Critical evaluation of buffering solutions for pKa determination by capillary electrophoresis

The performance of the most common and also some other less common CE buffers has been tested for the pKa determination of several types of compounds (pyridine, amines, and phenols). The selected buffers cover a pH ranging from 3.7 to 11.8. Whereas some buffers, like acetic acid/acetate, BisTrisH+/BisTris, TrisH+/Tris, CHES/CHES-, and CAPS/CAPS- can be used with all type of analytes, others like ammonium/ammonia, butylammonium/butylammonia, ethylammonium/ethylammonia, diethylammonium/diethylammonia, and hydrogenphosphate/phosphate are not recommended because they interact with a wide range of compounds. The rest of the tested buffers (dihydrogenphosphate/hydrogenphosphate, MES/MES-, HEPES/HEPES-, and boric acid/borate) can show specific interactions depending on the nature of the analytes, and their use in some applications should be restricted.     

Capillary zone electrophoresis of cationic and anionic drugs in methanol

The actual mobilities and dissociation constants of acidic and basic pharmaceuticals were determined in methanol. Actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH of the methanolic background electrolyte solution (pH(MeOH)). The pKa values of the pharmaceuticals in methanol (pK(a,MeOH)) were calculated by non-linear curve fitting to the measured mobility values. It was found that the shift in pKa value (when compounds were transferred from water to methanol) increased with the acidity of the analyte. The average pKa shift for compounds exhibiting acidic properties in water was ca. 5.5 units, and the shift for basic compounds about 2 units. As was shown for a mixture of beta-blockers, the calculated actual mobilities and pKa values can be utilised in the optimisation of pH conditions for separation. The practical value of the method was illustrated by the analysis of urine samples.     

Determination of the dissociation constants (pKa) of secondary and tertiary amines in organic media by capillary electrophoresis and their role in the electrophoretic mobility order inversion

Non-aqueous capillary electrophoresis (NACE) may provide a selectivity enhancement in separations since the analyte dissociation constants (pKa) in organic media are different from those in aqueous solutions. In this work, we have studied the inversion in mobility order observed in the separation of tertiary (imipramine (IMI) and amitryptiline (AMI)) and secondary amines (desipramine (DES) and nortryptiline (NOR)) in water, methanol, and acetonitrile. We have determined the pKa values in those solvents and the variation of dissociation constants with the temperature. From these data, and applying the Van't Hoff equation, we have calculated the thermodynamic parameters deltaH and deltaS. The pKa values found in methanol for DES, NOR, IMI, and AMI were 10.80, 10.79, 10.38, and 10.33, respectively. On the other hand, in acetonitrile an opposite relation was found since the values were 20.60, 20.67, 20.74, and 20.81 for DES, NOR, IMI, and AMI. This is the reason why a migration order inversion is observed in NACE for these solvents. The thermodynamic parameters were evaluated and presented a tendency that can be correlated with that observed for pKa values.     

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 solvent's 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+.     

Rapid screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis combined with short-end injection

A method applying pressure-assisted capillary electrophoresis combined with short-end injection has been developed for the rapid screening of the pKa values of pharmaceuticals. The electrophoretic separation is performed on a short capillary length with short-end injection under an applied pressure, and the effective mobility is measured in a series of 10 different buffers with constant ionic strength (I = 0.05). The application of pressure not only reduces migration times, particularly in lower pH buffers, but also improves the repeatability of effective mobility measurements. The influence of pressure on the effective mobility was investigated at various pH values. It was observed for the first time that an increase in pressure resulted in a slight decrease in the effective mobility when the pH was above the pKa for acidic analytes, whereas an increased effective mobility with increasing pressures was observed when the pH was below the pKa. However, the observed effective mobility shift by the applied pressure did not significantly affect the determined pKa values. The determined pKa values were in good agreement with published data. Furthermore, a stacking condition was applied to increase the sensitivity, and a concentration down to 2 microM could readily be detected with UV detection using a 50 microm I.D. capillary. This technique is particularly suitable for measurement of pKa values for compounds with poor aqueous solubility. The method also omits the commonly used preconditioning steps with sodium hydroxide and water. The exclusion of excessive preconditioning steps and the use of pressure reduces the total cycling analysis time, and makes it possible to determine the pKa in less than 40 min per compound without loss of accuracy.     

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis

The intra- and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra- and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra-instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8-2.6% and interinstrument reproducibilities were between 3.2-3.9%.     

pKa-dependent formation of amides in water from an acyl phosphate monoester and amines

Acyl phosphate monoesters are readily prepared biomimetically activated anionic derivatives of carboxylic acids that react rapidly with amines in water to form amides. A plot of the logarithms of the rate constants for the reactions of a series of primary amines with benzoyl methyl phosphate depends on the pKa of the conjugate acids of the amines (beta(nuc) approximately 0.9). This provides a simple and quantitative basis for regioselective acylation with these reagents.     

Kinetics and mechanism of pH responsive cationic desorption from poly(vinyl alcohol)-borate hydrogel

Desorption of potassium chloride and calcium chloride from poly(vinyl alcohol)-borate hydrogel was studied at different pH from 3 to 10 using phthalate, phosphate and borate buffers. The rate of desorption from hydrogel was evaluated using conductance method for analysis. It was observed that rate of desorption process decreases on increasing pH of solution from acidic medium to basic medium. The mechanism responsible for the desorption of interacted K⁺ and Ca²⁺ ions in hydrogel moiety in acidic, neutral and basic medium were analyzed. The pseudo first order rate constants for desorption process at different pH were summarized and the rate of desorption of monovalent and divalent cations at various pH compared on the basis of standard deviation and linear regression constant values.     

Differential elution of sodium or potassium dihydrogen- and hydrogenphosphate ions from a sephadex G-15 column with sodium or potassium chloride solution

When a mixed solution of sodium or potassium dihydrogenphosphate and disodium or dipotassium hydrogenphosphate was eluted from a Sephadex G-15 column with either a sodium or potassium chloride solution, the elution profiles of ions showed that the hydrogenphosphate ion was eluted more rapidly than the dihydrogenphosphate ion. When the sample solutions containing potassium dihydrogenphosphate and/or dipotassium hydrogenphosphate, all of which were supplemented with phosphorus-32-labelled potassium dihydrogenphosphate, were eluted with sodium chloride solution, the elution profiles of radioactivity showed that the dihydrogenphosphate ion changed to hydrogenphosphate ion and vice versa, depending on the pH values of the sample solution and the availability of the cation of the eluent during elution for the phosphate ion to pair with.     

Chromatographic evaluation using basic solutes of the silanol activity of stationary phases based on poly(methyloctylsiloxane) immobilized onto silica

The chromatographic behaviors of some basic solutes were evaluated on stationary phases based on poly(methyloctylsiloxane) immobilized onto silica (PMOS-SiO(2)). The test solutes present both hydrophobic and hydrophilic properties. Evaluations of the pH effect used 80:20 v/v methanol/buffered mobile phase over the pH range of 5-11.5 with inorganic buffers such as borate, carbonate and phosphate and with organic buffers such as citrate, tricine and triethylamine. Evaluations in acidic mobile phases used 50:50 v/v and 30:70 v/v methanol/buffer (pH 2.5; 20 mmol/L) mobile phases. The buffer concentration effect used 65:35 v/v methanol/phosphate (pH 7; 20 and 100 mmol/L) mobile phases. The results are compared with those obtained with two chemically bonded stationary phases. The immobilized phases show greater contributions from an ion-exchange mechanism than do the commercial phases. The results indicate that the silanol activity of PMOS-SiO(2) stationary phases can be adequately evaluated by using appropriate basic probes and mobile phases having different pH, using different buffers.     

Monolayer studies of single-chain polyprenyl phosphates

The monolayer properties of some single-chain polyprenyl phosphates (phytanyl, phytyl, and geranylgeranyl phosphates), which we regard as hypothetical primitive membrane lipids, were investigated at the air-water interface by surface pressure-area (pi-A) isotherm measurements. The molecular area/ pressure at various pH conditions dependence revealed the acid dissociation constants (pKa values) of the phosphate. The pKa values thus obtained at the air-water interface (pKa1 = 7.1 and pKa2 = 9.4 for phytanyl phosphate) were significantly shifted to higher pH than those observed in the bilayer state in water (pKa1 = 2.9 and pKa2 = 7.8). The difference in pKa values leads to a stability of the phosphate as both monolayer and bilayer states in a pH range of 2-6. In addition, the presence of ions such as sodium, magnesium, calcium, and lanthanum in the subphase significantly altered the stability of the polyprenyl phosphate monolayers, as shown by the determination of monolayer collapse and compression/expansion hysteresis. Although sodium ions in the subphase showed only a weak effect on the stabilization of the monolayer, addition of magnesium ions or of a small amount of calcium ions significantly suppressed the dissolution of the monolayer into the subphase and increased its mechanical stability against collapse. In contrast, the presence of larger amounts of calcium or of lanthanum ions induced collapse of the monolayers. Based on these experimental facts, a plausible scenario for the formation of primitive cell membrane by transformation of a monolayer to vesicle structures is proposed.     

Amino acid analysis using amine-sensitive membrane electrodes

A method for determining specific amino acids is described, based on the detection of the amine formed by the enzymatic reaction of an amino acid with decarboxylase, using an amine-sensitive membrane electrode. l-Tyrosine and l-phenylalanine are considered as examples. The corresponding amines tyramine and phenethylamine, respectively, were selectively detected by using a poly(vinyl chloride)-based membrane electrode containing sodium tetrakis[3,5- bis(trifluoromethyl)phenyl]borate as an ion exchanger and tricresyl phosphate as a solvent mediator. The detection limits of l-tyrosine and l-phenylalanine were 20 and 50μM, respectively. The response characteristics of electrodes were compared by changing ion exchangers and solvent mediators.     

Determination of dissociation constants of amino acids by capillary zone electrophoresis.

A method is proposed for the determination of dissociation constants of amino acids by capillary zone electrophoresis. According to the dissociation equilibrium of amino acids and the basic theory of electrophoresis, the nonlinear relationship between the pH value of the buffer and the effective electrophoretic mobilities of the analyte was obtained. The dissociation constants can be calculated from the pH values and the corresponding effective electrophoretic mobilities using the program written in C++. The dissociation constants, pKa1 and pKa2, of 11 kinds of amino acids were determined successfully by the proposed method. The determined dissociation constants were compared with values in the literature; the differences between them are in the range of -0.03 to 0.06. No significant differences were observed between the determined dissociation constants and the corresponding literature values.     

Medium-throughput pKa screening pharmaceuticals by pressure-assisted capillary electrophoresis

A fast screening method for the determination of the dissociation constants (pKa) of acidic, basic, and multivalent compounds was developed by using pressure-assisted capillary electrophoresis (PACE). External air pressure was applied to shorten the analysis time. The separation efficiency decreases as air pressure increases. However, it was found that air pressure does not affect the measurement of electrophoretic mobility and pKa significantly when it is less than 2 psi. The method was evaluated in terms of accuracy, precision, and ruggedness by using a set of 48 compounds with literature pKa values ranging from 2 to 10. The difference between the measured pKa values and literature values is less than 0.2 units. The throughput is approximately 20 compounds per day with a 12-point measurement ranging from pH 2.5 to 11. It was demonstrated that this method is applicable for pKa screening of pharmaceuticals with diverse chemical structures.     

Reactive interaction of aromatic amines with dialdehyde cellulose gel

A new chromatographic method was developed for separation of amines based on their interaction with aldehyde groups in stationary phase. Expecting specific interaction with aldehyde groups through imine formation (Schiff base), we introduced dialdehyde groups to a commercial cellulose packing by periodate oxidation and examined eluting behavior of various aromatic amines. Primary amines with acid dissociation constants (pKa) greater than 6 showed no delay at pHs of 4.0–5.5, indicating the lack of interaction because of complete protonation. Primary amines with pKa less than 6 showed remarkable delays according to the amount of aldehyde groups on cellulose. The delay was dependent on the pH of eluent. The amines with pKa of 4–5.3 eluted faster at lower pH, apparently because of the change in proportion of free and protonated species. Amines with pKa less than 3.4 also showed delays but they eluted slower at lower pH. The latter behavior can be ascribed to the change in the ratio of free/protonated species of imines formed. Certain degree of steric effect was also noted, that is, compounds with a primary amino group adjacent to bulky substituents (ortho compounds) showed weaker interaction with aldehyde groups than meta- and para-isomers.     

Comparison of atmospheric pressure photoionization and ESI for CZE-MS of drugs

The performance of atmospheric pressure photoionization (APPI) and ESI for CZE was compared using a set of seven drugs (basic amines, quaternary amines and steroids) and four different BGEs. The influence of volatile and nonvolatile BGEs of acidic and neutral pH on the MS responses of test compounds was evaluated by infusion of test solutions into the respective ion sources, and by actual CZE-MS experiments. The infusion experiments indicate that sodium phosphate buffers cause ionization suppression in ESI-MS, although for the amines the suppression was modest (25-60% signal reduction). By contrast, APPI-MS responses were not affected by nonvolatile BGEs. With phosphate buffers, ESI-MS responses for the basic amines were still a factor 3-13 higher than the APPI-MS signals, whereas the steroids yielded similar responses in ESI-MS and APPI-MS. The quaternary amines could readily be detected in ESI-MS, but detection in APPI-MS required specific interface conditions. Using typical CZE-APPI-MS settings, quaternary amines remained undetected. Remarkably, the S/Ns observed in CZE-ESI-MS for the test compounds, were generally similar when using volatile and nonvolatile BGEs. For basic compounds, the S/Ns obtained in CZE-ESI-MS were a factor 2-5 higher than in CZE-APPI-MS, whereas steroids yielded equal S/Ns in both methods. Overall, it is concluded that when using relatively low BGE concentrations, the sensitivity of ESI-MS detection in CZE is more favorable than APPI-MS detection, even when nonvolatile BGEs are employed.     

Determination of pKa values of anthraquinone compounds by capillary electrophoresis

In this paper, the dissociation constants (pKa1, pKa2) of five anthraquinones were determined from the relation between the effective mobility at different pH values and the buffer pH value, which was derived from the basic electrophoresis theory and the dissociation equilibrium of a binary acid. In addition, the changes of pKa values of the five compounds were also investigated when organic modifiers were added to the buffer system.     

Explanation for the enhanced dissolution of silica column packing in high pH phosphate and carbonate buffers

It has been reported that at high pH, the rate of bonded phase packing degradation in methanol/water mobile phases is greater for carbonate and phosphate buffers than for amine buffers. This conclusion was based on buffer pH determined in the aqueous buffer before dilution with methanol. Changes in buffer species pKa, and therefore buffer pH, upon methanol dilution are consistent with the observed degradation results. Measurements of pH in the methanol/water solutions confirm that the carbonate and phosphate buffers were considerably more basic than the amine buffer, even though all the buffers were pH 10 before dilution with methanol. These results demonstrate that it can be misleading to extrapolate aqueous pH data to partially aqueous solutions. Measurements of pH in the mixed solvent provide more reliable predictions of column and sample stability.     

Separation of diadenosine polyphosphates by capillary electrophoresis

The influence of buffer composition and pH on the electrophoretic behavior of diadenosine polyphosphates with a phosphate chain ranging from two to five phosphate groups has been examined. The electrophoretic mobility in carbonate buffer increases according to the number of phosphates, whereas in borate buffer the mobility changes in an irregular way as a function of pH. This finding can be rationalized by a well-known interaction of borate with ribose rings, which modifies the charge and the hydrodynamic radius of each diadenosine polyphosphate in a different way. Our study shows that the best separation of diadenosine polyphosphates can be achieved at the highest pH values of the range examined both in borate and carbonate buffers.     

Acid-base equilibria in nonpolar media. Absolute pK(a) scale of bases in tetrahydrofuran

The acidity constants (pKa) of 11 bases (amines, anilines, pyridines, pyrrolidines, and iminophosphoranes) have been determined in tetrahydrofuran by potentiometry, complemented by conductometric measurements. The pK(a) values of the studied bases cover a wide absolute pH range of acidity in tetrahydrofuran, from 7.4 to 21.7. From the pK(a) values obtained, a scale of absolute acidity in tetrahydrofuran has been established, which has allowed calculation of the absolute pKa values of 77 bases from literature relative pK(a) data.