Abstract
A high-performance liquid chromatographic assay for monitoring traces of d-alanine and d-glutamic acid in peptide and protein hydrolysates is presented, which uses a two-column set-up with column switching. The main advantage of the proposed method is the higher reliability of the results, compared with the analysis by derivatization and single-column chromatography of the diastereomers. A non-chiral alkylsilica reversed-phase column is combined with a second column, in which β-cyclodextrin is used as a chiral mobile phase additive. The amino acids of the hydrolysate are dansylated, and the amino acid of interest is separated from the others on the first column and transferred to the second column where chiral resolution is performed. The transfer volume of ca. 200 μl is small enough not to cause any peak distortion or dilation in the second column.
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