Electrophoresis conventional

The structure of these gel-like systems of micelles is very different from that of conventional electrophoresis media made from chemically and physically cross-linked polymers of polyacrylamide and agarose [75], The absence of chemical or physical cross-links in the Pluronic gel-like phases may allow a larger degree of freedom for macromolecular transport around the obstacles that make up the medium than occurs in conventional electrophoresis media. 

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... 

What is capillary electrophoresis and what advantages does it have over other conventional electrophoresis techniques. 

Capillary zon p.ec-trophoiobis is based on conventional electrophoresis of the nn.ilyte. 

CE is a family of techniques similar to those found in conventional electrophoresis zone electrophoresis, displacement electrophoresis, isoelectric focusing (IEF), and sieving separations. Other modes of operation unique to CE include micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). 

The most widely used separation medium in CE is water, that is, an aqueous medium. If an organic solvent has been used instead, the term nonaqueous CE (NACE) is used in order to make the difference. Nonaqueous solvents were first applied to conventional electrophoresis in the early 1950s [12,13]. The first NACE experiments were carried out in 1984 by Walbroehl and Jorgenson [14] and since then they have served as alternative media to the water environment in many electrophoretic applications [15-17]. 

Good descriptions of practical experimental techniques in conventional electrophoresis can be found in Refs. [81,253,259]. For the most part, these techniques are applied to suspensions and emulsions, rather than foams. Even for foams, an indirect way to obtain information about the potential at foam lamella interfaces is by bubble electrophoresis. In bubble microelectrophoresis the dispersed bubbles are viewed under a microscope and their electrophoretic velocity is measured taking the horizontal component of motion, since bubbles rapidly float upwards in the electrophoresis cells [260,261]. A variation on this technique is the spinning cylinder method, in which a bubble is held in a cylindrical cell that is spinning about its long axis (see [262] and p.163 in Ref. [44]). Other electrokinetic techniques, such as the measurement of sedimentation potential [263] have also been used. 

As an instrumental approach to conventional electrophoresis, capillary electrophoresis offers the capability of on-line detection, micropreparative operation and automation (6,8,45-47). In addition, the in tandem connection of capillary electrophoresis to other spectroscopy techniques, such as mass spectrometry, provides high information content on many components of the simple or complex peptide under study. For example, it has been possible to separate and characterize various dynorphins by capillary electrophoresis-mass spectrometry (33). Therefore, the combination of CE-mass spectrometry (CE-MS) provides a valuable analytical tool useful for the fast identification and structural characterization of peptides. Recently, it has been demonstrated that the use of atmospheric pressure ionization using Ion Spray Liquid Chromatography/ Mass Spectrometry is well suited for CE/MS (48). This approach to CE/MS provides a very effective and straightforward method which allow the feasibility of obtaining CE/MS data for peptides from actual biological extracts, i.e., analysis of neuropeptides from equine cerebral spinal fluid (33). 

Capillary electrophoresis has been demonstrated to be useful in monitoring the identity and purity of hGH. CZE is capable of discrimination between hGH and several closely related impurities and degradation products, either as the intact species (previous work) or as the trypsin digests (this work). Thus, it is an important adjunct to conventional methods, such as RP-HPLC of digests or conventional electrophoresis of intact proteins, for the identification of hGH. CZE possesses adequate sensitivity to monitor minor impurities it is comparable to RP-HPLC with respect to linearity and precision. Samples with a volume of at least 10 nanoliters will provide acceptable precision those that contain an internal standard will provide the best precision. The peak area response is linear it is possible to extend linearity by increasing concentration as long as the contribution of the sample and its matrix to field inhomogeneities is minimized. The work shows that CZE has potential to be useful in the quality control of proteins such as hGH. 

Further DNA analysis It is also possible to conduct further steps beyond conventional electrophoresis to allow more advanced analyses of the separated DNA bands. Of these steps, blotting is arguably the most versatile tool for further analysis. As outlined earlier, there are various types of blotting, but the most popular... 

In this section, instrumentation, general electrophoretic operations, technical and practical considerations, and types of conventional electrophoresis are discussed. 

General operations performed in conventional electrophoresis include separation, detection and quantification, and a number of blotting techniques. 

In contrast to the cumbersome and time-consuming tasks of conventional electrophoresis, CE is well suited to automation. Samples are easily applied to the capillary, a variety of detector types can be used, and the resulting electrophore-tograms can be analyzed and manipulated in much the same manner as chromatograms. Commercial instruments resemble many HPLC instruments in terms of automated sample loading and data analysis. Traditional serum protein electrophoresis, for example, can be fully automated with CE. 

One problem associated with conventional electrophoresis of serum proteins is its proclivity for point-of-application artifacts. These are bands that result from the fact that electrophoretic mobility (e.g., with AGE) is bidirectional from the point of application. Consequently the point of application remains part of the scanned area of interest. The fact that these must be immunotyped to distinguish real monoclonal proteins from artifacts is costly and time consuming. 

Figure 5-8 CE-based identification of uncommon hemoglobin (Hb) variants by clEF. Analysis of blood from a patient with Hb S/Aida trait detected the presence of seven different normal and abnormal structural Hb variants, some of which are not detectable by conventional electrophoresis because of a lack of sensitivity or inadequate resolution.The four abnormal variants include Hb S, Aida, S/Aida hybrid, and A2/Aida. Identifying a-variants of Hb A by cfEF helps discriminate between a- and b-globin gene mutations in samples containing unknown Hb variants. Glycated HbA (HbAlc) is also apparent in the electropherogram.

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