Strategies for pH gradients' optimization.

A few problems arising when optimizing the composition of the mixtures to be used for IPG casting with a two chambers mixer are discussed. The behavior in the composition space of a number of objective functions is described by simulation. This allows the formalization of rules to be followed for finding the initial guess required to start the optimization algorithm and for directing and limiting the search of the objective's minimum. the results of the simulation show that a fully automatic gradient computation and optimization is possible.

On the computational approach to immobilized pH gradients.

The unified treatment for computing the pH of complex mixtures of mono- and polyprotic buffers, including ampholytes, as utilized in the gradient simulation program PGS, is presented. Its ability to compute pH, buffering power and ionic strength is shown by discussing a few simulations. The problems arising in the automatic formulation of optimal mixtures are presented, as well as the merits and limits of several target functions utilized in such optimizations. It is shown that no universal target function exists and that a proper optimization method should account for the fact that more than one formulation is possible for a given pH range.

Formulations for immobilized pH gradients including pH extremes.

Formulations are given both for narrow (less than 2 pH units) and for wide range (up to 8 pH units) immobilized pH gradients, spanning between pH 2.5 and pH 11. The contribution from water to the buffering power (beta) at these pH extremes requires the recipes to be optimized (in terms of gradient linearity) for each desired level of beta av.

Ion retention by immobilized pH matrices.

The effect of the interaction between polyacrylamide matrices containing covalently bound acidic and basic residues and free ions was indirectly evaluated from the width of the residual salt fronts after electrophoresis under standard conditions. Around neutrality the front width is larger than at alkaline, and much larger than at acidic pH, and in all instances much higher for anions than for cations. The width decreases faster with the buffering power of the matrix than with its ionic strength. After about 5000 V x h, the migration of the salt front is very slow, and becomes negligible when diffusion is prevented. When inadequately buffered by the matrix, H+ and OH(-)--comigrating with anions and cations--increase the conductivity within the salt front and drastically lower the electrical field strength.

pH gradients generated by polyprotic buffers. II. Experimental validation.

The experimental validation refers to the computer program reported in the companion paper, able to simulate the course of pH, buffering power (beta) and ionic strength (I) of polyprotic buffers (either singly or in a mixture) titrated over any pH range. With simple oligoamines (up to five nitrogens) it is shown that it is impossible to generate linear pH gradients in the pH 4-10 interval, unless they are mixed in appropriate ratios. With pentaethylene hexamine, when used alone, it is possible to create a linear pH 4-10 interval, provided the molarity ratios are altered in the two chambers of the gradient mixer. The general rule operating for generation of linear pH intervals is constancy of buffering power throughout the titration. Local minima of beta produce steeper gradients, while local beta maxima flatten it. The ideal delta pK to arrange for linear pH gradients during titration is centred around 1 pH unit; thus polyprotic buffers with very large delta pK values (e.g., EDTA) appear to be totally useless for this purpose. The present computing algorithms should be quite efficient for optimizing existing buffer recipes for chromatofocusing or ampholyte displacement chromatography or for creating new, properly tailored, buffer mixtures.

pH gradients generated by polyprotic buffers. I. Theory and computer simulation.

This paper presents the general equations for computing pH, dissociation coefficients, buffering power and ionic strength of pure polyelectrolyte solutions (polyacids, polybases and zwitterionic species with any number of dissociable groups) and mixtures of any number of these species. A program has been written for simulating the behaviour of mixtures containing up to 50 species (including buffers and titrants), each of them with up to 10 dissociable groups. This allowed one to check the equations with the available data on a few oligoprotic species.

Isoelectric focusing in immobilized pH gradients: generation and optimization of wide pH intervals with two-chamber mixers.

A new technique for generating extended pH gradients (5 pH units) in Immobiline gels is reported. The previously described (J. Biochem. Biophys. Methods 7, 1983, 123-142) five-chamber gradient mixer has been replaced by a two-vessel device. A single mixture of the available Immobilines (pK 3.6, 4.6, 6.2, 7.0, 8.5 and 9.3) is made, with relative concentrations adjusted so as to produce the most uniform buffering power throughout the desired pH interval. This mixture is then divided into two portions, which are titrated to the extremes of the required pH span with an acidic titrant (Immobiline pK approximately 1) and a basic species (Immobiline pK 9.95). Highly reproducible pH gradients (pH 4-9) are thus generated, which appear extremely useful for the first dimensioned of 2-dimensional techniques. Our previously reported computer program has been implemented with an optimization algorithm which, given any cocktail of Immobilines, automatically adjusts the relative initial concentrations until the smoothest possible beta power is found. For the first time it is possible to perform IEF under controlled physico-chemical parameters: pH span and linearity, beta power, ionic strength and molarity of the buffering species.

Isoelectric focusing in immobilized pH gradients: generation of extended pH intervals.

A new technique for generating extended pH gradients (3-4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the 'Varigrad'-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857-862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4-8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in the pH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (beta) and ionic strength (I) courses.

A method for the in situ measurement of the electrical quantities in frog skin.

A method allowing the measurement of the electrical quantities related to the physiological functions of the frog skin in situ is presented. The method allows the performance of several experiments on the same pithed animal, which remains alive for a number of days. The preparation is very stable, and the electrical potential difference and short-circuit current values are higher than in isolated skin. The theory of measurement and the possible systematic errors are discussed. The possibilities of the method are evaluated on comparing the pH and temperature dependence of the electrical quantities in situ with previous measurements on isolated skin.

Membrane water permeability alterations induced by pollutants.

Lead ions and the surfactant nonylphenolethoxylate are found to inhibit the active transport-coupled water flow in the leg skin of Rana esculenta. The water permeability for osmotically driven flows remains unchanged for high flows, while it is remarkable lowered near zero osmotic pressure difference. The possible mechanism of the permeability change is discussed in connection with the available data on the electrophysiological effects of the pollutants.

Non linear volume flow dependence on osmotic pressure difference in frog skin.

The volume flow dependence upon the osmotic pressure difference of both impermeant (sucrose) and permeable (NaCl) species has been investigated in leg skin bags of Rana esculenta. It is concluded: 1. The hydration-dehydration error in the flow measurement with leg skin bags is negligible. 2. The flow-force relationship is non-linear. 3. Unstirred layers and solute permeation have little, if any, influence on non linearity. 4. Structural modifications of the skin induced with hypertonic solutions have been observed and may contribute to non linearity, as well as the multiple-barrier effect.

Sodium chloride reflection coefficient in rabbit gall bladder.

By means of the Kedem-Katchalsky thermodynamic description of active transport, a relationship has been derived between the apparent reflection coefficient and the Staverman reflection coefficient for passive transport of a solute which is both actively and passively transported. The relationship between volumetric flow and its driving forces, containing the Staverman reflection coefficient, was tested for sodium chloride in rabbit gall bladder and the reflection coefficient was evaluated.