Open and Toroidal Electrophoresis. Tarso B. Ledur Kist

(2) Toroidal (2) Microchip (2) Electrochromatography (3) Slab (3) End-labeled free-solution electrophoresis (4) Free-solution electrophoresis (5) Isoelectric focusing (6) Isotachophoresis (7) Microemulsion electrokinetic chromatography (8) Micellar electrokinetic chromatography (9) Sieving electrophoresis

      The full names and acronyms of these 54 combinations are presented in Appendix A. However, the feasibility of some of these techniques is not obvious. For instance, running the isotachophoresis mode in the toroidal layout may be difficult because the leading and terminating electrolytes must be exchanged before each high voltage rotation (see Chapter 6).

      Free flow electrophoresis (FFE) is a special layout mainly used for preparative separations in special macro- and microchip platforms (shallow two-dimensional chambers). Many separation modes can be performed in this setup. The sample is continuously fed as a narrow streak into the stream of the separation medium and an electric field is orthogonally applied to this flow direction, driving the components of interest to specific collection points of the outlet. This is also discussed in Appendix A.

      It must be remembered that, for some applications, gel slabs can be used for at least two purposes; merely as an anticonvective agent (to prevent band spreading) and/or as a sieving agent. In the latter case a high rate of analyte collisions against the polymer net is observed and plays a fundamental role in the separations. All of these techniques and their respective underlying phenomena are discussed in relation to the open and toroidal layouts throughout this book.

Electrophoresis and electroosmosis are the most commonly used phenomena that drive separations in the ESTs. Moreover, a large quantity of additional basic phenomena can be directly observed and studied in the many ESTs. Some examples of these additional phenomena include fluid flow, analyte adsorption, phase partitioning, and the degree of acid–base ionization, as well as the interactions of ion–ion, ion–molecule, target–ligand, antibody–antigen, and many others. Moreover, the quantity of phenomena that can be mathematically modeled using well established theories is remarkable. Most of these phenomena can be modeled from first principles, without the need to add empirical or ad hoc parameters. Even so, as in all areas of science, there are a few experimental observations for which good models do not currently exist, and some of them even lack an adequate theory. It is an interesting field for developing models, testing theories, making predictions, and testing hypotheses involving quantifiable predictions and measurements. Finally, but no less importantly, the resulting separation techniques have an impressive number of applications within diverse fields such as clinical analysis, pharmaceutical analysis, genetic analysis, food analysis, environmental analysis, and proteome analysis, to mention only a few.

      The present book is written in such a way as to – hopefully – make it an interesting read for experts in the field as well as for users of these technologies, non-specialists, and students. The whole book is richly illustrated and presents a large number of very useful equations showing the relationships between important operational parameters and other fundamental variables. These are important tools for both readers who are interested in the theories of the field and those who are interested in the practical applications of the ESTs. Mathematical deductions are shown only in the appendices because the intent is not to bother readers who are not very familiar with mathematical methods. Students interested in becoming familiar with the modeling of the electrokinetic phenomena are encouraged to read these appendixes.

      In Chapter 1 the fundamental concepts and definitions related to liquids that may contain a background electrolyte are reviewed, paying specific attention to their applications to the ESTs. Phenomena and definitions studied in this chapter include the relative permittivity of water, dissolution, solvation, dissociation, ionization, Gibbs free energy, ionization constants, both

-pH and p

-pH diagrams, the Henderson–Hasselbalch equation, and the buffer capacity of aqueous solutions.

      Chapter 2 is dedicated to the fundamentals of ESTs. This includes the electrophoresis of single molecules, ionic limiting mobility, bands, peaks, zones, isoelectric points, both turbulent and laminar flow, electroosmosis, suppression of electroosmosis, the Joule effect, heat dissipation, temperature profiles, molecular diffusion, band broadening, sample stacking, band compression, and the separation modes.

The focus of Chapter 3 is the open (common) electrophoresis layout. The relationships between independent variables (or users' operational parameters) and the performance indicators are given for this type of layout (open). Examples of such performance indicators are: number of theoretical plates, number of theoretical plates per unit time squared, plate height, resolution, resolution per unit time, peak capacity, band capacity, and both peak and band capacity per unit time.

      In Chapter 4 the toroidal layouts of the three platforms (capillary, microchip, and slab) are presented in detail. The microholes (capillary), microconnections (microchip), and connections (slab) that function as the hydrodynamic and electrical communication between the toroid's internal lumen and the external environment (reservoirs and electrodes) are examined in detail. The concept of the passive and active modes of operation are also presented. Active modes are used to prevent the bands from leaking out of the toroids and into the reservoirs.

      Chapter 5 gives a summary of the performance indicators presented in Chapters 3 and 4. Tables comparing the performance indicators as functions of the operational parameters are shown in this chapter. The performance indicators of the open and toroidal layout are contrasted and the pros and cons of each layout are examined.

      The high voltage setups used in the open and toroidal layouts are discussed in Chapter 6. One important aspect that differentiates the toroidal layout from the open layout is the way the high voltages are connected and operated. Conventional positive and/or negative high voltage modules can be used; however their output must be quickly redistributed (rotated) in a cyclic manner to keep the set of bands running until the desired resolutions are achieved. This is performed using high voltage distributors and the pros and cons of each are shown using didactic illustrations.

      Heat removal and temperature control in both open and toroidal layouts are presented in Chapter 7. In addition to the side effects of the temperature gradients on band dispersion, many unexploited potentials and advantages of a rational cooling design are presented and discussed for all platforms (capillary, microchip, and slab). This is examined with the aid of dozens of figures and a solid theoretical basis.

      Most