Free solution CE

In this group of methods the sample constituents can migrate differentially in zones through the capillary in a medium that can be either a gel (CGE) or an electrolyte (free solution CE). The experimental setup of commercial CE systems for zone electrophoresis is similar to the one presented in Figure 1. 

Fig. 6 Free-solution CE separation of PNA/DNA hybrid from excess PNA probe. M13 mpl8 ssDNA 4.2 X 10-8 M, and PNA probe 1.3 X 10-7 M. Detection LIF 488/520 nm. Buffer TBE, 7 M urea (pH 8.0). CE conditions 50-mm-i.d. polyacrylamide-coated capillary (27 cm in length and 20 cm to detector), 10 s gravity injection, separation voltage — 10 kV. Laser-induced fluorescence detection with excitation at 488 nm and emission at 520 nm. The buffer contained Triszborate (pH 8.0) with 7 M urea buffer. (From Ref. 37.)...

Why cannot oligonucleotides of different lengths be resolved in free-solution CE, and a gel must be used for their separation (2 marks)... 

Capillary zone electrophoresis (CZE), also known as free-solution CE, is the most widely used mode of CE essentially because of its versatility. Protein separation in CZE is based on the differential electrophoretic mobility of the analytes. This mobility is primarily dependent on a protein s size and net charge, the charge-to-mass ratio. Solvent properties that influence the size and charge of a protein include pH, ionic strength, viscosity, and dielectric constant.67 Manipulation of these properties, most notably pH, dictates the selectivity in CZE. Maximizing the charge difference between two proteins via pH modification optimizes their separation. 

Even though free-solution CE is most commonly used for neuropeptides and neuroproteins, other forms of CE have also been employed. For instance, as an alternative to conventional slab-gel electrophoresis, a method using sodium dodecyl sulfate (SDS) capillary gel electrophoresis was developed. It was applied to low-molecular-mass proteins (j8-trace protein, ft-microglobulin, -trace protein, and myelin basic protein) in cerebrospinal fluid [4], Advantageous features of capillary gel electrophoresis over slab-gel electrophoresis are compatibihty with small sample volumes, shorter analysis times, and more accurate quantihcation of the analytes. 

The first applications of CDs as chiral selectors in CE were reported in capillary isotachophoresis (CITP) [2] and capillary gel electrophoresis (CGE) [3]. Soon thereafter, Fanali described the application of CDs as chiral selectors in free-solution CE [4] and Terabe used the charged CD derivative for enantioseparations in the capillary electrokinetic chromatography (CEKC) mode [5]. It seems important to note that although the experiment in the CITP, CGE, CE, and CEKC is different, the enantiomers in all of these techniques are resolved based on the same (chromatographic) principle, which is a stereoselective distribution of enantiomers between two (pseudo) phases with different mobilities. Thus, enantioseparations in CE are commonly based on an electrophoretic migration principle and on a chromatographic separation principle [6]. 

Free Solution-CE, FSCE, also called Capillary Zone Electrophoresis, CZE), molecular size (SDS-CGE in capillaries filled with an acrylamide gel), or isoelectric point (CIEF). 

There are numerous variations on free solution CE (FSCE), such as micellar electrokinetic capillary chromatography (MECC or MEKC), where a moving, pseudostationary phase is added to the CE buffer, and secondary chemical equilibria or interactions ensue that effect separations of even neutral compounds, as well as ionic analytes. However, in general, CE utilizes truly homogeneous, solution phase separation approaches, without a stationary (permanent, fixed) phase, making it perhaps ideally suited for molecular recognition in searching combinatorial libraries. 

The pAfa values of pharmaceutical compounds can be determined from migration time data obtained by running the compound with free solution CE electrolytes at a range of pH values. The mobility of the solute at each pH can be calculated from its migration time and the EOF (measured against a neutral marker such as methanol), and a plot of mobility versus pH can be constructed (see Figure 4.6). The pAfa value can be calculated mathematically or obtained from the plot. 

The CE is quite suited for analysis of these ions too. Because of their relative charge to the small mass, they tend to migrate rapidly in this technique giving fast separation with very high theoretical plate numbers at a low cost per test. Both cations and anions can be analyzed in the same run. The separation can be based on simple free solution CE or based on a suitable chelating additive. In general, cations are measured in a low pH electrolyte containing a UV active species (imidazole... 

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