Abstract:

A novel chiral stationary phase (CSP) based on library of small peptides for high performance liquid chromatography (HPLC) is described. The conditions for digestion of bovine serum albumin (BSA) with trypsin were optimized, and the results of which were: enzymolysis time 6 h, enzymolysis pH 8.2, enzymolysis temperature 37 ℃. The obtained library of small peptides, which held many chiral recognition sites, could enhance the interaction between enantiomers and CSP. Then it was ultrafiltrated with membrane (Mr=6000) to discard Mr＞6000 molecules, and was bonded to aminopropyl-silica gel activated with 1,1′-carbonyldiimidazole. The proposed CSP held higher density of peptides’ ligand and was more steady than intact BSA-CSP. D,L-Tryptophan and D,L-tyrosine were well chiral separated on the novel peptides-CSP. The resolution (Rs) for D,L-tryptophan reached 10.48 with gradient pH buffer from 4.0 to 7.5. The Rs for D,L-tyrosine reached 13.50 with gradient pH buffer from 5.6 to 6.3. In the enantioseparation procedure of D,L-tryptophan and D,L-tyrosine, pH gradient elution was applied. The results could be ascribable to that the variation in pH of mobile phase can change dissociation of both enantiomers and peptides on CSP, which influenced the interaction between them by the conformation of peptides and/or the electrostatic situation and the hydrophobicity of solutes.

Key words: chiral separation , bovine serum albumin (BSA), chiral stationary phase (CSP), peptides, digestion with trypsin