Hydrophobic interaction chromatography mobile phase effects

Wu, S. L., Figueroa, A., and Karger, B. L. (1986). Protein conformational effects in hydrophobic interaction chromatography. Retention characterization and the role of mobile phase additives and stationary phase hydrophobicity. J. Chromatogr. 371, 3-27. 

Adsorption chromatography The process can be considered as a competition between the solute and solvent molecules for adsorption sites on the solid surface of adsorbent to effect separation. In normal phase or liquid-solid chromatography, relatively nonpolar organic eluents are used with the polar adsorbent to separate solutes in order of increasing polarity. In reverse-phase chromatography, solute retention is mainly due to hydrophobic interactions between the solutes and the hydrophobic surface of adsorbent. Polar mobile phase is used to elute solutes in order of decreasing polarity. 

Size-exclusion chromatography (SEC) is a method in which molecules are separated by size due to differential permeation into a porous support. It requires complete solubility of the analytes in the mobile phase and elimination of all interactions with the bonded phase. In these respects, SEC is not as useful for the separation of peptides as it is for proteins because peptides vary drastically in solubility, charge, and hydrophobicity. Peak capacity in SEC is fairly low compared to other HPLC methods because all separations must occur in the internal volume (Vi) of the support, which is generally less than half the volume of mobile phase in the column. Despite these deficiencies, SEC can be very effective for separating peptides from dimers, aggregates, small molecules, proteins, and other molecules which differ by size. 

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