Reversed phase chromatography purification

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. 

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. 

Kessel D, Thompson P (1987) Purification and analysis of hematoporphyrin and hematoporphyrin derivative by gel exclusion and reverse-phase chromatography. Photochem Photobiol 46 1023-1025. 

The moderate yield may be due to the purification by reverse-phase chromatography, because 1 contains a tertiary amine. Preparation of other derivatives of 2 has shown that the radical annulation normally proceeds with 50-60 % yield and that many functional groups are tolerated (free alcohols, amines, esters, chlorides and terminal alkenes). Also (Me3Si)4Si may be a useful substitute for hexamethylditin, because tin residues are toxic and difficult to separate from the products. 

Hearn MTW (1998), Fligh-resolution reversed-phase chromatography, In Janson JC, Ryden L (Eds), Protein Purification Principles, High-Resolution Methods, and Applications, 2nd Ed., Wiley-VCH, Weinheim, pp. 239-282. 

Non-polar and moderately polar solutes generally present the least difficulty, and these extracts are amenable to reversed phase partition chromatography followed by final purification of separated fractions by adsorption chromatography. Reversed phase chromatography is a practical first step because it is effective for a very wide range of compounds, and secondly because it has less tendency to be "fouled" by irreversible absorption of highly polar contaminants. 

Hodges, R. S., Burke, T. W. L., and Mant, C. T. (1988). Preparative purification of peptides by reversed phase chromatography—sample displacement versus gradient elution modes. ]. Chromatogr. 444, 349-362. 

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