Development of an affinity silica monolith containing human serum albumin for chiral separations

Abstract

An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3-2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: d/l-tryptophan and R/S-warfarin. The separation of R- and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase.

Figure 1

Reactions involved in the preparation of a HSA silica monolith. Further details on these reactions can be found in the text. Abbreviations: HSA, human serum albumin.

Figure 2

(a) Representative chromatograms obtained for the injection of racemic tryptophan onto an HSA silica monolith, and (b) plots of total plate height (H_total) versus linear velocity for D- and L-tryptophan when using the HSA silica monolith (◆) or a column containing HSA immobilized to 300 Å pore size, 7 μm silica particles (■). The mobile phase was pH 7.4, 0.067 M KPB. Other conditions are given in the text. The average retention factors for D-tryptophan in (b) were 1.16 for the silica monolith and 0.82 for the column containing silica particles; the average retention factors for L-tryptophan on these same columns were 13.80 and 8.65, respectively.

Figure 3

(a) Representative chromatograms obtained for the injection of racemic warfarin onto an HSA silica monolith, and (b) plots of total plate height versus linear velocity for R- and S-warfarin when using the HSA silica monolith (◆) or a column containing HSA immobilized to 300 Å pore size, 7 μm silica particles (■). The mobile phase was pH 7.4, 0.067 M KPB. Other conditions are given in the text. The average retention factors for R-warfarin in (b) were 119 for the silica monolith and 56 for the column containing silica particles; the average retention factors for S-warfarin on these same columns were 163 and 73, respectively.

Figure 4

Separation impedance (E) versus linear velocity for HSA columns using D/Ltryptophan or R/S-warfarin as the analytes. The supports used in case were (◆) a silica monolith or (■) 300 Å pore size, 7 μm silica particles. Other experimental conditions are given in the text.

Figure 5

Representative chromatogram obtained for the injection of racemic ibuprofen onto an HSA silica monolith at 2.5 mL/min. The mobile phase for this separation was pH 7.0, 0.067 M potassium phosphate buffer containing 5% isopropanol and 5 mM octanoic acid. Other experimental conditions are given in the text.