Preparation And Evaluation Of Novel Amide Hydrophilic Chromatographic Stationary Phase

The term"Hydrophilic Interaction Chromatography?HILIC?"is not long,but its history is long.As early as the 1930s and 1940s,people have used hydrophilic interaction chromatography to separate carbohydrates.HILIC is a combination of normal phase chromatography and reversed phase chromatography,providing a new choice for the separation of polar solutes.The core of the HILIC separation is the stationary phase,and the target of separation is determined by the type of polar group on the surface of the stationary phase.Based on the above reasons,this paper investigates the research status of HILIC at home and abroad,the composition and retention mechanism of HILIC chromatography,and discusses the way of modifying the surface of silica gel,and compares the advantages and disadvantages of each method.Finally,the sugar-containing polymer was grafted onto the surface of silica gel by atomic radical polymerization as a new chromatographic stationary phase and chromatographically evaluated.The main research ideas of the article are as follows:?1?Based on the previous studies,the synthesis method of the precursor2-aminoethyl methacrylamide hydrochloride?AEMA?was improved.The method of natural concentration of chromatographic grade methanol and crude extract was used to improve the AEMA's yield.Then,during the preparation of the sugar-containing monomer,we heated the raw material D-glucoside lactone at 60°C for half an hour,greatly shortening the reaction time,and successfully prepared the sugar-containing monomer 2-glycyl propylamine based on ethylformamide hydrochloride.?GAEMA?.?2?The modification process of the surface of the silica gel in this paper,one is the amination of the surface of the bare silicon sphere,and the macroinitiator is directly synthesized with the bromoisobutyryl bromide;the other is the combination with the commercially available amino silica gel.The 2-bromoisobutyryl bromide reaction of glycidol was used to synthesize a silica gel initiator.Then,under the condition of CuBr/ligand catalysis,the surface of the brominated silicon hydride sphere obtained in two ways was initiated to polymerize,and the sugar-containing monomer GAEMA was graft polymerized.According to the elemental analysis,the graft density obtained by the former is twice as large as that of the latter,and the preparation procedure of the latter is complicated.Therefore,the first scheme was selected as the main synthetic route.The GAEMA grafting density of the silica gel surface was optimized by changing the reaction temperature,initiator concentration and reaction time.The optimal graft density of SiO₂-g-GAEMA was obtained as HILIC chromatographic packing.Column loading experiment.?3?We selected two base pairs,two kinds of nucleus and six kinds of reducing sugars,and carried out a series of chromatographic performance evaluation on SiO₂-g-GAEMA,which showed good separation effect with respect to the above polar solute..The effects of mobile phase ratio,column temperature and buffer ratio on the retention factor of SiO₂-g-GAEMA were investigated.The stationary phase was found to have typical HILIC retention characteristics.SiO₂-g-GAEMA was initially used for the separation of four proteins?zein,bovine serum albumin,trypsin,lysozyme?,which can be rapidly separated by gradient elution within 10 min.This stationary phase has potential applications in the separation of serum-enriched glycopeptides and polar highly isomerized solutes.