Dissertationen zum Thema „Ion-pair chromatography“

Dosage des impuretes contenues dans l'acide amino-11-undecanoique, puis diminution de la teneur en silice des eaux utilisees dans les centrales thermiques ou nucleaires par deux types de procedes differents : copolymerisation en billes poreuses du formol et du pyrocatechol et impregnation de polymeres hydrophobes par un derive du catechol

Synthetic oligonucleotides, which are short strings of DNA or RNA, are a grooving area of importance for the pharmaceutical industry and for companies that manufacture diagnostic components. The manufacturing process of synthetic oligonucleotides involves many complex processes that use separation and purification techniques like ion-exchange chromatography, ion-pair reversed phase chromatography and ultra-performance liquid chromatography. In this study, the focus lies on the purification process, where the main aim is to develop a separation and purification method for modified oligonucleotides that can be applied on different scales, from an analytical to a preparative scale. Three modified oligonucleotides, and one unmodified with 44 bases, provided by Scandinavian Gene Synthesis (Västerås, Sweden), were analysed and purified on an ultra-performance liquid chromatography and on a preparative-system. Several parameters were investigated, e.g. mobile phase composition, gradients and concentration. Practical analysis and purification were made in two scales; analytical and semi-preparative.  The results showed that the samples contained impurities that were hard to separate from the main sample. The scaling-up tests showed that, with increasing concentration, the impurities become more aggregated with the main product. Fraction analysis showed that several pure fractions were collected from the semi-preparative purification, and therefore some amount of pure sample were collected from the semi-preparative run. In conclusion, the method developed in this master thesis worked well as a significant amount of samples were purified in the semi-preparative purification, and the method worked on modified and unmodified oligonucleotides, containing different amount of modifications.
Syntetiska oligonukleotider, vilket är korta strängar av DNA eller RNA, är ett framväxande område i läkemedelsindustrin och för företag som tillverkar diagnostiska komponenter. Tillverkningsprocessen för syntetiska oligonukleotider involverar många komplexa processer som använder separation- och reningstekniker som jonbyteskromatografi, jonparskromatografi och ultra-performance kromatografi. I denna studie ligger fokus på reningsprocessen där det huvudsakliga syftet är att utveckla en separation- och renings metod för modifierade oligonukleotider som kan appliceras på olika skalor – från analytisk till preparativ skala.  Tre modifierade oligonukleotider, samt en omodifierad med 44 baser, tillhandahållet av Scandinavian Gene Synthesis (Västerås, Sverige), analyserades och renades på ett ultra-performance kromatografi system och ett preparativt reningssystem. Flertal parametrar undersöktes, bland annat mobilfasens komposition, gradienter och koncentration. Analys och rening utfördes i två skalor; analytisk och semi-preparativ skala.  Resultatet visade att proverna innehöll föroreningar som var svåra att separera från huvudkomponenten. Uppskalningstesterna visade att föroreningarna blandade sig mer med huvudkomponenten då koncentrationen ökade. Fraktionsanalyser visade att flera rena fraktioner blev ihopsamlade från den semi-preparativa reningen, som därav visade att en betydelsefull mängd rent prov blev renat i den semi-preparativa reningen. Sammanfattningsvis, den metod som utvecklats i denna uppsats fungerade bra då betydelsefulla mängder oligonukleotider kunde renas till olika grad vid den semi-preparativa reningen, samt att metoden fungerade för både modifierade och icke-modifierade oligonukleotider som innehöll olika mängder modifikationer.

In this study, the effects of certain chromatographic conditions on various modified and unmodified oligonucleotides were investigated. At the forefront of this study was the investigation of a new Ion-pair Reversed-phase liquid chromatography (IP-RPLC) method, that had the potential to replace a previously established triethylammonium acetate (TEAA) IP-RPLC method developed for oligonucleotide separations. This method, utilising the counter ion dibutyl amine (DBA) and a Tris-buffer at pH 8, produced promising results indicating that the strong binding strength of DBA creates a hybrid IEX/RPLC separation method – the separation of oligonucleotides is dynamically based on both charge and length. Higher concentrations of DBA appear to produce better results that include improved efficiency, increased retention and even the potential discovery of hidden impurities. In conjugation with Ultra-high-pressure liquid chromatography (UHPLC) systems, sub-2 µm particle columns and gradient optimisations, separations of complex oligonucleotides could be achieved in short analysis times. Furthermore, effective separations at the analytical level can be applied and adapted to larger scale Prep-LC, potentially also improving the purification process of crude oligonucleotide samples. Further development and validation are, however, required for any future work with this method.
I denna studie har effekten av vissa kromatografiska förhållanden på olika modifierade och icke-modifierade oligonukleotider undersökts. I framkanten av denna studie var undersökningen av en ny IP-RPLC metod, vilken har potential att ersätta den tidigare etablerade trietylammonium acetat (TEAA) IP-RPLC metoden, vilken utvecklats för separationen av oligonukleotider. Denna metod, vilken använder dibutylamin (DBA) som motjon och en Tris-buffert vid pH 8, gav lovande resultat vilka indikerar att den starka bindningsstyrkan av DBA skapar en hybrid IEX/RPLC separationsmetod – separationen av oligonkuleotider styrs både av dess laddning och dess längd. Höga koncentrationer av DBA verkade ge bättre resultat som inkluderar hög effektivitet, ökad retention och även den potentiella upptäckten av gömda föroreningar. I samband med UHPLC systemer, kolonner med mindre än 2µm i partikelstorlek och optimiserade gradienter, separationer av komplexa oligonukleotider erhölls på korta analystider. Effektiva separationer vid den analytiska nivån kan appliceras och adapteras till storskalig preparative-LC, med potential att kunna förbättra reningsprocessen för syntetiserade oligonukleotider. Vidare utveckling och validering krävs för framtida användning av denna metod.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: In this work a robust reversed phase ion-pairing high performance liquid chromatographic (RP-HPLC) method has been developed for the separation, characterization and quantification of all possible [PtCl6-nBrn]2- (n = 0 – 6) and [PtCl4-nBrn]2- (n = 0 – 4) complex anions using UV-Vis detection. High resolution 195Pt NMR of more concentrated PtII/IV solutions served to validate the relevant species assignments, particularly those of the stereoisomer species, cis- and trans- [PtCl4Br2]2-, [PtCl2Br4]2- and mer- and fac-[PtCl3Br3]2-. Quantification of the PtII/IV species was achieved by means of IP-RP-HPLC coupled to either ICP-MS or ICP-OES, and together with the UV-Vis absorption spectra obtained by photodiode array (PDA) recording of all eluted species, allowed for the determination of the photometric characteristics (λmax and ε) of all the PtII/IV species. This data enables practical speciation studies of such PtII/IV complex anions using standard analytical equipment. The hyphenation of ion-pairing RP-HPLC to ICP-OES allows for the successful determination of the Pt to halide mole ratios of individually separated species in order to characterize these species in a novel manner. The Pt to chloride and/or Pt to bromide mole ratio of the [PtCl4]2- and the series of [PtCl6-nBrn]2- (n = 0 – 6) complexes were determined using HPLC-ICP-OES based on the 177.708 nm Pt, 134.724 nm Cl and 148.845 nm Br emission lines and served as a technique for the unambiguous chemical speciation of such complexes. An increase in sensitivity of the developed method was achieved by the use of an ion-pairing reversed phase ultra high performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS) method. This method proved capable of separating and characterizing the homoleptic and heteroleptic [PtIVCl6-nBrn]2- (n = 0 – 6) and mono-aquated [PtIVCl5-nBrn(H2O)]- (n = 0 – 5) complex anions in well defined acidic aqueous solutions. Ion-pairing ultra high performance liquid chromatography separation based on the volatile ion-pairing reagent, tributylamine, provided adequate chromatographic resolution as well as sufficiently low background noise for high resolution ESI-Q-TOF-MS detection. The wealth of structural information contained in the mass spectra obtained for each PtIV species simplified the identification of individual species. Moreover, the general fragmentation trends encompassing a constant incremental change of 44 Da (79/81Br - 35/37Cl) resulting from the successive substitution of Cl- by Br-, in combination with the observed elution order, facilitated the relevant species assignments. The developed method enabled the relative rapid (<13 min) characterization of all 22 [PtCl6-nBrn]2- (n = 0 – 6) and mono-aquated [PtCl5-nBrn(H2O)]- (n = 0 – 5) species. Quantification of each individual [PtCl6-nBrn]2- (n = 0 – 6) species by means of ion-paring HPLC-UV-Vis allowed for the determination of all 17 stability constants for the PtIV chloridobromido halide exchange reaction network. Determination of the associated Gibbs free energies for each ligand exchange reaction step, o rxnK ΔG n (n = 1 - 17), together with energy conservation relationships, served to validate the accuracy of the experimentally calculated stability constants. The experimentally determined overall formation constant, or ΔGo rxn, and those calculated using the standard reaction half cell reduction potentials of [PtCl6]2- and [PtBr6]2- were in good agreement, further confirming the experimentally obtained thermodynamic parameters. The thermodynamic driving force for the PtIV chloride-bromido exchange reactions is attributed to the hydration of the halide ligands, which drives the reaction towards the bromido PtIV species in aqueous solutions, even though the chlorido PtIV complexes are energetically favoured in this reaction network. Evaluation of other metal cation halido exchange reactions shows that all metal halido complexes exhibit the F- >> Cl- > Br- > I- order of thermodynamic stability and is only inverted due to the solvation of the relevant halide ligands. Furthermore, density functional theory (DFT) was used to predict the thermodynamic stabilities with respect to the isodesmic reactions involving chlorido-bromido PtIV stereoisomer pairs and chlorido-bromido PtIV ligand exchange reactions of the [PtCl6-nBrn]2- (n = 0 – 6) species and confirm the F- >> Cl- > Br- > I- order of thermodynamic stability as well as determining the ΔΔGo rxn within the range of 8 - 20 kJ.mol-1 to the experimentally determined ΔΔGo rxn.
AFRIKAANSE OPSOMMING: Tydens hierdie studie is „n robuuste “reverse-phase” ioonparing hoë-verrigting vloeistof chromatografie, RP-IP-HPLC, metode ontwikkel vir die skeiding, karakterisering en kwantifisering van alle moontlike [PtCl6-nBrn]2- (n = 0 – 6) en [PtCl4-nBrn]2- (n = 0 – 4) kompleks anione waar UV-Vis as detektor gebruik word. Die relavante spesies toedelings wat gemaak is, veral ten opsigte van die cis- en trans-[PtCl4Br2]2-, [PtCl2Br4]2- en mer- en fac-[PtCl3Br3]2- stereo-isomeerpare, is deur middel van hoë-resolusie 195Pt KMR van meer gekonsentreerde PtII/IV oplossings bevestig. Die PtII/IV spesies was gekwantifiseer deur die IP-RP-HPLC aan of „n ICPMS of „n ICP-OES te koppel. Daarenbowe was dit moontlik om die fotometriese eienskappe (λmax en ε) van elke individuele PtII/IV komplex anion te bepaal deur die UV-Vis absorpsie spektrum van elke elueerende spesies met PDA op te neem. Die nuwe metode wat tydens hierdie studie ontwikkel is het dit dus moontlik gemaak om sulke PtII/IV komplek sanione met standaard analitiese toerusting prakties te skei. Verder is gevind dat deur IP-RP-HPLC aan ICP-OES te koppel dit moontlik is om die Pt tot halied mol verhoudings van elke individueel geskeide spesies te bepaal en dus hierdie spesies op „n oorspronklike, nuwe manier te karakteriseer. Die Pt tot chloried en/of Pt tot bromied mol verhoudings van die [PtCl4]2- en die reeks van [PtCl6-nBrn]2- (n = 0 – 6) kompleks anione, soos bepaal deur gebruik te maak van HPLC-ICP-OES, is gebasseer op die 177.708 nm Pt, 134.724 nm Cl en 148.845 nm Br emissie lyne. Hierdie metode kan gebruik word vir die eenduidige chemiese skeiding van hierdie komplekse. Die sensitiwiteit van hierdie metode was egter verder verbeter deur gebruik te maak van ioonparing “reverse-phase” ultra hoë-verrigting vloeistof chromatografie gekoppel met elektrosprei ionisasie quadropool “time-of-flight” massa spektrometrie (UHPLC-ESI-Q-TOFMS). Deur dit te doen is dit nou selfs moontlik om die homoleptiese en heteroleptiese [PtIVCl6-nBrn]2- (n = 0 – 6) spesies, asook die “mono-aqauted” [PtIVCl5-nBrnH2O]- (n = 0 – 5) spesies in „n goed gedefinieërde aangesuurde waterige oplossings te skei en te karakteriseer. Die vlugtige ioon-paringsreagent, tributielamien, is vir die skeidingsproses op die IP-UHPLC gebruik om te verseker dat voldoende chromatografiese resolusie, so wel as lae genoeg agtergrondgeraas, verkry word vir hoë-resolusie ESI-Q-TOF-MS deteksie. Die rykdom informasie vervat in die massaspektrum van elke PtIV spesies het die indentifikasie van elke spesies vergemaklik. Daarenbowe het die fragmentasie tendens, aanduidend van „n konstante inkrementele verandering van 44 amu (71/81Br – 35/37Cl) weens die opeenvolgende substitusie van Cl- met Br-, tesame met die elusie volgorde, die spesies-toedelings gefasiliteer. Met hierdie nuut ontwikkelde metode is dit nou moontlik om al 22 [PtCl6-nBrn]2- (n = 0 – 6) en “mono-aquated” [PtCl5-nBrnH2O]- (n = 0 – 5) spesies in „n relatiewe kort tydperk (< 13 min) te karakteriseer. Deurdat elke [PtCl6-nBrn]2- (n = 0 – 6) spesies nou individueel met IP-HPLC-UV-Vis gekwantifiseer kan word, is dit moontlik om al 17 stabiliteitskonstantes vir die PtIV chloridobromido halied uitruilingsreaksienetwerk te bepaal. Die geassosieerde Gibbs vrye energie, ΔG°rxnKn (n = 0 – 17), wat vir elke stap in die uitruilingsreaksienetwerk bepaal is, tesame met die energiebewaring verhoudings, was gebruik om die akkuraatheid van die eksperimenteel bepaalde stabiliteitskonstantes te bekragtig. Verdermeer was die waarde van die algehele formasie konstante wat eksperimenteel bepaal is, ΔG°rxn, in goeie ooreenstemming met dit wat bereken is deur die standaard reaksie halfsel reduksie potensiale van [PtCl6]2- en [PtBr6]2-. Dus is die eksperimenteel verkrygde termodinamiese parameters bevestig. Die termodinamiese dryfkrag vir die PtIV chloried-bromied uitruilingsreaksies is toegereken aan die hidrasie van die halied ligande, wat in waterige oplossings die reaksie na die bromied PtIV spesies dryf, al is die chloried PtIV spesies energeties bevoordeel in hierdie reaksienetwerk. Evaluering van ander metaalkatioon- halied-uitruilreaksies wys dat alle metaal-halied komplekse die F- >> Cl- > Br- > I- orde van termodinamiese stabiliteit volg en dat hierdie volgorde slegs omgekeer sal word weens solvasie van hierdie halied ligande. Darenbowe digtheids funksionele teorie (DFT) gebruik om die termodinamiese stabiliteit met betrekking tot isodesmiese reaksies wat chloried-bromied PtIV stereoisomeer pare behels te voorspel, sowel as van chloried-bromied PtIV liganduitruilingsreaksies van die [PtCl6-nBrn]2- (n = 0 – 6) spesies, en bevestig die F- >> Cl- > Br- > I- volgorde van termodinamiese stabiliteit. Verder was dit ook moontlik om met DFT die ΔΔG°rxn tot so naby as 8 – 20 kJ.mol-1 te bereken.

Per- and polyfluorinated substances (PFAS) are compounds that consist of a carbon chainbackbone that is partially or entirely fluorinated, with an addition of a functional group. SomePFAS are known as persistent organic pollutants (POPs) and have therefore been drawing a lot ofattention as well as increased concerns. PFAS have been detected in humans, wildlife and theenvironment and some have exhibited toxic effects such as hepatotoxicity, immunotoxicity,reproductive toxicity and endocrine disruption as well as being persistent and bioaccumulative.Serum, plasma and whole blood have been used as biomonitoring matrices in many studies toevaluate human exposure to PFAS. Restrictions have been applied to some PFAS, but thesecompounds are still ubiquitous. This study will investigate the performance (recovery, matrixeffect (ME) in terms of intra-/inter-day repeatability) of ion-pair extraction (IPE) and solid phaseextraction with weak anion exchange (SPE-WAX). The extraction methods were adapted fromliterature and 13 PFAS were selected for this work based on prior biomonitoring studies. Thetarget PFAS content was analyzed with liquid chromatography coupled with tandem massspectrometry (LC-MS/MS). The extraction methods were also compared for extractableorganofluorine (EOF) extraction in terms of blank levels as well as the amount extracted withdifferent methods; the EOF content was measured with combustion ion chromatography (CIC).The EOF levels were used to estimate the amount of unidentified organofluorine (UOF), to avoidunderestimating potential health hazards. Samples extracted using IPE had an average ionizationenhancement of 9%, while SPE-WAX showed an average ionization suppression of -1%. SPEWAXshowed higher average recoveries for procedural blanks (78%), horse serum (96%) andhuman serum (95%) in comparison to IPE (69%, 36%, 88%, respectively). The CIC analysis forEOF content was observed to be below MDL (<50 ng/mL F) with some contaminations observedin the procedural blanks.

Une nouvelle méthode, simple et rapide, a été développée pour isoler dans le miel différents antimicrobiens usuellement recherchés en contrôle sanitaire et appartenant à quatre classes différentes: les sulfamides, les tétracyclines, les macrolides et lincosamides associés et les aminoglycosides. Ces molécules antimicrobiennes sont analysées par chromatographie liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM) après avoir été extraites de l’échantillon de miel par une méthode d’extraction unique. Afin de définir les conditions optimales de séparation et de détection, l’influence de la nature et de la concentration d’un agent d’appariement d’ions tel que le HFBA ou le PFPA, introduit dans la phase mobile, a pu être évaluée sur une colonne analytique en chromatographie de phase inversée de type C18. Plusieurs paramètres ont été pris en compte et étudiés lors de l’élaboration de la méthode d’extraction tels que la nature du solvant d’extraction, le pH, l’étape d’hydrolyse acide, l’efficacité de l’extraction par ultrasons et enfin la purification de l’extrait avant injection. La méthode développée a ensuite été validée suivant les recommandations de la Décision de la Commission Européenne (CE) No 2002/657 puis a subi une étape de validation supplémentaire en participant à une comparaison inter-laboratoire organisée sur des matériaux de miel contaminés et gérée par un organisme extérieur accrédité suivant la norme ISO17043. Par la suite, une démarche de transfert de la méthode analytique validée en CL-SM/SM a été mise en place pour son utilisation en chromatographie liquide couplée à la spectrométrie de masse à haute résolution (CL-SMHR). Une validation de cette démarche a été menée par l’application d’une étude statistique descriptive basée sur la notion de profil d’exactitude. Finalement, un programme expérimental de surveillance a été entrepris sur une série d’échantillons de divers miels collectés sur des marchés locaux pour tester la qualité des produits commercialisés au Liban. Contrôlés au regard de leur contamination en résidus d’antimicrobiens en CL-SM/SM parmi la trentaine de molécules prédéfinies dans l’étude, la positivité et/ou la non-conformité de certains échantillons ont pu être confirmées par l’utilisation de la CL-SMHR
A new, simple and rapid method has been developed for the determination of multiclass antimicrobial residues in honey (sulfonamides, tetracyclines, macrolides, lincosamides and aminoglycosides). All the compounds were extracted from honey within single extraction method and analyzed by liquid chromatography - tandem mass spectrometry (LC-MS/MS) operating in positive electrospray ionization mode. In our study, we examined the behavior of volatile perfluorinated carboxylic acids (HFBA and PFPA) used as ion-pairing reagents for the separation of multiclass of antibiotic residues by reversed phase Zorbax SB C18 column. Furthermore, the extraction and clean-up steps were investigated and optimized by using ultrasonic-assisted extraction and dispersive solid phase extraction (d-SPE). Different parameters affecting the extraction efficiency including type of solvent, pH, breaking efficiencies of N-glycosidic linkage by hydrochloric acid, ultrasonic extraction and its duration compared to shaking technique, along with dispersive SPE clean-up were examined prior sample injection. The method was then validated according to European Commission Decision (EC) No 2002/657. Furthermore, the method was tested for its validity through participation in proficiency testing scheme organized by FAPAS for the analysis of tetracycline in honey. Afterwards, a transfer of the validated LC-MS/MS analytical method has been applied for the determination of antimicrobial residues in honey from low resolution to High Resolution Mass Spectrometry (HRMS). For that purpose, descriptive statistical approach was performed to assess the performance of the method based on simultaneous evaluation of the trueness and the intermediate precision. Finally, the method was applied for the determination of antimicrobial residues in honey collected from local markets at different regions in Lebanon. Positive samples were then analyzed by the LC-HRMS to confirm the presence of analytes detected by LC-MSMS

碩士
國立中興大學
化學系
91
The solid phase extraction is a conventional pre-treatment method for determination of paraquat and diquat in water matrix. However, it need complicated procedure and takes much time in order to concentrate trace compounds. The conventional HPLC methods for analyze those compounds waste a lot of ion pair reagent and suffer a worse separation. In this study, a reversed-phase ion pair liquid chromatography coupling to microdialysis method has been developed to analyze paraquat and diquat in water matrix. Conditions for obtaining optimum dialysis efficiency such as flow-rate of perfusion stream, pH in sample solution and in perfusion stream, ion-pair reagent, added-salt in the sample solution, as well as chromatographic parameters were investigated. The experiments results indicated that the dialysis achieved at sample matrix of pH 6, and the perfusion with 0.01M sodium 1-Hexanesilfonate (pH 3, flow rate 5μl/min) offered the optimum dialysis efficiency. A reverse-phase C-18 column was used to separate the paraquat and diquat with an eluent of 0.01M sodium 1-hexanesilfonate, 0.01M triethylamine and 0.3M potassium bromide at pH 3. The measurement of analytes method was carried out with an UV detector operated at 287nm.The enrichment reached more than 100 times with RSD less than 5.68%. The paraquat and diquat compounds in waste water were well-separated with a reversed-phase ion pair liquid chromatography coupling to micro-dialysis method. It indicated the proposed method, provided a very simple, fast and good recovery method for the determination of paraquat and diquat in water matrix.

碩士
國立中央大學
化學研究所
92
Abstract The purpose of this study is to develop a method for the analysis of four stilbene-type disulfonate and one distyrylbiphenyl-type fluorescent whitening agents (FWAs) in different matrixes: laundry detergents, papers and infant clothes, as well as river waters. The sample pretreatment methods involve: the laundry detergent using dissolved, filter and oscillation; the paper and infant cloth samples using a hot-water extraction with SPE; the water sample using the C18 SPE cartridge. The contents of FWAs were then determined by an isocratic ion-pair chromatography with C18 reversed- phase column, applying TBA as the ion-pair reagent, and equipped with fluorescent detection. No further sample cleanup procedures were necessary due to the sensitive and selective fluorescence detection. The accuracy and precision of the method was validated and was successfully applied to determine the contents of FWAs in laundry detergents, papers, infant clothes and surface waters. The quantitation limits were 0.1 to 0.01 �慊/L. The linear response was performed in the concentration covering the range from 0.05 to 500 ng/mL for DSBP, and 0.5 to 5000 ng/mL (in five-level) for other four FWAs. The calibration curve was linear with coefficients of determination r2 > 0.999. The precisions of the curves as indicated by R.S.D. of calibration factors (CF = peak area/amount) were 4.5 ~ 8.7%. For laundry detergents, the results show that the DSBP (0.5 to 2700 �慊/g) and DAS1 (29 to 980 �慊/g) are the two most added FWAs in detergents. For paper and infant cloth, recovery of FWAs in spiked paper and infant cloth samples was between 76 to 92% and precision (RSD) ranging from 2.0 to 4.0%. Analysis of six paper materials and three infant clothes found concentrations of FWAs ranging between 0.01 to 3.8 �慊/g and n.d. to 24 �慊/g, respectively. For river water, recovery of FWAs in spiked water samples was between 73 to 89% and precision (RSD) ranging from 2.4 to 8.9%. Analysis of for water samples found concentrations of FWAs ranging between 0.02 to 3.5 �慊/L. The results demonstrated that the developed method is reliable, and offering a convenient analytical technique for trace-level determination of FWAs in complex environmental samples.

碩士
靜宜大學
應用化學系
82
Since metals and their complexes play an important role in both biological chemistry and in enviromental chemistry, the analysis of metals is rather important. In recent years, high performance liquid chromatography coupled with various detectors has become one of the best methods for metal analysis. In this study, we used 4-（2-pyridylazo）resorcinol (PAR） as a pre-column reagent, Cu-PAR and Co-PAR were separated and quantitatively determined by reverse phase high performance liquid chromatography with UV-VIS detector. We also investigated the absorption and chromatographic characteristics of these metal chelates. The optimum condition established in this work was:acetonitrile/methanol /tartrate buffer (pH=6.5)= 6/30/64 including 1.0×10▋▋ ion-pair reagent( tetrabutylammonium bromide) The separation was done with C▋▋ eversed phase column. The proposed method was appilied to determine Cu and Co in a relatively complex substance, namely, nickel-base superalloy. The precision and accuracy of the proposed chromatographic method were evaluated。

The thesis is focused on application of ion chromatography in pharmaceutical analyses of organic ions. Ion chromatography is increasingly used in the field of pharmaceutical analysis. This includes the analysis of impurities and metabolites. In the first part of this thesis, ion chromatography is compared with common separation techniques used in pharmacy, such as gas chromatography and high performance liquid chromatography. In the second part development and validation of methods of ion chromatography for purity evaluation and quality control of active pharmaceutical substances Rivastigmine hemitartrate and Pramipexole hydrochloride were carried out. Key words: ion chromatography, reversed-phase chromatography, ion-pair chromatography, ion-exclusion chromatography, ion-exchange chromatography, GC, HPLC

碩士
靜宜大學
應用化學系
89
Abstract The exceptional organic acid in body fluid was related as closely as physiological metabolism. This study will be established qualitative and quantitative analysis aimed at abnormal metabolic markers-organic acids (orotic acid, hippuric acid, uric acid, fumaric acid, a-ketoglutaric acid, and pyruvic acid) in body fluid. HPLC method with adding ion-pair reagent has a good stability and the consuming material used in long time. The optimum mobile phase for HPLC was 22% methanol and 10mM TBA (pH 4) aqueous in gradient elution. The better reproducibility (RSD<2.75%), linear range (0.1~100.0ppm), correlation coefficient (r>0.997), and detection limit (0.099~0.98ppm) were obtained. The optimum condition for CZE was 5% acetonitrile, 0.5mM cetyltrimethylammonium broide, 10mM/30mM phosphate buffer, and fused silica capillary (75cm×75mm) by applying electric field strength -160V/cm. The quantitative results were obtained with acceptable reproducibility (RSD<5.43%), linear range (0.7~80.0ppm), correlation coefficient (r>0.994), and detection limit (0.52~2.39ppm). The optimum methods of CZE and HPLC were applied to assay human urine with direct analysis and standard addition. The comparison between the results of two methods showed that CZE method had more stable data. By the way, the recoveries were above 84.6% in standard addition by CZE method. Keywords: abnormal metabolism, organic acids, ion-pair chromatography, capillary zone electrophoresis, analysis of human urine.

碩士
國立中興大學
化學系所
97
In this study, we have developed a novel green route analytical method for the determination of phenoxy acid herbicides (2,4-D, 2,4,5-T, and MCPP) in water sample using on-line microdialysis extraction coupled with subcritical water ion-pair chromatography. Microdialysis extraction liquid (1-Octanol) and Cellulose Acetate dialysis membrane were employed to extract the analytes from the agricultural water in Yunlin city, Taiwan. The Cellulose Acetate dialysis membrane was directly connected to the injection valve of subcritical water ion-pair chromatography system. After the microdialysis extraction, the extracted analytes were on-line introduced into the subcritical water separation column (Hamilton PRP-1) and detected by a UV detector. Under the optimal microdialysis extraction and subcritical water ion-pair chromatography conditions, the linear range for 2,4-D, MCPP and 2,4,5-T were 10–500 μg/L. The correlation coefficients were between 0.9960-0.9992. The limit of detection were 2 μg/L for 2,4-D, 4 μg/L for MCPP and 6 μg/L for 2,4,5-T. The recoveries of phenoxy acid herbicides in real sample (agricultural water) were between 70-85%. This method was using no organic solvent and could achieve good separation. The limits of detection were acceptable. So the proposed method provides a simple and eco-friendly procedure for isolating and determining phenoxy acid herbicides in agricultural water.

碩士
東吳大學
化學系
89
Azo dyes are a class of synthetic dyes, which are widely used as coloring agents in a variety of products, such as textiles, paper, leather and foodstuffs. However, some synthetic dyes may be hazardous because of their toxicity. Hence, a sensitive and reliable method of determining dyestuffs is required. A method based on ion-pair liquid chromatography coupled with diode-array detector (DAD) and mass spectrometric (MS) detection has been developed to determine several azo dyes, including acid yellow 23, acid red 27, acid red 18, food yellow 3, food red 17, acid red 26, food red 6, acid orange 20, acid orange 7 and acid yellow 36. A RP-ODS stationary phase is used, and the mobile phase contains 3.0 mM triethylamine as an ion-pair reagent. Good separations were obtained using gradient elution in 35 minutes. The linearity of the ten dyes is from 0.05-10ppm and the detection limits is lower than 10μg/l for an injection volume of 5μl. Mass spectrometry is suitable for confirmation of the identify of azo dyes in foodstuffs. The sulfonated group is strongly ionic species and therefore completely dissociated in the aqueous solution. So that, negative-ion MS with electrospray ionization (ESI) is much more sensitive than positive-ion MS. Selecting ion monitoring (SIM) was employed for quailtative determination of all compounds. A simple extraction method to extract analytical compounds in foodstuffs was developed. The application of the LC/DAD/MS method was demonstrated for several food samples.

碩士
朝陽科技大學
應用化學系碩士班
92
The potential of 3,5-dinitrobenzoyl quinidine as chiral selector added to a non-aqueous background buffer for the capillary electrophoretic separation of the enantiomers of N-derivatized amino acids and non-steroidal anti-inflammatory drugs are evaluated. Separation is based on chiral analytes of R or S of negatively charged and positively charged quinidine-derived chiral selector to form temporary ion-pair, because of different molecular interactions and mobiling. As a result, the differences in the overall migration of analytes can be achievd. To suppress one problems associated with the high UV absorption of the chiral selector and thus the high detecting background in the ‘total filling technique’, the ‘partial filling technique’ has been adopted. Several parameters including selector filling time and length of selector zone, selector concentration, type of non-aqueous background electrolyte and length of column have been evaluated. Optimun experimental conditions were found with a electrolyte made of 80 mM acetic acid, 20 mM triethylamine and 150 mM 3,5-dinitrobenzoyl quinidine in an EtOH-MeOH (60:40, v/v), and at voltage of –30kV. Under these conditions, (R) and (S) enantiomers of 3,5-dinitrobenzoyl leucine could be separated with α=1.10, Rs=5.16. (R) and (S) enantiomers of ketoprofen could be separated with α=1.06, Rs=1.13. Similar resolution, and efficiencies were observed for other analytes. 3,5-dinitrobenzoyl quinidine as chiral selector can be utilized to separate several kinds of compound induding aromatic π acid, aromatic π base and ascorbic acid.

(EN) In the first part of this work, analytical methods for determination of impurities of active pharmaceutical ingredients (API) in combined pharmaceutical dosage forms were developed and validated. Development of the methods covered both the optimization of sample preparation procedure and chromatographic conditions. The methods were validated according to International Conference on Harmonization guideline and both of them were confirmed to be able to analyze stability samples. Impurities in paracetamol, codeine phosphate hemihydrate and pitophenone hydrochloride in the presence of fourth API fenpiverinium bromide were separated by using ion-pair reversed phase chromatography with gradient elution. Symmetry C18, 250 x 4,6 mm, 5 µm heated to 35 řC was used as a separation column. A diode array detector was used. The detection wavelengths were set as follows: 220 nm for paracetamol impurity K, 245 nm for paracetamol and its other impurities and 285 nm for codeine, pitophenone and their impurities. Impurities in valsartan, amlodipine besylate and hydrochlorothiazide were separated by reversed phase UHPLC method with gradient elution. Chromatographic column Zorbax Eclipse C8 RRHD, 100 x 3,0 mm, 1,8 µm heated to 30 řC and spectrophotometric detection were used. The detection wavelengths were set as...

碩士
嘉南藥理科技大學
化粧品應用與管理系
101
The purpose of this study is to separate simultaneously seven water soluble whiten agents by ion pair chromatographic method. These components include ascorbic acid and its three derivative; arbutin, hydroquinone and kojic acid. The parameters of IPC for separation, such as pH value of buffer, organic modifier composition and ion pair reagent concentration, were studied in this research. The results showed these seven compounds could be well separated under optimum condition by using C18 column as stationary phase. The regression coefficient of all compounds calibration curve are larger than 0.999. The stability of these components in water of different pH values were also investigated. This method is applied for determination of these compounds in lotions by simple dilution. Recoveries of the six analytes were between 97.61-104.78%.