Preparation Of Two Novel β-Cyclodextrin Chiral Stationary Phases And Study On Their Chromatographic Performance By HPLC

With the development of science and technology,a variety of newly synthesized chiral compounds have been increasingly used in targeted therapeutic drugs,highly effective pesticides and biological pheromones.Many scientific studies have confirmed that the pharmaceutical activity,biotoxicity,metabolic pathway and environmental migration of chiral compounds are closely related to their stereo structure.Usually,one enantiomers has high activity,while the other has low or no activity,even affecting the health and reproduction of human beings.Chiral compounds not only bring high activity,but also bring new problems to drug safety,food safety and environmental safety.Therefore,the development of enantiomeric analysis is of great significance to the quality control of chiral drugs,the determination of chiral pesticide residues and the tracking of chiral environmental pollutants.Because the physical and chemical properties of enantiomers are almost the same,chiral separation is usually difficult.Choosing suitable methods and using highly selective separation materials is the key to solve the problem.In this paper,aβ-cyclodextrin bonded phase containing benzamide single spacer and an azobenzamide bridged bis-β-cyclodextrin-bonded phase were prepared,respectively.The former modified the bonding arm,while the latter introduced a new bridged cyclodextrin ligand,which improved the chiral chromatographic performance.The following research work was carried out around the preparation,evaluation,resolution mechanism and separation application of the new stationary phases:The first chapter introduced the structural characteristics of chiral compounds,the difference of enantiomers and the significance of chiral separations,summarized the basic principles and application scope of chiral resolution methods,and reviewed the important progress of chiral stationary phases in high performance liquid chromatography.The preparation methods of cyclodextrin chiral stationary phases were summarized systematically,including portal derivatization and ligand bonding,traditional cyclodextrin stationary phases and developing bridged cyclodextrin stationary phases.Take this as the basis of the topic selection and the starting point of this research.In chapter 2,a novel β-cyclodextrin-bonded chiral stationary phase(TCDP)containing benzamide single spacer was prepared,and the chiral chromatographic performance of the new stationary phase was evaluated by separating a series of chiral pesticides and drugs.TCDP successfully resolved 11 triazoles,8 flavanones and 5 β-blockers chiral compounds,among which 18 compounds were completely resolved.The resolution ranges of enantiomers of the above three chiral compounds were 1.45～3.33,0.35～2.45 and 1.26～1.58,respectively.The best resolution of tebuconazole enantiomer was 3.33,and the separation time was less than 13 min.Triticonazole with cis/trans isomers and bitertanol with two chiral centers were separated into four peaks.Especially,by using methanol-water or acetonitrile-water as mobile phase,the enantiomers of myclobutanil could be completely separated by TCDP,with the resolution reaching 1.68,while myclobutanil had no sign of resolution on common cyclodextrin stationary phases(CD-CSPs).Through comparative study,under the optimized conditions,ordinary cyclodextrin stationary phase(CDCSP)could resolve a small amount of analytes,while TCDP could resolve more widely.Chromatographic data analysis showed that the thiocarbamoyl benzamide bond arm of TCDP might provide hydrogen bond and coordination synergy for solute,which enhanced the chiral separation ability of the stationary phase.Therefore,the chiral separation function of the bonding arm should be fully developed when designing the stationary phases.In chapter 3,a new rapid and simple HPLC method for the determination of myclobutanil enantiomer residues in fruits and vegetables was established by using the above-mentioned self-made TCDP column.A new method for rapid detection of myclobutanil enantiomers in apples and tomatoes was established.The measuring conditions were as follows: samples were extracted by common Qu ECh ERS method,purified by N-propylethylenediamine adsorbent and graphitized carbon black,and quickly recovered by magnetic particles,and acetonitrile-water(12: 88,v/v)was used as mobile phase at a flow rate of 0.5 m L/min.The enantiomers of myclobutanil were completely separated on TCDP and detected at 221 nm.The results showed that the linear rangess of the two myclobutanil enantiomers were 0.5～50 μg/m L,the average recoveries of the two enantiomers in apples and tomatoes were 93.62% and 93.34%,and 95.43% and 94.80%,respectively,and the RSDs of the determination results was1.27%～2.01%(n=5).The lowest detectable concentration of myclobutanil enantiomers were 0.04 μg/m L(S/N=3).At present,only cellulose stationary phase can resolve myclobutanil enantiomers.Compared with cellulose stationary phases,the self-made β-cyclodextrin stationary phase(TCDP)has better solvent resistance,is more suitable for reversed-phase chromatography at lower preparation cost.It has certain practical value in enantiomeric analysis of this pesticide in fruits and vegetables.In chapter 4,a novel azobenzamide bridged bis-β-cyclodextrin-bonded stationary phase(AZCDP)was prepared and characterized for the first time.The new stationary phase was a multi-mode chiral stationary phase,and 41 chiral compounds were successfully separated by reversed-phase chromatography and polar organic mode.For example,AZCDP could resolve 8 flavanone compounds,among which the resolution of 2’-hydroxyflavanone and 4’-hydroxyflavanone was 3.42 and 2.72.Their resolutions on CDCSP were relatively low,and some solutes(6-hydroxyflavanone,7-hydroxyflavanone and hesperidin)could not be separated.This was because the cavity of a single cyclodextrin of CDCSP was small,and it was difficult to include a large volume of hydroxyl benzopyran ring in flavanone structures,while the bridged bis-β-cyclodextrin could identify a larger volume of guest(analytes)more finely through synergistic inclusion.AZCDP successfully resolved 11 triazole pesticides,and the resolution of tebuconazole was 2.35.Bitertanol containing two chiral carbons was resolved into four peaks,and myclobutanil,which was difficult to separate in the past,was also partially resolved.The 12 kinds of dansyl amino acids were successfully resolved by reversed-phase chromatography,which was especially suitable for the separations of substituted phenylalanine enantiomers(Rs,1.56～3.93).The analysis time was generally short(15～25 min),and it also had strong resolution ability for basic amino acids(arginine,Rs=1.52)and acidic amino acids(aspartic acid,Rs=1.35).In addition,AZCDP successfully resolved 10 kinds of β-blocker drugs in polar organic mode,7 of which were completely separated,the resolution of arotinolol reached 2.40,and the first resolution of betaxolol and bevanolol on the CD-CSPs was realized.The multi-recognition function of the "pseudo-cavity" was composed of the two synergetic inclusion cavities of the bridged cyclodextrin,the abundant hydrogen bonds at the ports and the flexible azobenzamide bridge group,which endowed the azobenzamide bridged bis-β-cyclodextrin stationary phase(AZCDP)with excellent chiral chromatographic performance,thus realizing efficient chiral separation and wider application range.Using LC-MS/MS method,based on the new stationary phase(AZCDP)made by ourselves,taking orange as an example,the separation and detection of the enantiomers of hexaconazole and paclobutrazol were successfully realized,which indicated that the new stationary phase had a certain application prospect in the analysis and determination of pesticide enantiomer residues in fruits and vegetables.At present,most expensive chiral columns depend on imports,so it is urgent to develop new high performance liquid chromatography separation materials with independent intellectual property rights.