Amino Acid Enantioseparation via Protein Based Stationary Phases

The protein-bonded stationary phases were some of the first to be developed and usually consist of natural proteins (e.g. bovine serum albumin, a1-acid glyco-protein, ovomucoid, etc.) bonded to a silica matrix. Proteins contain a large number of chiral centres of one configuration and are known to interact strongly with small chiral compounds for which they can exhibit strong enantiomeric selectivity. Some specific interactive sites on the protein provide the chiral selectivity, but there are many others that generally contribute to retention. Protein columns based on bovine serum albumin have been employed for the separation of the enantiomers of certain aromatic amino acids and various derivatives, including dansyl, N-(9-fluorenylmethoxycarbonyl)- (FMOC), N-(fluorescein thiocarbamoyl- (FITC) N-(2,4-dinitro-phenyl) and N-benzoyl. The use of the reagent N-(chloroformyl)carbazole to provide highly fluorescent derivatives has enabled the resolution of the enantio-mers of all of the protein amino acids often with high separation factors. Proteins have also been described as showing remarkable enantioselectivity towards N-acylated amino acids.

The mobile phases employed for this type CSP are generally composed of phosphate buffers (0.1-0.2 M) modified with a limited amount of pro-pan-1-ol. The pH range normally employed is be tween 4.5 and 8.0 and for example, in the case of the N-benzoyl-derivatized amino acids, increasing pH results in decreased retention. In general the lower the buffer concentration (from 0 to 0.1 M) the better the retention; however, an effect of increased buffer concentration (above 0.2 M) has been observed for N-benzoyl derivatives. An increase in organic modifier concentration reduces the hydrophobic interactions of the solutes with the column resulting in shorter retention times. Whilst very useful for the determination of, for example, enantiomeric purity, protein phases tend to have rather limited sample loading capacity.

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