Capillary electrophoresis sample injection

There are two modes of injection in capillary electrophoresis hydrodynamic injection and electroki-netic injection. In hydrodynamic injection, pressure or vacuum are plaeed on the inlet sample vial or the outlet waste vial, respeetively. For electrokinetic injection, the voltage is activated for a short time with the capillary and electrode immersed in the sample. 

Two introduction methods are commonly employed in capillary electrophoresis. Hydrodynamic injection is based on siphoning, or gravity feeding the sample into the anodic end of the capillary. The anodic end is removed from the buffer reservoir and placed in the sample solution. The capillary end is then raised so that the liquid level in the sample vial is at a height Ah above the level of the cathodic buffer, and is held in this position for a fixed time t. This process has been automated for reproducibility, and the hydrodynamic flow rate has been shown to obey Eq. 12.9 ... 

Figure 23-14 Capillary electrophoresis. Sample is injected by elevating or applying pressure to the sample vial or by applying suction at the outlet of the capillary. 

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. 

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

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

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.

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. 

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. 

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. 

The hollow fiber was dipped into dihexyl ether for 5 sec and excess adhering solvent was washed away by ultrasonification in a water bath. Then, 25 fiL of lOmM hydrochloric acid (aqueous, acceptor phase) was injected into the lumen of the hollow fiber with a microsyringe. This activated fiber was placed in the vial containing the donor solution and the vial vibrated at 1500 rpm for 45 min. The entire acceptor solution was flushed into a 200-fJ.L micro insert and subjected to capillary electrophoresis or HPLC detection. For 2 mL extractions, 250 //I. of plasma sample treated with... 

Packed capillaries with a larger inner diameter may be useful in preparative separations. These will find an application in proteome research as a part of multidimensional separation systems that will replace 2-D gel electrophoresis. The preparative CEC will require solving of the problems related to heat dissipation since the radial temperature gradient negatively affects the separations, and sample injection. The fabrication of sintered frits in larger bore capillaries is also very difficult. However, in situ polymerized monolithic frits can be fabricated in capillaries of virtually any diameter [190]. 

Figure 9.1 Schematic representation of a basic capillary electrophoresis system. The main components include a capillary (commonly contained within a housing that allows for temperature control), a power supply, and a detector. Automation is achieved through the use of computer-controlled setting of solutions and samples, displacement forces (to replace capillary contents and for hydrodynamic injection), and automatic data collection. (Courtesy of Agilent Technologies.)...

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