Electrophoretic charge separation

ESI nebulization involves a variety of electrochemical processes at the needle and at the counter electrode [27, 30]. The ESI interface can be considered as a electrochemical cell, in which part of the ion transport takes place through the gas phase (Figure 6.1). In positive-ion mode, an emichment of positive electrolyte ions occurs at the solution meniscus as the result of an electrophoretic charge separation. The liquid meniscus is pulled into a cone which emits a fine mist of droplets with an excess positive charge. Charge balance is attained by electrochemical oxidation at the capillary tip and reduction at the counter electrode. The topic arose significant discussion in 2000 and the discussion partners continued to disagree on the role of electrochemistry inESI-MS [39]. 

Considering the requirements for charge balance in such a continuous electric current device and the fact that only electrons can flow through the metal wire supplying the electric potential to the electrodes, one comes to the conclusion that the electrophoretic charge separation mechanism [of droplet charging and formation] requires that the [positive-ion] electrospray process should involve an electrochemical conversion of ions to electrons [within the metal ES capillary[. 

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. 

Figure 13.4 Schematic illustration of the electrophoretic molecular separation, (a) The charged molecules drift according to the electric field direction, (b) Separation of each fraction by applying several separate laminar flows, (c) Alternatively, each fraction can be separated by scanning the stripping laminar flow across the sample channel. Adapted from Ref. [43] with permission.

The lateral mobility of proteins and lipids in natural and artificial lipid bilayer membranes was determined by different methods. For long-range mobility, fluorescence recovery after photobleaching (13-15) and electrophoresis of membrane components (16) were employed. We employed the electrophoresis method for determination of the eletrophoretic and diffusional mobilities of PSI in the plane of hypotonically inflated, spherical thylakoid vesicles. To monitor the redistribution of PSI particles, we made use of the spatial characteristics of the contribution of PSI particles to electrophotoluminescence (EPL) (17, 18). The contribution of PSII to EPL was eliminated by heat treatment of the chloroplasts (19). The EPL originates from the PSI particles at the hemisphere of the vesicles at which the induced electrical field destabilizes the photoinduced charge separation (18). The electrophoretic and diffusional mobilities were measured in vesicular suspensions to avoid immobilization for microscopic visualization (20). The photosynthetic membranes are devoid of cytoskeletal elements that might interfere with the lateral mobility. 

The equivalent conductance of salts or ions increases as the concentration decreases. This phenomenon is directly related to the interionic forces present in solution a given cation, for example, will have more anions in its vicinity than expected from a purely random distribution. This ionic atmosphere has two effects, electrophoretic and time of relaxation, both of which tend to decrease the ion s mobility. In the former effect, the solvent molecules associated with the ionic atmosphere are moving in a direction opposite to that of the central ion. In the latter, the ionic atmosphere moves slower than the central ion, causing a charge separation (electrostatic retarding force) on the central ion. 

While we have only focused here on applications for DNA electrophoresis, colloidal crystals have also been used as a stationary phase for separating hydrophobic dyes and proteins [40]. The arrays may prove especially interesting for electrophoretic protein separations, since the pore size provides a steric constraint, no pressure drop is required for transport through the small pores and the bead surfaces serve as a substrate for reversed-phase adsorption. These arrays thus offer a tunable variety of separation mechanisms. Indeed, proteins with similar hydrophobic groups, which cannot be separated by high performance liquid chromatography (HPLC) due to their similar adsorption properties, are rapidly separated by charge in colloidal arrays [40]. 

Figure 4 Simultaneous chiral separation of the isomeric tripeptides Gly-a-Asp-PheNH2 and Gly- -Asp-PheNHa. Experimental conditions 40/47cm polyacrylamide-coated capillary, 50 im, 50 mmol I sodium phosphate buffer, pH 5.25, 60mgmr car-boxymethyl- -cyclodextrin, -20 kV, UV detection at 215 nm. (Reprinted with permission from Sabah S and Scriba GKE (1998) Electrophoretic stereoisomer separation of aspartyl dipeptides and tripeptides in untreated fused-siiica and polyacrylamide-coated capillaries using charged cyclodextrins. Journal of Chromatography A 822 137-145 Elsevier.)...

Sabah S, Scriba G (1998) Electrophoretic stereoisomer separation of aspartyl dipeptides and tripeptides in untreated fused-silica and polyacrylamide-coated capillaries using charged cyclodextrins. J Chromatogr A 822 137-145... 

A short-term electrophoretic-type separation of water and hydrocarbon oil occurs in the external electric field in the vicinity of the electrode surface. Under even a moderate electric field the mass transport of small water dipoles with dielectric constant of 80 is much more facile than that of polymers with dielectric constant of 2. High local concentration of water dipoles may be created in the vicinity of the electrode interface as a result of water redistribution and percolation in an external electric field. Relatively high water conductivity and the facile nature of charge-transfer reactions carried out through water dipoles located in the interfadal region explain the dramatic decrease in the low-frequency impedance immediately following the water injection. Deposition of conductive layers of water on the electrode/lubricant interface and significant presence of water dipoles in the diffusion layer replace both specifically adsorbed and electroactive lubricant additive species at the lubricant-electrode... 

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). 

Cross-flow-elec trofiltratiou (CF-EF) is the multifunctional separation process which combines the electrophoretic migration present in elec trofiltration with the particle diffusion and radial-migration forces present in cross-flow filtration (CFF) (microfiltration includes cross-flow filtration as one mode of operation in Membrane Separation Processes which appears later in this section) in order to reduce further the formation of filter cake. Cross-flow-electrofiltratiou can even eliminate the formation of filter cake entirely. This process should find application in the filtration of suspensions when there are charged particles as well as a relatively low conduc tivity in the continuous phase. Low conductivity in the continuous phase is necessary in order to minimize the amount of elec trical power necessaiy to sustain the elec tric field. Low-ionic-strength aqueous media and nonaqueous suspending media fulfill this requirement. 

Charged macromolecules, such as proteins or polymers, are often separated elec-trophoretically. The rate of migration through an electric field increases with net charge and field strength. Molecular size of analytes and viscosity of separation media both have inverse relationships with rate of migration. These variables must all be taken into account in order to optimize the conditions for an efficient electrophoretic separation. 

---