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11.1. INTRODUCTION Early experiments in the development of isoelectric focusing, a high-resolution steady-state electrophoresis method, occurred in 1912, with an electrolytic cell that was used to isolate glutamic acid from a mixture of its salts.1 A simple Ushaped cell, such as that used for moving-boundary electrophoresis ( 9), with two ion-permeable membranes equidistant from the center, created a central compartment that separated anodic and cathodic chambers, as shown in Figure 11.1. Redox reactions occurring in the anodic (Eq. 11.1) and cathodic (Eq. 11.2) electrolyte compartments generated H and OH ions in the respective chambers: ! 2 H2 O O2 4 H 4 e Anode ! 2 H2 O 2 e 2 OH H2 Cathode 11:1 11:2
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With this cell, glutamic acid placed in the central chamber can freely migrate across the ion-permeable membranes. As it crosses into the anodic chamber, the low pH causes protonation of the two carboxylates and the amine, so that a net charge of 1 is created on the molecule. This positively charged species then migrates toward the cathode, recrossing the membrane into the central chamber. When glutamic acid crosses the other membrane into the cathodic electrolyte compartment, the carboxylates and the amine group are deprotonated, and the molecule attains a net negative charge. This species migrates toward the anode, recrossing the membrane into the central compartment. The result of these migrations is that the glutamic acid becomes concentrated within the central compartment, at a pH close to its isoelectric pH, where it exists in the zwitterionic form with a net charge (and therefore mobility) of zero. The high concentration of glutamic acid at its pI value was the rst recorded demonstration of the isoelectric focusing (IEF) effect. A signi cant advance over this simple IEF experiment occurred in 1929,2 when the number of compartments separating the anodic and cathodic electrolytes was increased to 12. This work clearly demonstrated the concept of establishing a stepwise
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Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Corton ISBN 0-471-54447-7 Copyright # 2004 John Wiley & Sons, Inc.
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ISOELECTRIC FOCUSING
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Figure 11.1. The rst isoelectric focusing experiment, used to separate glutamic acid from a mixture of its salts.
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variation in pH from one electrode chamber to the other. This stepwise gradient results in a steady-state pH distribution and an accumulation of ampholytes at their isoelectric pH values. Ampholytes are chemical species capable of carrying net positive, zero, or negative charge, depending on the pH of the medium. Amino acids are common examples of ampholytes. At its pI, the net charge on an ampholyte is zero, so it will not migrate in an electric eld. Ampholyte species will therefore migrate in the eld until they reach a compartment in which pH pI; they then focus in that compartment. IEF was rst successfully applied to proteins in 1938, when it was used to separate the protein hormones vasopressin and oxytocin from tissue extracts.3 Twenty years later, ampholytes were rst focused in a continuous pH gradient, stabilized by a dense sucrose medium, as an alternative to the multicompartment method. The continuous pH gradient in the sucrose medium was established by allowing acid and base to diffuse into opposite ends of the sucrose medium, held in a U-cell, from their respective electrode chambers. The stabilization of this continuous pH gradient with carrier ampholyte species led to modern IEF methods. 11.2. CARRIER AMPHOLYTES The concept of carrier ampholytes in IEF was introduced in 1961.4 Carrier ampholytes are species that are amphoteric, so that they reach an equilibrium position along the separation medium, and are good electrolytes, possessing both ionic conductivity, to carry current, and buffering capacity, to carry pH. They are used to generate stable pH gradients in the presence of the electric eld, and are prefocused at their pI values before the sample is introduced. Ideally, a carrier ampholyte mixture consists of species that have identical diffusion coef cients and electrical mobilities, and differ in their pI values by only 0.05 pH unit, so that a linear pH gradient is generated. In practice, carrier ampholyte mixtures generate approximately stepwise changes in pH with distance along the separation medium. Figure 11.2 shows the pH gradients calculated for mixtures of eight carrier ampholyte species possessing pI differences of 0.05 and 0.10 pH units. The individual species focus at their pI values, where their buffering capacity is low. A linear
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