Call number: LD2668 .T4 1961 A43

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Chemical separations are a necessary component in many scientific analyses. Microfluidics, the use of micron-sized fluidic channels defined in glass or polymer blends, is a powerful branch of separation science that is developing rapidly. Miniaturized analytical devices offer important advantages compared to traditional bench-top techniques, most notably capillary electrophoresis (CE). This dissertation was focused on developing several novel methods to improve microfluidic based separations and techniques. The electrophoretic separation of small similarly charged analytes can be very difficult. Chapter 2 discusses a new buffer that has been developed for fast, high efficiency separations of amino acids by micellar electrokinetic chromatography (MEKC). This buffer is more environmentally friendly than the most commonly used surfactant containing buffers for MEKC separations. It uses a commercially available dish washing soap by Seventh Generation™ Inc. that contains three micelle forming agents; sodium lauryl ether sulfate (anionic), cocamidopropyl betaine (zwitterionic), and cocamide monoethanolamine (MEA) (non-ionic), and is completely void of organic solvents. Many biological samples contain analytes below the limit of detection of traditional detection systems; therefore, chapter 3 reports the fabrication of nanoporous membranes on microfluidic devices that are capable of analyte concentration enrichment. Donnan exclusion is responsible for the preconcentration of fluorescent dyes near a charged, porous titania membrane. The level of analyte enrichment was monitored, and enrichment factors greater than 4000 in 400 s were obtained for 2,7-Dichlorofluorescein. Chapter 4 describes the fabrication and characterization of paper based microfluidic devices. Mixtures of acrylate modified photocurable polymers were used to photolithographically define channels on multiple paper substrates. Flow characteristics are described and their use for monitoring complications associated with type 1 diabetes is demonstrated. Finally in Chapter 5, Sol-gel modified gold surfaces for preventing protein adsorption during surface plasmon resonance (SPR) detection are also presented.

Dissertation submitted in fulfilment of the requirements for the Masters Degree in Technology: Chemistry, Durban University of Technology, 2009. / The activity coefficients at infinite dilution ( γ13 ) were calculated for alkanes, alkenes, cycloalkanes, alkynes, ketones, alcohols and aromatic compounds from gas liquid chromatography (glc) measurements at three temperatures (303.15 and 313.15 and 323.15) K. The γ13 values were calculated from the retention data for two ionic liquids (ILs) with the same cation but different anions. The ionic liquids: [methyltrioctylammonium bis(trifluoromethylsulfonyl)imide and methyltrioctylammonium thiosalicylate] were used as the stationary phase. The γ13 data for methyltrioctylammonium thiosalicylate were higher than for methyltrioctylammonium bis(trifluoromethylsulfonyl)imide. For each temperature and each ionic liquid, γ13 values were determined for two columns and the average γ13 values were used for the calculation of the partial molar excess enthalpies at infinite dilution, H E 1 Δ , using the Gibbs-Helmholtz equation. Graphs of In γ13 vs 1/T were plotted to determine H E 1 Δ at T = 303.15 K. The selectivity, S12 , value for the hexane/benzene separation was calculated from the ratio of the avarage γ13 values to determine the suitability of the ILs as an entrainer for extractive distillation in the separation of aromatic and aliphatic compounds.The S12 values were also compared to the literature values for other ionic liquids. It was found that the longer chain alkyl group attached to the cation or anion gave lower S12 values. Both ILs have S12 values greater than one and in theory can be used as an entrainer for the hexane/ benzene separation. The capacities at infinite dilutions, k1 , were also calculated from the inverse of the avarage γ13 values for each IL at each temperature. / National Research Fund; Postgraduate Development and Support Directorate.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Advances in pharmacology, biochemistry and biotechnology are increasingly dependant upon affinity chromatography as a preferred separation technique for the purification and characterisation of specific biomolecules. In the past few years avidin-biotin technology has been widely and successfully used in the fields of medicine, pharmacy, biology and biochemistry. The avidin-biotin complex (ABC) has been used as a mediator for affinity chromatography, affinity cytochemistry, immunoassay, histopathology, bioaffinity sensors, erosslinking and immobilisation studies. The main reason for the popularity of the ABC and its growing usefulness in biotechnology is the exceptionally high affinity (1015 M-l) and stability of the noncovalent interaction between avidin and biotin. The use of the ABC is broadening as different biotin derivatives and avidin-containing conjugates are becoming commercially available. The aim of this work was to evaluate the usefulness of a plutonic" FI 08 and the ABC conjugate to effect affinity separation. Towards this aim, the adsorption of plutonic" F108 onto hydrophobic polysulphone membrane surfaces was studied. This information was used to determine the theoretical maximum amount of pluronic" FI08 that will adsorb onto a unit surface area of the membrane. It is known that the polypropylene oxide (PPO) centre block ofthe pluronic" F I08 surfactant molecule governs the concentration of pluronic" F I 08 molecules that will adsorb onto a given hydrophobic surface. If the maximum coating concentration of plutonic" FI08 is known, one can assume that the maximum coating concentration of any pluronic derivative, with the same PPO centre block size, will be the same. Adsorption studies were carried out, the Langmuir adsorption isotherm was determined, and subsequently the fractional coating was calculated. The end-groups of plutonic" FI08 were modified as follows and the substituted pluronic was adsorbed onto a membrane that was to act as the solid support matrix in the development of an affinity system: Amino pluronic was synthesised by first tosylating pluronic" FI08, followed by azidation with NaN3 then reduction with LiAI~. The synthesised amino pluronic was then biotinylated using N-hydroxysuccinimide biotin ester. The suitability of this synthetic route was first assessed on a model compound, 2-methoxyethylamine, and validated by NMR (Nuclear Magnetic Resonance) spectroscopy. The synthetic protocol was then used to derivatise the larger pluronic molecule. The affinity system was tested on two different hydrophobic surfaces: polystyrene and polysulphone membranes (PSMs). Avidin-conjugated horseradish peroxidase was obtained and used to interact with the immobilised biotin. The enzymatic reaction of the coupled peroxidase converted the substrate, 2, 2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) to a coloured product. The colour developed is proportional to the amount of biotin that was immobilised on the hydrophobic surfaces studied. Non-covalent immobilisation of the synthesised biotin-pluronic molecule was successfully obtained onto the hydrophobic polystyrene as well as the polysulphone membrane surfaces. / AFRIKAANSE OPSOMMING: Vooruitgang in die farmakologie, biochemie en biotegnologie word al meer afhanklik van affiniteits chromatografie as die verkose tegniek vir die suiwering en karaterisering van spesifieke biomolekules. Oor die afgelope jare het die avidien-biotien tegnologie homself as baie bruikbaar bewys in die mediese, farmakologiese, biologiese en biochemiese velde. Toepassings waar die avidien-biotien kompleks betrokke was sluit in die toepassing as 'n mediator vir affiniteits chromatografie, affiniteits sitologie, immuno bepalings, histopatologie, bioaffiniteits sensors sowel as kruisbinding en immobiliserings studies en vele meer. Die hoofrede vir die gewildheid van die avidien-biotien kompleks en die groeiende bruikbaarheid in die biotegnologie is die buitengewone hoë affiniteit (l015 M-I ) en stabiliteit van die nie-kovalente interaksie tussen avidien en biotien. Die toepassingsveld van die avidien-biotien kompleks word wyer met die verskeidenheid biotien derivate en avidien-bevattende konjugate wat kommersiëel beskikbaar is. Die doel van die werk wat hier gedokumenteer word is om die bruikbaarheid van Plutonic" FI08 en die avidien-biotien kompleks, vir gebruik in 'n affiniteits chromatografie sisteem, te evalueer. Om hierdie doel te bereik is die adsorpsie van Pluronic" FI08 aan hidrofobiese polisulfoon membraan oppervlaktes bestudeer. Die eksperimentele data wat gegenireer is, is gebruik om die teoretiese maksimum hoeveelheid Pluronic wat per eenheids oppervlakte membraan adsorbeer te bepaal. Dit is reeds bekend dat die polipropileen (PPO) middel blok van die Pluronic emulgant die konsentrasie van die geadsorbeerde Pluronic molekules op 'n gegewe hidrofobiese oppervlakte bepaal. Indien die maksimum bedekkingskonsentrasie VIr maksimum oppervlakbedekking van Plutonic" FI08 bekend is, kan teoreties aanvaar word dat die bedekkingskonsentrasie vir enige Pluronic derivaat met dieselfde grootte PPO blok dieselfde sal wees. Adsorpsiestudies was uitgevoer om die Langmuir adsorpsie isoterm te bepaal. Daaropvolgend was die fraksionele bedekking bereken. Amino-pluronic was gesintetiseer deur die eindpunte van Pluronic te derivatiseer. Hierdie Pluronic derivaat was gevolglik geadsorbeer aan 'n membraan wat gedien het as die soliede oppervlakte vir die ontwikkeling van 'n affiniteits chromatografie sisteem. Amino-pluronic was gesintetiseer deur Pluronic eers te tosileer en daarna te asideer met NaN3 en laastens te reduseer met LiAI~. Die produk was gebiotinileer deur gebruik te maak van N-hidroksisuksinimied-biotien-ester. Die bruikbaarheid van hierdie sintetiese roete is eers bepaal deur van 'n model verbinding, 2-metoksiëtielamien, gebruik te maak en dit met behulp van KMR (Kern Magnetiese Resonans) spektroskopie te karakteriseer. Die affiniteits sisteem is getoets op twee verskillende hidrofobiese oppervlaktes naamlik polistireen en polisulfoon membraan oppervlaktes. Avidien gekonjugeerd met 'n peroksiedase ensiem is gebruik om met die geïmmobiliseerde biotien te assosieer. Die ensiematiese reaksie van die gekoppelde peroksiedase het die substraat 2, 2' -azino-di-(3-etiel-benzthiazolien-6-sulfoonsuur) (ABTS) omgesit na 'n gekleurde produk, waar dit teenwoordig is. 'n Reeks wasstappe is gebruik om die gemodifiseerde peroksidase ensiem wat nie aan die hidrofobiese oppervlakte gekoppel nie, weg te spoel. Hierdeur is die mate van binding aan die hirofobiese oppervlakte gekwantifiseer deur die kleur te kwantifiseer wat ontwikkelomdat die kleurontwikkeling direk proporsioneel is aan die hoeveelheid peroksidase wat nog aan die membraan gekoppel is. Nie-kovalente immobilisasie van die gesintetiseerde biotien-pluronic molekule is suksesvolop beide die hidrofobiese polistireen oppervlakte sowel as die polisulfoon membraan verkry.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The separation of apple, cocoa and green tea phenolic compounds by comprehensive 2-dimensional liquid chromatography (2-D-LC) has been studied. In the first dimension, phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) on a diol stationary phase with a mobile phase containing acetonitrile, methanol, acetic acid and water. Gradient reversed-phase (RP) LC using a C18 column with fluorescence detection was employed in the second dimension to separate compounds according to hydrophobicity. Compounds were identified using negative electrospray ionisation mass spectrometry (ESI-MS) coupled to both HILIC and RP separations. The coupling of HILIC and RP separations proved to be especially beneficial since this provided simultaneous information on both the polarity and hydrophobicity of phenolics. The low degree of correlation (r2 < 0.21) between the two LC modes afforded peak capacities in excess of 3000 for the off-line method. An on-line method was also developed utilizing a short, small particle-packed column to provide fast separation in the second dimension. A 1 mm i.d. column was used in the first dimension for the on-line system to reduce injection volumes onto the second dimension column. A significantly lower practical peak capacity was measured for the on-line system, due largely to the reduction in second dimension peak capacity. On the other hand, analysis could be performed in an automated fashion using the online system reducing the risk of sample alteration and guaranteeing better operation reliability and reproducibility. Especially the off-line comprehensive HILIC × RP-LC method developed demonstrated its utility in the analysis of various groups of phenolic compounds including proanthocyanidins, phenolic acids, flavonols and flavonol conjugates in a variety of natural products. / AFRIKAANSE OPSOMMING: Die skeiding van fenoliese komponente in appel, kakao en groen tee is deur middel van ‘comprehensive’ 2-dimensionele vloeistof chromatografie (2-D-LC) bestudeer. Hidrofiliese interaksie chromatografie (HILIC) is gebruik om die fenoliese komponente in die eerste dimensie te skei op grond van polariteit, deur gebruik te maak van ‘n diol stationêre fase en mobiele fase bestaande uit asetonitriel, metanol, asynsuur en water. ‘n Gradiënt omgekeerde fase (RP) LC analisie op ‘n C18 kolom met fluorosensie deteksie is in die tweede dimensie gebruik om fenole volgens hidrofobisiteit te skei. Negatiewe elektrosproei-ionisasie massa spektometrie (ESIMS) gekoppel aan HILIC en RP skeidings is gebruik vir identifikasie van fenole. Die koppeling van HILIC en RP skeidings veral voordelig deurdat dit gelyktydige informasie verskaf het oor die polariteit sowel as die hidrofobisiteit van die fenoliese komponente. Die lae graad van korrelasie (r2 < 0.21) tussen die twee LC metodes was verantwoordelik vir piek kapasiteite bo 3000 vir die af-lyn metode. ‘n Aanlyn metode was ontwikkel deur gebruik te maak van ‘n kort, klein partikel gepakte kolom om vinnige skeiding in die tweede dimensie te verseker. 1 mm i.d. kolom was gebruik in die eerste dimensie vir die aanlyn sisteem om die inspuit volume op die tweede dimensie kolom te verminder. Aansienlike laer praktiese piek kapasiteit was gemeet vir die aanlyn sisteem, grootliks toegeskryf aan die reduksie in die tweede dimensie piek kapasitiet. Aan die ander kant, analise kan geoutomatiseerd uitgevoer word deur gebruik te maak van die aanlyn sisteem, wat monster alterasie, beter betroubaarheid en reproduseerbaarhied verseker. Veral die ontwikkelde af-lyn ‘comprehensive’ HILIC × RP-LC metode toon demonstreerbare voordele vir die analiese van verskeie groepe fenoliese komponente, insluitende proantosianiede, fenoliese sure, flavonole en gekonjugeerde flavonole in ‘n verskeidenheid natuurlike produkte.

Thesis (PhD (Chemistry and Polymer Science))--University of Stellenbosch, 2007. / This study describes the development of simplified analytical methods for the analysis of trace quantities of selected naturally occurring target compounds in complex biological matrices by stir bar sorptive extraction (SBSE) and gas chromatography/mass spectrometry (GC/MS). SBSE facilitates the direct extraction of organic compounds from aqueous samples by allowing the solutes to partition between the aqueous phase and a glass stir bar that is coated with a layer of polydimethylsiloxane (PDMS). The partitioning of polar compounds into the PDMS coating was enhanced by using different derivatization techniques in combination with SBSE. The derivatization of polar functional groups was performed with ethyl chloroformate, acetic acid anhydride, and O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine directly in the aqueous samples. Headspace derivatization of compounds containing a secondary alcohol group was performed directly on the stir bar coating in the presence of acetic acid anhydride vapors. The derivatized compounds were thermally desorbed (TD) and analyzed on-line by GC/MS. A number of experimental parameters, including salt addition, temperature and time were optimized to improve the recovery of the derivatized compounds by SBSE. The optimized methods were validated in terms of linearity, precision, and detection and quantitation limits prior to performing the quantification. Trace levels of tuberculostearic acid, a marker of tuberculosis, was detected in sputum samples that were decontaminated and concentrated before being analyzed by SBSE-TD-GC/MS. The method is sufficiently sensitive to detect the marker without the need to culture the organisms, namely M. Tuberculosis. The analysis of 4-hydroxynonenal has also been demonstrated by detecting trace levels of this oxidative stress marker in urine samples obtained from healthy volunteers. Furthermore, abnormally low testosterone/epitestosterone ratios were detected in a group of HIV positive patients by means of SBSE-TD-GC/MS. Further research is required to determine the clinical significance of this finding in the context of HIV infection. Finally, the excessive urinary excretion of estrone and 17β-estradiol following the administration of a high dose of the conjugated equine estrogens to a female volunteer has also been demonstrated.

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phenolic chemistry is quite complex; natural phenolic compounds vary widely in terms of size and chemical properties. The high structural diversity within this family presents severe analytical challenges. High performance liquid chromatography (HPLC) is the preferred method for phenolic analysis; however, conventional HPLC methods offer limited separation power and often provide incomplete separation of the large number of components present in natural phenolic extracts. Multi-dimensional chromatographic techniques have proven much more effective in the analysis of complex samples. The current study explored the potential of comprehensive two-dimensional liquid chromatography (LC×LC) for the characterisation of phenolic compounds in complex natural products, with the emphasis on proanthocyanidins (PACs). Initial work focused on the evaluation of the state of the art in phenolic analysis, to allow information which was used in the development of optimal 1-D separations for use in LC×LC. The combination of hydrophilic interaction chromatography (HILIC) in the first dimension with reversedphase liquid chromatography (RP-LC) in the second dimension afforded an orthogonal and powerful separation system for phenolics, providing separation on the basis of hydrophilicity and hydrophobicity, respectively. A detailed and systematic procedure was therefore developed to allow the optimisation and evaluation of on-line, off-line and stop-flow HILIC×RP-LC methods. Results showed that all three approaches provide much better separation performance than conventional one-dimensional LC (1-D LC) techniques. On-line HILIC×RP-LC offers automation, shorter analysis times, better reproducibility and minimal sample exposure. The off-line and stopflow methods are characterised by much higher peak capacities, but relatively long analysis times. It was also demonstrated that stop-flow operation results in negligible additional band broadening for procyanidins (PCs), implying that this method is an attractive alternative to the off-line method as it offers automation and minimal sample handling. Experimental verification of the predictions based on fundamental principles confirmed the validity of the optimisation procedure for cocoa PCs. The hyphenation of on-line HILIC×RP-LC separation with fluorescence (FL) and mass spectrometry (MS) detection methods provided enhanced resolution in a practical analysis time with the added benefit of selective detection and greater certainty in compound identification. This strategy proved much more powerful, as demonstrated by the identification of the highly complex PACs in grape seeds based on chromatographic retention data in two dimensions and accurate mass information. It was further shown that on-line coupling of HILIC×RP-LC separation with an optimised radical scavenging assay provides an improved approach for screening of individual radical scavengers in complex phenolic fractions, as demonstrated for cocoa, grape seed and green tea extracts. / AFRIKAANSE OPSOMMING: Fenoliese chemie is baie kompleks; natuurlike fenoliese verbindings varieer in terme van beide grootte en chemiese eienskappe. Hierdie hoë strukturele diversiteit binne die familie bied daadwerklike analitiese uitdagings. Hoëverrigtingvloeistofchromatografie (HPLC) is die voorkeurmetode vir fenoliese analises, maar konvensionele HPLC metodes bied egter 'n beperkte skeidingsvermoë en verskaf dikwels onvolledige skeiding van die groot aantal komponente teenwoordig in natuurlike fenoliese ekstrakte. Multi-dimensionele chromatografiese tegnieke is bewys om baie meer effektief te wees met betrekking tot die ontleding van komplekse monsters. Hierdie studie ondersoek die potensiaal van omvattende twee-dimensionele vloeistof chromatografie (LC×LC) vir die karakterisering van fenoliese verbindings in komplekse natuurlike produkte, met die fokus op pro-antosianidiëne (PAC’s). Aanvanklike werk het gefokus op die evaluering van moderne tegnieke vir fenoliese analise – inligting wat in die ontwikkeling van optimale 1-D skeidings vir die toepasing in LC×LC gebruik is. Die kombinasie van hidrofiliese interaksie chromatografie (HILIC) in die eerste dimensie met omgekeerde-fase vloeistof chromatografie (RP-LC) in die tweede dimensie verleen 'n ortogonale en kragtige skeidingsisteem vir fenoliese komponente en verskaf skeiding op grond van onderskiedelik hidrofiliteit en hidrofobiteit. ‘n Gedetailleerde en sistematiese prosedure is dus ontwikkel om die optimisering en evaluering van aan-lyn, af-lyn en stop-vloei HILIC×RP-LC metodes uit te voer. Resultate het getoon dat al drie benaderings baie beter skeidingsvermoë bied as konvensionele een-dimensionele LC (1-D LC) tegnieke. Aan-lyn HILIC×RP-LC bied outomatisering, korter ontledingstyd, beter herhaalbaarheid en minimale monster blootstelling. Die af-lyn en stop-vloei metodes word gekenmerk deur 'n veel hoër piekkapasiteit, maar relatief lang ontledingstye. Daar is ook getoon dat die stop-vloei prosedure geringe bykomende bandverbreding vir prosianodiniëne (PC’s) tot gevolg het, wat beteken dat hierdie metode 'n aantreklike alternatief is vir die af-lyn metode aangesien dit outomatisering bied en minimale monster hantering behels. Eksperimentele verifiëring van die voorspellings gebaseer op fundamentele beginsels bevestig die geldigheid van die optimalisering proses vir kakao PCs. Die koppeling van aan-lyn HILIC×RP-LC skeiding met fluoressensie (FL) en massaspektrometrie (MS) deteksie verskaf verbeterde resolusie binne 'n praktiese ontledingstyd saam met die bykomende voordeel van selektiewe opsporing en groter sekerheid betreffende die verbindings se identifikasie. Hierdie strategie was baie meer kragtig, soos gedemonstreer deur die identifisering van die hoogs komplekse PAC’s in druiwepitte gebaseer op chromatografiese behoud van die integriteit van die data in twee dimensies tesame met akkurate massa inligting. Daar is verder getoon dat aanlyn koppeling van HILIC×RP-LC skeiding met 'n geoptimiseerde radikale vangers deteksie-metode 'n beter benadering bied om die gedrag van individuele radikale vangers in komplekse fenoliese fraksies te bestudeer, soos bewys is vir kakao, druiwepitte en groen-tee ekstrakte.

The reaction between nitrogen-containing organic compounds (pyridines and amines) and alumina was studied through the use of chromatographic titrations and elemental analysis. The aluminum-nitrogen bond was the basis for the preparation of a series of new chromatographic adsorbents. A series of amines and pyridine homologs were bound to alumina and evaluated as chromatographic stationary phases. The surface coverage was determined using elemental analysis. The adsorption properties of the stationary phases were investigated by determining differential enthalpies, entropies, and free energies of adsorption for a selected group of organic moieties. Rate theory was used to examine further the interaction between the molecular probes and the tailored supports. Relative peak broadening was measured both as a function of flow rate and temperature to provide a chromatographic evaluation of molecular probe-adsorbent interaction occurring during the chromatographic process. Results indicated that the single-molecule moieties used as tailoring agents served as excellent deactivating agents but produced no significant changes in the selectivity of the adsorbents. Pellicular beads were synthesized by using 4-vinylpyridine as a linking agent between the alumina substrate and a series of polymers. The polymers used in this study were polystyrene, polymethylmethacrylate, and polyacrylonitrile. Each type of polymer-coated bead was prepared at different loading levels. Scanning electron microscopy was used to examine the gross change in the surface and elemental analysis used to determine the polymer loading. The adsorption properties of the polymeric pellicular supports were studied by determining the aforementioned thermodynamic quantities and by rate theory. The relative peak broadening was mentioned as a function of polymer type, loading, flow rate, and temperature. The chromatographic behavior of the selected molecular probes on the pellicular supports was sensitive to polymer type and loading. It was established that alumina can be modified with amines and pyridines and that a molecule possessing a nitrogen atom and a polymerization site can be used as an adhesive interface to chemically link polymeric phases to alumina. Currently, there are no commercially available modified aluminas. It has been demonstrated here that the preparation of such supports is feasible and can lead to chromatographically useful products.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

This study was conducted to set up a systematic approach utilizing advantages of both wet lab and bioinformatic methodologies to study biodegradation abilities and microbial bacterial-functional relationship within bioremediation communities. Firstly, 11pharmaceuticals and personal care products (PPCPs)were selected as target chemicals for establishing an effective determination process in analyzing trace-level concentrations in the environment, and understanding the removal routes during pollutants removal process in wastewater treatment process using activated sludge. Ultra performance liquid chromatography-tandem mass spectrometry was utilized to develop a rapid, sensitive and reliable method without solid phase extraction pre-concentration for trace analysis of 11 PPCPs in influent and effluent from municipal wastewater treatment plants. Shorten the detection time and significant reduction of detection cost were achieved due to the omitting usage of solid phase extraction (SPE)process and avoiding the consumption of hydrophiliclipophilic balancced (HLB)cartridge. Research on removal routes of ten selected PPCPs in activated sludge found activated sludge hardly removed carbamazepine. Biodegradation was the sole route to remove acyclovir, metronidazole, benzylparaben, ethylparaben, methylparaben and propylparaben. Both adsorption and biodegradation were involved in the removal of ranitidine and benzophenone-3, while fluoxetine could be totally removed by adsorption in activated sludge. Secondly, as the target microbial community, activated sludge community was used to set up the global bioinformatic analysis process. Both metagenomic and metatranscriptomic approaches were processed to characterize microbial structure and gene expression of activated sludge community. The taxonomic profile showed thatactivated sludge was dominated by Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobiaphyla. Gene expression annotation of nitrogen removal revealed that denitrification-related genes sequences dominated in both DNA and cDNA datasets while nitrifying genes were also expressed in relative high levels. Specially, ammonia monooxygenase and hydroxylamine oxidase demonstrated the high cDNA/DNA ratios, indicating strong nitrification activity. Ammonia-oxidizing bacteria present mainly belonged to Nitrosomonas and Nitrosospira species. A fast method to construct local sub-databases has been established for the quick similarity search and annotation of huge metagenomic datasets. The conducted tests showed sub-database annotation pipeline achieved a speedup of ~150-385 times, and got exactly the same annotation results with those of the direct NCBI-nr database BLAST-MEGAN method. This approach provides a new time-efficient and convenient annotation similarity search strategy for laboratories without access to high performance computing facilities. Thirdly, bisphenol A(BPA), which has a partially known biodegradation pathway and relevant bioremediating genes, was chosen as a model to establish a pipeline for systematical understanding the pathways and gene/bacteria relationships in an enriched microbial community. 11 new metabolites were detected during BPA degradation. Thereby, a novel pathway of degrading BPA metabolite was proposed. Sphingomonas strains were dominant taxa in initial degradation of BPA, while the other taxa were competing BPA metabolites during degradation. Metagenomic binning results showed a cytochrome P450 monooxygenase system, which was previously reported BPA mediator, was sharing by two Sphingomonas strains, showing the undergoing mechanism of competition of the two strains. The observations suggested bacterial specialization may occur in that community that each taxon was selected to degrade certain metabolite in a community economical way. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy