Capillary electrophoresis injection

In capillary electrophoresis the conducting buffer is retained within a capillary tube whose inner diameter is typically 25-75 pm. Samples are injected into one end of the capillary tube. As the sample migrates through the capillary, its components separate and elute from the column at different times. The resulting electrophero-gram looks similar to the chromatograms obtained in GG or HPLG and provides... 

Schematic diagram for capillary electrophoresis. The sample and source reservoir are switched when making injections.

An injection technique in capillary electrophoresis in which pressure is used to inject sample into the capillary column. 

Injecting the Sample The mechanism by which samples are introduced in capillary electrophoresis is quite different from that used in GC or HPLC. Two types of injection are commonly used hydrodynamic injection and electrokinetic injection. In both cases the capillary tube is filled with buffer solution. One end of the capillary tube is placed in the destination reservoir, and the other is placed in the sample vial. 

A means of concentrating solutes in capillary electrophoresis after their injection onto the capillary column. 

Source Adapted from Baker, D. R. Capillary Electrophoresis. Wiley-Interscience New York, 1995. "Concentration depends on the volume of sample injected. 

Luminol-based chemiluminescence methods have also been employed for detection of analytes in flowing stream analytical techniques such capillary electrophoresis (282), flow injection analyses, and hplc (267). AppHcations of the enhanced luminol methodology to replace radioassay methods have been developed for a number of immunological labeling techniques (121,283). 

DEVELOPMENT OF INFRARED DETECTION IN FLOW INJECTION ANALYSIS (FIA) AND CAPILLARY ELECTROPHORESIS (CE)... 

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.

Sulfonylureas are not directly amenable to gas chromatography (GC) because of their extremely low volatility and thermal instability. GC has been used in conjunction with diazomethane derivatization, pentafluorobenzyl bromide derivatization, and hydrolysis followed by analysis of the aryl sulfonamides. These approaches have not become widely accepted, owing to poor performance for the entire family of sulfonylureas. Capillary electrophoresis (CE) has been evaluated for water analysis and soil analysis. The low injection volumes required in CE may not yield the required sensitivity for certain applications. Enzyme immunoassay has been reported for chlorsulfuron and triasulfuron, with a limit of detection (LOD) ranging from 20 to 100 ng kg (ppt) in soil and water. 

As in HPLC, the coupling of MS detection with CE has provided an excellent opportunity for more selective analysis, but the much reduced flow rates, small injection volumes, limitations in the types of buffers used [since electrospray ionization (ESI) is used in capillary electrophoresis/mass spectrometry (CE/MS)], and need to... 

A number of developments have increased the importance of capillary electrophoretic methods relative to pumped column methods in analysis. Interactions of analytes with the capillary wall are better understood, inspiring the development of means to minimize wall effects. Capillary electrophoresis (CE) has been standardized to the point of being useful as a routine technique. Incremental improvements in column coating techniques, buffer preparation, and injection techniques, combined with substantive advances in miniaturization and detection have potentiated rugged operation and high capacity massive parallelism in analysis. 

Fang, Q., Wang, F.-R., Wang, S.-L., Liu, S.-S., Xu, S.-K., and Fang, Z.-L., Sequential injection sample introduction microfluidic-chip based capillary electrophoresis system, Anal. Chim. Acta, 390, 27, 1999. 

Capillary electrophoresis offers several useful methods for (i) fast, highly efficient separations of ionic species (ii) fast separations of macromolecules (biopolymers) and (iii) development of small volume separations-based sensors. The very low-solvent flow (l-10nL min-1) CE technique, which is capable of providing exceptional separation efficiencies, places great demands on injection, detection and the other processes involved. The total volume of the capillaries typically used in CE is a few microlitres. CE instrumentation must deliver nL volumes reproducibly every time. The peak width of an analyte obtained from an electropherogram depends not only on the bandwidth of the analyte in the capillary but also on the migration rate of the analyte. 

Hyphenation in capillary electrophoresis is still in its infancy. Critical aspects of CE hyphenation include the minute volumes of sample injected (typically a few nL) and small flow-rates (in the order of nLmin-1). Interfaces are not commercially available. CZE-UV can be used for the analysis of higher polyamide oligomers in HF1P solution [859]. A solvent elimination design with nebuliser has been described for CE-FTIR and CEC-FTIR coupling absolute detection limits are hundreds of pg [860]. An advantage of CE-FTIR is that analytes may be detected and identified without derivatisation. CE(C)-NMR [861-863] is advancing rapidly. 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

Fan et al. [106] developed a high performance capillary electrophoresis method for the analysis of primaquine and its trifluoroacetyl derivative. The method is based on the mode of capillary-zone electrophoresis in the Bio-Rad HPE-100 capillary electrophoresis system effects of some factors in the electrophoretic conditions on the separation of primaquine and trifluoroacetyl primaquine were studied. Methyl ephedrine was used as the internal standard and the detection was carried out at 210 nm. A linear relationship was obtained between the ratio of peak area of sample and internal standard and corresponding concentration of sample. The relative standard deviations of migration time and the ratio of peak area of within-day and between-day for replicate injections were <0.6% and 5.0%, respectively. 

Most of the reported methods of analysis of valproic acid and its sodium salt in biological fluids rely on the use of chromatography, especially gas chromatography, although high performance liquid chromatography (HPLC) is also reported. Other methods, such as flow injection analysis, enzyme-immunoassay, fluorescence-polarization capillary electrophoresis, and potentiometry are sometimes used. The reported methods can be classified as follows. 

Valproic acid has been determined in human serum using capillary electrophoresis and indirect laser induced fluorescence detection [26], The extract is injected at 75 mbar for 0.05 min onto a capillary column (74.4 cm x 50 pm i.d., effective length 56.2 cm). The optimized buffer 2.5 mM borate/phosphate of pH 8.4 with 6 pL fluorescein to generate the background signal. Separation was carried out at 30 kV and indirect fluorescence detection was achieved at 488/529 nm. A linear calibration was found in the range 4.5 144 pg/mL (0 = 0.9947) and detection and quantitation limits were 0.9 and 3.0 pg/mL. Polonski et al. [27] described a capillary isotache-phoresis method for sodium valproate in blood. The sample was injected into a column of an EKI 02 instrument for separation. The instrument incorporated a conductimetric detector. The mobile phase was 0.01 M histidine containing 0.1% methylhydroxycellulose at pH 5.5. The detection limit was 2 pg/mL. 

Parameters that should be tested in HPLC method development are flow rate, column temperature, batch and supplier of the column, injection volume, mobile phase composition and buffer pH, and detection wavelength [2], For GC/GLC methods, one should investigate the effects of column temperature, mobile phase flow rate, and column lots or suppliers [38], For capillary electrophoresis, changes in temperature, buffer pH, ionic strength, buffer concentrations, detector wavelength, rinse times, and capillaries lots and supplier should be studied [35, 36], Typical variation such as extraction time, and stability of the analytical solution should be also evaluated [37],... 

As can be seen in Table 3, a wide range of analytes derivatized with different labels have been detected using the POCL reaction. Most of these applications have employed flow injection or liquid chromatographic techniques. An area of growing interest is the combination of capillary electrophoresis with chemiluminescence. Several strategies have been used to detect analytes with fluorescent... 

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