18] High-pressure liquid chromatography and radioimmunoassay for the specific and quantitative determination of endorphins and related peptides

Abstract

Publisher Summary This chapter discusses high-pressure liquid chromatography (HPLC) and radioimmunoassay for the specific and quantitative determination of endorphins and related peptides. Several studies have shown that reversed-phase HPLC systems can be very useful for the separation of complex peptide mixtures. Retention on reversed-phase columns, which are nonpolar, is highly dependent on both molecular weight and polarity of the applied compounds. In general, for peptides retention, times appear to be proportional to their chain lengths and inversely proportional to their polarities as indicated by the solubilities of the constituent amino acids in water. The high resolving power of the HPLC system used allows the separation of peptides that differ in only one amino acid residue. The retention time enhancing effect of the introduction of apolar leucine as the C-terminus of the peptide sequence is remarkable. A potentially serious problem is the low recovery from a μBondapak C18 column after applying subnanogram amounts of peptides. Addition of glycylglycine to both the mobile phases at a final concentration of 1.5 × 10 −5 M can overcome this problem without interfering with the reversed-phase HPLC separation of enkephalins and endorphins, and will lead to almost complete recovery after the injection of peptide mixtures in picogram amounts.