with post-column derivatization. The second uses pre-column derivatization followed by reversed-phase HPLC. Derivatization is necessary because amino acids, with very few exceptions, do not absorb in the UV-visible region, nor do they possess natural fluorescence. 

As already mentioned the EP wants to replace old TEC tests with separation methods of higher efficiency for example, the purity of amino acids is currently evaluated by a TEC test for ninhydrin-positive substances that is only able to find and limit amino acids to 0.5%. However, this test is only valid in the case the amino acids are produced by the cleavage of peptides/proteins and purification. The ninhydrin method is also used in the amino acid analysis of peptides, utilizing a cation-exchange chromatography with a post-column derivatization and a subsequent UVA is detection. This method is often used in industries for purity evaluation of amino acids. 

Because of these ever-widening interests, the measurement of plasma tHcy is undertaken in many clinical chemistry and routine laboratories. Various methods are employed, including high-performance liquid chromatography (HPLC) assays, conventional amino acid analysis, capillary electrophoresis, gas chromatography with or without mass spectrometry, liquid chromatography with tandem mass spectrometry, and in many routine clinical chemistry laboratories immunoassays. In this chapter, those methods that are often available in laboratories involved in the investigation of inborn errors of metabolism are described, namely HPLC and tandem mass spectrometry. 

R.W.Zumwalt, K.C.Kuo and C.W.Gehrke, Amino Acid Analysis by Gas Chromatography, CRC Press Inc, Florida, 1987. 

Gas chromatography (GC) for amino acid analysis is the alternative to HPLC that has found the greatest acceptance. It requires the preseparation derivatization of the amino acids to render them volatile. For this purpose, amino acids are frequently converted into acylated esters. N-Trifluoroacetyl-n-butyl esters and /V-heptafluorobutyrylisobutyl esters are most commonly employed. There have been comparative studies (3,4) that document similar (if not equivalent) analytical results for GC and the classic ion-exchange chromatographic method applied to a variety of food samples. Comparison (5) of GC to the reversed-phase HPLC determination of amino acids (phenylisothiocyanate derivatized) also shows excellent agreement. 

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