CE analysis

PIXE detection limits for surface layers on bulk specimens are sufficiendy low to permit calibradon of true surfe.ce analysis techniques (e.g., Auger electron spectroscopy). 

The direct mode is used when the concentration of the SEE extract is enough for direct analysis in the CE instrument without the need for a pre-concentration step. In this case, the sample concentrator is by-passed and the SEE extract goes directly to the CE instrument. The extract is collected in a CE vial containing an appropriate solvent and is thus ready for the CE analysis (Eigure 6.12). 

Electropherograms of a urine sample (8 ml) spiked with non-steroidal anti-inflammatory drugs (10 p-g/ml each) after direct CE analysis (b) and at-line SPE-CE (c). Peak identification is as follows I, ibuprofen N, naproxen K, ketoprofen P, flurbiprofen. Reprinted from Journal of Chromatography, 6 719, J. R. Veraait et al., At-line solid-phase exti action for capillary electrophoresis application to negatively charged solutes, pp. 199-208, copyright 1998, with permission from Elsevier Science. 

Figure 11.19 SPME-CE analysis of urine samples (a) blank urine (a) directly injected and extracted for (b) 5 (c) 10 and (d) 30 min (b) Urine spiked with barbiturates, extracted for (e) 30 and (f, g) 5 min. Peak identification is as follows 1, pentobaitibal 2, butabarbital 3, secobarbital 4, amobarbital 5, aprobarbital 6, mephobarbital 7, butalbital 8, thiopental. Concenti ations used are 0.15-1.0 ppm (e, f) and 0.05-0.3 ppm (g). Reprinted from Analytical Chemistry, 69, S. Li and S. G. Weber, Determination of barbiturates by solid-phase microexti action and capillary electrophoresis, pp. 1217-1222, copyright 1997, with permission from the American Chemical Society.

As was indicated, there have not been a large number of publications on the CE analysis of methylxanthines in food systems. Analusis published a method using a 20-mm Borate buffer at pH 9.6 and UV detection at 254 nm and +22 kV applied voltage.41 Samples were diluted and prepared for analysis by filtration and analyzed using free solution electro-... 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

The main difficulties in CE analysis of cationic surfactants arise from their strong adsorption to the capillary wall and their ability to form micelles at low concentrations. The addition of organic modifiers in high amounts or separation in absolutely non-aqueous media disrupt micelle formation within the sample and also effectiveness of the organic modifier to disrupt micelles of alkylbenzyl dimethyl ammonium... 

There is a recent trend towards simultaneous CE separations of several classes of food additives. This has so far been applied to soft drinks and preserved fruits, but could also be used for other food products. An MEKC method was published (Lin et al., 2000) for simultaneous separation of intense sweeteners (dulcin, aspartame, saccharin and acesulfame K) and some preservatives (sorbic and benzoic acids, sodium dehydroacetate, methyl-, ethyl-, propyl- and isopropyl- p-hydroxybenzoates) in preserved fruits. Ion pair extraction and SPE cleanup were used prior to CE analysis. The average recovery of these various additives was 90% with good within-laboratory reproducibility of results. Another procedure was described by Frazier et al. (2000b) for separation of intense sweeteners, preservatives and colours as well as caffeine and caramel in soft drinks. Using the MEKC mode, separation was obtained in 15 min. The aqueous phase was 20 mM carbonate buffer at pH 9.5 and the micellar phase was 62 mM sodium dodecyl sulphate. A diode array detector was used for quantification in the range 190-600 nm, and limits of quantification of 0.01 mg/1 per analyte were reported. The authors observed that their procedure requires further validation for quantitative analysis. 

A major problem in CE analysis is the poor detection limits that are obtained with UV or DAD. Some potential exists for coupling biosensors to CE as a specific detection method (Bossi et al., 2000) although few published applications have appeared yet. 

Organic modifiers have been frequently employed in CE to increase the solubility of hydrophobic solutes in the aqueous buffer system. Unfortunately, many organic modifiers are UV absorbent and cannot be used without considerable loss of sensitivity of detection. A contactless conductivity detection system has been developed which extends the application range of UV-absorbing solvents [ 119]. As both natural pigments and synthetic dyes absorb in the visible part of the spectra, the application of UV-absorbing organic modifiers in their CE analysis does not cause detection problems. 

The application of an organic solvent in CE offers a new possibility to change the selectivity of CE systems. Moreover, the various ion-solvent interactions and ion-pair formations can be exploited for increased separation efficacy. Its application for CE analysis of ionic synthetic dyes may be expected in the future [121],... 

Samples collected during CE analysis can be used for off-line mass analysis by matrix-assisted laser desorption ionization (MALDI) and time-of-flight (TOF) MS. 

For high reproducibility of migration times and peak areas the sample matrix should be identical for all samples analyzed together. While this is challenging for forensic applications where analytes in whole blood or urine are determined, this requirement can be fulfilled easily in pharmaceutical analysis. After sample preparation of the drug product, the sample matrix is similar in most cases. The composition of blood or urine depends on its source. Thus, the changing sample matrix has more impact on the quality of the CE analysis. 

There are two parameters that need to be controlled during the separation step of any CE analysis, the temperature of the capillary and the voltage that is applied across the capillary. 

This chapter deals with the validation of capillary electrophoresis (CE) methods. It describes the various validation characteristics, namely accuracy, precision, specificity, detection limit, quantitation limit, linearity, and range in accordance with the official guidelines. Practical aspects related to the calculation of these parameters and factors affecting them in CE analysis have also been described. Validation requirements have been described according to the goal of the method. The chapter contains numerous tables and diagrams to illustrate these ideas. It also covers other related aspects such as instrument qualification, revalidation, and method transfer. 

The prerequisites for the development of a robust method are simple robust equipment, robust staff, and a robust development are needed. Even though the number of CE equipment manufacturers is low, the available instruments can be considered to be robust. In contrast, qualified staff for CE analysis is not often available. Since CE is based on absolutely different physics, i.e., electrophoretic and electroosmotic mobility,than HPLC,... 

Each of the chapters, written by selected experts in their respective fields, is designed to provide the reader with an in-depth understanding of CE theory, hardware, methodologies, regulations, and applications. The text includes state-of-the-art information on CE analysis of... 

Fig. 13.5 Steps involved in cloud point extraction (CPE) prior to HPLC, GC, and CE analysis.

Fig. 10 Formation of complexes between human rhinovirus HRV2 and neutralizing monoclonal antibody mAb 8F5 analyzed by CE. A fixed concentration of F1RV2 (15 nM) was incubated with an increasing concentration (indicated in the figure) of mAb 8F5 prior to the CE analysis at room temperature. O-phthalic acid was used as internal standard (IS). (Reprinted with permission from Ref. 34. Copyright 2000 American Chemical Society.)...

Fukushima T, Usui N, Santa T, Imai K. Recent progress in derivatization methods for LC and CE analysis. Journal of Pharmaceutical and Biomedical Analysis 30, 1655-1687, 2003. 

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