Comparison of short and ultrashort-chain alkylsilane-bonded silicas for the high-performance liquid chromatography of proteins by hydrophobic interaction methods

The optimization of the reversed-phase high-performance liquid chromatography of proteins has beene xamined using a series of maximum-coverage alkylsilane-bonded silica packings with different carbon chain-lengths (C₁-C₁₈). The greatest range of individual compounds that could be successfully chromatographed was obtained with < C₆ chain-length packings. Optimum recoveries and efficiencies were noted with a C₃ material (Ultrasphere SAC). No loss of performance or changes in selectivity were observed during prolonged use (> 200 h) of these columns under acid (pH 2.1) conditions. This system has been used to separate prolactin and growth hormone (22 kD) from biological samples by employing a trace-enrichment step on 10-μm tap-packed short alkyl-chain columns prior to gradient-elution chromatography on a 5-μm particle-size (8 nm pore-size) Ultrasphere SAC column.