Hydrophobic interaction chromatography mechanism

Various types of proteins have been purified using hydrophobic interaction chromatography including alkaline phophatase, estrogen receptors, isolectins, strepavidin, calmodulin, epoxide hydrolase, proteoglycans, hemoglobins, and snake venom toxins (46). In the case of cobra venom toxins, the order of elution of the six cardiotoxins supports the hypothesis that the mechanism of action is related to hydrophobic interactions with the phospholipids in the membrane. 

The mechanisms described above form the basis for the chromatographic modes described in Chapter 2, namely, normal-phase, reversed-phase, size-exclusion, ion-exchange, and affinity chromatographies. However, other modes that are variations of those mentioned above, such as hydrophobic-interaction chromatography (HIC), chiral, ion-exclusion, and ion-pair chromatographies are also used and will be mentioned. 

Investigate conditions, which may be solvent compatible with previous and subsequent unit operations. Identify the appropriateness of normal-phase, revcrsed-pha.se. lon-exchanec or hydrophobic interaction chromatography as the desired mechanism. 

Hydrophobic interaction chromatography (HIC) is a mode of separation in which molecules in a high-salt environment interact hydrophobically with a nonpolar bonded phase. HIC has been predominantly used to analyze proteins, nucleic acids, and other biological macromolecules by a hydrophobic mechanism when maintenanee of the three-dimensional structure is a primary eoneern [1-4]. The main applications of HIC have been in the area of protein purification because the reeovery is frequently quantitative in terms of both mass and biological activity. 

Hydrophobic interaction chromatography Mode of chromatography using as a retention mechanism the molecular interactions between the hydrophobic or nonpolar regions of the external surface of protein molecules and chains bonded to a siUca support. Although C18 alkyl chains would qualify, the name is reserved to polyether or polyamide chains which cause little degradation of the purified proteins. 

Besides afEnity chromatography, HPLC separations of four different modes are frequently used for proteins and peptides. These are size-exclusion, ion-exchange, reversed-phase, and hydrophobic interaction chromatographies. Each mode has a different separation mechanism with its own inherent advantages... 

The mechanical and chemiceil stability of these methacrylate based adsorbents makes them attractive alternatives for process chromatography where a robust column packing is essential. They have been used for size separations and hydrophobic interaction chromatography and after derivatisation as affinity matrices and for ion exchange chromatography. 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

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