Studies On Preparation And Applications Of Novel Chiral Monolithic Columns In Enantioseparation Of Amino Acids

Amino acids is one of the most important chiral molecular in nature. However, with the development in analytical sciences, several D-amino acids have been found in mammals and have been demonstrated to be naturally occrruing physiologically active substances or biomakers. The development of novel chiral sationary phase for chiral separation of trace amino acids in biological samples in HPLC is still hot topic. Currently, various CSPs have been widely used for the enantioseparation of N-derivatozed amino acids, such as those containing cellulose, cyclodextrins and quinidine as chiral selectors. Especially, CSPs in which quinidine are immobilized on silica particles have become popular because of their excellent enantioselectivity toward various kinds of N-derivatized amino acids in mammals. However, packed column has several disadvantages such as long anaylsis time, high back pressure and so on. Monolithic column has become a new popular chiral material for its good permiability and p H stability. In this research, two novel chiral monolithic columns has been well prepared and used for enantioseparation of trace D-amino acids in tea sample with offline 2D-HPLC.In the first chapter, the importance of enatioseparation of amino aicds, and the main chiral speration methods of amino acids and types of chial stationary phase has been reviewed. In the meantime, monolithic column has been introduced, the development of quinidine and cyclodextrin functionalized monolith has been described in detail.In the second chapter, the monolith is prepared by the in situ co-polymerization of monomer O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine(MQD), progens(dodecanol and cyclohexanol),crosslinker ethylene dimethacrylate(EDMA), and initiator Azobisisobutyronitrile(AIBN) within capillaries(100 μm I.D.). The the composition of polymerization mixture was optimized in order to obtain the best coloumn with good permeability and efficiency. In order to study the effect of the co-monomer HEMA on the enantioseparation ability of poly(MQD-co-EDMA) and poly(MQD-co-HEMA-co-EDMA) monolith in the micro- LC mode, 44 kinds of N-derivatized amino acids were analyzed. The results showed that the co- monomer HEMA has no effect on the enantioseparation of amino acids.In the third chapter, in order to improve the detection sensitivity on the monolith, a micro-LC/LIF system was set up and was used to separate ten N BD-amino acids. Five NBD-amino acids were baseline enantioseparated. It is worth noting that this is the first time to be obtain a baseline enantioseparation of NBD-Arg on quinidine based stationary phases. The LOD and LOQ values on UV and FLD were also compared. The RLOD(0.17%) and RLOQ(0.25%) for the D-enatiomer were determined on the micro-LC/LIF system. The results showed that the poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace D-amino acids in biological samples.In the fourth chapter, the methodology using offline 2D-HPLC for the analysis of trace D-amino acids in tea samples was established. A RP C18 column was used in the 1st D, while the poly(MBQD-co-HEMA-co-EDMA) monolith or a Chirakpak?IC-3 chiral column was used in the 2nd D. The D/L ratios of various amino acids in Oolong tea were studied. The results showed that the ratios of D/(D+L) of amino acids were Ile(1.09%)、Ser(0.30%)、Asp(0.25%)、Val(0.17%)、Ala(0.07%)、Pro(0.04%).In the fifth chapter, the monomer heptakis(2,3-di-O-acetyl)- mono(6-O- methacryloyl)–β-cyclodextrin(HDAM-β-CD) was firstly synthesized. A HDAM-β-CD modified polymer based monolith was then prepared through the in situ co-polymerization of the monomer(HDAM-β-CD), the porogens(1-propgyl alcohol(NPA)、1,4-butylene glycol(BDO) and H2O), the crosslinker(ethylene dimethacrylate(EDMA)), and the initiator(azobisisobutyronitrile(AIBN)) within capillaries(100 μm I.D.). The polymerization conditions were optimized in order to obtain the best column with good permeability and efficiency. The rentention mechanism and column characterization were also investigated.In the sixth chapter, the conclusions and prospects of this research was clearly described.