Hydrophobic Interaction Chromatography HIC

The differences in sizes and locations of hydrophobic pockets or patches on proteins can be exploited in hydrophobic interaction chromatography (HIC) and reversed-pha.se chromatography (RPC) discrimination is based on interactions between the exposed hydro-... 

Hydrophobic interaction chromatography (HIC) can be considered to be a variant of reversed phase chromatography, in which the polarity of the mobile phase is modulated by adjusting the concentration of a salt such as ammonium sulfate. The analyte, which is initially adsorbed to a hydrophobic phase, desorbs as the ionic strength is decreased. One application demonstrating extraordinary selectivity was the separation of isoforms of a monoclonal antibody differing only in the inclusion of a particular aspartic acid residue in the normal, cyclic, or iso forms.27 The uses and limitations of hydrophobic interaction chromatography in process-scale purifications are discussed in Chapter 3. 

Hydrophobic interaction chromatography (HIC) is a column chromatography technique which can determine particle hydrophobicity by interaction with a hydrophobic gel matrix [142,149,150]. Hydrophilic particles pass through the column without interaction, whereas particles with increased hydrophobicity show a retarded elution and are retained by the column. Hydrophobicity measurements are used to determine the hydrophobicity of nanoparticulate carriers and correlate this to their in vivo biodistribution [10, 149]. 

Hydrophobic interaction chromatography (HIC) occupies a unique niche in the field of analytical chromatography. A particular advantage of HIC is its unique selectivity. Whereas ion-exchange chromatography (IEC) principally reveals differences based on the surface charge of native proteins, HIC reveals differences based principally on their surface hydrophobicity. HIC is complementary to reversed-phase chromatography (RPC) in a different sense. Whereas HIC discriminates primarily on the basis of surface hydrophobicity, RPC principally reveals differences based on total hydrophobicity of all the hydrophobic residues of denatured proteins. 

Several researchers have used liquid chromatography in its different modes, such as reversed phase chromatography (RPC), hydrophobic interaction chromatography (HIC), lEC, SEC, and AC, to analyze and fractionate food proteins and peptides. 

The classification of chromatography as gel permeation chromatography (GPC size-exclusion chromatography, SEC gel filtration, GF), ion exchange chromatography (lEC), hydrophobic interaction chromatography (HIC), and affinity chromatography (AC) is... 

Because GFPuv exhibits stability to extreme conditions such as exposure to heat and chemical denaturants (disinfectants) in a wide pH range, its expression by prokaryotes, followed by extraction and purification, should be studied for its potential utility as a marker in validation procedures. In addition, the protein extracted from E. coli and further purified by hydrophobic interaction chromatography (HIC) resins should be analyzed qualitatively (2) by sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) to define the best purification method. SDS-PAGE with Coomassie or silver staining provides a sensitive method to determine the most appropriate HIC support for the purification of GFPuv. 

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. 

The surfaces of proteins are mostly hydrophilic. Although the majority of the hydrophobic residues tend to be buried in the interior of the protein, some hydrophobic regions are also found on the surface (Voet and Voet, 1995 Ladisch, 2001). The level of surface hydrophobicity differs from one protein to another, mainly as a consequence of the amino acid composition and sequence. Difference in surface hydrophobicity is the property exploited in hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC). 

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. 

There are other novel media with characteristics similar to functionalized membranes. Some of the commercially available media are listed in Table 2. These materials in many cases are at the cross-lines of definitions and are frequently compared in the MA literature. Organic separations in the reserve-phase (RP) and hydrophobic interaction chromatography (HIC) mode are not very common on filtration-based MA materials. However, the methacrylate copolymers can be used for this purpose. Also rodlike monolithic materials enable greater flexibility in these types of chemistry.13,14 The method of Tennikova and Svec15-17 has been used to commercialize a novel disk type separation media, called CIM (Convective interaction media, BIA, Ljubljana, Slovenia).18-21 Analytical-scale separations can be performed on... 

Hydrophobic interaction chromatography (HIC) Hydrophobicity High speed High capacity High resolution + + + + + +... 

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