The Amino Acid Pre-column Derivatization Hplc Method Development And Application

Amino acid, which is the basic unit of proteins, is one of the important compounds to human being. Thus, it has always been an important theme in the frontier of analytical chemistry to develop widely-suited HPLC analysis method for amino acids with high sensitivity, good separation as well as good reproducibility.The high performance liquid chromatographic methods with pre-column derivatization for the determination of amino acids were developed, while OPA and DNFB were used as the derivatization agents. Several factors, such as column temperature, the quantity of agents, reaction time and the pH of mobile phase were investigated, all of which had effect on the analysis results. As the results: the column temperature could largely affect the selectivity of separation, and sometimes even the peak order of amino acid's DNFB derivates(DNP-AA); while, the effect of derivatization reaction time had on the quantitative analysis of amino acid's OPA derivates was dramatically.To improve the poor selectivity of solo-packing columns, the Link Model and the Combined Model of two different packing materials were established, after the separation behavior of 5 different C18 packing materials investigated with DNP-AA. It proved that the link model using C18-B and C18-SA could greatly improve the selectivity, while the combined model with the two packings could achieve better peak shape and column efficiency.By the established methods above, the amino acids of some actual samples, such as beer and Cordyceps Sinensis, were measured. Meanwhile, the methods of sample pretreatment were improved. The results showed that the analysis methods could be widely suited.