Capillary electrophoresis theoretical plates

For enantioseparation on CSPs in CEC, nonstereospecific interactions, expressed as 4>K, contribute only to the denominator as shown in Eq. (1), indicating that any nonstereospecific interaction with the stationary phase is detrimental to the chiral separation. This conclusion is identical to that obtained from most theoretical models in HPLC. However, for separation with a chiral mobile phase, (pK appears in both the numerator and denominator [Eq. (2)]. A suitable (f)K is advantageous to the improvement of enantioselectivity in this separation mode. It is interesting to compare the enantioselectivity in conventional capillary electrophoresis with that in CEC. For the chiral separation of salsolinols using /3-CyD as a chiral selector in conventional capillary electrophoresis, a plate number of 178,464 is required for a resolution of 1.5. With CEC (i.e., 4>K = 10), the required plate number is only 5976 for the same resolution [10]. For PD-CEC, the column plate number is sacrificed due to the introduction of hydrodynamic flow, but the increased selectivity markedly reduces the requirement for the column efficiency. 

Efficiency The efficiency of capillary electrophoresis is characterized by the number of theoretical plates, N, just as it is in GC or ITPLC. In capillary electrophoresis, the number of theoretic plates is determined by... 

First, solutes with larger electrophoretic mobilities (in the same direction as the electroosmotic flow) have greater efficiencies thus, smaller, more highly charged solutes are not only the first solutes to elute, but do so with greater efficiency. Second, efficiency in capillary electrophoresis is independent of the capillary s length. Typical theoretical plate counts are approximately 100,000-200,000 for capillary electrophoresis. 

Because HPLC and HPCE are based on different physico-chemical principles, HPCE may be expected to address areas in which HPLC has shortcomings [884]. One such area is time of separation. In terms of speed of analysis, selectivity, quantitation, methods to control separation mechanism, orthogonality, CE performs better than conventional electrophoresis and varies from HPLC (Table 4.49). CE has very high efficiency compared to HPLC (up to two orders of magnitude) or GC. For typical capillary dimensions 105—106 theoretical plates are common in CE compared to 20 000 for a conventional HPLC column and... 

Other sources of broadening in real systems are mentioned later.) Capillary electrophoresis routinely produces 50 000-500 000 theoretical plates (Figure 26-19), which is an order-of-magnitude better performance than chromatography. 

There is much interest in high-efficiency- and high-speed separation media for liquid chromatography. The plate numbers available in practice have been in the range of 10,000-30,000 in HPLC for 20 years or so, but these are low compared to well over 100,000 theoretical plates in capillary gas chromatography or in capillary electrophoresis. This is caused by the limitation in the use of small-sized particles for HPLC, where a particle-packed column is commonly used under a pressure-drop of up to 40 MPa. An increase in column efficiency by using small particles, which is the approach taken in the past, is accompanied by an increase in the pressure-drop, as expected from Eqns. 5.2 and 5.3, below. Eqns. 5.1-3 describe the efficiency (plate height) and flow resistance of a column packed with particles [1-3], where N stands for the... 

The use of exceedingly thin capillary tubes ( 50 /im) in capillary electrophoresis not only reduces convection but it also provides rapid heat dissipation. Using up to 30,000 V, on the order of 106 or more theoretical plates are realized in such tubes (see Figure 8.2). 

At times, capillary electrophoresis is called high-performance capillary electrophoresis to reflect the high number of theoretical plates offered by this technique as a result, superior resolution is possible. The original contribu-tor(s) to high-performance capillary electrophoresis and the basis of separation in various modes are shown in Table 2.4 9... 

In capillary gel electrophoresis, one of the major contributors to band broadening, besides the injection and detection extra-column effects, is the longitudinal diffusion of the solute molecules in the capillary tube [14], The theoretical plate number (N) is characteristic of column efficiency ... 

Ruhr s group studied the separation of double- and single-stranded DNA restriction fragments in capillary electrophoresis with polymer solutions under alkaline conditions in epoxy-coated capillaries and found that at pH 11 the theoretical plate numbers exceeded several millions [96], At pH 12, single-stranded DNA molecules were still well separated in entangled hydroxyethylcellulose (HEC) solutions, but the resolution decreased significantly in dilute polymer solutions. 

There are two important equations which describe the resolution of capillary electrophoresis in terms of the number of theoretical plates, and the migration time for molecules through a column. 

Capillary electrophoresis and its most popular hybrid technique - capillary electrochromatography - are complementary to HPLC, offering rapid analysis, low consumption of sample and solvents, and usually a higher efficiency of separation (due to a larger number of theoretical plates). Similar to HPLC, enantioseparation with the use of electrophoretic methods can be conducted by direct (chiral phase... 

The separation of endogenous 17- or 18-hydroxylated corticosteroids of the 21-hydroxylated 4-pregnen series was obtained by capillary electrophoresis of their charged borate chelate complexes (323). Aldosterone, 18-hydroxycorti-costerone, 18-hydroxy-deoxycorticosterone, cortisone, cortisol and 11-deoxycor-tisol are separated and resolved with 400 mM borate buffer at pH 9.0. The corticosteroid/borate chelation complex as indicated by CE data correlated well with 11 B-NMR. The separation of corticosteroids and benzothiazin analogs were studied by MEKC and a comparison with CZE was made (324). Bile salts, which have a similar carbon skeleton to the corticosteroids, were used for the separation of these steroids. A short analysis time, 15 min, and a high number of theoretical plates (150,000-350,000) were obtained. Sodium cholate was found to be very effective. The MEKC method was applied to the determination of the drug substance in tablets and cream formulations. An internal standard method was used for quantitation. The purity of the drug substance was also determined. 

A relatively new separation technique that is capable of separating minute quantities of substances in relatively short time with high resolution is capillary electrophoresis (CE). It offers the ability to analyze a nanoliter (10 L) of sample, with over 1 million theoretical plates and a detection sensitivity of injected components at the attomole (10 mol) level or less ... 

CEC is a hybrid separation technique that combines capillary HPLC (50-pm capillary diameter) with capillary zone electrophoresis, and its main advantage is the enormous number of theoretical plates that can be achieved. This is a result of the uniform, plug-like flow that electro-endoosmosis generates and the fact that this allows the use of very small particles (3 pm diameter). The technology is at an early stage of development (as there are problems inherent in packing columns of such small particle size f9J), but has already resulted in efficiency values of up to 387,000 theoretical plates per meter (10). 

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