Preparation Of New Mixed Mode Chromatographic Stationary Phase And Its Application In Precise Separation Of Chitooligosaccharide

As the only positive alkaline oligosaccharide in nature,chito-oligosaccharide has attracted the attention of researchers in recent years.However,its strong polarity and insolubility in organic solvents limit its application in chromatographic separation technology,and it is difficult to obtain high purity chito-oligosaccharide with single degree of polymerization.In order to design an effective chromatographic stationary phase for the separation of chitosaccharides,a hydrophilic/ion-exchange mixed mode chromatographic stationary phase was prepared using silica gel as substrate and aminated amino acids as functional ligands.The main research contents are as follows:In order to explore the feasibility of preparing aminated amino acids by acyl chloride and ammonolysis,glycyryl-L-tyrosine was synthesized from L-tyrosine.The effects of water consumption,p H and dosage of chloroacetyl chloride on acylation reaction were investigated.The dosage of L-tyrosine 8 g,dosage ratio of L-tyrosine / water / chloroacetyl chloride 1/40/8,p H 9.5 to 10.5,reaction temperature 0-5 was the optimal reaction condition.Under this ℃condition,the purity of glycyryl-L-tyrosine was over 92%.The stationary phase of silica gel with 100 μm particle size was prepared using silica gel with 100 μm particle size as substrate.Through the characterization of infrared spectrum and elemental analysis,it is confirmed that the synthesis of alanyl-L-glutamine graft silica stationary phase is successful.The results showed that the graft reaction on silica gel surface could be promoted by temperature increase,but the specific adsorption of silica gel with large particle size to nitrogen was affected by nitrogen atmosphere,and the reaction ability of silica gel with functional ligands was weakened.On the basis of the 100 μm silica gel reaction,the coupling and grafting reactions of 40 and 10 μm silica gel were carried out.The results show that silica gel with small particle size has better stability and is not sensitive to nitrogen atmosphere and temperature change.The optimum reaction conditions were as follows: the reaction time was 48 h under the protection of 65 ℃ in nitrogen atmosphere,epoxy silane accounted for 20% of the coupling agent and toluene mixed solution,and the concentration of alanyl-L-glutamine aqueous solution was 0.033 g/m L.Bare silica gel and graft products with different particle sizes were used for chitosan oligosaccharide adsorption experiment,and the adsorption effect was tested.The silica gel with small particle size showed better elution ability than the silica gel with large particle size,and the graft product showed better adsorption ability than bare silica gel.A new type of chromatographic column was prepared with 10 μm particle size alanylL-glutamine grafted silica gel stationary phase.The effects of p H,water content and flow rate of mobile phase on the separation performance of the column were investigated.The results showed that the new column could provide hydrophilic and ion-exchange forces,and it was a kind of hydrophilic/ion-exchange mixed mode column.When the mobile phase flow rate was 1.0 m L/min,the optimal chromatographic separation was 3.30,3.72 and 2.62,respectively.The separation of chito-oligosaccharides and other sugars was compared between the new column and the commercial 5 μm amino hydrophilic column.The novel chromatographic column has low preparation cost,but exhibits similar separation ability to5 μm hydrophilic column,and can achieve rapid and complete separation of standard and crude chitosaccharides in 15 min.In addition,the new chromatographic column can realize the rapid detection of sucrose and maltose within 5 min,which has a good application potential in the separation of other sugars.