Acid-base equilibria of polyvalent electrolytes and theoretical description of polyelectrolyte behavior in electrokinetic separations.

The problem of the theoretical description of the dissociation of polyvalent electrolytes is considered. It is shown that the traditional model which assumes some constant microstate mobility values may have limited applications, since for each microstate the rate of conversion into other states is strongly dependent on pH and thus it may often become comparable with the characteristic times of other important processes (ionic atmosphere relaxation, biopolymer acceleration by electric field). The question of correct modelling of the electrophoretic flux, electrophoretic mobility and conductivity is discussed.

Capillary electrophoresis of peptides and proteins in isoelectric buffers: an update.

Capillary electrophoresis in acidic, isoelectric buffers is a novel methodology allowing fast protein and peptide analysis in uncoated capillaries. Due to the low pH adopted and to the use of dynamic coating with cellulose derivatives, silanol ionization is essentially suppressed and little interaction of macromolecules with the untreated wall occurs. In addition, due to the low conductivity of quasi-stationary, isoelectric buffers, high-voltage gradients can be applied (up to 800 V/cm) permitting fast peptide analysis with a high resolving power due to minimal diffusional peak spreading. Four such buffers are here described: cysteic acid (Cys-A, pI 1.85), iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). A number of applications are reported, ranging from food analysis to the study of folding/unfolding transitions of proteins.

Dissociation of polyvalent electrolytes.

Understanding of the dissociation mechanism for polyvalent electrolytes remains a matter of critical importance. Different theoretical approaches could result in different values for the microscopic parameters of the system under analysis, as well as for its integral characteristics (such as conductivity and buffering power). A unified classification of dissociation schemes is proposed here. Some examples of hybrid type schemes are considered and the expressions relating the macroscopic and microscopic constants are given. The possibility of applying the equation of Linderstrøm-Lang to multi-dissociating systems with non-constant total concentration is considered. The problem of proton binding curves (so-called titration curves) modeling is discussed.

Steady-state electrolysis of a solution of nonamphotheric compounds.

The problem of stationary electrolysis of a solution of nonamphotheric compounds (acids or bases) is considered. The analysis is performed by taking into account the mobility dependence on pH. The properties of such a system are also compared with the ones pertaining to the solution of an amphoteric substance. It is anticipated that steady-state electrolysis of free acids and bases, in a convection-free system, might be useful for creating narrow pH gradients in rather acidic and alkaline milieus, which might be adopted for focusing without resorting to conventional carrier ampholytes or immobilized pH gradients.

Steady-state electrolysis of an ampholyte solution and possibility of violation of the "law of pH monotony".

The problem of stationary electrolysis of a single-component ampholyte solution is analyzed. The effect of nonconstant relative mobility is taken into account. It is demonstrated that an incorrect dissociation model (i.e., expressing the resulting ampholyte flux by the arithmetic sum of the fluxes for "cationic" and "anionic" species) may lead to the violation of the so-called "law of pH monotony".

Steady-state concentration distribution of ampholytes in isoelectric focusing in a linear immobilized pH gradient.

The equation of balance between electrophoretic and diffusional mass flows in the steady state of isoelectric focusing is analyzed. To create the pH gradient, a model system composed of only one Immobiline is used. The solution is found for the case of a small sample concentration, but without assumptions about linear "focusing force" and constant (linear) conductivity profiles. The effect of sample concentration on the final concentration profile is also evaluated. At high sample concentrations, it is demonstrated that the steady-state distribution is essentially non-Gaussian and results in a considerably lower concentration maximum as compared with low sample levels.

On the concept of "normalized buffering power/conductivity ratio" of isoelectric buffers for capillary zone electrophoresis.

The introduction of a new physico-chemical parameter is proposed: the "normalized buffering power/conductivity ratio". It expresses the ratio of buffering power to conductivity (Rbeta/lambda), normalized by the electrolyte concentration, and gives an opportunity to calculate the properties of buffering ions typically used in capillary electrophoretic separations of biopolymers. This procedure of normalization is possible due to the fact that, in the concentration range practically used, the relationship of Rbeta/lambda on concentration is close to linear.

Human globin chain separation by capillary electrophoresis in acidic isoelectric buffers.

A simple and reliable method, utilizing capillary electrophoresis in uncoated capillaries in acidic isoelectric buffers, is reported for screening for thalassemia and other defects on the synthesis of human globin chains. A solution of 50 mM iminodiacetic acid (pI 2.23), containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme-free, denatured globin (alpha, beta and gamma) chains. Due to the low conductivity of such a buffer, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. It is additionally shown that inclusion of 2% surfactant (Tween 20) in the background electrolyte induces the splitting of the gamma chains into two zones, called Agamma and Ggamma, which represent the products of two genes coding for Ala or Gly as residue 136 of the chain.

Can amphoteric substances with very small (or negative) deltapK difference exist and what properties would their water solutions exhibit?

For calculating the buffer capacity of ampholytes an application of the stepwise and parallel dissociation models is considered. It is demonstrated that the scheme with stepwise dissociation possesses a "nonadditive" sum, i.e., the resulting buffer capacity of the system exceeds the sum of the separate contributions of the two ionogenic groups. On the other hand, the scheme of parallel dissociation is free from this disadvantage, and thus does not impose any restriction on the deltapK parameter.

Capillary zone electrophoresis of oligonucleotides in isoelectric buffers and against a stationary pH gradient.

Capillary zone electrophoresis of oligonucleotides in a background electrolyte of two different types of stationary buffers is proposed: single, isoelectric amphoteres and focused carrier ampholytes. In the first case, two zwitterionic molecules are evaluated: lysine and histidine. Although the former has a five times higher buffering power (beta) at the pI (9.74) than the latter (pI 7.47), due to the favorable delta pK value (1.6 vs. 3) and thus should be the preferred species, a new parameter for evaluating the performance of isoelectric buffers is proposed: the beta/lambda ratio, i.e., the ratio between the buffering power and its conductivity. Ideal buffers are those with the highest beta/lambda ratio, since this allows delivering very high voltage gradients with minimal Joule effects. Since the pI of Lys is situated in a pH region (9.74) where bulk water begins to conduct, whereas His has a pI close to neutrality, the beta/lambda ratio is more favorable for His than for Lys. In the second case (zone electrophoresis of oligonucleotides against a preformed pH gradient), it is shown that migration against a pH 6.5-10 Pharmalyte carrier ampholyte pH gradient offers a unique analyte resolution. This is possibly due to two effects: (i) When injected at the alkaline extreme (ca. pH 10) of the pH gradient, the oligonucleotide zones undergo a stacking effect, with consequent zone sharpening, due to modulation of their free mobility via protonation of the -OH group (enolate ion) in the hetero aromatic rings of G and T, which undergo a lactam-lactim transition. (ii) As the zones migrate down the pH gradient, they transit through a pH 6.5-8.5 zone where, for Pharmalytes, the beta/lambda ratio reaches a maximum and is constant as well. This last condition allows high voltage gradients (typically 1000 V/cm, even in 75 microm capillaries) to be delivered, thus greatly reducing the analysis time and maintaining peak sharpness, due to limited diffusion.

pH changes in Immobiline gels due to low-molecular mass ion adsorption and conditions for salt front formation during electrophoretic desorption.

A simple theoretical model of low molecular mass ion adsorption on a weak exchanger is proposed. For a system containing immobilized charges in uniform concentration the pH change values connected with neutral salt addition and followed by further washing and voltage application are evaluated. The question of stability of the moving boundary, arising in the area near the electrodes, is considered for the constant current stabilization at different values of Immobiline concentration, pK values of Immoboline and salt concentration. The threshold values of ion mobility capable of providing a sharp salt front formation are also evaluated for conditions of strong adsorption. We found that, with extremely acidic Immobilines, a sharp salt front does not arise under the latter condition, even in the case of very high Immobiline concentrations.

Generation of tryptic maps of alpha- and beta-globin chains by capillary electrophoresis in isoelectric buffers.

A novel method for generating peptide maps, following tryptic digests of proteins, is reported here: capillary zone electrophoresis in the presence of isoelectric buffers as the sole buffering species. A typical buffer composition comprises 50 mM aspartic acid (pH = pI = 2.77), 0.5% hydroxyethyl cellulose (added as a dynamic coating agent for preventing peptide adsorption to weakly ionized silanols), 5% trifluoroethanol and 1% zwitterionic detergent (CHAPS). With this buffer composition, a high-voltage gradient can be applied (typically 600 V/cm in 75 microns I.D. and 900 V/cm in 50 microns I.D. capillaries), thus drastically reducing the analysis times. The method is applied to the generation of peptide maps of alpha- and beta-globin chains from human adult hemoglobin. In the case of beta-peptides, at an operative pH of 2.77, which represents a cross-over point in the titration curve of peptides T2 and T9, the two analytes merge into a single peak. However it is shown that it is possible to change the pH of the zwitterionic buffer by adjusting its concentration in solution. In 30 mM Asp (pH 3.0) or 20 mM Asp (pH 3.1) resolution of these two peptides is fully restored. Isoelectric, amphoteric buffers thus seem to represent a novel, powerful buffer system able to offer high resolution and high selectivity.

Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: theory and methodology.

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 microm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion-driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (beta) as a function of delta pK, i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative beta value (= 2) is obtained for amphoteres possessing a delta pK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between the beta power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta-globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.

Ampholyte dissociation theory and properties of ampholyte aqueous solutions.

Two different approaches (stepwise and parallel mechanism) for describing the dissociation of amphoteric protolytes are described. Additionally, the classification of the possible model on the basis of the ampholyte lifetime state is proposed. The possibility of application of different schemes is discussed. It is suggested that the correct description may be based only on highly relaxing models. The formulas describing the properties of ampholyte solutions are derived for the case of two ionogenic groups. It is demonstrated that the incorrect choice of the dissociation model may result in essential mistakes in calculation of such values as buffer capacity, electrophoretic mobility, and conductivity. On the basis of the theory here developed, the interpretation of natural pH-gradient conductivity is given. Some terminology questions are also discussed.

Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers.

A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18-mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3-4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co-migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.

Dynamics of protein isoelectric focusing in immobilized pH gradient gels.

The protein migration velocity profile through the pH gradient in immobilized pH gradient gels (IPG) was investigated. Strong nonuniformities of migration velocity distribution, which could not be remedied by adding carrier ampholytes, were observed at the initial stages of a run (up to several thousand volt-hour products for wide pH gradients). The final (after a prerun) velocity distribution in IPG gels differs from the one containing carrier ampholytes, with maximum velocity being achieved not at the pH extremes but closer to neutrality. In some uniform pH media, created by either a single or by several Immobilines, essentially a nonconstant migration profile was also obtained immediately after a prerun. After a prerun, followed by a pause in the absence of current, the phenomenon of fast evolution of the final velocity profile was found.