Effect of Bead Size on the Analysis of Proteins and Peptides by High Pressure Liquid Chromatography

Abstract

The major thrust in the recent past has been on the development of column size and its packing material for the purification of proteins and peptides in low amounts by high pressure liquid chromatography (HPLC) to meet the requirement of amino acid sequence analysis, particularly by gas phase. This has resulted in the availability of smaller columns (microbore and mini) and packing material with different bead sizes (3 µM; 5 µM; 7 µM; 10 µM and 20 µM) and different pore sizes (75Å; 80 Å; 120 Å; 300 Å; 500 Å and 1000 Å). There are a number of reports available comparing the effect of pore size, mobile phase, temperature and column size (Freiser and Gooding, 1987; O’Hare et al. 1982; Liebra et al. 1984; Cohen et al 1984; Bhown and Bennett, 1984; Moore and Walters, 1984) on the separation of proteins, peptides, amino acids, and their derivatives. Use of the microbore column has also been advocated (Wilson, 1988). However, microbore column chromatography is limited, for example, by the need for a special type of solvent delivery system and restriction on the volume of the sample to be injected. In our efforts to search for an alternative to microbore column chromatography, we have investigated and compared the results of elution of protein, peptide and amino acid derivatives by microbore and macrobore columns packed with different bead sizes (3 µM; 5 µM; µM).