Hydrophobic interaction chromatography mobile phase

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. 

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. 

Fig. 21. Separation of cytochrome (peak 1), ribonuclease, (peak 2), carbonic anhydrase (peak 3), lysozyme (peak 4), and chymotrypsinogen (peak 5) by hydrophobic interaction chromatography on a molded poly(acrylamide-co-butylmethacrylate-co-N,AT,-methylenebisacry-lamide) monolithic column. (Reprinted with permission from [ 135]. Copyright 1998 Elsevier). Conditions column, 50 x8 mm i.d., 10% butyl methacrylate,mobile phase gradient from 1.5 to 0.1 mol/1 ammonium sulfate in 0.01 mol/l sodium phosphate buffer (pH 7) in 3 min, gradient time 3.3 min, flow rate 3 ml/min...

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. 

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

Liquid Chromatography The process by which the components of a liquid sample are physically separated based on their partitioning between a stationary phase and a moving (mobile) phase. Major modes include reverse phase, in which the stationary phase is non-polar, and normal phase, in which the stationary phase is polar. HILIC (Hydrophobic interaction chromatography) is a popular variant on the latter (Goodwin et al., 2007). 

In hydrophobic interaction chromatography (HIC)> nonpolar components are selectively expelled from an aqueous mobile phase due to the cohesive forces induced in water by hydrogen bonding. These forces can be modulated by the concentration of dissolved salts. The nonpolar species adsorb on or partition into a nonpolar stationary phase largely as a result of these forces. The nonpolar phase is often a bonded phase as noted above. 

Wu, S. L., Figueroa, A., and Karger, B. L. (1986). Protein conformational effects in hydrophobic interaction chromatography. Retention characterization and the role of mobile phase additives and stationary phase hydrophobicity. J. Chromatogr. 371, 3-27. 

HILIC uses a polar stationary phase to separate proteins and peptides. Samples are loaded onto a column using a low aqueous mobile phase and are eluted by increasing the water content of the mobile phase (152). HILIC is very suitable for separation of polar species such as sialic acid-containing glycopeptides, which have limited retention on most RP materials (153). A variant of HILIC is electrostatic repulsion-hydrophobic interactions chromatography and it utilizes IEX stationary phase and a high organic mobile phase (152,154). 

Pure aqueous mobile phases are only suitable for separations on weakly hydro-phobic 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. 1 M 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 Figure 10.16). 

Figure 10.16 Separation of a peptide mixture by hydrophobic interaction chromatography (reproduced with permission from A.J. Alpert, J. Chromatogr., 444, 269 (1988)]. Conditions column, 20cm x 4.6mm i.d. stationary phase, polypropyl aspartamide 5pm mobile phase, 1mlmin of 2M ammonium sulfate with 0.025M sodium phosphate, pH 6.5-0.025M sodium phosphate pH 6.5, linear gradient as indicated detector, UV 220 nm. 1 = substance Ml—9) 2 = [Arg ]-vasopressin 3 = oxytocin 4 = substance P, free acid 5 —[Try ]-substance P, 6 = substance P,1 [Tyr ]-somatostatin 8 = somatostatin 9 = [Tyr ]-somatostatin.

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