Study On Enantioseparation Abilities Of The Ligand-exchange Chromatography And Polysaccharide-based CSPs

It is well known that a pair of optically enantiomers often show quite difference inpharmacological and biological activities， especially in chiral drugs. Therefore, theenantioseparation is a continuously challenging task. Chromatography is one of the mosteffective methods for the enantioseparation. Among the chromatographic methods, highperformance liquid chromatography(HPLC) is fast, accurate and will not disrupt the structuraland biological activity of enantiomers, which is widely used in agriculture, biology and so on.Thus, the research and development on the HPLC chiral stationary phases(CSPs) has alwaysbeen the critical study. The ligand-exchange chromatography and polysaccharide-based chiralstationary phases are the most widely used among many chiral stationary phases. In this paper,the coated-type and the immobilized-type chiral stationary phases in ligand-exchangechromatography were synthesized and used in the separation of amino acids. Moreover, theseparation of amino acids and amino acid derivatives on the ChiralPak IA was alsoinvestigated. The major contents are as follows:The research was carried out on the ZORBAX SB-C18column,which coated with（R）-STC with dynamic coating method and the coating amount was calculated by UVspectrophotometer.The immobilized-type chiral stationary phases in ligand-exchange chromatography wereprepared by bonding the L-Phenylalanine onto the surface of the acidified silica gel whichwas modified by the γ-glycidyl propyl trimethoxy silane(KH-560). The structure of CSP wascharacterized by IR spectrum and the immobilized amount was calculated by TG. The CSPwas treated with screening and solvent deposition to determin the uniformity of particle sizeand ensure the stability of column pressure and the enatioseparation ability of the column.The separation of amino acids was evaluated on the chiral stationary phases inligand-exchange chromatography which the selectors were（R）-STC and L-Phenylalanine andthe influences of the chromatographic conditons such as the copper(Ⅱ) salt, concentration ofcopper(Ⅱ), pH and so on were investigated. The results showed that the concentration of anycomponent in the chromatographic system generated a great impact on the separation, so it’sneeds to moderate concentrations due to the constraint from the equilibrium in the process ofthe ligand-exchanged. The separations on the two kinds of CSPs obtained the highestseparation at25℃. The amino acids were nearly resolved in baseline on coated-type CSP using CuSO4solution as eluent. Besides, the baseline resolution of dopa on the coated-typeCSP provides a new way to obtain L-dopa. The immobilized-type CSP has the best separationefficiency in Cu(OAc)2-KH2PO4buffer solution. Compared the chiral recognition ability ofthe two kinds of CSPs, the coated-type was better for its chiral selector is S-trityl-(R)-cysteinewhich containing the aromatic ring in its structure and belongs to the rigid conformation whilethe immobilized-type adopted the L-Phenylalanine as the chiral selector which theconformational instability and easy to bend.The enantioseparation of the dopa, phenylalanine and N-substituted amino acids onChiralpak IA column were evaluated. The results showed that all N-substituted amino acidswere separated on Chiralpak IA column and most were baseline resolved while dopa andphenylalanine only obtained part solution. When the ethanol was used as the polar componentof the mobile phase instead of isopropanol, better results were obtained. As the concentrationof ethanol increased, the retention decreased and the separation factor reached the maximumwhen containing35%ethanol in elution, but the resolution factor changed irregularly.Increasing the additive concentration can minimize the peak broadening arising from theinteraction between the racemates and CSP and the results showed the mobile phase consistedof hexane-ethanol (75:25,v/v) containing0.5%TFA is ideal for enantioseparation. ComparedChiralpak IA column with the L-Phenylalanine ligand-exchange CSP, dopa and phenylalaninewere better resolved on the L-Phenylalanine ligand-exchange CSP while the Chiralpak IAcolumn showed a high chiral recognition ability on the separation of the N-substituted aminoacids due to the different enantioseparation mechanisms of the two kinds of CSPs.