Auswahl der wissenschaftlichen Literatur 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