Preparation Of Bovine Serum Albumin And Human Serum Albumin Chiral Stationary Phases And The Study Of Their Properties

In this paper, two protein chiral stationary phases (CSP) have been synthesized by immobilizing Bovine serum albumin (BSA) and human serum albumin (HSA) on aminopropyl silica gel which had been further activated with 1,1'- carbonyldiimidaole. Good separations of several racemic compounds were obtained on the above CSPs. Furthermore, character of thermodynamics and dynamics was studied. In chapter two, synthesis of BSA-CSP was developed on the CDI-activated silica gel; the methods used in this study provide numerous advantages over traditional methods, such as easy preparation, low cost, as well as timesaving. BSA was immobilized on the CDI-activated silica gel after packing column and before packing column. The results indicated that the former method is superior to the latter. Good separations of 7 racemic compounds were obtained on the BSA-CSP. The effects of the mobile phase, including the pH and the concentration of 1-propranol in the mobile phase, on the retention behavior of D-, L-tryptophan on BSA-CSP were investigated in detail. The ( of D-, L-tryptophan is 2.21 under the optimal chromatographic condition, mobile phase is 50mM Na2HPO4-NaH2PO4(pH=7.0).In chapter three, [6S]- and [6R]-calcium folinate(LV) were successfully separated on the BSA-CSP prepared in our lab. The effects of pH, the salt concentration, and column temperature on the separation were investigated in detail. Baseline separation was achieved in 10 min: 4.46 min and 7.59 min, α = 2.10,Rs = 2.78 under the optimal chromatographic condition, 50mM Na2HPO4-NaH2PO4(pH=6.5). Character of thermodynamics was studied, and we presume that LV has a single type of binding site on BSA. In chapter four, HSA-CSP was synthesized on the CDI-activated silica gel, 5 racemic compounds were successfully separated on HSA-CSP. The effects of the compsition of the mobile phase, the column temperature on the retention behavior of D-, L-tryptophan on HSA-CSP were also investigated. The ( of D-, L-tryptophan is 10.87 under the optimal chromatographic condition, temperature is 25℃, mobile phase is 50mM Na2HPO4-NaH2PO4(pH=7.0).In chapter five, the effect of column temperature on D-, L-tryptophan was investigated, and ΔΔH= -2.64 kJ/mol, ΔΔS= 8.382 J/mol·K respectively. Frontal analysis was used to determine the association constant and the active binding sites of L-tryptophan on HSA, Ka=3.23×104 L/mol, mL=250 nmol/g, adsorption capacity ranged from 244 ng to 2092 ng when the concentration of L-tryptophan ranged from 203 μmol/L to 324 μmol/L.