Hydrophobic interaction chromatography applications

Meliander, W. R., Corradini, D., and Horvath, C. (1984). Salt-medated retention of proteins in hydrophobic-interaction chromatography. Application of solvophobic theory. J. Chromatogr. 317, 67-85. 

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]...

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. 

Other methods that are related to affinity chromatography include hydrophobic interaction chromatography and thiophilic adsorption. The former is based on the interactions of proteins, peptides, and nucleic acids with short nonpolar chains on a support. This was first described in 1972 [113,114] following work that examined the role of spacer arms on the nonspecific adsorption of affinity columns [114]. Thiophilic adsorption, also known as covalent or chemisorption chromatography, makes use of immobilized thiol groups for solute retention [115]. Applications of this method include the analysis of sulfhydryl-containing peptides or proteins and mercurated polynucleotides [116]. 

Ion exchange chromatography, hydrophobic interaction chromatography (HlC), and affinity chromatography (AC) show some similarities concerning practical realization therefore, most of the hints given for lEC are applicable for HlC and AC. Examples for AC are given in Protocols 3.6.2.4 (biospecific desorption) and 3.6.2.5 (elution by partial denaturation). 

Eriksson KO (1998), Hydrophobic interaction chromatography, In Janson JC, Ryden L (Eds), Protein Purification Principles, High-Resolution Methods, and Applications, 2nd Ed., Wiley-VCH, Weinheim, pp. 283-310. 

Abbreviations for type of application IEX Ion Exchange, HIC Hydrophobic interaction chromatography, AF Affinity chromatography, RP Reverse phase, IMAC Immobilized metal affinity chromatography. 

Eriksson, K.-0. (1989) Hydrophobic interaction chromatography, in Protein Purification - Principles, Higfi Resolution Methods and Applications (Janson, J.-C., Ryden, L., Eds.) VCH, Weinheim. 

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 (HIC) and affinity chromatography (AFC) [29-31] have been used in lipase purification. The ligands are very specific and each ligand is only applicable for the separation of the lipase from a certain source. Polypropylene glycol is reported to be a suitable ligand for the fractionation of Chromohacterium viscosum lipase [29]. 

LPO, which is positively charged at neutral pH, can be isolated from milk or whey by ion-exchange chromatography which has been scaled up for industrial application. These methods isolate LPO together with lactotrans-ferrin (Lf) which is also cationic at neutral pH. LPO and Lf can be resolved by chromatography on CM-Toyopearl or by hydrophobic interaction chromatography on Butyl Toyopearl 650 M (see Fox and Mulvihill, 1992). 

A general difBculty in hydrophobic-interaction chromatography is the application of the sample. If the sample is dissolved in water or a low-buffer solution, only small injection volumes should be applied. Remember that water is the strong elution solvent in HIC On the other hand, the addition of salt to the sample can result in a predpitation of the protein. However, the addition of salt also allows a larger injection volume. The salt concentration in the sample therefore needs to be carefully optimized. This problem also limits the... 

The buffering capacity also depends directly on the concentration of the. . buffer. In all applications the maximum buffer concentration is constrained. In reversed-phase and hydrophilic interaction chromatography, the buffer conoen- tration is constrained by the solubility in the presence of organic solvents, and in ion-exchange and hydrophobic interaction chromatography retention is directly influenced by the buffer concentration. 

The support matrices used for ion-exchange chromatography have been modified by different means and successfully used for hydrophobic interaction chromatography (HlC), which also has a broad field of applications for the purification of... 

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