Capillary electrophoresis migration time

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... 

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

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. 

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. 

Anions and uncharged analytes tend to spend more time in the buffered solution and as a result their movement relates to this. While these are useful generalizations, various factors contribute to the migration order of the analytes. These include the anionic or cationic nature of the surfactant, the influence of electroendosmosis, the properties of the buffer, the contributions of electrostatic versus hydrophobic interactions and the electrophoretic mobility of the native analyte. In addition, organic modifiers, e.g. methanol, acetonitrile and tetrahydrofuran are used to enhance separations and these increase the affinity of the more hydrophobic analytes for the liquid rather than the micellar phase. The effect of chirality of the analyte on its interaction with the micelles is utilized to separate enantiomers that either are already present in a sample or have been chemically produced. Such pre-capillary derivatization has been used to produce chiral amino acids for capillary electrophoresis. An alternative approach to chiral separations is the incorporation of additives such as cyclodextrins in the buffer solution. 

Affinity capillary electrophoresis (ACE), reviewed by Shimura and Kasai,42 is a method for studying receptor-ligand binding in free solution using CE. The technique depends upon a shift in the electrophoretic mobility of the receptor upon complexation with a charged ligand. Pure receptor preparations or accurate concentration values are not required because only migration times are measured. 

Affinity capillary electrophoresis is an approach where the migration pattern of interacting molecules are used to identify and quantify specific binding and estimate binding constants. Therefore, the solutes are first separated conventionally by CE. In a second run, the run buffer is doped with a specific complex-forming substance, and the change in the retention time... 

In some cases the equilibration rate is very slow compared to the time scale of the analytical separation. The pre-equilibrated reaction mixture behaves indeed as a mixture of inert components and can be separated by capillary electrophoresis. The concentrations are directly derived from the peak areas or peak heights after calibration. This method is suitable if ligand and substrate are separable and the migration time does not exceed 1% of the half-life of complex decomposition. 

In the absence of EOF and separation mechanism other than electrophoresis, each analyte migrates with its own velocity which, according to Equation 6.8, is proportional to the strength of the electric field applied across the capillary tube. The constant of proportionality of the observed velocity of the charged analyte is defined as the observed mobility (p bs) and can be directly calculated by the migration time and the other experimental parameters, according to the following equation ... 

In capillary electrophoresis instruments, the electro-osmotic flow is used to impose, on all charged species in the sample, a direction of migration that is oriented from the anode towards the cathode. An increase in the electro-osmotic flow vEOS decreases, at the detector, the gap in migration times of ions travelling in the same direction. The use of fused silica capillaries partially deactivated by coating the inner wall allows modulation of the electro-osmotic flow. A voltage gradient can also be used to this end. 

Figure 26-38 Calibration curve for protein molecular mass in sodium dodecyl sulfate-capillary gel electrophoresis. The abscissa, fre, Is the migration time of each protein divided by the migration time of a small dye molecule. [Data from j. K, Grady, J. Zang, t. M. Laue, RArosio, and N. D, Chasteen, "Characterization of the H- and L-Subunlt Ratios in Ferritins by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis," Anal. Biochem. 2002,302.263.)...

Capillary electrophoresis is not used as much as liquid chromatography. Advantages of electrophoresis relative to chromatography include (1) higher resolution, (2) low waste production, and (3) generally simpler equipment. Drawbacks of electrophoresis include (1) higher limits of detection, (2) run-to-run irreproducibility of migration times, (3) insolubility of some analytes in common electrolyte solutions, and (4) inability to scale up to a preparative separation. 

Liquid chromatography is two decades more mature than capillary electrophoresis. As trained electropherographers become more common, more separations will be handled by electrophoresis. For example, electrophoresis displaced liquid chromatography as the preferred method for forensic analysis of alkaloids in opium and heroin.51 The enabling technology for this application was dynamic coating of the capillary between runs to eliminate adsorption of analytes on the silica surface and decrease variations in migration times to less than 0.5%. 

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