Electrophoretic methods

Electrophoretic methods are widely used alternatives for the analytical determination of the enantiomeric purity of chiral compounds [194]. Due to the high elTi-ciency of capillary electrophoresis, separations can be achieved even when very low selectivities are observed. At a preparative scale, these methods are well established for the purification of proteins and cells [195] but there is very little published on enantioselective separations. Only recently, some interest in chiral preparative applications has been manifested. Separation of the enantiomers ofterbu-taline [196] and piperoxan [197] have been reported by classical gel electrophoresis using sulfated cyclodextrin as a chiral additive, while the separation of the enantiomers of methadone could be successfully achieved by using free-fluid isotachophoresis [198] and by applying a process called interval-flow electrophoresis [199]. 

The feasibility of separating the enantiomers of chlopheniramine on a micropreparative scale was also recently shown by applying flow-counterbalanced CE using carboxymethyl (l-cyclodextrin as a chiral selector [200]. All these apphcations were performed at a mg scale or even less and cannot currently compete with the liquid chromatographic methods discussed previously. However, the scope and limitations of the electrophoretic approach still need to be challenged [201]. 

Erancotte, Preparation of drug enantiomers by chromatographic resolution on chiral stationary phases, in The Impact of Sereochemistry on Drug Development and Use, H.Y. Aboul-Enein, I.W. Wainer (Eds.), Chemical Analysis Series, 

Erancotte, Chromatography as a separation tool for the preparative resolution of racemic compounds, in Chiral Separations Applications and Technology, 

Dingenen, Polysaccharide phases in enantio-separations, in A Practical Approach to Chiral Separations by Liquid Chromatography, G. Subramanian (Ed.), VCH Publishers, Weinheim, 1994, 

In Chapter 7 we examined several methods for separating an analyte from potential interferents. For example, in a liquid-liquid extraction the analyte and interferent are initially present in a single liquid phase. A second, immiscible liquid phase is introduced, and the two phases are thoroughly mixed by shaking. During this process the analyte and interferents partition themselves between the two phases to different extents, affecting their separation. Despite the power of these separation techniques, there are some significant limitations. 

The advantages of CE and MEKC (small sample size, high separation efficacy and speed) have been exploited in the analysis of flavonoids too. The results obtained in the analysis of wines have been reviewed earlier [211]. 

As the separation characteristics of liquid chromatographic and electrophoretic techniques markedly differ from each other, combined methods using the advantages of both procedures have been successfully used for the analysis of flavonoids. Thus, the use of CZE-UV, HPTLC-UV and GC-MS for the measurement of flavonoids in seeds and root exudates of Lotus pedunculatus has been reported. The rooting solution and seed exudate were passed through cellulose acetate filters to bind the flavonoids. After extraction, 

ANALYSIS OF POLYPHENOLS IN REAL SAMPLES (FIGURES CORRESPOND TO CONCENTRATIONS IN pG/MLf 

Comparisons between this technique and neutral elution (see below) for detecting DSBs in irradiated DNA show good agreement, which is satisfying. Probably the TAFE system will become the method of choice. 

fig of supercoiled DNA in 20 fil of buffer (pH 7.4) are exposed to a source of radical species. The reaction is terminated by adding 5 fil of 0.1 M EDTA containing 50% sucrose and 0.1% bromophenol blue (to act as marker for electrophoresis) and the mixture subjected to electrophoresis through agarose gels (0.5 to 2%) (see Ueda et al., 1985). 

A limitation is that multiple SSBs will only give form II, unless two breaks occur close together in alternate strands, giving a DSB and hence form III. 

It is important to use a minimum of a non-reactive buffer such as phosphate, with no radical scavenging properties. 

Electrophoresis is the movement of charged particles under an electric field these particles can be small ions, proteins, DNA and other biomolecules, and colloidal 

FIGURE 5.14 (a) Representation of a positively charged particle under electrophoretic 

ft is the particle electrophoretic mobility. It should be noted that, whereas Equation 5.74 is valid for a spherical particle satisfying Stokes s law, the definition of p given by Equation 5.75 is more general for large nonspherical particles, other numerical coefficients would appear in Equation 5.73, and for smaller particles and ions, this law losses validity, but always a mobility can be measured. 

The mobility of particles is determined by electrophoretic experiments by several techniques, which are covered in numerous texts (Kruyt 1949 Lyklema 1995 Birdi 1997 Hiemenz and Rajagopalan 1997 Kosmulski 2009) and will not be discussed here. 

FIGURE 5.15 Electric field lines around a spherical particle of radius R the interaction with the diffuse layer (its thickness k is marked) causes a distortion of the field lines. 

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Although each capillary electrophoretic method has its own unique considerations, the following description of the determination of a vitamin B complex provides an instructive example of a typical procedure. 

The use of agarose as an electrophoretic method is widespread (32—35). An example of its use is in the evaluation and typing of DNA both in forensics (see Forensic chemistry) and to study heritable diseases (36). Agarose electrophoresis is combined with other analytical tools such as Southern blotting, polymerase chain reaction, and fluorescence. The advantages of agarose electrophoresis are that it requires no additives or cross-linkers for polymerization, it is not hazardous, low concentration gels are relatively sturdy, it is inexpensive, and it can be combined with many other analytical methods. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

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. 

E. Heftmann (Ed.), Chromatography Fundamentals and Applications of Chromatographic and Electrophoretic Methods, 2 volume set, Elsevier Science, New York, 1983. ISBN 0444420452 (set). 

The sulfate is obtained by evaporating the aqueous layer in vacuo. The hydrochloride can be obtained in the same way but using HCl instead of H2SO4. SAM-HCl has a solubility of 10% in H2O. The salts are stable in the cold at pH 4-6 but decompose in alkaline media. [Cantoni Biochem Prep 5 58 1957.] The purity of SAM can be determined by paper chromatography [Cantoni J Biol Chem 204 403 1953 Methods Enzymol 3 601 1957], and electrophoretic methods or enzymic analysis [Cantoni and Vignos J Biol Chem 209 647 1954]. 

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