Separation of phenylthiohydantoin amino acids by capillary electrochromatography

Capillary electrochromatography (CEC) was employed as a rapid and high‐efficiency method for the isocratic separation of all 20 important phenylthiohydantoin (PTH) amino acids, the end products of Edman degradation during N‐terminal protein sequencing. For this purpose, 75 μm ID fused‐silica capillaries were packed with standard 3 μm Hypersil octadecyl silica (ODS) particles using a two‐step column fabrication process, which represents a fast, reliable and efficient means of producing long‐term stable columns. The influence of solvent composition, pH, type of buffer cation, buffer concentration, and temperature on retention behavior of PTH amino acids was investigated. Same‐day and day‐to‐day reproducibility of the retention times (over a period of two months) were found to be better than 3%. When comparing this new technique with traditional reversed phase‐high performance liquid chromatography (RP‐HPLC) methods applied in automated protein sequenators, CEC shows essentially shorter separation times and superior resolution.