Synthesis Study Of Amino Acid Ligand-exchange Stationary Phase

Ligand-exchange chromatography (LEC) is a convenient and effective method for aminoacids racemate chiral separation. Immobilized type Ligand-exchange chiral stationary phase(CSP) is synthesized by two steps: First, KH-560react with silica for preparation of KH-560bonded silica (KBS). Afterwards, amino acids are used to react with epoxy group whichpresent in KBS for preparation of Ligand-exchange CSPs. So it is necessary to determineKH-560content in KBS and amino acid content in Ligand-exchange CSPs, respectively.Currently, only elementary analysis is used for these two parameters but element analyzer isextremely expensive and only few laboratories can afford it. There is an urgent necessity toestablish a convenient and effective method for these two parameters.KH-560bonded silica is the intermediate product for preparation of Ligand-exchangeCSPs. Traditionally, it’s synthesized in toluene, with the conditions of nitrogen protection andwaterless, high temperature heating under long time reflux. Meanwhile, toluene is harmful forhuman body. In this dissertation, preparation of KBS under hydrous condition and use thisKBS to prepare Ligand-exchange CSPs are carried on and studied. Main research contents areas follows:1. The study on determination of KH-560total content on KBS:Epoxy group and dihydroxy group present in KBS are turned to formaldehyde bychemical conversion. Quantitative determination of generated formaldehyde by acetylacetonespectrophotometry is discussed. Analysis conditions and results of two methods, solid-liquidmethod and dissolving method, are are investigated and compared. Influence factors forepoxy group and dihydroxy group to generate formaldehyde are investigated. Eventually,conditions and method of determination of KH-560total content on KBS are confirmed asfollows: dissolving method for sample processing, epoxy group and dihydroxy group is arehydrolyzed and oxidized to formaldehyde, acetylacetone spectrophotometry for determinationof generated methanal. RSD is0.5~1.0%, recovery is8.7~101.8%.2. The study on determination of amino acid content on Ligand-exchange CSPs:Organic nitrogen of amino acid is turned to nitrate radical by chemical conversion.Quantitative determination of generated nitrate radical by UV spectrophotometry and thymolspectrophotometry are discussed. Influence of consisting ions on the results of nitrate radicaldetermination is investigated, and it can be effectively eliminated by precipitation. The resultsof amino acid content determined by UV spectrophotometry and thymol spectrophotometryare compared; also the results are inspected by elementary analysis method. Eventually,conditions and method of determination of amino acid content on Ligand-exchange CSPs areconfirmed as follows: Organic nitrogen of amino acid is oxidized by potassium persulfate togenerate nitrate radical, UV spectrophotometry (consisting ions are precipitated) or thymolspectrophotometry for determination of generated nitrate radical, calculate amino acid content.Respectively, RSDs of two methods are1.0~1.3%and1.0~1.2%, recoveries are98.0%~104.0%and97.0~103.1%.3. The study on the reaction of KH-560with silica under hydrous conditions Under hydrous condition, epoxy group is active to hydrolysis, so two parameters,KH-560total content (epoxy group and dihydroxy group) and KH-560available conten(epoxy group), are carried on to investigate preparation of KBS. Each reaction conditions,such as water volume fraction, reaction temperature, KH-560concentration, reaction time, areinvestigated. The results of carbonation test, IR, TGA shows that KH-560is successfullybonded to silica surface. The effect of silica on epoxy group is investigated, the resultsindicate that silica promote epoxy group hydrolyze to dihydroxy. Eventually, the optimizedsynthesis conditions are confirmed as follows: water volume fraction5%(V%),90℃,KH-5600.25g/mL(DMF),3h4. The study on the addition reaction of amino acids with KBS:Benzene ring amino acids (L-Phe, L-Tyr) and penta-heterocycles amino acids(L-Pro,L-His) are selected for this research. Solubilities of four amino acids in solvent are inspected.Then the effects of reaction temperature, reaction time, molar ratio of amino acid and epoxygroup on amino acid content on Ligand-exchange CSPs are investigated. Also, the effect ofethanol and ethanol-sodium hydroxide on the L-proline content on Ligand-exchange CSP isinvestigated, the results shows that ethanol-sodium hydroxide is better than ethanol. Theinfluence of amino acid structure on amino acids bond yields are discussed, the resultsindicate that penta-heterocycles amino acids are better benzene ring amino acids. he influenceof available rate of KBS on addition reaction of amino acids with KBSs are investigated.Theresults shows that KBS with high available rate is suitable for synthesis Ligand-exchange CSP.Eventually, the optimized synthesis conditions are confirmed as follows: amino acid: epoxygroup(mol:mol)=1.2,75℃,3h.