Gradient and isocratic high-performance hydrophobic interaction chromatography of proteins on agarose columns

Abstract

Columns of pentyl and octyl agarose (6–8 cm × 6 mm I.D., bead size 5–7 μm) have been used successfully for high-performance hydrophobic interaction chromatography of both model proteins and the proteins in human serum. Interestingly, these columns can be eluted not only with a negative salt gradient but also isocratically. The efficiency of the columns is high (around 15 000 plates per metre for cytochrome c in isocratic elution). In the range pH 2.5–11.5 the capacity factor changed drastically for some, but not all, proteins (particularly below pH 5.5 and above 8.5), which means that pH is an important separation parameter in the optimization of resolution in hydrophobic interaction chromatography. The residence time of the proteins on the column has no observable influence on the appearance of the chromatograms. Some experimental conditions which are required for high resolution in isocratic elution are discussed.