Separation and determination of the amino acids by ion exchange column chromatography applying post-column derivatization

Abstract

The most perfect method for the determination of the amino acid composition of pure protein feeds or biological fluids is still the ion exchange column chromatography (IEC). With the help of the lithium buffer system, most of the problems in the field of free amino acid analysis of biological fluids can be solved. At IEC, most contaminants move rapidly through the post-column system and are discarded before the separation of amino acids begins, resulting in better performance. The time required for the sample preparation is minimal compared to pre-column methods, and the detection (with ninhydrin or OPA) is chemically specific for amino acids. A new off-line sample preparation method has been introduced before analysis by an ion-exchange chromatography and integrated amperometric detection, which eliminates carbohydrates from amino acid samples. However, the major problems remained in the hydrolysis of the protein, the de-proteinization of the biological fluids and the partial decomposition of methionine, cysteine and tryptophan during the sample preparation for analysis. At the moment the traditional IEC with post-column ninhydrin derivatization seems to be the best method to analyse both pure proteins and feeds and complex mixtures, but in some comparison the HPLC methods were found to be similar to that of the IEC.