Reversed phase and hydrophobic interaction 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. 

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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. 

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

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

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... 

Module 3, Column and Mobile Phase Design (CMP). This is the core module for ECAT. It can currently specify i) analytical column and mobile phase constituents for reverse phase chromatography of common classes of organic molecules ii) reverse phase, ion exchange phase and hydrophobic interaction chromatography of proteins and peptides iii) a limited set of specialty classes of molecules best treated by straight phase chromatography (e.g., mono- and disaccharides). The rules for selection of the HPLC detector are under development within Module 3. Some of the rules for detector mobile phase compatibility are already encoded. A set of rules for detector selection is ready but not yet encoded. 

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. 

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. 

Sorbent and Mobile-Phase Considerations for Reversed-Phase Chromatography and Hydrophobic Interaction Chromatography... 

This is a simple upgrade of the isocratic system with the facility for gradient elution techniques and greater functionality (Fig. 1.1(b)). The basic system provides for manual operating gradient techniques such as reversed phase, ion exchange and hydrophobic interaction chromatography. Any of the detectors listed above under the isocratic system can be used. 

Ion-interaction chromatography is an intermediate between reversed-phase and ion-exchange chromatography. Introduction of amphiphilic and Uo-philic ions into the mobile phase causes their adsorption on the hydrophobic surface of packing material with subsequent transformation into a pseudo ion-exchange surface. Ionic interactions with charged analytes can occur in the mobile phase and with counterions that may be adsorbed on the stationary-phase surface. 

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. 

Reversed phase (RPC) and hydrophobic interaction chromatography (HIC) are based on very similar types of interactions they have been regarded as different... 

Bonded silicas are fairly stable in an acidic pH range of I to 2. Such conditions are usually applied in the resolution of peptides and proteins by reversed-phase HPLC. Separation of biopolymers is performed in the pH range between 5 and 8 with buffers in ion-exchange and hydrophobic interaction chromatography. The type and concentration of buffer have a remarkable effect on the stability of the bonded silica. For instance, phosphate buffers at pH 7 are not recommended, particularly when separations are performed at elevated temperatures. The lifetime of the column in phosphate will be much shorter than in Tris buffer [31]. Buffers with a pH above 8 should not be applied due to the dissolution of the silica matrix. 

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