Junichi Koyama

Performance of polymer-coated silica C18 packing materials prepared from high-purity silica gel : The suppression of undesirable secondary retention processes

Abstract Coating with silicone polymer of silica surfaces was shown to be very effective in suppressing the undesirable peak tailing in reversed-phase liquid chromatography caused by hydrogen bonding, ion-exchange and chelate formation processes. Silica particles containing various amounts of metal impurities were derivatized to octadecylsilylated (ODS) silica phases with or without subsequent trimethylsilylation, and to a polymer-coated phase with subsequent introduction of octadecyl groups. The performance of endcapped ODS phases prepared from high-purity silica gel was satisfactory for hydrogen-bond acceptors and protonated amines, but not acceptable for chelating compounds. The extent of tailing seen with chelating compounds depends on the solute structure and the metal content of silica particles. Coating with silicone polymer was more effective than trimethylsilylation of ODS phases for the suppression of tailing for chelating compounds, and afforded alkyl-bonded silica packing materials free from undesirable secondary effects by using metal-free silica particles.

Effect of column length and elution mechanism on the separation of proteins by reversed-phase high-performance liquid chromatography

Abstract Different theories have been proposed for the elution of proteins in reversed-phase high-performance liquid chromatography. To establish the correct elution mechanism, the effects of column length and the concentration of the organic solvent on column efficiency and the elution of high- and low-molecular-weight compounds were examined. It was concluded that protein elution principally involves the same retention process as for low-molecular-weight compounds, although the influence of partition is small under steep gradient conditions. In accordance with this, wide-pore packings in a short column (35 mm) gave excellent separations of proteins and were usable with a wide range of gradients.

Heigh-performance liquid chromatographic analysis and characterization of proteins

In order to estimate the molecular weights of proteins the analytical conditions of GPC and HPLC were investigated. Under presence of 0.2% SDS and 0.5M sodium phosphate buffer (pH 7.0), the plots of molecular weight versus retention time were linear in the range of 3000 to 150000 molecular weights. Furthermore, rough coformation of proteins were surmisable by analysis of changes in GPC chromatogram after denatured with 2-mercaptoethanol.Retention times of small peptides on reversed-phase HPLC depend on their hydrophobisity. Molecular weights of peptides could be estimated by their retention times. This method is useful to estimate the molecular weights of small peptides and for pattern analysis of hydrolyzed peptides.Proteins could be classified by analysis of their amino acids composition because they are characteristic for each proteins.It is very effective for analysis of proteins in cosmetics and characterizing unknown proteins by combination of these methods.