Study On Novel Methods For Enantiomeric Separation Of Amino Acids And Dopa By Capillary Electrophoresis

It has long been presumed that amino acids accur solely as L-enantiomers in proteins and body fluids of mammals. However,D- enantiomers have been discovered in species of lower animals,mammalian organs and blood in recently. Moreover,some D-form amino acids are biosynthesized in body of mammals. Furthermore,some D-amino acids have been observed in mice,and in human plasma in relation to some diseases. Therefore,chiral analysis of D,L-amino acids is of great intrest and of increasing importance in life science and many other related fields. One of the most effect and most commonly used therapies for Parkinson's disease so far is levodopa. While L-dopa is pharmacologically active,D-dopa has also toxic properties. The use of racemic mixtures containing L-dopa,D-dopa may course serious side effects. Therefore,it is important to development a mathod for separation and deteramination of enantiomers of dopa in clinical analysis and other related fields.In this thesis,we studied the interation of aino acids and cyclodextins,amino acids and pesudo stationary phase,developed four novel methods on chiral separation of D,L-amino acids. Furthermore,we developed a new method for determination of enantiomeric purity of dopa. The research results obtained are as follows:1. The reaction of amino acids with 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl) was studied in detail. Enantioseparation of NBD-Amino acids (NBD-AAs) in CE has been studied with various chiral selectors such as α-CD ,β-CD ,HP-β-CD and γ-CD. With β-CD ,partial separation of all fourteen amino acids was observed. The baseline enantioseparation for NBD-AAs was observed only with HP-β-CD. Under optimum condition,the mixtures of seven NBD-AAs were enantiomerically separated less than 10 min. fourteen NBD-AAs were chiral separated totally. 2. Enantiomeric separation of amino acids derivatized with 1-chloro-2,4- dinitrobenze by capily zone electrophoresis using carboxymethyl-β-cyclodextrin (CM-β-CD) was studied. Mixtures of seven DNP-AAs and five DNP-AAs were chiral separated. Sixteen DNP-AAs were baseline enantioseparati on at all. 3. The enantioselective separation of three underivated aromatic acids was accomplished using hydroxyproply-α-cyclodextrin as a chiral selector. The simultaneous enantioselective separation of all three aromatic amino acids was obtained in a single run. 4. We applied dehydroabietyl chloride that is a new chiral reagent for the formation of diastereomers and resolved D,L-amino acid. The MEKC separation was performed at pH9.0 using a low SDS concentration (25mmol/L). 5. A new method for enantioseparation of dopa was established. Then,we applied this method to the optical purity testing of dopa. The linearity was investigated by adding the racemic compound to the standard L-enantiomer of dopa in the range of 0.1~2.0%. The concentration of D-dopa in levdopa is 0.20% by tested.