Preparation Of Monolith And Separation Performance Study

High performance liquid chromatography (HPLC) is widely used in bioseparations for advantages of speediness, high efficiency and high resolution. The key component involved in HPLC is assigned to column technique in which monolith is highlight in recent years. In this thesis, monolith based on poly-methacrylate was modified with various ligands for separation of proteins as well as small molecules, and a systematic study was listed below:Monolithic skeleton was prepared by in-situ polymerization in a stainless steel chromatographic tube with glycidyl methacrylate (GMA) as the functional monomer, ethylene dimethacrylate (EDMA) as the crosslinker,2,2’-azobis-(isobutyronitrile)(AIBN) as the initiator and n-heptane as the porogen. Focused on the specific surface area and pressure drop of the monolith, the porous structure was adjusted by variation of polymerization temperature, proportion of GMA and EDMA, content of AIBN, as well as time for polymerization The optimal conditions were:GMA:EDMA80:20(v/v), initiator content1%(w%, with respect to monomers), temperature65℃, and time for polymerization12h. The resulting monolithic skeleton was constructed by large pores with1.9μm average diameter and85%of them had the pore size over500nm which facilitated the following modifications with polymeric ligands. Fewer micropores were contained because no size exclusion phenomenon was observed for different molecular weight polymers on the column. The total porosity of the monolith was64%, and specific surface area was5.30m2·g-1.DEAE-dextran (500kDa) and polyethyleneimine (PEI,30kDa) were employed as anionic ligands to modify the monoliths with the evaluated ligand densities of9.93μmol·g-1and242.9μmol·g-1, respectively. The adsorption ability was enhanced with the increasing of PEI molecular weight. Dynamic binding capacities of BSA on DEAE-dextran and PEI monolith were23.8mg·g-1and41.5mg·g-1, not influenced by operating velocities. Hemoglobin and ovalbumin also showed the relative high capacities of17.5mg·g-1and 42.1mg.分’on PEI monolith.The lmmobilized DEAE.dextran afforded both strOllg and weak l’orl-exchange cheml’stries Of chromatography while only weak i’on-exchange property was fourld for PEl morlolith.Fast s印aratiotis of lysOzyme,hemoglObin,ovalbuml’n were achieved on both COlumrls wi’thout the changing of r-solution by Va叫ing flow rates,which demonstrated the cOlumns were prOral’Sing f10r fast analysis0r preparation of protel’ns. Crude lipase was als0subjected to PEl mollolith alld52.2％enzyme activity was recovered wim maximum purificali’on of4.5folders.FHrthermore,two monoIiths exhibited the excelIent permeabilities which WOlfe only one fourth0fthe repon.Amino terminated poly ethylerie glyccrol(AT-PEG)was prepared and used to modi母the morlolithic skeleton to afrord the hydrophobic interaction(HI)columrl.Adsorl)tion ability of AT-PEG COlumll was6.5mg BSA per gram media.10rl-eXChange property brought OHt by aml’rlO group013AT-PEG toutd be eliminated when(N1-14)2S04content in mobiIe phase was higher than0.12M,and hydrophobic adsorption was achieved when (NH4)2S04COntent is higher thall1.7M for BSA.AT-PEG monol汕proyided the baseline separation0f cytochrome C,RNase A and Iysozyme within2.5min at flow rate0f1445mI.min一. Altcrnati’vc way t0prepare Hl coIilmll was carl"ied out by n-butylaml’rle modificati013.This COlumrl,labeled aS C4morlol胁had the higher hydrophobic时alld ligand dellsit)r0f2.16mmol.g.,which brought the relative larger binding capacity0f14.4mg BSA per gram media.(NH4)2S04in mobile phase higher thall0.15M shOUld be pI’OVided t0cover t11e ion-exchange adsorptl’on caused by amine group Of the ligand,and BSA WaS complerely adsorbed013media in1.2M(NH4)2S04due t0hydrophobic int-l’aCtiOrl.Four proteins,eytochrome C,RNltse A,lys0巧me and ovalbamin were Separated on C4moIlol油w汕in3min,and recove叮of each protein WaS kept aS constant at vatiouS now rates.The Iigand dCrlSity0f C8mollolith,modified by n--OC锣lamine should be kept aS41.5"mol.g’.for protein separatiOn,alld adsorl)tiOrl capacity was7.15mg BSA per gram media.As well,(N1-14)2S04COiltent for separation shOHld be higher thlll30.15M t0aVOid the iOl’l-exchallge intcraeti011between sample and the amine伊oup in the ligand. Lysozy’me sufifered the extensiVe peak broadelling On C8monol油f01.too large hydrophobicit),of the ligand.The BSA recoveries orl AT-PEG C4and C8COIumn were higher thall90％.As expeeted,C8mollolith with high ligand derlSl’t),(2.3mmol.g.)could be employed t0separate pueralrin from pueraria navorle$aS the reversed phase chromatography and maximum loading was35mg..ml-’×20山.The mcdia was stable in an acidic mobile phase at least in a week.The puerarin fraetion with high purity was obtained It-C0rding t0UV-vis SCalllling and HPLC analysis.HEl7’equations for separationS0f both bimolecule and$1TIall molecule on ilrlonolim were deduced. Furthermore, a compression factor was introduced to evaluate HETP values of PEI monolith and C4monolith in linear gradient elution The HETP values maintained at5μm in spite of the variations of operating flow rates for protein separations on both PEI and C4monoliths. Due to the inhomogeneity of porous structure and convective flow model, eddy diffusion controlled the column efficiency Elution of toluene on C8monolith indicated that the HETP decreased to a plateau with the enhancement of flow rate. The lower molecular weight encouraged the axial dispersion of toluene, and deteriorated the column efficiency. In comparison to protein separation, the eddy diffusion was also extended.Preparative separation of puerarin from pueraria flavones on oligo-β-cyclodextrin bonded Sepharose HP media was investigated. Sample was pretreated with Al2O3to remove impurities which could foul the gel. The optimal condition for preparative run was:mobile phase of7%HAc, flow rate of10ml·min-1. The ethanol content in sample should be diluted to5%with distilled water, preventing the fingering effect which deteriorated the resolution of puerarin, and loading amount reached300mg crude extracts per run. Puerarin fraction was evaporated to dry, decolored with95%HAc and crystallized with water The final product afforded the purity higher than97%with the recovery of32.4%, which was remarkably improved in comparison to literature.