Hydrophobic-interaction chromatography

Hydrophobic interaction chromatography (HIC) uses hydrophobic interactions on weakly hydrophobic stationary phases combined with a negative ion strength gradient (starting with high salt concentration). The technique was developed for separation of proteins, which had too high retention with ordinary reversed-phase materials, but is not widely used in HPLC. 

With hydrophobic interaction chromatography (HIC), hydrophobic proteins interact with slightly hydrophobic surfaces (propyl, ether, phenyl) at high salt concentrations. For elution, a salt gradient from high to low salt concentrations 

The interaction between the sample and the stationary phase is so strong in reversed-phase chromatography that an aqueous eluent is too weak without the addition of an organic solvent. However, organic mobile phases are not allowed in certain cases of protein separation because of the risk of denaturation and subsequent loss of biological activity. 

Protein poorly retained Increase salt concentration. Change salt to one that increases the surface tension. Change pH towards the isoelectric point of the protein. Change stationary phase to one with smaller hydrocarbon- aceous moieties and/or lower ligate density, i.e. reduce phase ratio. 

Insufficient selectivity Change salt. Use additives that selectively affect protein, e.g. inhibitors or allosteric effectors. 

The composition of the mobile phase markedly influences the retention behaviour, as can be seen from Table 10.1. 

Pure aqueous mobile phases are only suitable for separations on weakly hydrophobic stationary phases hence materials containing one tenth to one hundredth of the carbon load of classical reversed phases have been developed. This is achieved by low coverages of short-chain groups such as butyl or phenyl. Proteins can then be retained when the eluent has a relatively high salt content (e.g. 1M or more) and eluted when the salt content drops. This mild method of protein separation, which is a variant of reversed-phase chromatography, is known as hydrophobic interaction chromatography (HIC) (Fig. 10.15). 

Retention and selectivity in HIC depend on temperature in addition to the nature of the mobile and stationary phases the strength of the hydrophobic interaction increases with increasing temperature.38 The primary stationary-phase variables are the ligand density, structure, and hydrophobicity. The more hydrophobic the stationary phase, the greater is the retention of the analytes. If the stationary phase is too hydrophobic, however, the analytes may become denatured. Some analytes may only be handled satisfactorily on hydrophilic stationary phases.12 

Group name Group structure Exchanger type 

The most popular hydrophobic interaction chromatographic beads (resins) are cross-linked agarose gels to which hydrophobic groups have been covalently linked. Specific examples include 

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. 

Reverse-phase chromatography may also be used to separate proteins on the basis of differential hydrophobicity. This technique involves applying the protein sample to a highly hydrophobic column to which most proteins will bind. Elution is promoted by decreasing the polarity of the mobile phase. This is normally achieved by the introduction of an organic solvent. Elution conditions are harsh and generally result in denaturation of many proteins. 

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Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

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 ... 

Salt Effects. The definition of a capacity factor k in hydrophobic interaction chromatography is analogous to the distribution coefficient, in gel permeation chromatography ... 

The recovery of recombinant chymosin from a yeast fermentation broth showed that large-scale hydrophobic interaction chromatography could... 

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-... 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

Step 2. Hydrophobic interaction chromatography on a column of Phenyl-Sepharose CL-4B. The sample was adsorbed on the column in the basic buffer containing 0.5 M (NH SC. The photoprotein adsorbed was first washed with the same buffer, then eluted with the basic buffer. 

Fig. 8. Preparative isolation of hexon antigen of EDS-76 by hydrophobic-interaction chromatography on Butyl-PG column (2x5 cm) (A) application of the allantoic fluid diluted (1 5) by 50 mM potassium acetate, pH 4,130 ml (B)0.01 mol/1 potassium acetate, pH 5.5 (C) 0.01 mol/1 potassium bicarbonate pH 8.0, 10% isopropanol (D) 0.01 mol/1 potassium carbonate pH 9.6, 10% isopropanol. EDS-0 — components of alantoic fluid eluted with buffer A, EDS-1 — desorbed hexon fraction eluted with buffer C, EDS-2 — fraction desorbed with buffer D [56]...

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. 

Figure 4.27 Flow chart for coluwi selection based on sample type (m - molecular weight). PLC precipitation-liquid chromatography SEC = size-exclusion chromatography lEC - ion-exchange chromatography HIC hydrophobic interaction chromatography LSC liquid-solid chromatography RPC - reversed-phase liquid chromatography BPC (polar) bonded-phase chromatography and IPC - ion-pair chromatography.

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. 

Gagnon, P., Grund, E., and Lindback, T., Large-scale process development for hydrophobic interaction chromatography, part I gel selection and development of binding conditions, BioPharm, 8, 21, 1995. 

Puma, P., Duffey, D., and Dawidczyk, P., U.S. Patent appl. 94,944, Purification of oligodeoxyribonucleotide phosphorothioates using DEAE-5PW anion ion-exchange chromatography and hydrophobic interaction chromatography, 1994. 

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... 

Perkins, T.W., Mak, D.S., Root, T.W., and Lightfoot, E.N., Protein retention in hydrophobic interaction chromatography modeling variation with buffer ionic strength and column hydrophobicity, J. Chromatogr. A, 766, 1, 1997. 

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