AMINO ACID ANALYSIS OF PEPTIDES USING HPLC WITH EVAPORATIVE LIGHT SCATTERING DETECTION

Abstract

A new procedure for the amino acid analysis of peptides has been devised utilizing high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). This procedure eliminates the need for complex derivatization schemes inherent of previous amino acid analysis procedures since the ELSD detects the amino acids directly. This quantitative method detects and separates 18 of the common amino acids in a one hour run time using cation exchange chromatography coupled with ELSD. The procedure was tested by analyzing the hydrolysate of human parathyroid hormone 1–34 (PTH), a synthetic polypeptide. A standard digestion consisting of 24 hour hydrolysis at 110°C in 6 N hydrochloric acid with 3% phenol was used. Validation data reveal this is an accurate and precise procedure for the amino acid analysis of peptides. The sensitivity of this technique is low compared to other state-of-the-art analytical techniques. Detection limits varied depending on the amino acid being analyzed and were as low as 200 picomoles for this new procedure. This approach yields a simple, universal method that is well suited for the amino acid analysis of peptides, of which sufficient quantity is available. Only basic HPLC instrumentation with an ELSD is necessary for this procedure.