Chapter 13 Amino acids and peptides

Abstract

This chapter discusses amino acids and peptides, and the detection methods used to identify them. All primary amino acids form the same complex following reaction with ninhydrin, making this reagent unsuitable for pre-column derivatization. Most cation-exchange high-performance liquid chromatography (HPLC) columns utilized for ninhydrin-based post-column amino acid analysis still employ sulfonated polystyrene/divinylbenzene resins. Silica-based ion-exchange packings, popular for peptide separations, have poor resistance to the high pH values, ionic strength, and elevated temperature required to elute free amino acids. Modern automated amino acid analyzers based on post-column ninhydrin derivatization are capable of providing highly reliable and reproducible analyses. Phenylisothiocyanate (PITC), also known as Edman's reagent, has long been used for the sequencing of polypeptides and proteins, and was introduced for the analysis of amino acids in the early eighties. It is currently the most extensively used reagent for pre-column derivatization. Derivatization following cation-exchange chromatography of free amino acids has distinct advantages, provided that the instrumentation yields reproducible flow rates and reaction temperatures [lo]. Moreover, there is no need for derivatization to proceed to completion, and problems of derivative stability are not significant. The distinction among a peptide, polypeptide, and protein, in terms of the number of peptide residues they contain, is somewhat arbitrary. However, peptides are usually defined as containing 50 amino acid residues or less. Although molecules of more than 50 residues usually have a stable three-dimensional structure in solution, and are referred to as proteins, conformation can be an important factor in peptides as well as proteins.