Sieving media

The following explanation of the behavior of analytes during CE-SDS in the presence of sieving media is found in a monograph by Guttman 135 the electric force (Fe) that a particle experiences when placed in an electric field depends on the net charge (Q) of the particle and on the intensity of the electric field (E) ... 

In the presence of a sieving media the motion of the ions is impeded by a frictional force (Ff). The frictional force is dependent on the translational friction coefficient (f) ... 

Novel sieving media and acceleration of electrophoretic runs coupled with suitable data processing are likely to provide excellent approaches to DNA sequencing by multiplexed CE. The technology can be readily scaled to 1024 capillaries, which is a convenient number for available array detectors. Such a system can be optimized to accelerate the sequencing process enormously. 

Initially, gels were prepared within the capillary by copolymerization of acrylamide with bisacrylamide [9] as in slab gel electrophoresis. The capillary wall was coated with an acrylate as described above to remove the EOE Thus the gel could also be chemically fixed on the wall. Agarose which can be thermally mobilized has also been used [10]. With these gels the problem discussed above arose. Therefore liquid gels were introduced [11] after they had already been proposed for classical electrophoresis [12]. Some remarks on the properties of the sieving media will be given as the information found in the literature can be very confusing. 

The problem with HEC solutions are their high viscosities. Therefore, dex-trans differing in molecular mass have been used as sieving media [26]. These solutions can easily be replenished due to their low viscosities and exhibit good separation properties. 

It should always be kept in mind that the analysis time increases with increasing concentration of the sieving media. The regime where a separation is no longer possible seems to drift to lower molecular masses. This has been observed with PEG and dextrans (cf. Fig. 10). 

As already discussed for DNA separation, the pore size of the sieving media should only be dependent on its concentration, and not on its molecular mass. 

Nongel sieving media have been applied to problems of forensic interest by McCord et al. (1993a), who used the following procedure for buffer preparation 0.1 mAf EDTA was added to 100 mAf Trisma base and 100 mAf boric acid pH was adjusted to 8.7 with cesium hydroxide. Hydroxyethylcellulose was dissolved in this buffer at a concentration of 0.5% (w/v), and the solution was filtered through a 0.45 mm cellulose acetate filter. Prior to analysis, ethid-ium bromide was added to a concentration of 1.27 mAf. Phenylmethyl-coated... 

The introduction of Sephadex in 1959 provided the biochemist with a new powerful tool for the separation of complex mixtures of biopolymers on the basis of their molecular size. The scope of the technique was further extended by the introduction of the bead-form agaroses which permit separation of particles and molecules up to 40000000 daltons. Early work showed that separation might be influenced by solute-matrix interactions rather than purely steric factors [171], Increasing work has been done on the nature and extent of these interactions and, more recently, these effects have been used to improve and even effect separations on gels such as Sephadex. These interactions have been reviewed recently [172, 173] and in this section we will briefly consider some aspects of solute-matrix interactions and their application in the separation procedure. Recent developments of new molecular sieve media and some new column techniques are also discussed. 

A change in entropy will thus have a significant effect on the selectivity when molecular sieving is considered. This is thoroughly discussed by Singh and Koros [9]. The flux may be described as in Equation 4.16 where Eq.ms is the activation energy for diffusion in the molecular sieving media. 

Different sieving polymers of identical molecular masses show, however, significant differences in their selectivity. This becomes obvious from Fig. 13, where the influence on selectivity for all concentrations and molecular masses for all the studied sieving media is summarized. It can be seen... 

Fig. 13 Superimposition of all investigated sieving media in one Dolnik plot. Corresponding sieving polymers carry identical symbols and line styles...

The interaction of the polymeric analyte molecules (charged or uncharged) with the polymeric sieving media (uncharged or charged) is a general and principal method for analytical separation of water-soluble polymers. Transfer of this technique to water-insoluble polymers by applying the technique of non-aqueous capillary electrophoresis (NACE) seems feasible. 

Size-based separations of homogeneous polyelectrolytes, such as DNA, are not possible in free solution electrophoresis [159]. This is due to the proportionality of the friction hydrodynamic force and total charge of the molecule to its length. The friction hydrodynamic forces exerted on the free-drained polymer coil while it moves as well as the accelerating electrostatic force both increase proportionally with the addition of a nucleotide to the chain. This is why one must typically use a sieving media, such as a gel or an entangled polymer solution, to obtain size-based separations of DNA using electrophoresis. 

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