Hydrophobic interaction HIC

HIC is a binding technique and therefore rather independent of sample volume, provided that conditions are chosen to strongly bind the target protein. 

The total amount of protein which is loaded and binds to the column should not exceed the total binding capacity of the column. For optimal separations when performing gradient elution, use approximately one fifth of the total binding capacity of the column 

Correct sample preparation ensures good resolution and extends the life of the column. To ensure efficient binding during sample application samples should be at the same pH as the starting buffer and in high ionic strength solution (e.g. 

5 M ammonium sulphate or 4M NaCl). Samples must be free from particulate matter, particularly when working with bead sizes of 34 pm or less (see Chapter 8 for details of sample clarification procedures). 

Fab fraction from STREAMLINE SP, 2 ml HiTrap HIC Test Kit (1 ml columns), 

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Hydrophobic interaction (HIC) Aqueous, usually buffered Apolar ligand (e.g. octylamino) bonded to support matrix. A form of AC ligand complexes with apolar (hydrocarbon) sites on protein solute. Usually proteins. 

Reversed-phase (RPC), ion-pair (IP), ion-exchange (IEC), hydrophilic interaction (HILIC), hydrophobic interaction (HIC), normal-phase (NP), gel permeation (GPC), size-exclusion (SEC), nonaqueous RP (NARP). 

Proteins are isolated and characterized with different chromatographic techniques. Depending on the protein, ion-exchange (lEX), size-exclusion (SEC), affinity (IMAC), hydrophobic interaction (HIC), and reversed-phase chromatography (RPLC) may be applied. Traditionally, separation materials with low mechanical stability and limited resolution were used. In recent years, HPLC materials were developed that offered the separation power required by the protein chemist. Now, pellicular ion-exchange resins as well as organic polymer monoliths exhibit high resolution at relatively short retention times, while... 

Hydrophobic interaction (HIC) Particles of well defined size, coated witii small hydrc hobic lig ds C2—C4 Saline aqueous solutions Well develr red fot protein separation No prior desalting necessary, grxxl after lEC... 

The application and preparation of afEnity resins was first described in a paper by Cuatrecasas in 1968 [1]. In this work, nearly aU features of this technique were explored. Affinity chromatography is the youngest of the four major purification methods used in biochromatography. In 1968, ion exchange (lEX), hydrophobic interaction (HIC), and gel filtration (GF) were already well established. What new opportunities in the purification of biomolecules were then added by affinity chromatography ... 

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-... 

In HIC, the hydrophobic interactions are relatively weak, often driven by salts in moderate concentration (I to 2 M), and depend primarily on the exposed residues on or near the protein surface preservation of the native, biologically active state of the protein is an important feature of HIC. Elution can be achieved differentially by decreasing salt concentration or increasing the concentration of polarity perturbants (e.g., ethylene glycol) in the eluent. 

Reversed-phase chromatography rehes on significantly stronger-hydrophobic interactions than in HIC, which can result in unfolding and exposure of the interior hydrophobic residues, i.e., leads to protein denaturation and irreversible inactivation as such, RPC depends... 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

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. 

Hydrophobic interaction chromatograph (HIC), while very attractive in principle, has proved difficult to scale up for processing. A recent series of articles explores some of the unique problems associated with process-scale HIC. Load sample preparation20 must be carefully examined to prevent protein aggregate formation in the presence of the relatively high salt concentrations used in this technique. Successful scale-up also requires the setting of wide specifications to accomodate routine variations in the feed.21 The effect of the salt concentration on capacity may be somewhat more... 

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. 

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