Hydrophobic interaction chromatography stationary phase

Hydrophobic Interaction Chromatography. Hydrophobic interactions of solutes with a stationary phase result in thek adsorption on neutral or mildly hydrophobic stationary phases. The solutes are adsorbed at a high salt concentration, and then desorbed in order of increasing surface hydrophobicity, in a decreasing kosmotrope gradient. This characteristic follows the order of the lyotropic series for the anions ... 

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Two mechanisms for retention in reversed-phase ion-pair liquid chromatography have been considered. One is the adsorption of the hydrophobic paired ion on the hydrophobic surface of stationary phase material. In the second mechanism, the hydrophobic counter-ion is held on the surface of the hydro-phobic stationary phase, and the analyte ion is retained by ion-ion interactions, as shown in Figure 4.16. In the latter case, of a dynamic ion-exchange... 

If the molecular masses of solutes are >2 000 and if they are soluble in organic solvents and their molecular diameter is >30 nm. Figure 25-14 tells us to try molecular exclusion chromatography.. Stationary phases for this type of separation are described in Chapter 26. If the molecular masses of solutes are >2 000, and they are soluble in water, but not ionic, and have diameters <30 nm, the decision tree says to use reversed-phase chromatography, or hydrophobic interaction chromatography. 

Minimized interaction between eluite molecules and column packing is operative in hydrophobic interaction chromatography where biological solutes are so mildly bound to a stationary phase that their delicate native tertiary structure is not spoiled. This separation of extreme mildness is also a separation of chemically differing species, i.e., separation by composition. 

Currently, the best way of preserving the native conformation under the conditions of elution chromatography is hydrophobic interaction chromatography with minimized adsorption forces. These can be adjusted by selecting the appropriate stationary phase, e.g., from a series of alkylated agarose gels with various alkyl chain lengths. 

Liquid Chromatography The process by which the components of a liquid sample are physically separated based on their partitioning between a stationary phase and a moving (mobile) phase. Major modes include reverse phase, in which the stationary phase is non-polar, and normal phase, in which the stationary phase is polar. HILIC (Hydrophobic interaction chromatography) is a popular variant on the latter (Goodwin et al., 2007). 

In hydrophobic interaction chromatography (HIC)> nonpolar components are selectively expelled from an aqueous mobile phase due to the cohesive forces induced in water by hydrogen bonding. These forces can be modulated by the concentration of dissolved salts. The nonpolar species adsorb on or partition into a nonpolar stationary phase largely as a result of these forces. The nonpolar phase is often a bonded phase as noted above. 

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. 

A further polymeric modification is the so-called tentacle type (Fig. 3.14c), where the polymer or oligomer does not encapsulate the bead. This stationary phase is useful for mild separation or purification of proteins by ion-exchange chromatography, as described by Muller (1986, 1990). Chang et al. (1985) and Chang and An (1988) reported a hydrophobic interaction chromatography (HIC) variant where long, flexible side-chains can surround the proteins and thereby interact only with their hydro-... 

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