Reversed-phase chromatography hydrophobic interaction

Polarity Adsorption chromatography Reverse-phase chromatography Hydrophobic interaction chromatography... 

M.T.W. Hearn, Reversed-phase and hydrophobic interaction chromatography of proteins and peptides, in HPLC of Biological Macromolecules, 2nd ed., K.M. Gooding and F. Regnier (Eds.), Marcel Dekker, New York, 2002, pp. 172-173. 

Reversed-phase HPLC Hydrophobic interaction chromatography Size-exclusion chromatography Ion-exchange chromatography... 

Many publications of the last decades reported the preparation, characterization, and performances of various types of adsorptive membrane. Ion exchange, affinity, reverse phase, or hydrophobic interaction membrane chromatography have been described. In the following, various methods involved in the preparation of different classes of adsorbers will be discussed. 

M. I. Aguilar and M. T. W. Hearn, Reversed-phase and hydrophobic-interaction chromatography of proteins. 

Hydrophobic interactions are important in liquid-liquid extractions as well as in reversed-phase chromatography. This interaction is driven entropically by the exclusion of water from the nonpolar species the inclusion of water in a nonpolar phase requires the molecular ordering of the water. 

Reversed-Phase and Hydrophobic Interaction Chromatography of Proteins and Peptides... 

When a protein is adsorbed to a hydrophobic surface, denaturation is often not an instantaneous phenomenon. Thus, the extent of unfolding will increase with increasing time of adsorption. Both Karger and Hearn et al. [40] have monitored this residency effect on reversed-phase and hydrophobic interaction columns using a combination of techniques including derivative spectroscopy. The Hearn group also studied denaturation kinetics in size exclusion chromatography in the presence of the powerful denaturant urea. 

Besides afEnity chromatography, HPLC separations of four different modes are frequently used for proteins and peptides. These are size-exclusion, ion-exchange, reversed-phase, and hydrophobic interaction chromatographies. Each mode has a different separation mechanism with its own inherent advantages... 

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. 

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.]... 

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. 

The mechanism of reversed phase chromatography can be understood by contrast with normal phase chromatography. Normal phase liquid chromatography (NPLC) is usually performed on a polar silica stationary phase with a nonpolar mobile phase, while reversed phase chromatography is performed on a nonpolar stationary phase with a polar mobile phase. In RPLC, solute retention is mainly due to hydrophobic interactions between the solutes and the nonpolar hydrocarbon stationary surface. The nonpolar... 

It is believed that retention in reversed-phase chromatography is a function of sample hydrophobicity, whereas the selectivity of the separation results almost entirely from specific interactions of the solute... 

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

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