Electrophoretic techniques

Electrophoretic techniques are well suited to separate charged compounds. Separation is due to migration induced by high voltage and takes place either in a buffer solution or in the pores of a gel filled with buffer solution. Several electrophoretic techniques are used here only the most important ones will be discussed. Most of these methods are used for analysis, but some (such as 2D gels) also for isolating macromolecules for further studies. Electrophoretic techniques are particularly important for studying macromolecules, especially proteins. 

It is perhaps an indication of the limited success of electrophoretic techniques for the determination of pesticide residues at trace levels that although many papers and reviews on the subject have been published, very few laboratories involved in the routine analysis of residues rely on such techniques for their work. Electrophoretic techniques have suffered because of poor flexibility and sensitivities compared with chromatographic techniques. 

Capillary electrophoresis (CE) or capillary zone electrophoresis (CZE) is the technique most often employed in pesticide residue analysis. In its most basic form, free zone electrophoresis, a fused-silica capillary is filled with electrolyte (running buffer or background electrolyte). A potential is applied across the capillary and the cations 

On-line sample-stacking techniques and, more recently, the use of isotacho-phoresis have added to the potential benefits of CE by permitting the concentration of analyte in a large volume by exploiting the difference in the electric field between the dilute sample and system buffer. The electric field is much stronger in the dilute buffer-sample and hence analyte ions move faster until they reach the border with the separation buffer. At this point they slow down, causing the analyte to concentrate as a sharp sample band at the interface. 

The principal limitation in the use of electrophoretic techniques is the lack of availability of suitable detection systems for quantitative analysis and unequivocal identification of pesticide analytes. Traditionally, either ultraviolet/visible (UVA IS) or fluorescence detection techniques have been used. However, as with chromatographic techniques, MS should be the detection system of choice. A brief comparison of the numbers of recent papers on the application of GC/MS and LC/MS with capillary elec-trophoresis/mass spectrometery (CE/MS) demonstrates that interfaces between CE 

Analyte matrix Mode Detection LOD Buffer Pesticides identified Reference 

Because of their high separation capacity, short analysis time, low reagent consumption and simplicity, various electrophoretic methods have found application in the separation and quantitative determination of anthocyanins in various complex matrices [267].The different techniques used for the measurement of anthocyanins in beverages [268], the application of capillary electrophoresis (CE) for the analysis of natural food pigments [269], the use of CE for the determination of anthocyanins in foods [270] and in medicinal plants [271] have been previously reviewed. 

The migration order of wine anthocyanins in CE has been studied in detail and the results have been compared with those obtained by RP-HPLC-MS. Wines were filtered and used for the analyses without any other pretreatment. Wine samples of 10 ml were freeze-dried, redissolved in methanol and applied for semi-preparative fractionation. CZE measurements were carried out in a fused-silica capillary (46 cm effective length, 75 /an i.d.). The capillary was conditioned with 0.1 M NaOH (2 min), water (2 min) and running buffer (5 min). The buffer consisted of 50 mM sodium teraborate (pH = 8.4) containing 15 per cent (v/v) 

INDIVIDUAL TOTAL MONOMER (TMA) AND TOTAL ACYLATED MONOMER (TAcMA) ANTHOCYANIN CONTENTS OF COMERCIAL ANTHOCYANIN-RICH EXTRACTS 

TMA and TAcMA are the sum of all monomer and all acylated monomer anthrocyanins. respectively, expressed as mg Mv-3-Gl/100 g dry matter, nd, not detected amounts. Mean value of at least three determinations SD. 

The retention data prove that the CZE method separates the pigments in a considerably shorter analysis time than HPLC does. It was supposed that the good separation capacity and low reagent consumption make CZE a valuable alternative to RP-HPLC [272], 

This mode of electrophoresis, in which the electrolyte migrates through the capillary, is the most widely used. In this mode, samples are applied as a narrow band that is surrounded by the electrolyte buffer. This electrolyte can be, depending upon the application, acidic (phosphate or citrate) or basic (borate) or an amphoteric substance (a molecule possessing both an acidic and an alkaline function). The electro-osmotic flow increases with the pH of the liquid phase or can be rendered inexistent. This procedure is also called, in contrast to CGE (cf. Section % A3), free solution electrophoresis. 

This technique represents the transposition of the classic agarose or polyacrylamide (PACE) gel electrophoresis into a capillary. The capillary is filled with an electrolyte impregnated into a gel. This produces a filtration effect, which decreases the 

CGE has been adapted to DNA sequencers. Special instruments fully automated (from sample loading to data analysis) have been designed with multiple capillaries that can simultaneously analyze many samples through a fluorescent-based detection. 

The modified technique is known as capillary array electrophoresis for the separation of the DNA or RNA fragments based on their size. 

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Distinction is also made among electrophoretic techniques in terms of the type of matrix employed for analysis. Matrices include polymer gels such as agarose and polyacrjiamide, paper, capillaries, and flowing buffers. Each matrix is used for different types of mixtures, and each has unique advantages. 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Various support media may be employed in electrophoretic techniques. Separation on agarose, acrylamide, and paper is influenced not only by electrophoretic mobiUty, but also by sieving of the samples through the polymer mesh. The finer the weave of selected matrix, the slower a molecule travels. Therefore, molecular weight or molecular length, as well as charge, can influence the rate of migration. 

Paper Electrophoresis. Paper (qv) as an electrophoretic matrix was employed in some of the first electrophoretic techniques developed to separate compounds. Paper is easier than a gel matrix because the paper matrix requires no preparation. Besides being easy to obtain, paper is a good medium because it does not contain many of the charges that interfere with the separation of different compounds. Two types of paper employed in this type of electrophoresis are Whatman 3 MM (0.3 mm) and Whatman No. 1 (0.17 mm). 

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

Electrophoretic techniques are based on the movement of ions in an electrical field. An ion of charge q experiences a force F given by T = Eq/d, where E is the voltage (or electrical potential) and dis the distance between the electrodes. In a vacuum, T would cause the molecule to accelerate. In solution, the molecule experiences frictional drag, iy, due to the solvent ... 

The flow profiles of electrodriven and pressure driven separations are illustrated in Figure 9.2. Electroosmotic flow, since it originates near the capillary walls, is characterized by a flat flow profile. A laminar profile is observed in pressure-driven systems. In pressure-driven flow systems, the highest velocities are reached in the center of the flow channels, while the lowest velocities are attained near the column walls. Since a zone of analyte-distributing events across the flow conduit has different velocities across a laminar profile, band broadening results as the analyte zone is transferred through the conduit. The flat electroosmotic flow profile created in electrodriven separations is a principal advantage of capillary electrophoretic techniques and results in extremely efficient separations. 

Electrodriven separation techniques are destined to be included in many future multidimensional systems, as CE is increasingly accepted in the analytical laboratory. The combination of LC and CE should become easier as vendors work towards providing enhanced microscale pumps, injectors, and detectors (18). Detection is often a problem in capillary techniques due to the short path length that is inherent in the capillary. The work by Jorgenson s group mainly involved fluorescence detection to overcome this limit in the sensitivity of detection, although UV-VIS would be less restrictive in the types of analytes detected. Increasingly sensitive detectors of many types will make the use of all kinds of capillary electrophoretic techniques more popular. 

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) An electrophoretic technique used for the separation of proteins. 

The separation methods routinely employed in the laboratory include the various chromatographic and electrophoretic techniques, whose selectivity is continually being increased by the introduction of new adsorbents, e.g. with chemically modified surfaces (Fig. 1). 

The validity of diagnosis by this technique has been examined by comparing more than 2,000 samples by the CM-Cellulose procedure, the original CM-Sephadex procedure, and by starch gel electrophoresis. It Is Interesting that occasionally the AS condition at birth Is not diagnosed by the electrophoretic technique. 

This introduction was intended to merely provide basic concepts and principles of electrophoretic separation. It should be noted that a wide variety of electrophoretic techniques, such as isoelectric focusing and two-dimensional gel... 

Moving-boundary electrophoretic techniques, originally demonstrated by Tiselius in 1937, employ a U-tube with the sample occupying the lower part of the U and the two limbs being carefully filled with a buffered electrolyte so as to maintain sharp boundaries with the sample. Electrodes are immersed in the electrolyte and direct current passed between them. The rate of migration of the sample in the electric field is measured by observing the movement of the boundary as a function of time. For colourless samples, differences in refractive index may be used to detect the boundary. Such moving-boundary techniques are used mainly in either studies of the physical characteristics of molecules or bulk preparative processes. 

Polyacrylamide gel (PAG) electrophoresis is performed either in cylindrical glass tubes or in flat beds (Figure 3.27). The method is comparable with other electrophoretic techniques but care must be taken to keep the current low to prevent any significant heating effect. 

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