| Capillary electrophoresis (capillary electrophoresis, CE) is a class ofelectrophoresis technology based on the variance of mobility or distribution coefficientbetween the components such as ions or charged particles in a sample, to achieve highefficiency and rapid separation, by using electric field in high voltage as a driving forceand capillary as separation channel. Compared with the traditional technology ofhigh-performance liquid chromatography, CE has the characteristics such as multipleseparation modes, high separation efficiency (two orders of magnitude), fast analysisspeed, reagent consumption, low cost, and has been widely used in inorganic ions,carbohydrates, amino acids, protein, nucleic acid, environmental samples, drugs, foodand enantiomers by chiral separation and analysis. The study on theory and applicationof CE in analytical chemistry is one of the most active areas. As so far, how to improvethe separation selectivity and sensitivity of detection of CE is still one of the concerns ofworkers. In this paper, on the basis of the previous work, we developed some newcapillary electrophoresis systems, based on the fast and efficient separation, highsensitivity detection; and applied them to the actual biological samples analysis."A highseparation efficiency, high sensitivity" as the main goal, the present paper mainly focuson the following exploratory work:Chapter one: the history of CE technology was reviewed, and the fundamentaltheory, basic separation models, application characteristics, as well as hyphenatedtechniques were introduced. The hot spots are the discussion of the development and themodification of CE separation model for the analysis of real samples. At the same time,we narrated the main research contents of this thesis, the methods for establishing,improving and optimizing separation system as a starting point.Chapter two: In this section, chiral fat-soluble enantiomers of1-phenyl-R,S-tetrahydrogen isoquinoline (ER, ES) were rapidly separated using β-cyclodextrin(β-CD) modified micellar capillary electrokinetic chromatography coupled with electrochemical detection (EC), and the possible oxidation mechanism of theenantiomers was discussed. The novel micellar system of35mmol/L phosphate buffersaline (PBS)(pH7.85) containing30mM sodium deoxycholate,20mM β-CD and20%(v/v) acetonitrile was developed as the running buffer. Among them, the surfactantsodium deoxycholate acted to form the micelles in the buffer, β-CD was employed topromote the separation, and the acetonitrile acted as an organic modifier. After theoptimization of the factors such as detection potential, separation voltage, sampling timeand the composition of running buffer, the baseline separation was obtained within12min at20kV of separation voltage. The R.S.D values (n=5) of migration times andpeak areas of the analytes were2.3%(ER),2.7%(ES) and2.0%(ER),3.5%(ES),respectively. The detection limit was0.5μmol/L for ER and0.2μmol/L for ES, also itwas found that trace ER could be detected at the limit proportion of ER to ES for1:500.This protocol was successfully applied for monitoring the amount of ER from ES insynthetic drug intermediates.Chapter three: Based on the micelle modified capillary electrophoresis forimproving separation efficiency, we explore new molecular membrane modifiedmicroemulsion electrokinetic chromatography to improve the separation efficiency ofchiral drugs. A capillary modified by assembling molecular film was presented forchiral separation of sertraline with microemulsion electrokinetic chromatography. Theassembling molecular film was constructed with the poly(diallyldimethylammoniumchloride) and β-cyclodextrin via inclusion complexation.The separation efficiency of cis-trans isomers and enantiomers of sertraline wasimproved with the running microemulsion which contained the acetonitrile, sodiumdodecyl sulfate, n-butanol and n-hexane buffered with sodium tetraborate. Baselineseparation of four sertraline cis-trans isomers and enantiomers was achieved under theoptimum conditions. The detection limit for isomers and enantiomers of sertraline(1S,4S,1R,4R,1S,4R,1R,4S) was0.15mg/L,0.15mg/L,0.30mg/L,0.30mg/L,respectively. The mechanism of chiral separation was studied and the method could beapplied for determination of commercial Zoloft tablet samples satisfactorily.Chapter four: Based on improving the detection sensitivity of drug metabolismand raw product in the life system, we reported a rutin-modified glassy carbon electrode(R/GCE) which was prepared through electrochemically bonding. The modifiedelectrode shows the ability of electrocatalytic oxidation for sertraline and is used for the detection of sertraline in pharmaceutical tablets. The modification mechanism andelectrochemical behavior of the R/GCE are systematically discussed. The GCE wasmodified by rutin film through reacting with the nucleophilic hydroxyl groups on thesurface of carbon as the Michael acceptor. The rutin film undergoes electrochemicaloxidation related to the two catechol hydroxyl groups, which carries out a two-electrontwo-proton reversible reaction resulting in electrocatalytic oxidation for sertraline. Alsothe oxidation mechanism of sertraline on R/GCE is investigated by infrared spectrum(IR) and electrochemical techniques. By the method of differential pulse voltammetry,the oxidation current of sertraline linearly depend on its concentration within the rangefrom3.0to90.0μM with a detection limit (S/N=3) of1.0μM in coexistence of AA andDA (20μM). On this basis, the contents of sertraline in Zoloft tablets are detected withsatisfactory results.Chapter five: Under specific experimental conditions, a polymer-capillaryelectrophoresis (CE) was developed for the rapid analysis of bacteria, the twoGram-positive bacteria: Staphyloccocus aureus Rosenbach（SAR） and Bacillus subtilis(BS) and one Gram-negative bacterium: Pseudomonas aeruginosa (PA). The coupleassistance of poly (ethylene oxide)(PEO) and β-cyclodextrin (β-CD) was used toseparate the bacteria in order to enhance the separation efficiency. The influences ofspecific operating parameters on the migration time and separation efficiency wereinvestigated such as the pH, concentration of PEO and β-CD, concentration ofphosphate buffered saline (PBS), separation voltage and sampling time. Because ofcatalytic effect of lysozyme to gram-positive bacteria, SAR and BS, the hydrolysis ofSAR and BS was studied by adding the commercial Hen egg-white lysozyme (HEWL)with PA at present. According to the different hydrolysis quantity of SAR and BS, therate constant of SAR and BS was calculated, respectively for first time. Results showthat, in addition to the modifier PEO and β-CD, the pH and concentration of buffer werealso the most important factors to influence the separation efficiency of the bacteria,also the not suitable pH and concentration value of running buffer will lead to thehydrolysis of cell membrane. At last the optimal condition of10mmol/L PBS (pH7.2),PEO0.02%and β-CD0.02%, injection time with2s and20kV separation voltage waschosen. The detection limit of BS, PA and SAR was1.2×10~2cells/mL,1.1×10~2cells/mLand0.8×10~2cells/mL, respectively. The HEWL has high catalytic activity to Gram-positive bacteria (SAR and BS) but not to PA, thus it provided the effectivechemical method to sieve the gram-speciesual bacteria.Chapter six: Reactive oxygen species (ROSs) in particular the hydroxyl radical(OH˙) may attack biological macromolecules therefore to result in oxidativestress-originated diseases. It is hence in significant importance to establish the efficientmethods to determine it and screen the efficient scavengers for antioxygenic therapy. Ahigh performance liquid chromatography (HPLC) approach was researched in this paperto determine hydroxyl radical with p-hydroxybenzoic acid as the probe which yielded3,4-dihydroxybenzoic acid (3,4-DHBA) as the product after attacked by OH˙during theincubation for30min at37C. A competition strategy is employed for valuation ofantioxygenic activity of scavengers such as flavonoids there hydroxyl radicals attackedboth probe and flavonoids. The different yielding of3,4-DHBA was then detectedwhich reflected the scavenging efficiency of those flavonoids. With the aid of thisreaction, the structure-activity relationship (SAR) of the antioxygenic activity of thoseflavonoids could also be assessed by comparing the hydroxyl radical scavengingefficiency and the rate constant. |
PDF Full Text Request