High-performance Hydrophobic Interaction Chromatography Research Articles

Effect of the type of salt on separation of proteins in high performance hydrophobic interaction chromatography on TSKgel phenyl‐5PW

Effect of the type of salt on separation of proteins in high performance hydrophobic interaction chromatography on TSKgel phenyl‐5PW

High-performance hydrophobic-interaction chromatography on ether-bonded phases : Chromatographic characteristics and gradient optimization

This paper is a continuation of the evaluation of hydrophilic ether columns for the separation of proteins by hydrophobic-interaction chromatography. In this approach, linear salt gradients of decreasing concentration of ammonium sulfate yield sharp chromatographic peaks with high mass recovery and maintenance of biological activity. Mild adsorption conditions are indicated by the minimal changes in chromatographic peak area of native proteins as a function of contact time by the biopolymer with the stationary phase. Further evidence of minimal kinetic processes under the chromatographic conditions are seen in the constantly of isocratic retention with mobile phase flow-rate and sample load up to 2 mg. Based on the well-behaved chromatographic characteristics, we have explored gradient optimization in terms of the Snyder model for gradient elution. It is shown that changes in retention, peak capacity and peak height follow predicted gradient time dependencies. Moreover, the influence of particle diameter and column length are found to be in agreement with expected behavior, based on the model. As a consequence of the agreement, prediction of conditions for optimum separation for a particular problem are possible. Other studies examine the influence of specific ion effects, e.g., Mg 2+ binding to protein, which can override retention based on predicted surface tension behavior. Some characteristics of the ether column for hydrophobic-interaction chromatography are shown, e.g., column stability at pH 8 and sample capacity.

Preparative high-performance hydrophobic interaction chromatography of proteins on TSKgel Phenyl-5PW

Preparative high-performance hydrophobic interaction chromatography of proteins on TSKgel Phenyl-5PW

High-performance hydrophobic interaction chromatography of proteins

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and β-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 μ sodium sulfate in 0.1 m potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of α-amylase and β-glucosidase ranging in size from 10 to 200 μg were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.

Operational variables in high-performance hydrophobic interaction chromatography of proteins on tskgel phenyl-5PW

The effect of operational variables on retention, recovery and resolution was investigated in hydrophobic interaction chromatography of proteins on TSkgel Phenyl-5PW with a linear gradient comprising a decreasing salt concentration. Sodium sulphate was more effective in retaining proteins than ammonium sulphate or potassium phosphate. The effect of the eluent pH on retention was not as great as reported on other supports. The addition of an organic solvent or chaotropic agent promoted the desorption of proteins. High recovery was observed in most separations where the flow-rate, gradient time, initial ammonium sulphate concentration, isopropanol concentration in the final buffer and eluent pH were varied. The resolution was affected to different extents by the column length, flow-rate, gradient time and addition of organic solvent or chaotropic agent in the final buffer.

High-performance hydrophobic interaction chromatography of proteins

High-performance hydrophobic interaction chromatography of proteins was investigated on Butyl-G3000SW and Phenyl-G3000SW prepared by coupling butyl and phenyl groups with G3000SW, which is a support for high-performance gel chromatography. It was possible to separate proteins with high efficienty under mild eluting conditions such as isocratic elution with 0 to 2 M ammonium sulphate in 0.1 M phosphate buffer (pH 6.0) and linear gradient elution with ammonium sulphate concentration decreasing from 1.5 or 2 M to 0 in 0. 1 M phosphate buffer (pH 6.0). The recovery of proteins or enzymatic activity was almost quantitative.