This work deals with development and optimization of a method for separation and detection of pro- tamines using capillary zone electrophoresis. The developed method uses a fused silica capillary with inner diameter of 50 µm and effective length of 41,5 cm. Driving voltage is 30 kV. Background electrolyte is aque- ous solution of 45 mmol dm−3 phosphoric acid. Analyte is detected spectrophotometrically at wavelength of 200 nm. Sample is injected hydrodynamically. The method allows for determination of protamines in the concentration range between 11 µg ml−1 to 1000 µg ml−1 , limit of detection is 4 µg ml−1 . Viablity of the method has been verified with a real sample of NPH insulin injection. For sufficiently sensitive detection of protamines in NPH insuline it is necessary to prepare the sample in acidic environment. For the pur- poses of this work the sample was acidified by addition of background electrolyte into the sample so that the background electrolyte concentration in the resulting solution of the sample was 18 mmol dm−3 . The main advantage of the method is the rapid analysis, migration time of the analyte is about 2 min. Disadvantage of the method in comparison to alternate methods using CZE or RP-HPLC is the inability to separate individ- ual protamine peptides. This disadvantage...

As cyanobacteria (also known as blue green algae) produce a range of cyclic peptides which are highly toxic, capillary electrophoresis and associated techniques have been investigated to assess their applicability for toxin monitoring in the water bodies of kwaZulu Natal, South Africa. Capillary electrophoresis (CE) is a technique in which charged molecules can be efficiently separated in a buffer solution within a capillary tube under the influence of a strong electric field. Two CE modes, namely capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) were initially evaluated using a laboratory-built CE instrument. The former mode lacked selectivity due to the similar charge to size ratio of the algal toxins. However, with the latter mode, incorporation of a surfactant (sodium dodecyl sulphate) into the buffer, produced sufficient resolution between components. Parameters including surfactant concentration, buffer ionic strength, buffer pH and operating voltage were systematically optimized to separate the four algal toxins under investigation (microcystin YR, microcystin LR, microcystin RR and nodularin). The optimum separation conditions were: 30 mM borax, 9 mM sodium dodecyl sulphate, pH 9.18, 30 kV applied voltage, 10 s hydrodynamic injection, 70 cm x 50 Ilm Ld. bare fused silica capillary (LEFF 40 cm) and UV detection at 238 nm. Under these conditions, typical detection limits were in the low ng/IlL range (14.13 ng/IlL for microcystin LR to 29.85 ng/ILL for nodularin). The MECC method was evaluated in terms of migration time precision, efficiency and resolution, peak area and normalised peak area precision. Standard deviation values for retention times acquired using replicate electrokinetic injections ranged from 0.018 to 0.054 and 0.069 to 0.148 for hydrodynamic injections. Normalised peak area precision for replicate hydrodynamic injections were in the range 84 to 97 % RSD, while improved % RSD values of 11.5 to 18.7 were achieved for electrokinetic injections. Due to poor precision resulting from the lack of automation on the laboratory built CE system, poor correlation between increasing concentration and a corresponding change in normalised peak areas were achieved. The MECC method developed was applied to the analysis of an algal scum extract to illustrate the technique. A general problem with CE is that it suffers from poor detection sensitivity. Hence in this study, alternative injection modes, sample concentration strategies and alternative detection techniques were investigated in an attempt to improve detection limits for algal toxins. Using optimized electrokinetic injection conditions, detection limits were five to ten times better than those obtained with hydrodynamic injections. On-line sample concentration methods were partially successful. Field amplified back and forth MECC in which analyte injected in the entire column volume and subsequently focused in a narrow band by manipulating the electric field, resulted in an enormous sensitivity enhancement that ranged from 197 times for microcystin RR to 777 times for microcystin YR when compared to hydrodynamic injections. Field amplified sample stacking (FASI) was ineffective for toxin preconcentration, while electro-extraction produced detection limits ranging from 0.27 ng/J.tL for microcystin YR to 1.08 ng/J.tL for microcystin RR. Solid phase extraction, in which analytes are first trapped and concentrated on HPLC material in a cartridge and then eluted in a more concentrated form for injection, was found to be practical only in the offline mode. A concentration detection limit of less than 0.002 ng/J.tL was obtained. Attempts with on-line solid phase extraction failed due to problems associated with coupling the cartridge with the separation capillary. Finally, laser induced fluorescence (LIF) detection was investigated as an alternative to UV detection. Unfortunately, the algal toxins were not amenable to LIF detection because tagging with the fluorescent moiety, fluorescein isothiocyanate (FITC), was prevented by the stereochemistry of these cyclic peptides. A comparative study between HPLC and MECC revealed that the former displayed poor efficiency peaks and long analysis times for toxin analysis. However HPLC was superior in terms of retention time precision (0.12 to 0.64 % RSD) and area precision (1.78 to 2.86 % RSD). Mass detection limits for MECC (0.0142 to 0.0603 ng) were far superior to those achieved by HPLC (0.55 to 1.025 ng). In addition to HPLC and MECC, a preliminary investigation of micro-high performance liquid chromatography (J.tHPLC) and capillary electrochromatography (CEC) for the analysis of algal toxins was made using 50 J.tm Ld. capillary columns packed in-house, with reverse phase HPLC packing material. / Thesis (M.Sc.)-University of Natal, 1997.

Isotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient concentrations without heart-cutting or splitting. Examples of comprehensive ITP-CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of individual ITP zones. Direct online coupling of ITP to CE in this comprehensive arrangement involved the use of columns having different diameters with one directly inserted inside the other. A counterflow was applied when the isotachophoretic sample stack reached the bifurcation point. Large volume (10 µL) injections were made using an electrically-insulated commercial polymeric rotary valve injector for increased reproducibility compared to previous comprehensive ITP-CE studies, with ITP and CE retention time RSD values ranging from 2-5%. An ultraviolet (UV) detector positioned at the bifurcation point was used to determine the beginning of CE injection. Application of a splitting voltage at the bifurcation point showed no affect on analyte transfer into the CE column. By using multiple injections of the ITP band(s), CE column overloading was not observed. Online capillary isotachophoresis (ITP) and comprehensive isotachophoresis-capillary electrophoresis (ITP-CE) were also coupled with electrospray ionization (ESI)-orthogonal acceleration time-of-flight mass spectrometry (TOFMS). Separations were performed using 200 µm I.D. and 50 µM I.D. polyvinylalcohol (PVA)-coated fused silica capillaries for ITP and CE, respectively. Both ITP and ITP-CE were coupled to TOFMS for analysis of sufficient (10-5 M) and insufficient (10-6-10-7 M) concentrations of angiotensins in mixtures. ITP-TOFMS of a single mixed zone of five angiotensins (3 x 10-7 M) showed that ion suppression due to the co-elution of angiotensin III in the electrospray significantly reduced the ionization of other analytes. A practical solution to the detection difficulties for ITP mixed zones involved the insertion of a CE separation between the ITP and TOFMS for online preconcentration, separation, and identification in one system.

Drugs used in the pharmaceutical industry often occur as a mixture of several isomers with a different biological activity. In a case that some isomer provides an undesirable side effect, it is important to separate it from the mixture and check the chiral purity of a drug. Capillary zone electrophoresis plays a significant role in chiral separations. A different affinity of isomers to complexation reagent is used for their separation from each other. The extent of their interaction is characterized by the complexation constant. Most commonly the cyclodextrins are used for the chiral separations of β-blockers and they could be in neutral or charged form. They probably interact with them through the creation of inclusion complexes. A successful baseline enantioseparation of all the β-blockers that have been studied, labetalol, pindolol, alprenolol and atenolol, was provided by using the background electrolyte containing charged cyclodextrins. The highest resolution of peaks was observed using sulfated cyclodextrins.

in English The concise summary of literary information about bilirubin and its structure derivative ranarubin is the topic of this thesis. The experimental part is dedicated to investigation of some properties of this substances and their comparison. The rate of degradation and acid- base behaviour was monitored under the laboratory conditions using capillary zone electrophoresis. The results point to differences in their behaviour.

Techniques of capillary electrophoresis are essential for both the quantitative and qualitative analysis of a large variety of compounds. They find application in many areas of chemistry, from pharmacological industry and food processing, up to highly specialized biotechnological laboratories. Over the last hundred years, their mathematical model has been described with precision. Thus, current research mainly aims for the development of more advanced and more specialized applications. During the greatest boom of affinity capillary electrophoresis within the nineties, many authors would describe similar phenomena under different names. The first part of this work focuses on the consolidation and unification of the problematics and terminology of this method. It also discusses the phenomena which can affect electromigration and separation during electrophoretic processes. We will focus on a specific subset of affinity capillary electrophoresis, partial filling capillary electrophoresis, which, to our knowledge, has not been fully theoretically explored yet, and present a mathematical model allowing the determination of complexation constants and state some limitations of this approach. The second part of this thesis focuses on the development and application of computer softwares, which are meant...

Surfactans are synthetically made surface-active ingredients contained in washing and cleaning products. They decrease the surface tension and remove dirt. Surfactans can be divided into four basic classes: anionic, cationic, non-inonic and ampholitic. The most commonly used are the anionic ones. Thanks to the massive use they penetrate into waste water and can disturb the environment. This thesis is focused on the optimization methods and the determination of three anionic surfactants by capillary zone electrophoresis.

Evaluating data in capillary zone electrophoresis usually involves many steps that require using several different programmes. Apart from evaluating the electrophoreogram itself, it is usual to process the obtained data in some other way. For example, a suitable model is fit to the data in order to obtain physical and chemical parameters of the separation (e.g. stability constant in case of complexation). It is also important to know the accuracy of the evaluation (the calculation error). In this work, new parts of the Eval programme, originally developed for electrophoreogram evaluation, were implemented. The programme now automatically estimates the Haarhoff-van der Linde function (solution of continuity equation in capillary) parameters for analyte peak. Complexing agents are often used to improve the separation in the capillary zone electrophoresis. Complexation in the capillary can be described by its physical and chemical parameters. A new part was added to the Eval programme that allows the user to fit a rectangular hyperbole function to the obtained data. Thus, the regression parameters of this dependence can be gained. The programme can also draw profile diagrams for these parameters, from which the confidence intervals can be read. An option that allows two dependencies to be fitted at...

Martina Riesová Abstract of Ph.D. Thesis Capillary electrophoresis is a method of choice in many analytical laboratories for its high separation efficiency, rapidity, low consumption of chemicals and therefore low costs. Inherent to each electrophoretic separation system are system peaks, which can significantly affect or confuse the electrophoretic results. In capillary zone electrophoresis, position of system zones can be predicted easily and reliably by means of prediction software based on a theoretical description of electromigration. However, the prediction of only position of a system zone may not be sufficient for identification of system peaks in obtained electropherograms. Therefore, an existing theoretical model was significantly extended and new version of PeakMaster software (PeakMaster 5.3) was introduced in the framework of this thesis. PeakMaster 5.3 enables to predict not only the positions of system zones, but also their shapes and polarity. Thus, PeakMaster 5.3 improves the prediction possibility of overlapping or interaction of system peaks with analyte peaks. Moreover, composition of the sample can be optimized in order to obtain convenient shapes and amplitudes of system peaks. The applicability of capillary zone electrophoresis can be extended by addition of a complexation agent into...

A determination of corrosion inhibitors in engine coolants represent a difficult analytical problem due to their different physicochemical properties. Nowadays a lot of instrument methods are needed to determine them. The aim of this work was the development of methods for their determination using only one instrumentation. Capillary zone electrophoreses was chosen as a suitable technique. Three electrophoretic methods for three groups of corrosion inhibitors were developed. Firstly, method for determination of inorganic anions was developed in range from 5 to 50 ppm with limit of detection around 1 ppm. Background electrolyte contains sodium chromate, CTAB and CHES solution. Analytes were detected indirectly at 450 nm. Another method was for determination of organic acids anions. They were determined in range from 5 to 500 ppm. Limit of detection for each analyte was around 1 ppm. Measurement was made with PDC a CTAC water solution. Indirect detection was used for this determination at 350 nm. Last developed method can be use for determination of aryltrizoles in range from 5 to 500 ppm. Limit of detection was around 1 ppm. As a background electrolyte was used sodium tetraborate solution. The developed methods were validated and their suitability for determination of corrosion inhibitors in real...