Influence of methanol as a buffer additive on the mobilities of organic cations in capillary electrophoresis

The mobilities of a series of aromatic ammonium ions, ranging in charge from +1 to + 3, were investigated by capillary electrophoresis using buffers consisting of 0-75% v/v methanol. This is an extension of our previous studies involving anion mobility in methanol-water media [1]. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. For all of the buffer compositions studied, the ionic strength effect increased with increasing cation charge, and varied as a function of solvent 1/eta epsilon (1/2) as predicted by the electrophoretic term within the Pitts' equation. In the presence of methanol, the ionic strength effects became more dramatic. The absolute mobilities of the cations were altered by the addition of methanol to the electrophoretic media. For example, at 75% MeOH, a migration order reversal was observed between the + 2 and + 3 ammonium ions. These solvent-induced selectivity changes are attributed to dielectric friction. As predicted by the Hubbard-Onsager dielectric friction model, dielectric friction increased with increasing methanol content and with increasing analyte charge. Further, the changes in cation mobility correlated to the changes in solvent relaxation time (tau), epsilon and eta. Although not predicted by the Hubbard-Onsager theory, the + 3 ammonium ion experienced more dielectric friction than the - 3 sulfonate and - 3 carboxylate investigated previously [1]. This apparent failure of the Hubbard-Onsager model results from its continuum nature, whereby ion-solvent interactions are not taken into account.     

Dielectric friction as a mechanism for selectivity alteration in capillary electrophoresis using acetonitrile-water media

The mobilities of a series of aromatic carboxylates and sulfonates, ranging in charge from -1 to -4, were investigated as a function of acetonitrile concentration in the electrophoretic buffer. Absolute mobilities were determined by extrapolation of the effective mobilities to zero ionic strength according to the Pitts' equation. In general, anions of higher charge were more strongly influenced by ionic strength, with similarly charged anions experiencing ionic strength effects that were not significantly different at the 95% confidence level. Furthermore, the relative magnitudes of the Onsager slopes varied with acetonitrile content according to the z/(etaepsilon(1/2)) dependence in the electrophoretic effect of the Pitt's equation. Addition of acetonitrile to the electrophoretic media resulted in changes in the absolute mobilities of the anions. These acetonitrile-induced selectivity alterations were attributed to dielectric friction. As predicted by the Hubbard-Onsager model of dielectric friction, changes in sulfonate mobility were shown to correlate to changes in solvent viscosity (eta), dielectric constant (epsilon), and relaxation time (tau). The combined effects of ionic strength and dielectric friction caused analytes with higher charge-to-size ratios to be slowed to a greater extent upon addition of acetonitrile compared to those with lower charge-to-size. For example, at 75% acetonitrile and 20 mM ionic strength, a migration order reversal occurred between the triply and singly charged sulfonates.     

Determination of synthetic polypeptide conformations and molecular geometrical parameters by nonaqueous CE

The aim of this work was to study changes in homopolypeptide chain conformation as a function of the number of residues by the modeling of the electrophoretic mobility. For this purpose, the frictional coefficients of poly(N(epsilon)-trifluoroacetyl-L-lysine) with different number of residues (up to 11) were determined from the absolute ionic mobilities and modeled by the hydrodynamic frictional coefficient of an equivalent cylinder. This approach allowed determination of geometrical parameters of the polypeptide chain in a liquid phase (nonaqueous solution of the BGE). The fact that the BGE and analyte are dissolved in mixed (methanol-ACN) organic solvent implied to take into account different effects and corrections that are generally not considered in aqueous solvent: namely, the effect of ion-pairs between constituents of the BGE for the calculation of the ionic strength, the effect of ion-pairs between the solutes and the electrolyte counterions and the correction due to the dielectric friction (Hubbard-Onsager equations). In addition, the influence of the ionic strength on the electrophoretic mobility was corrected using the Pitts equation, and the effect of lateral charges due to a slight deprotonation of the -NH- group in the lateral chain was also considered. From this modeling, molecular geometrical parameters relative to the linear and helico?dal conformations were obtained with very good correlation coefficients. Interestingly, this work also points out that the use of ionic mobility modeling for extracting molecular geometrical parameters can also be applied to end-charged polypeptides with slightly charged lateral chains (3% of elementary charge per residue).     

Determination of ζ‐potential,charge, and number of organic ligands on the surface of water soluble quantum dots by capillary electrophoresis

The number of charges and/or organic ligands covalently attached to the surface of CdTe quantum dot nanoparticles has been determined from their electrophoretic mobilities measured in capillaries filled with free electrolyte buffers. Three sizes of water soluble CdTe quantum dots with 3‐mercaptopropionic and thioglycolic acids as surface ligands were prepared. Their electrophoretic mobilities in different pH and ionic strength values of separation buffers were measured by capillary electrophoresis with laser induced fluorescence detection. The ζ‐potentials determined from electrophoretic mobilities using analytical solution of Henry function proposed by Ohshima were in the range from ?30 to ?100 mV. Charges of QDs were calculated from ζ‐potentials. As a result, numbers of organic ligands bonded to QDs surface were determined to be 13, 14, and 15 for the sizes of 3.1, 3.5, and 3.9 nm, respectively. The dissociation constants of organic ligands bonded on QDs surfaces estimated from the dependence of QDs charge on pH of the separation buffer were 7.8 and 7.9 for 3‐mercaptopropionic acid and 6.9 for thioglycolic acid.     

Reliable electrophoretic mobilities free from Joule heating effects using CE

Ionic electrophoretic mobilities determined by means of CE experiments are sometimes different when compared to generally accepted values based on limiting ionic conductance measurements. While the effect of ionic strength on electrophoretic mobility has been long understood, the increase in the mobility that results from Joule heating (the resistive heating that occurs when a current passes through an electrolyte) has been largely overlooked. In this work, a simple method for obtaining reliable and reproducible values of electrophoretic mobility is described. The electrophoretic mobility is measured over a range of driving powers and the extrapolation to zero power dissipation is employed to eliminate the effect of Joule heating. These extrapolated values of electrophoretic mobility can then be used to calculate limiting ionic mobilities by making a correction for ionic strength; this somewhat complicated calculation is conveniently performed by using the freeware program PeakMaster 5. These straightforward procedures improve the agreement between experimentally determined and literature values of limiting ionic mobility by at least one order of magnitude. Using Tris-chromate BGE with a value of conductivity 0.34 S/m and ionic strength 59 mM at a modest dissipated power per unit length of 2.0 W/m, values of mobility for inorganic anions were increased by an average of 12.6% relative to their values free from the effects of Joule heating. These increases were accompanied by a reduction in mobilities due to the ionic strength effect, which was 11% for univalent and 28% for divalent inorganic ions compared to their limiting ionic mobilities. Additionally, it was possible to determine the limiting ionic mobility for a number of aromatic anions by using PeakMaster 5 to perform an ionic strength correction. A major significance of this work is in being able to use CE to obtain reliable and accurate values of electrophoretic mobilities with all its benefits, including understanding and interpretation of physicochemical phenomena and the ability to model and simulate such phenomena accurately.     

Relation between electrophoretic behavior and molecular shapes of aromatic anions.

The capillary electrophoretic behavior of 44 aromatic organic ions was investigated. The observed ionic radii (r(obs0)) for the aromatic organic ions were obtained from the electrophoretic mobilities of sodium tetraborate (pH 9.2), potassium tetraborate (pH 9.2), ammonium borate (pH 9.2), and trisodium phosphate (pH 11.7) buffers with zero ionic strength. The linear relationships between the r(obs0)) values and the ionic radii (r(calc)), calculated by either the AM1 or PM3 method, were determined for benzyltrialkylammonium and aromatic sulfonate ions. However, the r(obs0)) values were constant for the aromatic carboxylate ions in buffers, in spite of the different r(calc) values. This indicates that aromatic carboxylate ions, such as benzenecarboxylate, pyridinecarboxylate, naphthalenecarboxylate, and anthracenecarboxylate ions, migrate as planar ions in buffers, whereas aromatic sulfonate ions could migrate as approximately spherical ions.     

Electrophoretic mobilities of large organic ions in nonaqueous solvents: determination by capillary electrophoresis in propylene carbonate, N,N-dimethylformamide, N,N,-dimethylacetamide, acetonitrile and methanol

The mobilities of the monocharged permanent tertraphenylphosphonium cation and tetraphenylborate anion are determined by capillary zone electrophoresis in different organic solvents as a function of the ionic strength, I, of the background electrolyte. The nonaqueous solvents are propylene carbonate (PC), N,N-dimethylformamide (DMF), N,N,-dimethylacetamide (DMA), acetonitrile (MeCN) and methanol (MeOH). The ionic strength is between 5 and 50 mmol/L. The mobility as a function of I is in good agreement with the theory of Debye, Hückel and Onsager (DHO), extended by the ion size parameter as introduced by Falkenhagen and Pitts. The values of the limiting DHO slopes of the mobility vs. I curves (the slopes express the influence of the solvent on the reduction of the mobility with increase of I) decrease in the order MeCN > MeOH > DMF > DMA > PC. Absolute mobilities (obtained by extrapolation to I = 0) of a particular ion differ by a factor of about 7 between the solvents. However, constancy within 10% is observed for their Walden products (the absolute mobility multiplied with the solvent's macroviscosity). The role of dielectric friction on the mobility of the present monocharged, large analyte ions is discussed according to the theory of Hubbard and Onsager. Based on the radii of the ions, the static permittivity of the solvent and its permittivity at infinite frequency, and the relaxation time of polarization, an equal contribution of dielectric and hydrodynamic friction is predicted in MeOH as solvent. Experimental data are in contrast to this prediction, indicating the overestimation of dielectric friction, and the dominance of hydrodynamic friction on the migration of the analyte ions in all solvents under consideration.     

Capillary electrophoresis applied for the determination of acidity constants and limiting electrophoretic mobilities of ionizable herbicides including glyphosate and its metabolites and for their simultaneous separation

Thermodynamic acidity constants and limiting ionic mobilities were determined for polyprotic non-chromophore analytes using capillary electrophoresis with capacitively coupled contactless conductivity detection. It was not necessary to work with buffers of identical ionic strength as ionic strength effects on effective electrophoretic mobilities were corrected by modeling during data evaluation (software AnglerFish). The mobility data from capillary electrophoresis coupled to conductivity detection were determined in the pH range from 1.25 to 12.02 with a high resolution (36 pH steps). With this strategy, thermodynamic acidity constants and limiting ionic mobilities for various acidic herbicides were determined, sometimes for the first time. The model analytes included glyphosate, its metabolites, and its acetylated derivates (aminomethyl phosphonic acid, glyoxylic acid, sarcosine, glycine, N-acetyl glyphosate, N-acetyl aminomethyl phosphonic acid, hydroxymethyl phosphonic acid). The obtained data were used in simulations to optimize separations by capillary electrophoresis. Simulations correlated very well to experimental results. With the new method, the separation of glyphosate from interfering components like phosphate in beer samples was possible.     

Ion-pair association and acid-base equilibria in nonaqueous capillary electrophoresis of weakly basic compounds

CE in nonaqueous media was used to study the migrating behavior of two weakly basic s-triazine pesticides and one of their metabolites. The target pesticides were selected to be representative for each of the two main groups: propazine and deethylatrazine for the chloro-s-triazines group and ametryn for the methylthio-s-triazines group. To elucidate the phenomena involved, systematic studies were carried out in the different organic media studied. Absolute mobilities were determined in 50% v/v methanol (MeOH)/ACN by extrapolation of the effective mobilities to zero ionic strength in the presence of different concentrations of perchloric acid. Conductivity measurements performed in MeOH and 50 and 20% v/v methanol/ACN permitted the evaluation of the associations of the components of the BGE. The effects of ionic strength on the actual mobilities of the compounds were determined in the presence of perchloric acid and SDS in different organic media. Two different ion-pair equilibria were considered: one due to the presence of perchlorate anions present in the BGE and second that from the added dodecyl sulfate anions. Bearing in mind that these weakly basic compounds can exhibit ion-pair and acid-base equilibria, the acid-base and ion-pair parasite reaction coefficients were determined. Finally, the effects of ionic strength, ion-pair interactions and acid-base properties on the effective electrophoretic mobilities of the analytes are discussed.     

Nitromethane as solvent in capillary electrophoresis

Nitromethane has several properties that make it an interesting solvent for capillary electrophoresis especially for lipophilic analytes that are not sufficiently soluble in water: freezing and boiling points are suitable for laboratory conditions, low viscosity leads to favourable electrophoretic mobilities, or an intermediate dielectric constant enables dissolution of electrolytes. In the present work we investigate the change of electrophoretically relevant analyte properties - mobilities and pKa values - in nitromethane in dependence on the most important experimental conditions determined by the background electrolyte: the ionic strength, I, and the pH. It was found that the mobility decreases with increasing ionic strength (by, e.g. up to 30% from I = 0 to 50 mmol/L) according to theory. An appropriate pH scale is established by the aid of applying different concentration ratios of a buffer acid with known pKa and its conjugate base. The mobility of the anionic analytes (from weak neutral acids) depends on the pH with the typical sigmoidal curve in accordance with theory. The pKa of neutral acids derived from these curves is shifted by as much as 14 pK units in nitromethane compared to water. Both findings confirm the agreement of the electrophoretic behaviour of the analytes with theories of electrolyte solutions. Separation of several neutral analytes was demonstrated upon formation of charged complexes due to heteroconjugation with chloride as ionic constituent of the background electrolyte.     

Change in electrophoretic mobility of HL-60RG cells by apoptosis

We measured the electrophoretic mobilities of HL-60RG cells and their apoptotic cells triggered by Actinomycin D as a function of the ionic strength of the suspending medium at pH 7.4. Both types of cells showed negative mobilities. The apoptotic HL-60RG cells exhibited larger mobility values in magnitude than intact HL-60RG cells in the whole range of the electrolyte concentration measured. The obtained data were analyzed via a mobility expression for soft particles, that is, colloidal particles with ionpenetrable surface layers. The observed mobility difference between the intact and apoptotic HL-60RG cells was found to be due mainly to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells rather than the difference in charge density in their surface layers. A possible explanation for this mobility change by apoptosis is given.     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Accurately describing weak analyte-additive interactions by capillary electrophoresis

When modeling analyte-additive interactions in capillary electrophoresis (CE), it is necessary to correct for all changes in the apparent electrophoretic mobility of an analyte that are not due to specific binding. Current models based on dynamic complexation have corrected for bulk viscosity changes in the background electrolyte (BGE) when additives are used, while assuming negligible changes in the dielectric constant and other physicochemical properties of the solution. In this report, a study of weak interactions between deoxyribonucleotides and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) revealed significant nonideality in binding isotherms. Changes in the dielectric properties of the solution due to the addition of high concentrations of HP-beta-CD to the BGE was observed to alter the electrophoretic mobility of analytes. A relative dielectric correction factor was required to normalize analyte mobilities to a reference state of zero additive concentration. The use of both a relative dielectric factor and a viscosity correction factor was found to increase the accuracy of the model, reflected by a higher degree of correlation between predicted and measured analyte mobilities. This type of correction is particularly relevant when studying weak analyte binding interactions or when using high concentrations of additive in the BGE. This work is vital for accurate determination of weak binding constants and mobility values, as well as providing a deeper understanding of the fundamental parameters influencing a separation in CE.     

Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population.     

From small charged molecules to oligomers: a semiempirical approach to the modeling of actual mobility in free solution

According to Stokes' treatment, the ionic mobility of particles, which are small with respect to Debye length, is usually considered to be proportional to the nominal charge and inversely proportional to the hydrodynamic radius. Experimentally, it is well known, however, that the ionic mobility of a small multicharged molecule does not depend linearly on its nominal charge in a wide range. This behavior can be accounted for by a condensation of the charge or a modification of the friction coefficient with the charge. This paper presents a semiempirical modeling of the actual mobility based on the assumption of additivity of frictional contributions pertaining to the uncharged molecular backbone and to each charged or uncharged moiety. Condensation of the charge was not considered. The model first appeared to be suitable for multicharged analytes having a characteristic dimension smaller than the Debye length, such as benzene polycarboxylic acids and polysulfated disaccharides. This approach was then adapted to account for the actual mobilities of singly and evenly charged oligomers (N-mers) having a dimension smaller than or similar to the Debye length. Rather good experimental agreement was obtained for polyalanines and polyglycines (N < or = 6), fatty acid homologs, fully sulfonated polystyrene oligomers (N < or = 13), and polycytidines (N < or = 10). Especially the influence of the polymerization degree on the mobility of oligomers having identical charge densities was clarified. It is also shown that the electrophoretic contribution to the overall friction coefficient increases linearly with the nominal charge but hardly depends on the chemical nature of the charged moieties. This model should be of interest to evaluate the role of various physicochemical phenomena (hydrodynamic and electrophoretic frictions, hydrodynamic coupling, charge condensation) involved in the migration of charged oligomers.     

Capillary electrophoresis separation of vinpocetine and related compounds: prediction of electrophoretic mobilities in partly aqueous media

Offord's equation, a relationship between electrophoretic mobility and charge, size and shape of peptides, has been extended to quantitate the electrophoretic mobility of vinca alkaloids. Partly aqueous protonation constants and the derived theoretical mobilities have been proven to be able to predict experimental electrophoretic mobilities. In practice, seven vincamine derivatives of very low water-solubility were separated by capillary electrophoresis. Buffer total concentration, apparent pH and methanol content, the three most important parameters of the running buffer, were used in triangular resolution mapping to characterize separation. Even though electrophoresis is well known to slow down in partly aqueous media, under our optimized circumstances a baseline separation was achieved within 8 min in each case.     

Preparative Isoelectric Focusing

Electrophoretic mobility has been used for years as a basis for separation and characterization of proteins. The resolving power of conventional electrophoresis is limited, and the characterization by mobility is not unique because of the dependence of the protein mobilities on the pH and ionic strength of the buffers.     

Characterization of carboxylated nanolatexes by capillary electrophoresis

Poly(styrene-co-acrylic acid) (St/AA) and poly(styrene-co-methacrylic acid) (St/MA) nanolatexes with different acid contents were prepared by emulsion copolymerization and were analyzed by capillary electrophoresis (CE) and by laser doppler velocimetry (LDV). Due to the intrinsic differences in the methodologies, CE (separative technique) and LDV (zetametry, nonseparative technique) lead to very different electrophoretic mobility distributions. Beyond these differences, the variation of the electrophoretic mobility is a complex and nonlinear function of the hydrodynamic radius, the ionic strength, and the zeta potential. To gain better insight on the influence of the ionic strength and the acid content on the electrophoretic behavior of the nanolatexes, the electrophoretic mobility data were changed into surface charge densities using the O'Brien, White, and Ohshima modeling. This approach leads to the conclusion that the surface charge density is mainly controlled at high ionic strength (～50 mM) by the adsorption of anionic surfactants coming from the sample. On the contrary, at low ionic strength, and/or in the presence of neutral surfactant in the electrolyte, the acid content was the main parameter controlling the surface charge density of the nanolatexes.     

Nonaqueous CE using contactless conductivity detection and ionic liquids as BGEs in ACN

N,N'-Alkylmethylimidazolium cations have been separated in NACE when one of the N,N'-dialkylimidazolium salts (ionic liquids (ILs)) was used as an electrolyte additive to the organic solvent separation medium. The separated species were 1-methyl-, 1-ethyl-, 1-butyl-, 1-octyl-, 1-decyl-3-methylimidazolium and N-butyl-3-methylpyridinium cations and BGE composed of 1-ethyl-3-methylimidazolium ethylsulfate or 1-butyl-3-methylimidazolium trifluoroacetate [BMIm][FAcO] (A6; B2) diluted in ACN. It was demonstrated that contactless conductivity detection (CCD) may be applied to monitoring the separation process in nonaqueous separation media, allowing to use the UV light-absorbing imidazolium-based electrolyte additives. There could be marked three concentration regions of added ILs; at first ionic strength of BGE below 1-2 mM, and then the actual electrophoretic mobility of analytes rises from 0. At concentrations above 1-2 mM, the added IL facilitated separation. In concentration region of 1-20 mM, the actual electrophoretic mobility of analyzed imidazolium cations was increasing with decrease in separation medium ionic strength. At higher concentrations of BGE (above 30 mM), the conductivity of the separation media became too high for this detector. Some organic dyes were also successfully separated and detected by contactless conductivity detector in a 20 mM A6 separation electrolyte in ACN.