Studies On The Thermodynamics Of Chiral Drugs During The Process Of Chiral Separation On The BSA Chiral Column And The Application Of The Two-dimensional Liquid Chromatography By A Single Column

Chiral stationary phase based on high performance liquid chromatography is one of the most important ways of chiral separation. Protein-based chiral stationary phases have been widely used for the separation of drug enantiomers because natural protein exhibited the broadest range of enantionselectivity of drugs. In this paper the thermodynamics of six kinds of chiral drugs and four kinds of small molecular drugs during the chiral separation or retention process on the BSA chiral stationary phase (BSA-CSP) were investigated in detail, and the separation and purification of egg white using two-dimensional liquid chromatography by a single column (2DLC-1C) was also studied. The thesis included the following five parts:1. Literature ReviewThe significance of the chiral separation, preparation of chiral stationary phases based on a protein and recent progresses in protein-based chiral stationary phases for enantionseparations were introduced in this part. In addition, the applications of ion exchange chromatography (IEC) and hydrophobic interaction chromatography (HIC) to protein separation were also summerized. Finally, the new technology of two-dimensional liquid chromatography by a single column (2DLC-1C) was introduced.2. Synthesis and characterization of BSA-CSPThe bovine serium albumin chiral stationary phase (BSA-CSP) was synthesized by immobilizing BSA on aminopropyl silica gel activated with1,1’-carbonyldiimidaole. The enantiomeric separation of six kinds of chiral drugs, such as D,L-tryptophan, warfarin, benzoin, mandelic acid,2-phenyl cyclohexanone and2-naphthalene cyclohexanone enantio-mers were achieved successfully with bovine albumin chiral column. The results showed that the BSA-CSP synthesized by this method had the capability of chiral separation for enantiomers. The effect of the pH, injection volume, ionic strength in the mobile phase and temperature on the retention behavior of D-,L-tryptophan on BSA-CSP were investigated in detail. In addition, the interaction mechanism about D,L-tryptophan, warfarin and benzoin on BSA-CSP was studied by stoichiometric displacement theory for retention (SDT-R). With the comparison of both logI and Z values between D-and L-typed enantiomers, the results indicated that the values of logI and Z of D-isomers were greater than that of L-ones, which showed that the contact area and affinities between the D-isomers and BSA-CSP were greater than that of L-ones.The greater of△1gI between the isomers, the greater of the separation factor was.3. Thermodynamics of chiral drugs and small molecular drugs separated with BSA-CSPThermodynamics of six kinds of chiral drugs and four kinds of small moleculuar drugs separated with BSA-CSP were studied in this chapter. During the range of column temperature from283.15K to308.15K, the good linear relationship of logk’ to1/T of these drugs could be obtaind. All△H obtained of these compounds were negative, which indicated that it was exothermic reaction during the enantioseparation of these chiral drugs. The absolute values of△H of L-isomers were less than D-ones. The result showed that the former could be eluted more easely than the latter. In addition, the obtained△H values of different chiral compounds during the separation process with BSA-CSP were different, the order of the absolute△H values of these chiral drugs was mandelic acid<2-phenyl cyclohexanone<2-naphthalene cyclohexanone<benzoin<warfarin<tryptophan,which indicated that the interactions between the chiral compounds and BSA also followed the same order. It was that as same as the order obtained from the values of k’of these compounds. The thermodynamic parameters were calculated in order to promote an understanding of the relationship between enthalpy and entropy for enantioseparation.The chiral separation of these drugs with BSA-CSP was enthalpy-controlled, and the enthalpy-entropy compensation existed resulting that the same separation mechanism of these drugs on BSA-CSP was followed.4. The separation and purification of hen egg white using SCX/HIC2DLC-1CThe egg white were separated and purified by using two-dimensional liquid chromatography with a single SCX/HIC dual-functional mixed-mode column (2DLC-1C). Four kinds of proteins, such as avidin, lysozyme, ovalbumin and transferring could be purified completely. The egg white were separated in SCX mode firstly, and the lysozyme and advin can be purified with the purities of95%and98%,respcetively.The fractions which were not retained in SCX mode on the2D column were separated again in HIC mode with the same column. The ovalbumin and ovotransferrin were purified with the purities of96%and98%, repectively. Because of a salt-water system as the mobile phase, the molecular structure and the bioactivity of protein can be maintained during the separation process,2DLC-1C can be used to be a powerful tool for fast separation of intact protein.5. Weak anion-exchange/hydrophobic interaction chromatography (WAX/HIC) for the separation and purification of hen egg whiteThree proteins from egg white were separated and purified by using WAX/HIC dual-function mixed-mode column. The egg white were separated in WAX mode firstly,and the ovalbumin can be purified, The fractions which were not separated in WAX mode on the2D column were separated again in HIC mode with the2D column. The G3globulin and ovotransferrin were purified. Because the same mobile phase can be used under the two separation modes with a single dual-function column, the a strong and a weak eluent solution only needed to be exchange for2DLC-1C.