Dissertation
Dissertation > Mathematical sciences and chemical > Chemistry > Analytical Chemistry > Instrument analysis ( physics and physical chemistry ) > Chromatographic analysis > Liquid chromatography analysis

Chemiluminescence Detector with On-line Electrogenerated Reagents for High Performance Liquid Chromatography

Author ZhangZuoTu
Tutor ZhangZhuJun
School Shaanxi Normal University
Course Analytical Chemistry
Keywords On-line Electrogenerated Reagents Chemiluminescence Detector High Performance Liquid Chromatography
CLC O657.72
Type PhD thesis
Year 2007
Downloads 797
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High Performance Liquid Chromatography (HPLC) as a powerfulmodern separation technique has been widely used for the analysis of many analytes indiverse fields. Detectors are one of the most important components of HPLC systemsince they produce a wealth of information about the separated components which canbe stored in computers and manipulated as desired to assist solute identification. Inrecent years, chemiluminescence (CL) as a detection technique of HPLC is veryattractive due to higher sensitivity, wider linear dynamic ranges and simplerinstrumentation. The advance of CL detection has greatly catalyzed the growth andpopularity of HPLC-CL application, and made trace analysis possible owing to itscapability of measuring pictogram or femtogram quantities of compounds in thecolumn eluate. The investigations and applications of HPLC-CL technology arecurrently also an important subject, and a hot point in analytical science.Chemiluminescence analysis with electrogenerated reagents, a novel methodbased on direct CL mechanism or energy-transfer mechanism, coupled with flowinjection analysis (FIA), has been applied in many analytes in certain fields, in whichunstable and nascent regents required for the CL reactions can be generatedelectrochemically on-line.However, a drawback of low selectivity of this method remains to be furtherstudied for better application and a wider use.For this purpose, such unstable and nascent reagents as BrO-, [Cu(HIO62]5-,B(OH)3·OOH-, Mn(Ⅲ), Co(Ⅲ) and Ag(Ⅱ) for CL reactions have been on-line orin-situ electrogenerated, coupled with HPLC to provide a novel methodology for bothgood sensitivity and selectivity in detecting analytes in complex matrices. The effectsof several parameters on the HPLC resolution and CL emission were studiedsystematically. Several novel methods based on HPLC with chemiluminescencedetectetor using on-line or in-situ electrogenerated reagents have been developed forthe determination of Sudan dyes in hot chilli products, glucocorticoid andβ2-agonistresidues in animal tissues, tetracyclines residues in milk and some drugs in pharmaceutical and biological samples.This dissertation consists of five chapters. Chapters 1 is a review and Chapters 2to 5 are research reports including 11 research works.In Chapter 1, the development and tendency of the HPLC with CL detection arereviewed. It covers the principles of the HPLC with CL detector, the design of CLdetectors, CL detection systems (including luminol, peroxyoxalatcs, Ru (bpy)32+,KMnO4, K3Fe(CN)6 and Ce4+) combined with HPLC, the applications of analyticalmethods for many kinds of inorganic, organic and biologic samples, and the trends ofHPLC-CL in environmental, life science, pharmacy and clinical medical science.In Chapter 2 to 5:(1) Development and optimization of an analytical method for thedetermination of Sudan dyes in hot chilli pepper by high-performance liquidchromatography with on-line electrogenerated BrO--luminol chemiluminescencedetection.The determination of four Sudan dyes by means of high-performance liquidchromatography (HPLC) with chemiluminescence (CL) detection was proposed. Themethod was based on the enhancement effect of Sudan dyes on the chemiluminescencereaction between luminol and BrO-, which was on-line electrogenerated by constantcurrent electrolysis. The separation was carded out on Nucleosil RP-C18 column(250mm×4.6mm i.d., 5μm, pore size, 100 (?)) at 35℃. The mobile phase consisted ofa V (methanol): V (0.2% aqueous formic acid)=90:10 solution. At a flow-rate of 1.0mL min-1, the total run time was 25 min. The effects of several parameters on theHPLC resolution and CL emission were studied systematically. For the four Sudandyes, the limits of detection (LOD) at a signal-to-noise of 3 ranged from 4 to 8μg kg-1and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 13 to 27μg kg-1. The relative standard deviations (R.S.D.) of intra- and inter-day precision werebelow 4.4%. The average recoveries for all four Sudan dyes (spiked at the levels of1.0 and 1.5 mg kg-1) in chilli tomato sauce and hot chilli pepper ranged from 94% to105%, and the relative standard deviations of the quantitative results were from 2.5 to4.2%. The proposed method had been successfully applied to the determination of fourSudan dyes in hot chilli products.(2) Determination of tetracyclines residues in milk using high performanceliquid chromatography with on-line electrogenerated BrO--luminol chemiluminescence detection.The determination of four tetracyclines (TCs) by means of high-performanceliquid chromatography (HPLC) with on-line electrogenerated BrO--luminolchemiluminescence (CL) detection is proposed. The procedure is based on theenhancement effect of TCs on the chemiluminescence reaction between luminol andBrO- in alkaline medium. The oxidant BrO- was on-line electrogenerated by constantcurrent electrolysis. The separation was carried out on Nucleosil RP-C18 column(250mm×4.6mm i.d., 5μm, pore size, 100 (?)) at 25℃. The mobile phase consisted ofa V(acetonitrile):V(0.05 molL-1 potassium dihydrogen phosphate buffer, pH 2.5)=30:70 solution. At a flow-rate of 1.2 mL min-1, the total run time was 11 min. Theeffects of several parameters on the HPLC resolution and CL emission were studiedsystematically. For the four TCs, the detection limits at a signal-to-noise of 3 rangedfrom 0.002 to 0.008μg mL-1. The relative standard deviations for the determination ofTCs ranged from 2.0 to 3.6% (n=11, C=0.01μg mL-1). The method has beensatisfactorily applied to the analysis of spiked raw milk samples.(3) Detection of glucocorticoid residues in pig liver by high-performanceliquid chromatography with on-line electrogenerated [Cu(HIO62]5--luminolchemiluminescence detection.A novel method was developed for the simultaneous determination ofglucocorticoid residues such as triamcinolone(TR), prednisolone(PR),hydrocortisone(HC), cortisone(CO), methylprednisolon(MP), dexamethasone (DE)and triamcinolone acetonide(TA) by high-performance liquid chromatographycoupled with chemiluminescence detection. The procedure was based on theenhancement effect of glucocorticoids on the chemiluminescence reaction betweenluminol and the complex of trivalent copper and periodate ([Cu(HIO62]5-), which wason-line electrogenerated by constant current electrolysis. The HPLC separation used aNucleosil RP-C18 column (250mm×4.6mm i.d., 5μm, pore size, 100 (?)) with a mobilephase consisting of acetonitrile and 1.0 mmol L-1 ammonium acetate (pH 6.8,40:60, v/v) at a flow rate of 0.8 mL min-1.The effects of several parameters on theHPLC resolution and CL emission were studied systematically. Liver samples werehydrolyzed with Helix pomatia juice followed by a solid-phase extraction procedure.Under optimum conditions, the limits of detection (LOD) at a signal-to-noise of 3ranged from 0.08 to 1.0 ng g-1 and the limits of quantification (LOQ) at a signal-to-noise of 10 ranged from 0.27 to 3.33 ng g-1 for seven glucocorticoids. Therelative standard deviations (R.S.D.) of intra- and inter-day precision were below 6.8%.The average recoveries for glucocorticoids (spiked at the levels of 5 to 50 ng g-1) in pigliver ranged from 88 to 106 %, and the relative standard deviations of the quantitativeresults were from 2.0 to 6.9 %. The proposed method had been successfully applied tothe determination of glucocorticoid residues in pig liver.(4) Development of an analytical method for the determination ofβ2-agonistresidues in animal tissues by high-performance liquid chromatography withon-line electrogenerated [Cu(HIO62]5--luminol chemiluminescence detection.A novel method was developed for the simultaneous determination ofβ2-agonistresidues such as terbutaline(TB), salbutamol(SB) and clenbuterol(CB) byhigh-performance liquid chromatography (HPLC) coupled with chemiluminescence(CL) detection. The procedure was based on the enhancement effect ofβ2-agonists onthe chemiluminescence reaction between luminol and the complex of trivalent copperand periodate ([Cu(HIO62]5-), which was on-line electrogenerated by constant currentelectrolysis. The HPLC separation used a Nucleosil RP-C18 column (250mm×4.6mmi.d., 5μm, pore size, 100(?)) with a mobile phase consisting of 90% acetonitrile and10% aqueous ammonium acetate (20 mmolL-1, pH 4.0) at a flow rate of 1.0 mLmin-1.The effects of several parameters on the HPLC resolution and CL emission werestudied systematically. Liver samples were hydrolyzed withβ-glucuronidase followedby a solid-phase extraction procedure using Waters OasisMCX cartridges. Underoptimum conditions, the limits of detection (LOD) at a signal-to-noise of 3 rangedfrom 0.007 to 0.01 ng g-1 and the limits of quantification (LOQ) at a signal-to-noise of10 ranged from 0.023 to 0.033 ng g-1 for threeβ2-agonists. The relative standarddeviations (R.S.D.) of intra- and inter-day precision were below 4.5%. The averagerecoveries forβ2-agonists (spiked at the levels of 0.05 to 5.0 ng g-1) in pig liver rangedfrom 84% to 110%, and the relative standard deviations of the quantitative results werefrom 1.6 to 7.2%. The proposed method had been successfully applied to thedetermination ofβ2-agonist residues in pig liver samples.(5) Simultaneous determination of levodopa and benserazide byhigh-performance liquid chromatography with on-line electrogenerated nascentB(OH)3·OOH--luminol chemiluminescence detection. The nascent perborate was on-line electrogenerated on the surface of platinumelectrode with constant current electrolytic method in the Na2BO4-Na2CO3-NaOHaqueous medium, and it was found that the strong chemiluminescence (CL) ofluminol reacting with nascent perborate (B(OH)3·OOH-) could be greatly inhibited bylevodopa and benserazide. Based on this finding, a novel chemiluminescence methodusing nascent electrogenerated perborate coupled with high-performance liquidchromatography for the simultaneous determination of levodopa and benserazide hasbeen developed. The effects of several parameters on the HPLC resolution and CLemission were studied systematically. Under the optimum experimental conditions,the calibration graphs were linear over the range 0.1-50μg mL-1 for levodopa and0.05-20μg mL-1 for benserazide. The limits of detection (LOD) (3s) for levodopa andbenserazide were 0.04μg mL-1, 0.01μg mL-1, respectively. The proposed HPLC-CLmethod had been applied to the determination of levodopa and benserazide inpharmaceutical and human urine samples.(6) Detection of metoclopramide by high-performance liquid chromatographywith on-line electrogenerated nascent B(OH)3·OOH--luminol chemiluminescencedetection.The determination of metoclopramide(MCP) in human serum samples by means ofhigh-performance liquid chromatography (HPLC) with on-line electrogeneratedB(OH)3·OOH- chemiluminescence (CL) detection was proposed. The method wasbased on the inhibition effect of MCP on the chemiluminescence reaction betweenluminol and B (OH)3·OOH-, which was on-line electrogenerated by constant currentelectrolysis. Under the optimal conditions, a linear range from 5 to 500 ngmL-1(R2=0.9991), and a detection limit of 0.6 ng mL-1 (3s) for MCP were achieved.The relative standard derivations (R.S.D.) for 50 ng mL-1 MCP were 3.4% within a day(n=11) and 4.2% on five consecutive days (n=6), respectively. The recovery of MCPfrom serum samples was more than 94%.(7) Detection of indomethacin by high-performance liquid chromatographywith in-situ electrogenerated Mn (Ⅲ) chemiluminescence detection.The determination of indomethacin (INM) in pharmaceutical and biologicalsamples by means of high-performance liquid chromatography (HPLC) with in situelectrogenerated Mn (Ⅲ) chemiluminescence (CL) detection was proposed. Themethod was based on the direct CL reaction of INM and Mn (Ⅲ), which was in-situ electrogenerated by constant current electrolysis. The chromatographic separation wascarded out on Nucleosil RP-C18 column (250mm×4.6 mm i.d., 5μm, pore size, 100(?))at 20℃. The mobile phase consisted of methanol: water: acetic acid=67:33:0.1solution. At a flow-rate of 1.0 mL min-1, the total run time was 10 min. The effects ofseveral parameters on the HPLC resolution and CL emission were studiedsystematically. Under the optimal conditions, a linear range from 0.01 to 10μgmL-1(R2=0.9991), and a detection limit of 8 ng mL-1 (signal-to-noise ratio=3) for INMwere achieved. The relative standard derivations (R.S.D.) for 0.1μg mL-1 INM were2.2% within a day (n=11) and 3.0% on five consecutive days (n=6), respectively. Therecovery of INM from urine samples was more than 92%. The applicability of themethod for the analysis of pharmaceutical and biological samples was examined.(8) Determination of puerarin in healthy food by high-performance liquidchromatography with on-line electrogenerated Mn(Ⅲ) chemiluminescencedetectionBased on the direct chemiluminescence reaction of puerarin(PU) with on-lineelectrogenerated Mn(Ⅲ) in H2SO4 medium, a novel HPLC-CL detection method fordetermination of PU was developed. The proposed method has been appliedsatisfactorily to the determination of PU in healthy food. The chemiluminescenceintensity was linear with PU concentration in the range of 2×10-3-1.0μg mL-1(R2=0.9991) and the limits of detection (LOD) were 7×10-4μg mL-1. The relativestandard deviations (RSD) (n=11) for PU was 2.3%. The recoveries of PU fromhealthy food samples were 93-108%.(9) Determination of captopril in human serum and urine samples by highperformance liquid chromatography with on-line electrogenerated Mn (Ⅲ)chemiluminescence detection.The separation and determination of captopril in human serum and urine samplesby means of high performance liquid chromatography with on-line electrogeneratedMn (Ⅲ) chemiluminescence detection was proposed. The method was based on thedirect chemiluminescence reaction between captopril and Mn (Ⅲ), which was on-lineelectrogenerated by constant current electrolysis. The chromatographic separation wasperformed on a nucleosil RP-C18 (250mm×4.6mm i.d., 5μm) column with an isocraticmobile phase consisting of acetonitrile-1% aqueous acetic acid (60:40, v/v) at aflow-rate of 1.2 mL min-1. The temperature was 25℃. The effects of several parameters on the HPLC resolution and CL emission were studied systematically.Under the optimal conditions, the linear range and detection limit for captopril were5~800 ng mL-1 and 0.9 ng mL-1, respectively. The relative standard derivation for10.0 ng mL-1 captopril was 2.2% (n=11). The average recoveries for captopril inhuman serum and urine ranged from 94.8% to 103.2%, and the relative standarddeviations of the quantitative results were below 3.4%. The proposed method had beenapplied to the determination of captopril in human serum and urine samples.(10) Detection of celecoxib in human serum by high-performance liquidchromatography with on-line electrogenerated Ag(Ⅱ) chemiluminescencedetection.The determination of celecoxib (CEL) in pharmaceutical and biological samplesby means of high-performance liquid chromatography (HPLC) with on-lineelectrogenerated Ag(Ⅱ) chemiluminescence (CL) detection was proposed. The methodwas based on the direct CL reaction of CEL and Ag(Ⅱ), which was on-lineelectrogenerated by constant current electrolysis. The effects of several parameters onthe HPLC resolution and CL emission were studied systematically. The applicability ofthis method for the analysis of pharmaceutical and biological samples was examined.(11) Detection of naphazoline hydrochloride in pharmaceutical preparationsby high-performance liquid chromatography with on-line electrogenerated Co(Ⅲ)chemiluminescence detection.A novel chemiluminescence detector of the high performance liquidchromatography for the determination of the naphazoline hydrochloride (NH) wasdeveloped. Based on the direct chemiluminescence reaction of NH and Co(Ⅲ) whichwas on-line electrogenerated by constant current electrolysis in H2SO4 medium.. Toobtain the highest NH sensitivity, the CL reaction conditions (including the design offlow cell, the effects of H2SO4 concentration in electrolyte, CoSO4, electrolytic currentand flow rate) and HPLC mobile phase composition in the HPLC-CL system wereoptimized. Under the optimal conditions, the linear range and detection limit for NHwere 0.1~80.0μg mL-1 (R2=0.9992) and 0.05μg mL-1, respectively. The relativestandard derivation for 1.0μg mL-1 NH was 1.8% (n=11). The proposed method hadbeen applied to the determination of NH in in pharmaceutical preparations.

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