Electrophoresis peak capacity

This book is organized into five sections (1) Theory, (2) Columns, Instrumentation, and Methods, (3) Life Science Applications, (4) Multidimensional Separations Using Capillary Electrophoresis, and (5) Industrial Applications. The first section covers theoretical topics including a theory overview chapter (Chapter 2), which deals with peak capacity, resolution, sampling, peak overlap, and other issues that have evolved the present level of understanding of multidimensional separation science. Two issues, however, are presented in more detail, and these are the effects of correlation on peak capacity (Chapter 3) and the use of sophisticated Fourier analysis methods for component estimation (Chapter 4). Chapter 11 also discusses a new approach to evaluating correlation and peak capacity. 

Although classic two-dimensional gel electrophoresis provides exquisite peak capacity, it suffers from several limitations. First, the technology is labor-intensive and difficult to automate, which hampers applications to large-scale proteomics analyses (Hanash, 2000). 

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

This chapter illustrates possible applications of capillary electrophoresis in impurity profiling. Due to the large peak capacity of the technique, it is extremely well suited to separate the main drug compound from its possible impurities that often have a very related chemical structure. Moreover, the high efficiencies obtained, as well as the low reagent consumption make it a viable alternative to liquid chromatography in many cases of drug analysis. 

JC Giddings. Generation of variance theoretical plates, resolution and peak capacity in electrophoresis and sedimentations. Separ Sci 4 181-189, 1969. 

A substantial gain in peak capacity can be made by utilizing two-dimensional systems, as noted in Section 6.5. This approach has been successfully implemented in the form of two-dimensional electrophoresis systems, described in Section 6.4, but effective technology for two-dimensional column chromatography is still to be developed [13,14]. 

Peak capacity nc, like resolution, can be calculated in a straightforward manner for both isoelectric focusing and isopycnic sedimentation. However, it is most interesting to cast ne in a form such that these steady-state methods can be related to the analogous transient methods, namely, zone electrophoresis and rate-zonal sedimentation. To do this we substitute a from Eq. 8.42 into the basic peak capacity expression nc = L/4o-, Eq. 5.59. Then using D = 9177/, we get... 

Generation of Variance, Theoretical Plates, Resolution, and Peak Capacity in Electrophoresis and Sedimentation, J. C. Giddings, Sep. Sci., 4, 181 (1969). 

A new topic is now included Chapter 20 about quahty assurance. Part of it could be found before in chapter 19 but now the subject is presented much broadly and independent of Analytical HPLC . Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now chapter 25) and troubleshooting (now chapter 26). Some new sections were created 1.7, comparison of HPLC with capillary electrophoresis 2.11, how to obtain peak capacity 8.7, van Deemter curves and other coherences 11.3, hydrophilic interaction chromatography 17.2, method transfer 18.4, comprehensive two-dimensional HPLC 23.3, fast separations at 1000 bar 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added. 

Shadpour et al. [81] and Osiri et al. [82] employed SDS micro-capillary gel electrophoresis (SDS p-CGE) and micellar electrokinetic capillary (MEKC) electrophoresis in the first and second dimensions, respectively, to sort intact proteins using a poly (methylmethacrylate), PMMA, microchip. A diagram of the microchip is shown in Fig. 4. The electrophoresis commenced in the first dimension for a prescribed amount of time and, then, the bands from the first dimension were sequentially injected into the second dimension for development. The 2D electrophoresis system could generate a peak capacity of 2,600 for proteins isolated from fetal calf serum (see Fig. 4). 

Giddings, C. J., Generation of variance, theoretical plates , resolution, and peak capacity in electrophoresis and sedimentation, Sep. ScL, 4, 181,1969. 

The rapid separations offered by capillary electrophoresis have made it amenable as a detector in hyphenated techniques. For LC-CE, the total analysis time is usually governed by the LC separation, which generally takes minutes. However, capillary electrophoresis detection adds more peak capacity because of a second and orthogonal dimension for separation, and shorter separation conditions for LC can often be tolerated. For example, a 2.5 min reversed-phase liquid chromatography gradient was used in conjunction with 2.5 s CE separations for the detection of a tryptic digest of cytochrome c. ... 

Second, separation efficiency must be improved. Current two-dimensional electrophoresis systems are generating a spot capacity of a few hundred components an order of magnitude improvement is desirable. Spot capacity is given by the product of the peak capacity in each dimension, so that a threefold improvement in separation efficiency in each dimension will generate the desired improvement. MECC is not an ideal separation mode for proteins, and would be replaced with a technique... 

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