Hydrophobic high-performance liquid chromatography of hormonal polypeptides and proteins on alkylsilane-bonded silica

Abstract

Thirty-two hormonal polypeptides and nine proteins (8-65 kD) have been used to evaluate the potential of high-performance liquid chromatography on alkylsilane-bonded silica for separating and recovering biologically active compounds of this type. The basic method used was gradient elution with acetonitrile in an acid phosphate buffer. Variation of key chromatographic parameters demonstrated that low pH (less than 4.0) and high buffer molarity (greater than 0.1 M) are mandatory for reproducible high efficiency polypeptide chromatography. Simple NaCl-HCl mixtures of appropriate acidity and molarity could be substituted for the acid phosphate buffer, with the advantage of minimising non-physiological ion contributions to eluted materials. Minor selective effects were noted with different organic modifiers, but variation of other parameters, including choice of specific alkylsilane packings, did not materially influence separations. Under optimal conditions all of the polypeptides tested could be efficiently chromatographed, and many simultaneously resolved, as could most of the proteins tested. Three of the more hydrophobic proteins could not, however, be eluted from the alkylsilane packings. Retention orders of smaller compounds (less than 15 residues) generally correlated with the sum of the Rekker fragmental constants of their strongly hydrophobic residues. Larger polypeptides showed numerous anomalies when ranked by this means, however, limiting its predictive value. The separation of at least eighteen discrete components from a partially-purified posterior pituitary extract has demonstrated the capability of alkylsilane-type reversed-phase packings for the hydrophobic high-performance liquid chromatography of complex biological mixtures.