Isolation of pituitary peptides by reversed-phase high-performance liquid chromatography : Expansion of the resolving power of reversed-phase columns by manipulating pH and the nature of the ion-pairing reagent

Abstract

The reversed-phase liquid chromatographic behaviour of pituitary peptides at various pH values was compared. The retention times of peptides at pH 1, 3 and 4.5 were essentially as expected for acidic solvent systems. However, at pH 5.5 or 7, radically different elution characteristics were observed. This is probably brought about by the increase in polarity that accompanies ionization of the peptide carboxyl groups at higher pH values. Basic hydrophobic ion-pairing reagents, such as tetrabutylammonium ion, can be used to maximize differences in polarity. We have previously shown that the use of acidic hydrophobic ion-pairing reagents at low pH (e.g. trifluoroacetic and heptafluorobutyric acids) enhances the basic polarity of peptides. The combination of both approaches facilitated the reversed-phase purification of posterior pituitary glycopeptide and α-N-acetyl-β-endorphin1–27 from an extract of 200 rat neurointermediate pituitaries. The separation of these two peptides at low pH, which is particularly difficult, was achieved by the sequential use of solvent systems containing trifluoroacetic acid (0.1%), heptafluorobutyric acid (0.13%) and finally triethylamine acetate (0.01 M, pH 5.5).