Wide-Bore Columns of Poly(Glycidyl Methacrylate-Co-Divinylbenzene)-Based Monolithic Beds for Reversed-Phase and Anion-Exchange Chromatographic Separation of Biomolecules.

Abstract

Three monoliths based on poly(glycidyl methacrylate-co-divinylbenzene) were prepared in the confines of borosilicate glass columns (100 × 3 mm I.D.). The first monolith was applied for analysis of proteins by reversed-phase high-performance liquid chromatography. It furnished a fast base-line separation for four proteins (ribonuclease A, cytochrome c, α-lactalbumin and myoglobin) in <80 s, with optimum resolution range of 2.11-2.84, and extremely small values of peak width at half height with a range of 1.0-1.6 s. The second and third monoliths were surface-modified into weak and strong anion-exchangers, respectively, and were investigated for anion-exchange (AE) high-performance liquid chromatography of four proteins with acidic isoelectronic points (bovine carbonic anhydrase, conalbumin, ovalbumin and soybean trypsin inhibitor) and of 5-phosphorylated oligodeoxythymidylic acids fragments [d(pT)12-18]. The weak AE monolith experienced complete elution of the four proteins applying a basic Tris-HCl buffer (0.02 M, pH 8.9); however, the strong AE monolith established a base-line separation of these proteins in ~14 min. Both monoliths showed base-line separation of the seven fragments of d(pT)12-18 in ~6 min. The ion-exchange capacity determined by frontal and elemental analyses was comparable for the weak AE monolith (0.75 and 0.80 meq/g) and for the strong AE monolith (0.81 and 0.87 meq/g), respectively. Finally, a run-to-run and monolith-to-monolith reproducibility showed a relative standard deviation in retention time of d(pT)12-18 fragments of <2%.