Hydrophobic-interaction chromatography protein separation

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

Xie, S., Svec, F., and Frechet, J.M.J., Rigid porous polyacrylamide-based monolithic columns containing butyl methacrylate as a separation medium for the rapid hydrophobic interaction chromatography of proteins,. Chromatogr. A, 775, 65, 1997. 

Protein separation by hydrophobic interaction chromatography is dependent upon interactions between the protein itself, the gel matrix and the surrounding aqueous solvent. Increasing the ionic strength of a solution by the addition of a neutral salt (e.g. ammonium sulfate or sodium chloride) increases the hydrophobicity of protein molecules. This may be explained (somewhat simplistically) on the basis that the hydration of salt ions in solution results in an ordered shell of water molecules forming around each ion. This attracts water molecules away from protein molecules, which in turn helps to unmask hydrophobic domains on the surface of the protein. 

Since the hydrophobicity of styrene- or alkyl methacrylate-based monolithic matrices is too high to make them useful for hydrophobic interaction chromatography, porous monoliths based on highly hydrophilic copolymers of acrylamide and methylenebisacrylamide were developed [70,135]. The hydrophobicity of the matrix required for the successful separations of proteins is controlled by the addition of butyl methacrylate to the polymerization mixture. The suitability of this rigid hydrophilic monolith for the separation of protein mixtures is demonstrated in Fig. 21, which shows the rapid separation of five proteins in less than 3 min using a steeply decreasing concentration gradient of ammonium sulfate. 

Hydrophobic interaction chromatography is a form of reverse-phase chromatography most appropriate foi protein separations. 

Klostermeyer (1975) used an activated thiol-Sepharose 4B column with Tris-HCl buffer containing dithiothreitol to separate the K-and aa2-caseins from the aai-and /3-caseins in whole casein. More recently, Creamer and Matheson (1981) studied the fractionation of casein by hydrophobic interaction chromatography on octyl- or phenyl-Sephar-ose CL-4B columns. The whole casein was adsorbed onto the column from dilute phosphate buffers. A gradient of 0 to 40% ethylene glycol followed by 6 M urea was employed to desorb the protein. Optimum separation was obtained with an increasing urea gradient. Under all conditions, the major /3-casein component was eluted more readily than the asi-casein in spite of its higher hydrophobicity. 

Analytical Properties Separation by hydrophobic interaction chromatography, using aqueous salt solutions near pH = 7 used primarily in protein work Reference 18... 

Fractionation is less specific and essentially requires separation efficiency. This is generally performed with ion-exchangers and hydrophobic interaction chromatography, which enable efficient separation by salt or pH gradients. Hydroxyapatite is also a good tool for fractionation where proteins can be separated by both phosphate gradients and sodium chloride gradients. 

Chromatography is one of the most important tools in protein purification. Chromatographic purification techniques include affinity chromatography (AC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and gel filtration (GF). These techniques separate proteins according to differences in specific protein properties. The protein property used for separation, the attributes of each technique, and its suitability for different purification steps are summarized in Table 32.7. 

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