Tarso B. Ledur Kist - Open and Toroidal Electrophoresis

Chapter 2is 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 3is 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 4the 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 5gives a summary of the performance indicators presented in Chapters 3and 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 of the detectors that are compatible with the open layout are also compatible with the toroidal layout. They are presented in Chapter 8, which also includes the following detection systems: absorption, contactless conductivity, fluorescence, thermal lens, and mass spectrometry. Many advantages of fluorogenic and labeling reactions in both the open and toroidal layouts are presented. In addition, the use of these reactions to increase the detection limits, performance indicators, and separation selectivities is also discussed.

In Chapter 9a few examples of the applications of the toroidal layout are presented. These include the analyses of amino acids, stereoisomers, and isotopomers, among others. The potential of toroidal electrophoresis to obtain separation efficiencies of over one hundred million theoretical plates is shown and discussed. This is the only chapter that omits the open layout, as it would be almost impossible to present all of its applications (across all platforms and separation modes) in a single book.

Important mathematical deductions pertaining to the ESTs are made in the appendixes. These deductions have been left for the appendixes to make the main text more fluidic and light.

Water is by far the most widely used solvent in the preparation of gels, linear polymer solutions, and buffer solutions for electrokinetic separation techniques (ESTs). This is because of its unrivaled capacity to dissociate and ionize a great variety of substances (salts, acids, and bases). Ultimately, due to the large number of reactions that occur within water, it is the substance that makes life possible. Therefore, there are many reasons to take a closer look at the interesting properties of this unique liquid.

The temperature of a solvent, for instance water, acetonitrile or methanol, is related to the average energy per degree of freedom that these molecules have. Average energy is given by , where represents the Boltzmann constant and represents temperature in Kelvin.[1] The degrees of freedom consist of the translation velocity, which occurs in the three space coordinates, and both the vibrational and rotational degrees of freedom of the molecules. Raising the temperature of liquid water at 1 atm from 278.15 K (5 C or 41 F) to 348.15 K (75 C or 167 F) increases the average velocity ( ), indeed , by about 25%. The consequential increase in thermal energy is important for the collision rate and momentum transfer among the molecules within the liquid. Such collisions with fast-moving molecules cause random movements of the microscopic particles that are suspended in the liquid (or in a gas), which can be observed under the microscope ( Brownian motion ). This is the basis for the denaturation and renaturation of nucleic acids, as well as the denaturation of proteins (disruption of both the secondary and tertiary structures). This same process also plays a role in dissolution, dissociation, ionization, and in maintaining the equilibrium of reactions. The Gibbs free energy change quantitatively gives the role of enthalpy, temperature and entropy for chemical reactions in the general case (see Section 1.1.8).

Water is a remarkable solvent for the dissolution, dissociation, and ionization of many substances. There are a few ways to predict these unique properties of water. The predictive models belong either to the classical theory of electromagnetism, statistical mechanics, thermodynamics, or the molecular dynamics simulations that use dedicated software to predict the physical movements of atoms and molecules (which are based on the fundamental Newton equations of mechanics). Above all, it must be remembered that oxygen has the second highest electronegativity of the whole periodic table [2] (see Figure 1.1). Only fluorine has a higher electronegativity; it forms only one covalent bond with a hydrogen atom (hydrogen fluoride) and is not liquid at room temperature (above 19.5 C). Nitrogen, the other neighbor of oxygen in the periodic table, forms three covalent bonds with hydrogen and undergoes sp 3hybridization, producing three sp 3bonding orbitals and one sp 3non-bonding orbital (forming ammonia). It is also a gas at room temperature. In water molecules, on the other hand, oxygen establishes covalent bonds with two hydrogen atoms. The resulting sp 3structure contains two bonding orbitals (with a partial positive charge at each hydrogen position) and two non-bonding orbitals (with a partial negative charge on each). This produces a large electric dipole moment, especially considering that this is a small molecule, which explains the high relative electric permittivity of water (a small molecule with a large electric dipole moment), as shown in Table 1.1. The large amount of hydrogen bonds per unit volume within bulk water plays an important role in many of its unique properties. For example, the relatively high values of surface tension, boiling point, thermal conductivity, and latent heat of evaporation, to name a few. The strength and directional feature of hydrogen bonds explains a few additional odd properties, e.g., the solid (ice) has a lower density than the liquid (water).