Hydrophobic stationary phase

Interactions between proteins and salts in the binding buffer are also a major determinant of selectivity. Salts that are strong retention promoters in HIC are excluded from protein surfaces by repulsion from their hydrophobic amide backbones and hydrophobic amino acid residues.8,9 This causes the mobile phase to exert an exclusionary pressure that favors the association of proteins with the column, regardless of stationary-phase hydrophobicity.1(W2 Because this mechanism involves the entire protein surface, the degree of exclusion is proportional to average protein hydrophobicity, regardless of the distribution of hydrophobic sites. 

The balance between stationary-phase hydrophobicity and solvent exclusionary forces is the major determinant of selectivity on nonphenyl columns. On strongly hydrophobic columns, hydrophobic interactions between the proteins and the column are most likely to be the dominant determinant of selectivity. The low levels of binding salts required to achieve retention exert relatively less influence. On weakly hydrophobic columns, the high levels of binding salts cause solvent exclusionary effects to exert a greater influence. 

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. 

Even if chain length is the key parameter, the ligand bonding density (usually above 2.5 umol/m ) may be very influential in determining overall stationary phase hydrophobicity. When the monolayer capacity, theoretically estimated by [L]j, increases as a result of increased bonding density, adsorption competitions are less operative and enhanced retention is expected. It should be noted that ligand bonding density can be calculated on the basis of the column carbon load and the total surface area of the column. 

F ig y re 5.1 Solute-micelle and solute-stationary phase hydrophobic ( 4) and electrostatic... 

The effects from molecular size are often related to hydrophobic interactions with nonpolar parts of the stationary phase. Hydrophobic interactions can be reduced by including 10-20% of methanol/acetonitrile in the mobile phase. This is often done when cation exchangers are used in the first dimension of two-dimensional separation of peptides, with reversed phase in the second dimension. 

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 ... 

In reversed-pViase chromatography (RPC), the mobile phase modulator is typically a water-miscible organic solvent, and the stationary phase is a hydrophobic adsorbent. In this case, the logarithm of solute retention factor is commonly found to be linearly related to the volume fraction of the organic solvent. 

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

There is usually an ionic strength above which there is no more effect on hydrodynamic size or, worse yet, there are hydrophobic interactions of the polymer with the stationary phase. Thus, the optimum 1 is usually at the low 1 end of the plateau of size vs 1. This concentration will minimize ionic strength effects while also minimizing wear on the pump seals and pistons. 

A general phenomenon observed with chiral stationary phases having hydrophobic pockets is that a decrease of flow rate results in an increase in resolution. This change has significant impact mostly in reversed-phase mode (see Fig. 2-10). 

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. 

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

By far the most popular phase system at the present time is the one in which R n-octadecyl. Since the stationary phase is hydrophobic and nonpolar, while the mobile phase is relatively polar, this mode of operation is frequently called reverse phase... 

Not only in HPLC, but also in modem thin-layer chromatography, the application of reversed-phase stationary phases becomes increasingly important. The advantage of the hydrophobic layers in comparison with the polar, surface-active stationary phases is the additional selectivity and a reduced hkehhood of decomposition of sensitive substances. 

Compared with liquid column chromatography, in PLC there is a certain limitation with respect to the composition of the mobile phase in the case of reversed-phase chromatography. In planar chromatography the flow of the mobile phase is normally induced by capillary forces. A prerequisite for this mechanism is that the surface of the stationary phase be wetted by the mobile phase. This, however, results in a Umitation in the maximum possible amount of water applicable in the mobile phase, is dependent on the hydrophobic character of the stationary RP phase. To... 

The popularity of reversed-phase liquid chromatography (RPC) is easily explained by its unmatched simplicity, versatility and scope [15,22,50,52,71,149,288-290]. Neutral and ionic solutes can be separated simultaneously and the rapid equilibration of the stationary phase with changes in mobile phase composition allows gradient elution techniques to be used routinely. Secondary chemical equilibria, such as ion suppression, ion-pair formation, metal complexatlon, and micelle formation are easily exploited in RPC to optimize separation selectivity and to augment changes availaple from varying the mobile phase solvent composition. Retention in RPC, at least in the accepted ideal sense, occurs by non-specific hydrophobic interactions of the solute with the... 

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