Hydrophobic interaction chromatography sorbents

Sorbent and Mobile-Phase Considerations for Reversed-Phase Chromatography and Hydrophobic Interaction Chromatography... 

Kleinmann, I., Plicka, J., Smidl, P. and Vins, I. 1994. Hydrophobic interaction chromatography of proteins on HEMA-based sorbents. Am. Lab. April, 34H-34L. 

Hydroxyapatite Hydroxyapatite, which has the formula of Cajo(P04)6 (0H)2, is widely used as a sorbent for hydrophobic interaction chromatography. Hydroxyapatite is an underivatized matrix with a specific inherent surface functionality. In the 1980s, it had been introduced as ceramic beaded material with good physical strength (Kato, Nakamura, and Hashimoto, 1987). The interactions of the surface phosphate and caldum-ions with the amino and carboxyl groups of proteins are complex and not fully understood. The elution of proteins is typically... 

Hydrophobic interaction chromatography (HIC) was used for comparative analysis of TSPC [ 10]. A hydrophilic pol5meric sorbent, HEMA BIO Phenyl-1000 (particle size 10 mm) modified by phenyl groups, served as the stable phase. The mobile phase was phosphate buffer (pH 7.4) with ammonium sulfate. Elution was performed with the mobile phase in molar concentration range from 2.0 M to 0.0 M (pure buffer) with respect to (NH4)2S0. For coelution of hydrophilic and hydrophobic components of protein fractions, Brij-35 polyoxyethylene dodecyl ether with increasing concentration from 0% to 3% was added to the mobile phase. UV detection was usually set at 230 nm. 

Hydrophobic-interaction chromatography (HIC) is a powerful method for protein purification based on the reversible interaction between the protein surface and a hydrophobic chromatographic sorbent [27]. This technique is used in protein purification as a complement to other separation techniques and as a next step after ammonium sulfate precipitation of the sample or after lEX separation. 

All individual methodologies described in the following, such as ion exchange, hydrophobic interaction, and affinity chromatography, are potentially usable in packed-bed mode as well as in fluid-bed mode if the solid phase sorbent meets the specific requirements of density and particle size. The only exception today is gel filtration, which requires a large number of plates for an acceptable resolution that the fluid bed cannot guarantee. 

The nonmodified sorbents discussed so far show polar surface characteristics. But there are many chromatographic separation problems that can be solved using hydrophobic interactions of the stationary phase with compounds of appropriate molecular structure. Sorbents that are suitable for this task are the so-called reversed-phase materials (RP phases). In this connection, reversed phase means that the relative polarities of the stationary and mobile phase are reversed compared with the already described situation in adsorption chromatography i.e., the RP stationary phase is less polar... 

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