Electrophoresis chromatographic separation

Electrophoresis. Chromatographic Separation, and Gel Filtration Electrophoresis, chromatography, and gel filimtion all separate components in dilute solution. They must be followed by concentration steps if a dissolved product is satisfactory, and addilioanl processing steps if pure prodnets are desired. 

Principles and Characteristics In electrophoresis the separation of electrically charged particles or molecules in a conductive liquid medium, usually aqueous, is achieved under the influence of a high electric field. This differs from chromatographic separations... 

Bog-Hansen, T.C., Prahl, P., and Lowenstein, H. (1978) A set of analytical electrophoresis experiments to predict the results of affinity chromatographic separations. Fractionation of allergens from cow s hair and dander./. Immunol. Meth. 22, 293. 

A variety of microscale separation methods, performed in capillary format, employ a pool of techniqnes based on the differential migration velocities of analytes under the action of an electric field, which is referred to as capillary electromigration techniques. These separation techniques may depend on electrophoresis, the transport of charged species through a medium by an applied electric field, or may rely on electrically driven mobile phases to provide a true chromatographic separation system. Therefore, the electric field may either cause the separation mechanism or just promote the flow of a solution throughout the capillary tube, in which the separation takes place, or both. 

Another recent development is the advent of pulse amperometry in which the potential is repeatedly pulsed between two (or more) values. The current at each potential or the difference between these two currents ( differential pulse amperometry ) can be used to advantage for a number of applications. Similar advantages can result from the simultaneous monitoring of two (or more) electrodes poised at different potentials. In the remainder of this chapter it will be shown how the basic concepts of amperometry can be applied to various liquid chromatography detectors. There is not one universal electrochemical detector for liquid chromatography, but, rather, a family of different devices that have advantages for particular applications. Electrochemical detection has also been employed with flow injection analysis (where there is no chromatographic separation), in capillary electrophoresis, and in continuous-flow sensors. 

Following the discovery of DNA structure and the genetic code, various associated techniques involving the manipulation and separation of DNA fragments have been developed. Several of the physical methods are useful in nucleic-acid biochemistry these include, centrifugation, electrophoresis and chromatographic separation of DNA fragments. 

Chromatographic and electrophoretic separations are truly orthogonal, which makes them excellent techniques to couple in a multidimensional system. Capillary electrophoresis separates analytes based on differences in the electrophoretic mobilities of analytes, while chromatographic separations discriminate based on differences in partition function, adsorption, or other properties unrelated to charge (with some clear exceptions). Typically in multidimensional techniques, the more orthogonal two methods are, then the more difficult it is to interface them. Microscale liquid chromatography (p.LC) has been comparatively easy to couple to capillary electrophoresis due to the fact that both techniques involve narrow-bore columns and liquid-phase eluents. 

Many groups have used electrophoresis to enhance a primary chromatographic separation. These techniques can be considered to be two-dimensional, but they are not comprehensive, usually due to the loss of resolution in the interface between the two methods. For instance, capillary electrophoresis was used in 1989 by Grossman and co-workers to analyze fractions from an HPLC separation of peptide fragments. In this study, CE was employed for the separation of protein fragments that were not resolved by HPLC. These two techniques proved to be truly orthogonal, since there was no correlation between the retention time in HPLC and the elution order in CE. The analysis time for CE was found to be four times faster than for HPLC (12), which demonstrated that CE is a good candidate for the second dimension in a two-dimensional separation system, as will be discussed in more detail later. 

Pieper R, Gatlin CL, Makusky AJ, Russo PS, Schatz CR, Miller SS, et al. The human serum proteome Display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Prote-omics 2003 3(7) 1345 1364. 

The widely used strategy for protein identification is depicted in Figure 8.15. This strategy thus is to cleave the protein either by trypsin, V8 protease, Lys-C endoprotease or by a reactant such as BrCN. The mixture then is analysed by mass spectrometry to obtain the molecular masses of the largest possible number of peptides. The two ionization methods, MALDI and ESI, are used. MALDI is best to use if one wants to avoid chromatographic separation, because it yields very simple spectra, has a better sensitivity and is not so sensitive to the presence of contaminants. However, ESI can be coupled directly with HPLC or capillary electrophoresis (CE) if a separation is wanted. Furthermore, ESI is often used with mass spectrometers that allow MS/MS data to be easily obtained. 

---