Preparation And Application Of Novel Azolyl Bridged Cyclodextrin Chiral Stationary Phases For HPLC

Due to the unique stetreochemical structure of chiral drugs and pesticides can better match proteins,enzymes,nucleic acids and other basic life substances,so that their activity is much higher than that of non-chiral similar substances,and they are increasingly widely used in medicine and agricultural production.But usually only one enantiomer shows high activity,other enantiomers activity is low or inactive,and even will pollute the environment and food,directly or indirectly affect people’s health.Therefore,it is of great significance to establish a method for the analysis of harmful enantiomers in related products.The separation of enantiomers with almost identical physical and chemical properties is a very difficult task,and the separation materials play a key role in the chiral separation process.At present,it mainly relies on foreign patented brands of cellulose polysaccharides chiral stationary phases.Bridged cyclodextrins are a class of novel supramolecular compounds which are composed of two or more cyclodextrins connected by bridging groups.Because its adjacent cyclodextrin cavity can co-bind the guest,and the bridge provides a variety of action sites,its binding constant is similar to that of biological enzymes,so it is often used as artificial simulation enzymes.At present,the potential of this extraordinary molecular recognition ability in chiral separation needs to be further exploited.In this paper,two new bridged cyclodextrin chiral stationary phases were obtained by the design and synthesis of azolyl bridged cyclodextrin chiral stationary phases.On the basis of chemical structure characterization,a series of chiral probes with different structures were used to systematically study the chiral chromatographic properties of the two new stationary phases,and the related separation mechanism was discussed.It has been preliminarily used in the separation and content determination of chiral pesticide enantiomers.It is expected to be applied in practice in the future.The first chapter mainly describes the basic concept of chirality and the difference of enantiomers of chiral compounds,and summarizes the separation and analysis methods of chiral enantiomers,as well as the preparation,properties and application of common chiral stationary phases.The research results of derivative cyclodextrin stationary phase and the potential of bridged cyclodextrin with multiple molecular recognition ability as chiral stationary phase are summarized,which provides a theoretical basis for the design of bridged cyclodextrin stationary phase in this paper.In the second chapter,imidazoledicarboxylic acid and 6-amino-β-cyclodextrin were reacted with DCC assisted dehydration and condensation at room temperature,and a novel imidazolediamide bridged bis-（β-cyclodextrin）chiral stationary phase（IMCDP）was synthesized and characterized.Some triazoles,flavanones,amino acids andβ-blockers were used as chiral probes to systematically evaluate the chiral chromatographic properties of the new stationary phase.The results showed that the new stationary phase could resolve 56 chiral compounds in reversed-phase and polar organic mode,among which the resolution of hexaconazole,2’-hydroxyflavanone,DNS-lysine and metoprolol were 3.11,2.12,3.01 and 1.62,respectively.Most solutes could be completely separated,and the resolution ability of the new stationary phase was much stronger than that of the ordinary cyclodextrin CSPs.By optimizing the composition of the mobile phase and buffer p H value,as well as changing the size of amino acid derived groups,IMCDP was able to resolve 33 derivatives of 18 amino acids,The enantiomers of DNS-Lue,DNS-Phe,PITC-Pro and DNP-Lys were 2.76,2.87,2.01 and 3.01,respectively.Therefore,the new stationary phase had obvious advantages over others in the separation of amino acid derivatives.The polar triazoles and flavanones were also resolved well in the reversed-phase mode.A variety ofβ-blockers were successfully resolved in polar organic mode.It could be concluded that the synergistic inclusion of adjacent cavities of bridged cyclodextrin,hydrogen bonds andπ-πprovided by imidazolediamide bridge group,and acid-base sites similar to pyrrole and pyridine should improve the chiral chromatographic property of IMCDP.In the third chapter,the bridged cyclodextrin was synthesized by"click chemistry"reaction between azide-β-cyclodextrin and 1,6-heptadiyne,and then it was bonded to the surface of ordered mesoporous SBA-15 by using isocyanate-propyl siloxane coupling agent,and a novel bis-triazolyl bridgedβ-cyclodextrin bonded phase（BCDP）was prepared.The chromatographic performance of the new stationary phase was systematically evaluated by using various solute probes.The results showed that BCDP could well separate a series of aromatic homologues and positional isomers,showing good hydrophobicity and reversed-phase chromatographic performance.Eleven triazole pesticides（Rs=1.33～3.15）could be separated by reversed-phase chromatography mode,even though the bridged cyclodextrin port was not derivatized.Tebuconazole（Rs=1.77）,paclobutrazol（Rs=1.62）and metconazole with two chiral centers those were difficult to separate on the same type of stationary phases were completely separated by BCDP,which showed strong resolution ability for triazoles.It could completely separate flavanone parent,2’-,3’-,4’-hydroxy flavanone and its glycoside（Rs=1.49～2.62）.It exhibited strong resolution abilities for hydrophobic DNS-LEU（Rs=1.99）,polar DNS-THR（Rs=1.73）,alkaline DNS-ARG（Rs=1.33）and acidic DNS-ASP（Rs=1.18）.In polar organic mode,BCDP had a strong effect and selectivity onβ-blocker drugs.For example,the resolution of arotinolol and atenolol were 2.78 and 2.22,respectively.Propranolol and pindolol with large aromatic groups were usually difficult to be separated,but they could be separated on BCDP（Rs=1.47 and Rs=1.14）.Obviously,the synergistic inclusion effect of bridged cyclodextrin stationary phase was helpful to broaden its chiral resolution range and make up for the shortage of limited inclusion due to the small cavity（～0.65 nm）of a single cyclodextrin.In addition to the inclusion effect,the bis-triazole bridge group generated by click reaction could also provide hydrogen bonding,π-πand hydrophobic interaction,and participate in molecular recognition.The hydrophobic and flexible propyl bridge group on BCDP could also assist the synergistic inclusion effect,thus improving the chiral chromatographic performance of BCDP.In chapter 4,based on the evaluation of chromatographic performance,BCDP was packed into the chromatographic column for HPLC resolution and enantiomeric determination of triazole pesticides residues in fruits and vegetables,showing the preliminary analytical application.The Qu ECh ERS method assisted by magnetic nanoparticles was used to pretreat the samples,and the enantiomers and internal standard were quickly separated（～30 min）by optimal gradient elution of Me OH-H₂O as mobile phase.A new HPLC method was established for the simultaneous determination of the residues of six enantiomers in fruits and vegetables（apples,tomatoes and cucumbers）after moniting by PDA at 210 nm.The experimental results showed that the enantiomers of triazole pesticides exhibited a good linear relationship with the response values in the concentration range of 2.5～50 mg/L（R²=0.9986～0.9999）,the high average recoveries（91.70～109.92%）of enantiomers in fruits and vegetables were obtained.The detection limit LOD（S/N=3）was low（≤0.03mg/kg）,and the reproducibility of the samples was good（RSDs:0.97～1.32%for intra-day,1.30～3.03%for inter-day）.Compared with the Maximum Residue Limits of Pesticides（MRLs）in the National Standard for Food Safety（2021 edition）,the LOD and LOQ of this method were lower than those in the standard method.It showed that this method had high sensitivity,which was suitable for the detection of pesticide residues with low content of triazole pesticides,and served for the scientific evaluation of pesticide enantiomer safety analysis in food.