The Synthesize Of High Performance Liquid Affinity Chromatographic Stationary Phase With Dentritical Spacer Arms Used As Biomacromoleculer Separation

A preparative method of high performance liquid affinity chromatographic (HPLAC) stationary phase used as biomacromolecular separation is described. The separation performance of stationary phase is preliminary inspected.The 3-5m non-porous composite microspheres of ziroconia and urea-formaldehyde(UF) resin was synthesized by the reaction of ziroconyl chloride with hexamethylenetetraamine (HMTA) and urea, and used as the matrix of HPLAC stationary phase. For the non-porous matrix, retention time is shorted and high bioactivity and rate of recovery of biomacromolecular are remained, because of few mass tranfer in the porous of microsphere. The diameter and construction of the composite microspheres are judged by electron microscope. In methanol medium the starburst dendriner spacer arms are linked with the imido-groups on the surface of matrix by the addition reaction with methylacrylate and amination reaction with ethylenediaine. After repeating three times of these steps, amine-terminated dentritical spacer arms(PAMAM) with generation=3.0 can be obtained. The topological structure of the spacer arm was inspected by solid 13C NMR. RNA and DNA ligands are coupling to matrix with spacer arm by two different methods. For RNA: 8-Br-substituted RNA ligand is obtained by the reaction of liquid bromine with RNA, and bonded to the dentritical spacer arms of matrix in the solution of NaOH(pH=l 1.0). The amount of 8-Br-RNA coupled was measured by UV spectrophotometry.. A new type [8-Br-RNA-(PAMAMHZirconia-UF resin)] stationary phase of HPLAC is synthesized. For DNA: The spacer arms which have been succinylated are activited by N-hydroxysuccinimide, then DNA ligand is coupled to the spacer arms in the present ethyldimethylaminopropyl carbodiimide(EDAC). The amount of DNA coupled is determined by UV spectrophotometry. A new type [DNA-(PAMAM)-(Zirconia-UF resin)] stationary phase of HPLAC is prepared. At last, performance of affinity chromatography columns is inspected by selecting operation parameter of biomacromoleculer separation.