Application Of Biporous Matrix To High-Speed Chromatography

In this article, Flow-through chromatography was applied to the chiralresolution and the purification of plasmid. At first, a rigid spherical biporouspoly (glycidyl methacrylate-co-ethylene dimethacrylate) matrix was prepared byradical suspension-polymerization using superfine granules of calcium carbonateas solid porogen and the mixture of cyclohexanol and dodecanol as liquidporogen. The results from scanning electron microscopy and mercury intrusionporosimetry measurements revealed that the matrix contained two families ofpores that were, micropores (10-180 nm) and macropores (180-7000 nm).A novel protein chiral stationary phase (CSP) was prepared by couplingbovine serum albumin (BSA) to the biporous resin with triazine as a linker.The biporous CSP was applied to the resolution of d, l-tryptophan (Trp). Theexperiments reveal that pH value and flow velocity of the mobile phase have alittle effect on the resolution of Trp. When the flow velocity was as high as1800 cm h-1 and the load of d, l-Trp was 0.1 mg, the resolution of d, l-Trp wasstill above 1. By comparison of the resolution of d, l-Trp on this CSP at highflow velocities with that predicted theoretically for conventional supports weconcluded that the increased flow velocity had little effect on the resolution ofenantiomers with biporous packings. The results indicate that the proteinbiporous CSP was promising for high-speed resolution of enantiomers.The use of ammonium sulfate and calcium chloride to precipitate RNAand protein in plasmid DNA purification process was investigated. The resultshows that ammonium sulfate was by far the best precipitant with high RNA andprotein removal. When the concentration of the ammonium sulfate reached 2.5 Min the lysate, the purification factor for plasmid was up to 10.2 and the yield wasabove 90%. Although calcium chloride can remove the impurities mentionedabove, a certain mount of plasmid was lost in this procees.A novel hydrophobic absorbent with wide pores was prepared by coupledphenol to the biporous resin (Ph-BiPR). The macropores in the biporous resinprovided not only convective flow channels for the mobile phase, but also a largesurface for biomolecules binding. With a loading up to 2.6 mg of 5.4 kb plasmid(pcDNA3) in 8 mL feedstock and operated at a flow velocity as high as 20cm/min, nearly 100% of plasmid was recovered with a purity of 100%. Theresults indicate that the hydrophobic medium is promising for high-speedpurification of plasmid DNA.Another chromatographic packing with 2-mercaptopyridine as ligands wasprepared and used to purify plasmid from lysate. The experimental resultsrevealed that the throughput of the chromatograhic process with this packing wasrelative higher than that of Ph-BiPR at high flow rate.