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 constancy 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., Mg2+ 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.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004355:Drug Stability; D004798:Enzymes / Q000032:analysis; D004987:Ethers / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000032:analysis; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*; D012343:RNA, Transfer / Q000032:analysis; D012997:Solvents; D013500:Surface Tension; D013997:Time Factors
D004798:Enzymes; D004987:Ethers; D011506:Proteins; D012997:Solvents; D012343:RNA, Transfer; D008274:Magnesium