Capillary electrophoresis (CE) has a wide range of applications in the field of analytical chemistry. In general the most expensive part in a CE system is the detector due to the fact that the detector must have a high sensitivity for small detection volumes and low concentrations. Building portable instruments is one way to make the instruments cheaper and has the advantage that they can be used virtually everywhere. However, downscaling of CE instruments puts some extra demands on the detector. This report describes the design and building of two homemade light-emitting diode (LED) based detectors; a LEDabsorbance photometric detector (LED-AP) and a LED-induced fluorescence (LED-IF) detector. The main goal was to install them inside a portable CE and make a simple separation. The performance of the two detectors had to be evaluated before the main goal could be achieved. p-Nitrophenol was used to create a sensitivity graph for the LED-AP detector, calculating the upper linearity to 5.6 mM when the sensitivity had dropped 10 % caused by non-linearity. The sensitivity graph also showed that the detector had an effective pathlength of 74.2 µm and a stray light of 4.5 % for a 75 µm i.d fused-silica capillary. The LED-IF detector was evaluated by determining the limit of detection (LOD) for fluorescein, at a signal to noise ratio of 3. The LOD was 0.72 µM ± 0.01 µM when immersion oil was used to limit the light scattering from the optic fibres in to the capillary and 0.58 µM ±0.02 µM when silicone oil was used. Without doing any improvements only the LED-AP detector could be used in the portable CE. As a common application area for portable CE instruments is environmental analysis, indirect detection using p-nitrophenol as a probe for separating anions was done to test the system. All analytes were eluted in less than 4 minutes.

Two types of fluorescent molecules had been designed and synthesized to improve the analytical infrastructure of capillary electrophoretic separations. First, a hydrophilic version of the permanently cationic acridine-based fluorophore, HEG2Me2-DAA was synthesized. HEG2Me2-DAA has a lambda^ex max of 490 nm which matches the 488 nm line of the commonly used argon ion laser. The emission spectra of HEG2Me2-DAA are pH-independent. HEG2Me2-DAA was used in capillary electrophoresis with an aqueous background electrolyte and was found to be free of the detrimental peak tailing of the acridine orange-based fluorophore that was caused by adsorption on the inner wall of the fused silica capillary. Bovine serum albumin was labeled with excess of the designed amine reactive reagent and the lowest concentration at which the tagged bovine serum albumin was tested was 15 nM. Chicken ovalbumin was also labeled with FL-CA-PFP and analyzed by capillary isoelectric focusing (cIEF) with LIF detection. The pI values of the tagged proteins shifted in the alkaline direction by about 0.02 compared to the pI values of the non-tagged proteins. A tri-functional probe intended to enable selective enrichment and selective detection of a variety of molecules (e.g., natural products, pharmaceuticals, inhibitors, etc.) was also designed and synthetized by combining FL-CA with biotin and an azide group in a "proof-of-principle" level experiment. In cIEF, the profile of the pH gradient can only be determined with the help of pI markers. A large set of pyrene-based fluorescent pI markers was rationally designed to cover the pI range 3 to 10. To prove the feasibility of the proposed synthetic approach, the subgroup of the pI markers having the greatest structural complexity was synthesized and characterized. The classical zone electrophoretic pI determination methods failed due to severe chromatographic retention of the APTS based pI markers on the capillary wall. Exploratory work was done to design a new pI value determination method that combines the advantages of the immobilized pH gradient technology of the OFFGEL instrument and the carrier-ampholyte-based IEF technology. The method aspects of cIEF have also been improved in this work. The new segmented loading method yielded a more linear pH gradient than the previously known cIEF methods. To exploit a unique property of the newly developed fluorescent pI markers, we used them as pyrene-based ampholytic carbohydrate derivatizing reagents. The pI4 carbohydrate derivatization reagent proved advantageous over 8-aminopyrene-1,3,6-trisulfonic acid (APTS): the pI4 conjugates have higher molar absorbance at 488 nm than the APTS conjugates and become detectable in positive ion mode of MS affording better detection sensitivity.

Capillary electrophoresis (CE) is an analytical technique that is very useful for investigating processes that modify the charge and mass of proteins and polypeptide pharmaceuticals. This report explores the ability of CE to determine the aggregation constant between insulin molecules. Bovine insulin is a polypeptide (Mw=5733, pI = 5.3) that has two α-amino groups (Gly and Phe) and one ε–amino group (Lys). Analysis of concentration dependence of electrophoretic mobility of insulin at different conditions yields the association constant for dimerization of insulin. The association constant estimates how tight the peptide molecules are associated. The association constant is a useful factor to evaluate the purity of a peptide or protein sample. The association reaction of bovine insulin molecules was found to be favoured by temperature. The association constants were 7200 M -1, 8000 M -1, and 36000 M -1 at 15 oC, 25 oC and 35 oC, respectively. The interactions between the peptide molecules increase at higher temperature, resulting in stronger association. The association constant was estimated to be 3000 M -1in the presence of dioxane (5%, w/v %) at 25 oC. However, the interaction sites remain to be explored.

The research presented in this thesis involves the application of chiral cationic and anionic surfactants for simultaneous enantioseparation of structurally similar compounds in capillary electrophoresis (CE) and CE coupled to mass spectrometry (CE-MS). The first chapter briefly introduces the fundamentals of CE and CE-MS, emphasizing the micellar electrokinetic chromatography (MEKC) and MEKC-MS techniques, as well as ionic liquids (ILs) and affinity CE (ACE). In chapter 2, a mixture of five racemic profen (PROF) drugs are simultaneously separated with the combined use of 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) and IL-type surfactant, N-undecenoxycarbonyl-L-leucinol bromide (L-UCLB). Enantioseparations of these PROFs are optimized using a standard recipe containing 35.00 mM TM-β-CD, 5.00 mM sodium acetate at pH 5.0, and varying the concentration as well as chain length of the IL surfactants. The batch-to-batch reproducibility of L-UCLB is found to be acceptable in terms of enantiomeric resolution, and migration time. A competitive inhibition mechanism is proposed to investigate the ternary interactions among TM-β-CD, ILs, and PROFs. The apparent binding constant of TM-β-CD to L-UCLB is estimated by nonlinear and linear plotting methods. The binding constants of one representative PROF (e.g., fenoprofen) to TM-β-CD and to L-UCLB are estimated by a secondary plotting approach. The R- and S-fenoprofen having different binding constant values, resulting in the enantioseparation due to the synergistic effect of TM-β-CD and L-UCLB. The R- and S-configurations of barbiturates display differences in potency and biological activity. In Chapter 3, a multivariate MEKC-ESI-MS approach for the simultaneous analysis of the racemic mixture of three barbiturates is presented. The chiral selector employed is the polymeric surfactant polysodium N-undecenoxycarbonyl-L-isoleucinate. The central composite design is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise ratio for these barbiturates. In preliminary experiments, the ranges of the factors investigated in the multivariate approaches are determined. Then the multivariate optimizations are conducted to determine the best overall chiral resolution with shortest possible run times for barbiturates. The limit of detection of ESI-MS is several folds higher compared to the UV detection. The predicted optimum results are in good agreement with the experimental data.

Sulfonated porphyrins and phthalocyanines have been under consideration as microbicides, compounds which, when used in a topical formulation, can prevent transmission of the human immunodeficiency virus. Our studies have been directed toward the characterization of members of these classes. For the sulfonated phthalocyanines, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was helpful in determining the extent of sulfonation. We present the first report of spectroscopic characterization of a pentasulfonated phthalocyanine. Capillary electrophoresis data were sensitive to the concentration of the compounds (Chapter 1). Mass spectrometry was also very useful for establishing the extent of sulfonation in series of sulfonated porphyrins. Capillary electrophoresis was very useful in separating mixtures of these species. A study on sulfonation of a series of tetra(difluorophenyl)porphyrins showed that species with red-shifted Soret peaks were being formed. Data were consistent with an intramolecular sulfone bridge from the phenyl substituent to the porphyrin core. Sulfonation of the tetranaphthylporphyrins ring readily gave more than one sulfonic acid group per naphthyl side chain (Chapter 2). In cancer chemotherapy of solid tumors, it is desired to kill the tumor cells with minimal damage to the surrounding tissue. Brachytherapy seeds have been a considerable help in this regard for some tumors. In further developing approaches to selective tumor damage, we have evaluated a technique, Auger Electron Therapy (AET) in which one introduces a compound that is expected to bind to DNA, absorb the radiation, and then catalyze clustered DNA damage via release of a series of Auger electrons. We chose a series of metals (silver, indium, molybdenum, palladium, platinum, ruthenium, silver and zirconium) with appropriate energy levels to absorb an x-ray photon from the brachytherapy seed and used the tetracationic porphyrin 5,10,15,20-tetrakis(1-methylpyridinium-4-yl) porphyrin (TMPyP4) as a scaffold. The amount of clustered DNA damage was quantitated by a plasmid assay. Experiments evaluated the effect of buffer, concentration of glycerol, irradiation time, and concentration of the porphyrin. No metal studied gave significant double stranded (localized) DNA damage. Significant single stranded DNA damage was observed, however, in the order zirconium >> ruthenium > palladium > platinum > silver ~ indium (Chapter 3).

University of Macau / Institute of Chinese Medical Sciences

Microfluidics emerged during the early 1990s with channel networks in silicon or glass. Microprocessing of these materials is labor-intensive and time-consuming, it requires sophisticated equipment in a clean room, and often involves hazardous chemicals. The subsequent use of polymer greatly simplified the fabrication of microchips and led to the rapid development of the field. Polymer such as poly(dimethylsiloxane) (PDMS), has other attractive properties, such as being elastic (easy to make efficient microvalves), permeable to gases, and compatible with culturing biological cells. Despite these advantages, applications of PDMS chips are severely limited by a few drawbacks that are inherent to this material: (i) strong adsorption of molecules, particularly large biomolecules, onto its surface; (ii) absorption of nonpolar and weakly polar molecules into PDMS bulk; (iii) leaching of small molecules from PDMS bulk into solutions; and (iv) incompatibility with organic solvents. To overcome all these problems, Teflon plastics seem to be the perfect solution. They are well-known for their superior inertness to almost all chemicals and all solvents; they also show excellent resistance to molecular adsorption and molecule leaching from the polymer bulk to solutions. However, Teflon has a high chemical inertness of the surface, which is restricted the bonding temperature (>260¢XC).It is not conducive to the low-temperature packaging process. This study presents a simple and rapid process for sealing Teflon-based microfluidic chip at a temperature of 140oC which is lower than typical bonding temperature of 260oC. A simple ammonium plasma treatment is used to enhance the surface energy of Teflon substrates such that the bonding temperature can be greatly reduced. Results indicate that the ammonium plasma treated Teflon substrates can be sealed using hot press bonding at a temperature of 140oC for 20 min. The measured iv bonding strength for the Teflon-based microfluidic devices is higher than those bonded at a reported temperature of 260oC for 60 min. It shows the measured contact angle for the Teflon substrates treated with different plasmas. Results indicated that the ammonium hydroxide plasma exhibited the best wettability property and the contact angle reached the minimum value of 45o after 5 min of treatment. The ESCA analysis showed the best Defluorination by ammonium plasma. The fluorine/carbon atomic ratio degraded from 1.96 to 1.10 by 5 minutes. The measured bonding strength for the Teflon substrates bonded with different surface activation protocols. Results showed that the bonding strength was enhanced upto 93% after the plasma treatment. The plasma treatment not only enhanced the bonding strength but also reduced the bonding temperature and time. The measured surface roughness only increased 15¡Ó5 nm (Ra) after the plasma treatment, which is acceptable for most applications in microfluidic systems. Finally, the fluorescence optical architecture and cross-chip successfully detected and isolated £XX-174 fragment of DNA samples confirmed the Teflon substrate for the emerging microfluidic plastic chip. The developed method provides a simple and rapid way to fabricate Teflon-based microfluidic devices.

This high-resolution biogeochemical study investigated spatial and temporal variability in the mixing interface zones within a wetland-aquifer system near a municipal landfill in the city of Norman, Oklahoma. Steep biogeochemical gradients indicating zones of enhanced microbial activity (e.g. iron/sulfate reduction and fermentation) were found at centimeter-scale hydrological and lithological interfaces. The small resolution study was achieved by combining passive diffusion samplers with capillary electrophoresis for chemical analysis. The spatial and temporal variability of biogeochemical processes found at the interfaces was evaluated in a depth profile over a period of three years. Correlations between geochemical parameters were determined using Principal Component Analysis (PCA) and the principal factors obtained were interpreted as a dominant biogeochemical process. Factors scores were mapped by date and depth to determine the spatial-temporal associations of the dominant processes. Fermentation was the process controlling the greatest variability in the dataset followed by iron/sulfate reduction, and methanogenesis. The effect of seasonal and hydrologic changes on biogeochemistry was evaluated from samples collected in a wet/dry period from three locations exhibiting upward, downward, and negligent hydrologic flow between aquifer and wetland. PCA was used to identify the principal biogeochemical processes and to obtain factor scores for evaluating significant seasonal and hydrological differences via analysis of variance. Iron and sulfate reduction were dominated by changes in water table levels and water flow paths, whereas methanogenesis and bacterial barite utilization were dominated by season and associated with a site with negligible flow. A preliminary study on microbial response to changes in geochemical nutrients (e.g. electron acceptors and electron donors) was conducted using in situ microcosms with the purpose of quantifying iron and sulfate reduction rates. Problems encountered in the experiment such as leaks in the microcosms did not allow the determination of respiration rates, therefore the experiments will be repeated in the future. The results suggest that iron and sulfate reduction were stimulated with the addition of sulfate and ferrihydrite (electron acceptors) and acetate and lactate (electron donors). This research demonstrates the importance of assessing biogeochemical processes at interface zones at appropriate scales and reveals the seasonal and hydrological controls on system processes.

Disulfide bonds play important roles in establishing and stabilizing three-dimensional protein structure, and mass spectrometry (MS) has become the primary detection method to decipher their biological and pathological roles. Several experimental methods before or after MS detection have been developed to aid in disulfide bond assignment, such as tandem MS followed by database searching or modification of the disulfide bond via chemical reduction or oxidation. Despite these technological advancements, the detection and proper assignment of disulfide bonds have remained experimentally difficult. Therefore, we have developed an alternative method for disulfide bond elucidation using capillary electrophoresis-mass spectrometry (CE-MS) combined with an on-target performic acid oxidation method for matrix assisted laser desorption/ionization (MALDI) deposited samples. An information rich CE-MS method that results in distinct charge-state trends observed in two-dimensional plots of log(mu eff) versus log (MW) was developed to enhance the confidence of peptide and protein identifications. The charge-state trends provide information about the number of basic amino acid residues present within each peptide. This information can be used to develop methods to screen for posttranslationally modified peptides (e.g., phosphorylation, disulfide bonds, etc.). In the case of disulfide bonds, the highly charged peptides (i.e., 3, 4 or greater charge states) have a high probability of being disulfide-linked peptides, owing to charge contribution of both peptides forming the disulfide bridged peptide. However, intra-linked disulfide bridged peptides can also be present at lower charge states. Therefore, a chemically selective method to rapidly locate disulfide-linked peptides that have been separated by CE-MS must be developed. An on-target performic acid oxidation method was developed to provide the chemical selectivity towards disulfide bonds, i.e., converting the cystine bond to form two peptides modified with a cysteic acid (SO3H) side chain. The on-target oxidation method offers (i) no post-oxidation sample cleanup, (ii) improved throughput over solution-phase oxidation methods, and (iii) easily adapted to CE separations coupled offline with MALDI-MS. The evaluation of the on-target oxidation experimental parameters, the fragmentation behavior of cysteic acid-containing peptides and an alternative method for disulfide bond elucidation, using CE-MS combined with the ontarget oxidation method, are discussed within.

Fluorescent labeling can improve the detection sensitivity in capillary electrophoretic (CE) separations down to attomolar concentrations. However, most fluorescent labels are not compatible with CE because their fluorescence properties and charge states are pH-dependent, they are often hydrophobic and they have a tendency to significantly change the properties of the analytes after labeling. A group of fluorescent labeling reagents have been prepared whose fluorophores have properties that are optimized for CE separations. These fluorophores have fluorescence properties and charge states that are independent of pH in the 2 < pH < 11 range. Their excitation maxima are also compatible with the 488 nm line of the Argon ion laser. A mono-cationic acridine-based fluorescent label was prepared and was found to not shift the pI of a labeled model protein in capillary isoelectric focusing separation (cIEF). Lower loading, due to increased sensitivity, led to better resolution of closely spaced isoform peaks having a pI = 0.05. A tri-anionic pyrene-based fluorescent labeling reagent was also synthesized and was used in the sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) separation of proteins. The fluorophore led to an LOQ in the nM range, and did not alter the migration behavior of proteins in the sieving matrix. A third fluorescent labeling reagent was developed as a solid phase reagent (SPR) where the fluorophore was immobilized on a solid surface through a cleavable anchor. The fluorophore is di-anionic and is based on pyrene. The SPR was designed to allow the simultaneous capture and labeling of an analyte and the efficient release of the label-analyte conjugate under mild acidic conditions. The use of the SPR allowed the labeling of a diamine whose concentration was in the low nanomolar range. The SPR opens up the possibility for mono-labeling and proportional multiple labeling of proteins.