Applications of CZE

Applicability of CZE to the Edman phenylthiohydantion derivatives of amino acids (140) is limited because the neutral amino acids cannot be resolved by this method and by the reduced thickness of the sample requiring relatively high concentrations of the fluorescent material for detection. These limitations may be overcome by a micellar technique that confers mobility to neutral 140 species and by application of thermotropic detection that allows one to detect a few tens of fmol of the derivative, obtained after injecting ca 0.5 nL, at a concentration of ca 1 p-M330. 

An interesting application of CZE was the study of the interaction of human serum transferrin and fluorescein isothiocyanate (FITC). Measurements were carried out in an uncoated fused-silica capillary (total length 59 cm 75 pm i.d. effective lengths for... 

Busch et al. studied the applicability of CZE to the examination of hapten-antibody complex formation (11). The catalytic antibodies examined have been used to accelerate a Diels-Alder reaction. Association constants of two hapten-antibody complexes were investigated and compared to the ELISA method. The samples contained buffer, hapten, and antibody. The constants obtained with CZE are a factor of 3-5 larger than those found with the ELISA method. The free-hapten concentration is measured directly this allows confirmation of the stoichiometric model. Because of the poor concentration sensitivity of UV detection, the application of an extended optical path length such as a bubble cell is necessary to obtain reliable binding parameters. 

Chromatographic and related electrophoretic methods for the separation of transition metal complexes or their ligands were reviewed . Micellar electrokinetic chromatography (MEKC) presents a new development in the field of capillary zone electrophoresis (CZE). The use of micellar solutions expands the application of CZE to electronically neutral solutes, as well as charged ones. Thus, electrically neutral / -diketonates Cr(dik)3, Co(dik)3, Rd(dik)3, Pt(dik)2 and Pd(dik)2 were separated by CZE in micellar solutions of sds. A linear log-log relationship was found between the distribution coefficient and the partition coefficient of the complex between dodecane and water, which was used for prediction of both the distribution coefficients and the migration times of different metal complexes . 

The CE analysis of phenolic acids in complex matrices such as beer was iuvestigated. The voltammetric end determination required separation of interfering components and optimization of pH at the various stages of the procedure ". Application of CZE and MEC with DA-UVD to the analysis of antioxidants was investigated. Gallic acid (8) and some of its derivatives (33a-c, the amide of 8 and its trimethyl ether), BHA (31) and BHT (32a) were only partially resolved by CZE, whereas full resolution was achieved by MEC l... 

Applications of cze include the detection of trace amounts of DNA and the separation of peptide fragments. Furthermore, this technique is beneficial to forensic scientists because restriction mapping can be performed, allowing assays for DNA to be carried out at the scene of a crime (see Forensic testing). It is also possible to interface capillary electrophoresis on-line with a mass spectrometer as a sample introduction technique in the analysis of amino acids and proteins (70). Further improvements in capillary electrophoresis include the need to increase column capacity. Most reported separations involve the resolution of only 20—30 components, whereas high resolution hplc is capable of resolving several hundred components in a mixture (see Chromatography). 

Other Techniques. A growing technique related to lc/ms and regarded as complementary to it is that of capillary zone electrophoresis/mass spectrometry (cze/ms) (22). Using cze/ms, high resolution separation of water-soluble compounds is accompHshed by the principles of electrophoresis (qv). The sample is then coupled to the mass spectrometer by electrospray ionization (23) or a fast atom bombardment interface (fab) to produce molecular ions (24). Biotechnology applications of cze/ms have great potential (25). 

The CZE analysis of Tf seems to be an alternative to CDT measurements kits, which are prone to inaccurate diagnostics. Normal CDT ranges are method-dependent and, consequently, results must be interpreted by taking into account method-specific cutoff values [176,184], In any case, several authors support the application of CZE for CDT determination in the clinical laboratory [191] and claim that it represents an alternative to HPLC and that it should be taken into account as a reference method for CDT [67], In comparison to HPLC, CZE methods have the advantages of easier sample preparation, faster analysis times, higher isoforms resolution, and faster column reconditioning [197],... 

The literature comprises a larger number of methodologies employing electromigration principles, diverse preconcentration strategies, and a variety of detection schemes for pollutant standards combined as test mixtures and for real samples. Tables 31.1 through 31.9 compile representative applications of CZE and EKC methods to the most important classes of pollutants in different environmental compartments. 

CZE separation of synthetic dyes has been approached by simple (borate and citrate) and volatile buffers (ammonium acetate) modified by solvents as well as nonaqueous systems (ammonium acetate/acetic acid in MeOH). Environmental applications of CZE methodologies include the analysis of spent dyebaths and wastewater samples and the monitoring of groundwater migration, where eosin was used as a fluorescent tracer (details in Table 31.8). ... 

Table 6.10 reports the main areas of application of the various ionisation methods and the principal ions detected. A breakdown of MS techniques applied to various types of analytes is as follows thermally stable, low-MW Cl, El thermally instable, low-MW APCI (FLA, LC-MS), ESI and high-MW DCI, FD, FAB, LD, ESI (FLA, LC-MS, CZE-MS). Soft ionisation techniques such as FL, FAB and LD are useful for the detection of non-volatile, sometimes oligomeric, polymer additives. Recent developments in ionisation techniques have allowed the analysis of polar, ionic, and high-MW compounds, previously not amenable to mass-spectrometric analysis. Figure 6.4 shows the applicability of various atmospheric pressure ionisation techniques in terms of molar mass and polarity. 

A number of CZE applications exist for the separation of proteins and other molecules in purity analysis, structural studies, binding and equilibrium determinations, in-process product analysis, and mobility measurements. The following applications illustrate the use of CZE for both research and routine QC analysis. 

The concept of impurity profiling is very important for antibiotics, since most of them are still produced by fermentation or by semisynthesis starting from fermentation products. Antibiotics are typically complex mixtures of several components and their composition depends on the fermentation conditions. Impurities due to degradation occur frequently. Commercial samples usually contain significant amounts of impurities with only minor structural differences among them, but differing widely in their pharmacological activities. These impurities can exhibit antibiotic activity, but in many cases they are inactive and sometimes even toxic. The applicability of CE in the analysis of antibiotics has been reviewed elsewhere. The use of CZE in impurity analysis of antibiotics is discussed in detail below. 

Dedicated applications of capillary zone electrophoresis (CZE) coupled to MS are discussed, particularly in the field of drug analysis. Development of other capillary-based electrodriven separation techniques such as non-aqueous capillary electrophoresis (NACE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC) hyphenated with MS are also treated. The successful coupling of these electromigration schemes with MS detection provides an efficient and sensitive analytical tool for the separation, quantitation, and identification of numerous pharmaceutical, biological, therapeutic, and environmental compounds. 

Since all electrophoretic mobility values are proportional to the reciprocal viscosity of the buffer, as derived in Chapter 1, the experimental mobility values n must be normalized to the same buffer viscosity to eliminate all other influences on the experimental data besides the association equilibrium. Some commercial capillary zone electrophoresis (CZE) instruments allow the application of a constant pressure to the capillary. With such an instrument the viscosity of the buffer can be determined by injecting a neutral marker into the buffer and then calculating the viscosity from the time that the marker needs to travel through the capillary at a set pressure. During this experiment the high voltage is switched off. 

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

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