Electrophoresis formats

A schematic representation of a CE system is presented in Figure 9.1. In this diagram, the CE components have obvious counterparts to those found in slab gel electrophoresis. Instead of buffer tanks there are two small buffer reservoirs, and the capillary takes the place of the gel (or more accurately, a gel lane). The capillary is immersed in the electrolyte-filled reservoirs, which also make contact with the electrodes connected to a high-voltage power supply. A new feature to the conventional gel electrophoresis format is the presence of an online detection system. 

The first work on pKa determination by zone electrophoresis using paper strips was described by Waldron-Edward in 1965 (15). Also, Kiso et al. in 1968 showed the relationship between pH, mobility, and p/C, using a hyperbolic tangent function (16). Unfortunately, these methods had not been widely accepted because of the manual operation and lower reproducibility of the paper electrophoresis format. The automated capillary electrophoresis (CE) instrument allows rapid and accurate pKa determination. Beckers et al. showed that thermodynamic pATt, (pATf) and absolute ionic mobility values of several monovalent weak acids were determined accurately using effective mobility and activity at two pH points (17). Cai et al. reported pKa values of two monovalent weak bases and p-aminobenzoic acid (18). Cleveland et al. established the thermodynamic pKa determination method using nonlinear regression analysis for monovalent compounds (19). We derived the general equation and applied it to multivalent compounds (20). Until then, there were many reports on pKa determination by CE for cephalosporins (21), sulfonated azo-dyes (22), ropinirole and its impurities (23), cyto-kinins (24), and so on. 

II. CAPILLARY ELECTROPHORESIS FORMATS A. Capillary Zone Electrophoresis... 

Other applications of CE to analyze food additives include the determination of vitamin C and preservatives (benzoate and sorbate) by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection. The separation was optimized by adjusting the pH value of the buffer and the use of hydroxypropyl- -CD (HP- -CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-f)-CD and 0.25 mM CTAB with a LOD ranging from 0.5 to 3 mg/L, whereas a histidine/tartrate buffer with 0.06% HP-fl-CD and 0.25 mM CTAB gave a LOD ranging from 3 to 10 mg/mL. By using a microchip electrophoresis format, a considerable reduction of analysis time was accomplished. ... 

Recently, the MECC technique has been extensively investigated by many scientists [86-89] as a means of obtaining selective separations of neutral and ionic compounds while retaining the advantages of the capillary electrophoresis format. 

Capillary SDS-PAGE has several advantages over the more conventional electrophoresis formats [27], including nanogram sample capacity, prospects for automation, ease of quantification, and sensitivity. Capillary gel electrophoresis with fraction collection has also been used for micropreparative purification of macromolecules [38, 93]. 

Section 28 14 The nucleotide sequence of DNA can be determined by a technique m which a short section of single stranded DNA is allowed to produce its complement m the presence of dideoxy analogs of ATP TTP GTP and CTP DNA formation terminates when a dideoxy analog is incorporated into the growing polynucleotide chain A mixture of polynucleotides dif fermg from one another by an incremental nucleoside is produced and analyzed by electrophoresis From the observed sequence of the comple mentary chain the sequence of the original DNA is deduced... 

Paperboard materials PAPERCHEM Paper chromatography Pap er-co a ting pigmen t Paper colorants Paper coloring Paper electrophoresis Paper fiber Paper formation aids Paper grades Paper industry... 

Electrophoresis and electro osmosis can be used to enhance conventional cake filtration. Electrodes of suitable polarity are placed on either side of the filter medium so that the incoming particles move toward the upstream electrode, away from the medium. As most particles carry negative charge, the electrode upstream of the medium is usuaHy positive. The electric field can cause the suspended particles to form a more open cake or, in the extreme, to prevent cake formation altogether by keeping aH particles away from the medium. 

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Most electrophoretic methods have been tried in a free-flow format, including isoelectric focusing, native zone electrophoresis, and isotachophoresis. Most free-flow electrophoresis equipment has very low (ca 1 g/(L-h)) capacity, and resolution is reduced by heating and electroosmotic considerations. 

Polyacrylamide gel electrophoresis results suggest that p-LG undergoes a greater conformational loss as a fimction of extrusion temperature than a-LA, presumably due to intermolecular disulfide bond formation. Atomic force microscopy indicates that texturization results in a loss of secondary structure of aroimd 15%, total loss of globular structure at 78 °C, and conversion to a random coil at 100 °C (Qi and Onwulata, 2011). Moisture has a small effect on whey protein texturization, whereas temperature has the largest effect. Extrusion at or above 75 °C leads to a uniform densely packed polymeric product with no secondary structural elements (mostly a-helix) remaining (Qi and Onwulata, 2011). 

A variety of formats and options for different types of applications are possible in CE, such as micellar electrokinetic chromatography (MEKC), isotachophoresis (ITP), and capillary gel electrophoresis (CGE). The main applications for CE concern biochemical applications, but CE can also be useful in pesticide methods. The main problem with CE for residue analysis of small molecules has been the low sensitivity of detection in the narrow capillary used in the separation. With the development of extended detection pathlengths and special optics, absorbance detection can give reasonably low detection limits in clean samples. However, complex samples can be very difficult to analyze using capillary electrophoresis/ultraviolet detection (CE/UV). CE with laser-induced fluorescence detection can provide an extraordinarily low LOQ, but the analytes must be fluorescent with excitation peaks at common laser wavelengths for this approach to work. Derivatization of the analytes with appropriate fluorescent labels may be possible, as is done in biochemical applications, but pesticide analysis has not been such an important application to utilize such an approach. 

Gysler, J., Mazereeuw, M., Helk, B., Heitzmann, M., Jaehde, U., Schunack, W., Tjaden, U.R., and van der Greef, J., Utility of isotachophoresis-capillary zone electrophoresis, mass spectrometry and high-performance size-exclusion chromatography for monitoring of interleukin-6 dimer formation, /. Chromatogr. A, 841, 63, 1999. 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

The difficulty with protein arrays is that proteins do not behave as uniformly as nucleic acid. Protein function is dependent on a precise, and fragile, three-dimensional structure that may be difficult to maintain in an array format. In addition, the strength and stability of interactions between proteins are not nearly as standardized as nucleic acid hybridization. Each protein-protein interaction is unique and could assume a wide range of affinities. Currently, protein expression mapping is performed almost exclusively by two-dimensional electrophoresis and mass spectrometry. The development of protein arrays, however, could provide another powerful... 

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