Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography

Summary

An improved HPLC method for the quantitative determination of the amino acids from hydrolysed cheese proteins and peptides is described. Some important improvements to the existing method are suggested. The addition of piperidine-4-carboxylic acid (PICA) as a hydrolysis-resistant internal standard enabled the quantification of secondary amino acids. The following analytical parameters have been determined: repeatability, detection limit and linearity in the measuring range. A statistical comparison with the classical ion chromatographic method gave an excellent correlation for all determined amino acids. Both methods are free of artifacts and systematic errors. Compared with ion chromatography, HPLC shows the following advantages: faster equilibration of the column, shorter retention times, more stable baseline, narrower peaks and more sensitive fluorescence detection. A drawback is the slightly lower repeatability for some amino acids.