Simo P. Porras

Non-aqueous capillary electrophoresis

The benefits of non-aqueous capillary electrophoresis have been described in a number of recent publications. The wide selection of organic solvents, with their very different physicochemical properties, broadens our scope to manipulate separation selectivity. The lower currents present in non-aqueous solvents allow the use of high electric field strengths and wide bore capillaries, the latter in turn allowing larger sample load. In many cases detection sensitivity can also be enhanced. The potential of non-aqueous capillary electrophoresis is discussed throughout the paper, and the feasibility of capillary electrophoresis under non-aqueous media is demonstrated with reference to several applications.

Electrophoretic mobilities of cationic analytes in non-aqueous methanol, acetonitrile and their mixtures : Influence of ionic strength and ion-pair formation

The mobilities of cationic analytes in organic solvents and water are compared, and the reasons for differences in the mobilities are discussed in detail. Actual mobilities (at background electrolyte concentration 10 mmol/l) of anilinium ions were determined by capillary zone electrophoresis in water, methanol, acetonitrile and mixtures of methanol and acetonitrile (in volume ratios 1:1, 1:3 and 3:1). The actual mobilities correlated with the viscosity of the organic solvent: the products of actual mobility and viscosity were constant within 7%. However, these products were significantly larger in water. Larger products of mobility and viscosity in water were also found for unsubstituted anilinium when the absolute mobility (at zero ionic strength) was taken into consideration. Thus, ion-solvent interactions must be responsible for the seemingly high mobility in water compared with that in organic solvents. This finding can be explained by the effect of the ion on the water structure. Based on equilibrium constant for ion-pair formation given in the literature, about 20% of the main background electrolyte constituent (tetrapropylammonium perchlorate) is associated at 10 mmol/l concentration in acetonitrile. Comparison of the plot of the measured mobilities of the analytes vs. the square root of the corrected ionic strength of the background electrolyte in acetonitrile with the prediction based on the Debye-Hückel-Onsager theory showed the measured mobilities deviate negatively from the theoretical line. This is apparently due to ion pairing, which takes place for the analytes as well.

Capillary zone electrophoresis of basic analytes in methanol as non-aqueous solvent mobility and ionisation constant.

The electrophoretically relevant properties of monoacidic 21 bases (including common drugs) containing aliphatic or aromatic amino groups were determined in methanol as solvent. These properties are the actual mobilities (that of the fully ionised weak bases), and their pKa values. Actual mobilities were measured in acidic methanolic solutions containing perchloric acid. The ionisation constants of the amines were derived from the dependence of the ionic mobilities on the pH of the background electrolyte solution. The pH scale in methanol was established from acids with known conventional pK*a values in this solvent used as buffers, avoiding thus further adjustment with a pH sensitive electrode that might bias the scale. Actual mobilities in methanol were found larger than in water, and do not correlate well with the solvents viscosity. The pK*a values of the cation acids, HB-, the corresponding form of the base, B, are higher in methanol, whereas a less pronounced shift was found than for neutral acids of type HA. The mean increase (compared to pure aqueous solution) for aliphatic ammonium type analytes is 1.8, for substituted anilinium 1.1, and for aromatic ammonium from pyridinium type 0.5 units. The interpretation of this shift was undertaken with the concept of the medium effect on the particles involved in the acid-base equilibrium: the proton, the molecular base, B, and the cation HB+.

Capillary Zone Electrophoresis of Basic Drugs in Non-Aqueous Acetonitrile with Buffers Based on a Conventional pH Scale

SummaryThe pKa* values of 10 nitrogen-containing basic drugs in non-aqueous acetonitrile were determined from the pH* dependence of their electrophoretic mobilities. The pH* scale in the organic solvent was established using background electrolytes with known conventional pKa* values, making further calibration with reference pH electrodes unnecessary. In acetonitrile the pKa* values of analytes (or their conjugated cation acids, BH+, respectively) were 5.2±8.9 pK units>those in water. The observed change in pKa* values of cationic analytes was, however, much less than the known respective change for neutral acids type HA. From the pKa* values and the actual mobilities, it is possible to predict pH* conditions to enable separation of analytes, and this was demonstrated for two pairs of common drugs.

Effect of electrolyte and solvent composition on capillary electrophoretic separation of some pharmaceuticals in non-aqueous media.

Non-aqueous capillary electrophoresis was used to study the separation selectivity of positively charged drug substances and negatively charged diuretics. Study was made of the effects of organic solvent composition and the background electrolyte on the separation. The separation selectivity could be altered considerably by varying the methanol/acetonitrile composition. In addition, the migration order and the resolution of the pharmaceuticals could be altered merely by changing the electrolyte cation or the anion. The electrolytes tested were alkali metal acetates, ammonium acetate, ammonium chloride and ammonium bromide. As with aqueous background electrolyte solutions, the electroosmotic flow was decreased with increasing size of the alkali metal cation of the electrolyte in methanol/acetonitrile 50:50 (v/v).

Extremely high electric field strengths in non-aqueous capillary electrophoresis

The influence of high electric field strength on the separation of basic analytes in non-aqueous alcohol background electrolyte (BGE) solutions was investigated. Increasing the separation voltage in capillary electrophoresis (CE) may be advantageous if the conductivity of the BGE solution is low enough to allow fast separations without excessive Joule heating or band broadening. The voltage range tested was 20-60 kV with methanol and ethanol, and 25-60 kV with propanol and butanol as solvent for BGE. The resulting electric field strengths ranged from 660 V cm(-1) to 2000 V cm(-1). Experiments were made with a special laboratory constructed CE instrument. The separation efficiency vs. voltage curve was found to vary with the alcohol BGE solution. The increase in voltage decreased the separation efficiency in the case of methanol BGE solution, but with the other BGEs a clear efficiency maximum was obtained above 30 kV. The highest separation efficiencies were achieved with propanol BGE solution, where the efficiency maximum was reached at 45 kV. However, reasonable efficiency was achieved even at 60 kV. The extent of Joule heating was determined by calculating the temperature inside the capillary and the observed plate heights were interpreted in terms of the Van Deemter equation. The decrease in the separation efficiency with higher voltage was attributed mainly to Joule heating in the case of methanol and ethanol BGE solution and to the analyte adsorption on the capillary wall with propanol and butanol BGE solutions.

Capillary electrophoresis with wide-bore capillaries and non-aqueous media

Abstract A major drawback of capillary electrophoresis is that its use for semi-preparative purposes is problematic. To overcome the problems associated with wide-bore capillaries, non-aqueous background electrolyte instead of aqueous buffers was used. The effect of the capillary diameter on capillary electrophoretic separation was investigated in ethanol–acetonitrile–acetic acid (50:49:1, v/v) containing 20 m M ammonium acetate. This buffer allowed the use of wider capillaries and higher electric field strength than the corresponding buffer containing water in place of ethanol and acetonitrile. Increasing the internal diameter of the capillary from 50 to 200 μm allowed a 16-fold increase in the sample load at the same ratio of injected volume to total volume. In wide-bore capillaries the difference between the inlet and outlet buffer level leads to siphoning, which has a marked effect on the apparent electroosmotic velocity and the efficiency of the separation. The effect of the physical properties of the solvent on siphoning is discussed. Our results indicate that non-aqueous media may have a major role in semi-preparative capillary electrophoresis.

Capillary electrophoresis of anionic analytes in methanol: effect of counter-ions on electrophoretic mobility.

Mobilities of 11 substituted benzoates and 3 nitrophenolates were determined in non‐aqueous methanol with Li+, Na+, K+, Rb+, and tetrabutylammonium (Bu4N+) as counter‐ions of the background electrolyte. The influence of the ionic concentration of the background electrolyte on the mobility of the analyte anions is more pronounced compared to aqueous solutions. The deviation from the dependence of the mobilities on the ionic strength from the Debye‐Hückel‐Onsager theory indicates the occurrence of ion‐pair formation. For a given ion concentration (10 mmol/L), the decrease of the analyte mobility follows the counter‐ion sequence Li+ < Na+ < K+ < Rb+, which is the inverse order of their Stokes radii. Bu4N+ as counter‐ion has a similar effect on the analyte mobility than Li+ (which has the same Stokes radius, but a six times smaller crystal radius). Exceptions are some di‐ and trihydroxybenzoates. The mobilities in methanol and in water with the same counter‐ion (Na+) at a given ionic concentration show very low correlation.

Alcohols and wide-bore capillaries in nonaqueous capillary electrophoresis

The feasibility of using C1‐C5 alcohols as electrolyte solutions in nonaqueous capillary zone electrophoresis was investigated. The separation of basic narcotic analgesics and acidic diuretics was modified by changing the alcohol in an electrolyte solution containing alcohol‐acetonitrile‐acetic acid (50:49:1, v/v) and 20 mM ammonium acetate while other experimental conditions were kept constant. The alcohols studied were methanol, ethanol, 1‐propanol, 2‐propanol, 1‐butanol, 2‐butanol, and 1‐pentanol. The results indicate that even longer‐chain alcohols can be used in nonaqueous capillary zone electrophoresis and, because of the lower currents they allow, they are especially advantageous in wider capillaries. Basic analytes were separated in 200 μm and 320 μm ID capillaries with 1‐butanol‐acetonitrile‐acetic acid (50:49:1, v/v) containing 20 mM ammonium acetate as electrolyte solution. Problems related to the use of wide‐bore capillaries are discussed.

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: Separation efficiency under high electrical field strengths

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1‐propanol, and 1‐butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory‐built CE instrument suitable for high‐voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm–1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.