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.     

Determination of acid-base dissociation constants of amino- and guanidinopurine nucleotide analogs and related compounds by capillary zone electrophoresis

CZE has been applied for determination of acid-base dissociation constants (pKa) of ionogenic groups of newly synthesized amino- and (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonate, acyclic nucleoside phosphonate diesters and other related compounds. These compounds bear characteristic pharmacophores contained in various important biologically active substances, such as cytostatics and antivirals. The pKa values of ionogenic groups of the above compounds were determined by nonlinear regression analysis of the experimentally measured pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE performed in series of BGEs in a broad pH range (3.50-11.25), at constant ionic strength (25 mM) and temperature (25 degrees C). pKa values were determined for the protonated guanidinyl group in (amino)guanidino 9-alkylpurines and in (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonates and acyclic nucleoside phosphonate diesters, for phosphonic acid to the second dissociation degree (-2) in acyclic nucleoside phosphonates of amino and (amino)guanidino 9-alkylpurines, and for protonated nitrogen in position 1 (N1) of purine moiety in acyclic nucleoside phosphonates of amino 9-alkylpurines. Thermodynamic pKa of protonated guanidinyl group was estimated to be in the range of 7.75-10.32, pKa of phosphonic acid to the second dissociation degree achieved values of 6.64-7.46, and pKa of protonated nitrogen in position 1 of purine was in the range of 4.13-4.89, depending on the structure of the analyzed compounds.     

pH gradient high-performance liquid chromatography: theory and applications

pH gradient high-performance liquid chromatography (HPLC) is a method of reversed-phase high-performance liquid chromatography suitable for ionogenic substances. It consists in programmed increase during the chromatographic process of the eluting strength of eluent with respect to the analytes separated. On the analogy of the conventional organic modifier gradient reversed-phase HPLC, in the pH gradient approach the eluting strength of the mobile phase increases due to its changing pH: increasing in case of acids or decreasing in case of bases. At the same time the content of organic modifier remains constant. A theory of the pH gradient HPLC has been elaborated. The resulting mathematical model is easily manageable. Its ability to predict changes in retention and separation of analytes following the changes in chromatographic conditions is demonstrated. The pH gradient method is uniquely suitable to determine pKa values of analytes. An equation is presented allowing to calculate pKa values basing on appropriate retention data. The effects on pKa are discussed of the concentration of methanol in the mobile phase. The RP HPLC-derived pKa data correlate to the reference pKa values (w(w)pKa) but are not identical. That may be explained by the effects on the chromatographically determined pKa of the specific interactions of analytes with stationary phases. The proposed pH gradient RP HPLC procedure offers a fast and convenient means to get comparable acidity parameters for larger series of compounds, like drug candidates, also when the analytes are available only in minute amounts and/or as complex mixtures.     

Effect of temperature on the chromatographic retention of ionizable compounds. II. Acetonitrile-water mobile phases

The retentive behavior of weak acids and bases in reversed-phase liquid chromatography (RPLC) upon changes in column temperature has been theoretically and experimentally studied. The study focuses on examining the temperature dependence of the retention of various solutes at eluent pH close to their corresponding pKa values, and on the indirect role exerted by the buffer ionization equilibria on retention and selectivity. Retention factors of several ionizable compounds in a typical octadecylsilica column and using buffer solutions dissolved in 30% (v/v) acetonitrile as eluent at five temperatures in the range from 25 to 50 degrees C were carefully measured. Six buffer solutions were prepared from judiciously chosen conjugated pairs of different chemical nature. Their pKa values in this acetonitrile-water composition and within the range of 15-50 degrees C were determined potentiometrically. These compounds exhibit very different standard ionization enthalpies within this temperature range. Thus, whenever they are used to control mobile phase pH, the column temperature determines their final pH. Predictive equations of retention that take into account the temperature effect on both the transfer and the ionization processes are evaluated. This study demonstrates the significant role that the selected buffer would have on retention and selectivity in RPLC at temperatures higher than 25 degrees C, particularly for solutes that coelute.     

Fluorescence spectrometry for quantitative characterization of cobalt(II) complexation by Leonardite humic acid

Quenching of the fluorescence of a Leonardite humic acid by Co(II) has been studied at different pH. The interaction was monitored by emission fluorescence and by synchronous fluorescence with two different offsets (deltalambda=20 and 80 nm). It was found that synchronous fluorescence performed with the smaller offset resolves the individual components of the heterogeneous material better than emission or synchronous fluorescence performed with the larger offset. Enhancement of the signal induced by Cobalt(II) complexation resulted in more complex behavior for measurements performed by synchronous fluorescence with an offset of 20 nm, however. The quenching profiles obtained for pH 5.0, 6.0, and 7.0 ([KNO(3)]=0.1 mol L(-1); [LHA]=3.3 mg(C) L(-1); [Co(II)]=1.0 x 10(-6)-1.6 x 10 (-3) mol L(-1)) by emission and synchronous (deltalambda=80 nm) fluorescence were analyzed by two methods: 1. a non-linear least-squares procedure that leads to conditional constants; and 2. a pH-dependent discrete logK spectrum model that leads to stability constants. The first method resulted in poor fitting and unreasonable values for maximum capacities. The second procedure resulted in smooth fitting that accounted well for the pH changes when results for pH 6.0 and 5.0 were predicted by use of the four values of logK(Co)(i) (4.31, 3.76, 7.32, and 7.67 corresponding to the four sites (i) of the respective pKa(i) values 4, 6, 8, and 10) calculated at pH 7.0 for the equilibrium     

Prediction of retention behaviour and evaluation of pka values of peptides and quinolones in liquid chromatography.

The present paper examines the effect of the solute ionisation on the retention behaviour in liquid chromatography of a series of peptide and quinolone compounds of biological interest, using acetonitrile-water media as mobile phases and a polymeric-based stationary phase. Polymeric columns with polystyrene-divinylbenzene (PS-DVB) polymer show advantages over silica-based reversed-phase packings since the former are stable in a wide pH range. (s)(s)pKa values have been evaluated using chromatographic data in acetonitrile-water mixtures with acetonitrile percentages of 30, 35, 40 and 50% (v/v) for quinolones and 12.5 and 20% (v/v) for peptides. The quinolones show great retention on PS-DVB phase stationary. It was thus necessary to work with a higher acetonitrile content in the mobile phase than for the less retained peptides. The pH values were measured in the hydroorganic mixtures, used as mobile phases, instead of in water and account was taken of the effect of activity coefficients. The derived equations permit the chromatographic determination of (s)(s)pKa. values of the peptides and quinolones in acetonitrile-water mixtures by fitting it to the experimental data in a nonlinear least-square procedure and also permit the prediction of the effect of (s)(s)pH on their chromatographic behaviour. We have also compared the obtained (s)(s)pKa values with those previously obtained in acetonitrile-water mixtures from potentiometric measurements.     

A new approach based on a combination of direct and headspace cold-fiber solid-phase microextraction modes in the same procedure for the determination of polycyclic aromatic hydrocarbons and phthalate esters in soil samples

This study describes a new approach to cold-fiber solid-phase microextraction (CF-SPME) based on a combination of different extraction modes in the same extraction procedure. Also, the high quantity of water required to facilitate both the desorption of analytes from the matrix and their transport to the fiber coating is reported. The extraction mode was changed from the direct to the headspace mode in a single extraction while manipulating the extraction times and coating temperature to improve the extraction of compounds with different volatilities. Compounds with low volatility were better extracted in the direct mode, while the headspace mode was more appropriate for volatile compounds. Polycyclic aromatic hydrocarbons (PAHs) and phthalic acid esters (PEs) in sand or soil samples were used as model compounds and matrices in this study. The optimized conditions were: sample pH in the range of 4-7, addition of 12 mL of 194 g L(-1) aqueous NaCl solution in a 15 mL vial, and 80 min total extraction time with a sample temperature of 90°C (50 min in direct mode with coating at 90°C followed by 30 min in headspace mode with coating at 30°C). The proposed procedure was compared with conventional CF-SPME (with and without addition of water) and was found to be more effective for all the analytes, since it is capable of extracting both heavier and lighter compounds from soil samples in a single extraction procedure. The use of an excess of water and a combination of extraction modes in the same CF-SPME procedure are the main factors responsible for this enhancement. The proposed method was applied to the extraction of PAHs and PEs in spiked soil samples and excellent results were obtained for most of the compounds evaluated.     

乙酸和一氯乙酸电离常数的高效毛细管区带电泳法测定

首次建立了测定一氯乙酸和乙酸的电离常数的高效毛细管区带电泳新方法.该方法利用中性标记物和电流突跃两种方法来标记电渗流,通过测定乙酸和一氯乙酸在一定pH的缓冲溶液中的电泳淌度,结合数据回归分析拟合,求得乙酸和一氯乙酸的电离常数;所得数据和文献报道值较为接近.总体而言,毛细管区带电泳法可简单、快速、可靠地用于测定待测化合物的电离常数.     

Electronic absorption study on acid-base equilibria for some keto and thioketo pyrimidine derivatives. Experimental and theoretical evidence of enolization and solute-solvent interactions

The UV-vis spectra of recently synthesized 1-amino-5-benzoyl-4-phenyl-1H-pyrimidine-2-one, (I), and 1-amino-5-benzoyl-4-phenyl-1H-pyrimidine-2-thione (II), were studied in aqueous methanol (5%, v/v, methanol) and pure methanol. The nature of the electronic transitions and the role of carbonyl oxygen of I and thiocarbonyl sulfur of II in the behavior of the observed UV-vis spectra were discussed. The carbonyl group at position 2 of I and the thiocarbonyl group of II were found to be enolized instead of protonation. Quantum chemical calculations showed agreement with the experimental evidence. However, the carbonyl group of the benzoyl moiety at position 5 of both compounds underwent neither enolization nor protonation. Acid-base equilibria of the compounds against varying pH have been examined in detail. The pKa values of all related equilibria were determined at room temperature and an ionic strength of 0.10 M from the pH-dependence of the absorbance values using the Henderson-Haselbalch equation and graphical logarithmic analysis. The mean acidity constants for the protonated forms of the compounds were determined as pKa1=4.214 and pKa2=6.678 for I and pKa1=3.739 and pKa2=6.258 for II. The mean acidity constants (pKa3) for the enol form of I and the thioenol form of II were determined as 11.278 and 11.063, respectively. The preferred dissociation mechanisms were discussed based on the data of UV-vis spectroscopy and a mechanism was proposed for each compound. The formation of intramolecular and intermolecular hydrogen bonding were found with I but not with II. The intramolecular bonding stabilizing the enol form was favoured at pH values corresponding to pKa1 and above. On the other hand, the intermolecular hydrogen bonding stabilizing the free form of the carbonyl group was favoured at all pH values.     

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.     

Evaluation of calibration data in capillary electrophoresis using artificial neural networks to increase precision of analysis

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.     

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.     

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.     

Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.     

Pyridylthiazoles: highly luminescent heterocyclic compounds

Absorption, fluorescence, and fluorescence excitation spectra of two substituted [(5-methyl-2-pyridine-2'-yl-1,3-thiazole-4-yl)oxy]acetic acid and its methyl ester (2,2'-pyridylthiazoles) are studied at various pH values in aqueous solution. The acid exhibits pKa(1)=2.10+/-0.07 and pKa(2)=3.45+/-0.03, whereas the ester pKa=1.93+/-0.03. The protonation site is the pyridyl-nitrogen. When protonated, the cisoid conformer is the most stable; however, the transoid conformer is more stable in the deprotonated form. Fluorescence quantum yields close to unity are found. Large Stokes shift values are explained by the shortening of the inter-ring bond in the excited state. These compounds may be useful for metal sensing and as laser dyes.     

Linear-scaling molecular orbital calculations for the pKa values of ionizable residues in proteins

In this report, we present a computational methodology for the pKa prediction of proteins, based on linear-scaling molecular orbital calculations for their solution-conformations obtained from NMR measurements. The method is used to predict the pKa values of five carboxylic acids (Asp7, Glu10, Glu19, Asp27, and Glu43) in turkey ovomucoid third domain (OMTKY3), and six aspartates residues (Asp 22, Asp 44, Asp 54, Asp 75, Asp 83, and Asp 93) in barnase. For OMTKY3, all the predicted pKa values are within 1 pH units from the available experimental ones, except for the case of Glu 43. For barnase, the root-mean-square deviation from experiment is 1.46 pH units. As a result, the proposed pKa calculation method correctly reproduces the relative order of the pKa values among the carboxylic acids located in different sites of the proteins. The calculated pKa values are decomposed into the contributions of short- and long-range structural difference effects. The results indicate that in both proteins the pKa value of the given carboxylic acid is partially influenced by long-range interactions with distant charged residues, which significantly contribute to determining the relative order of the pKa values. The current methodology based on LSMO provides us useful information about the titration behavior in a protein.     

Accurate pKa determination for a heterogeneous group of organic molecules.

Single-molecule studies that allow to compute pKa values, proton affinities (gas-phase acidity/basicity) and the electrostatic energy of solvation have been performed for a heterogeneous set of 26 organic compounds. Quantum mechanical density functional theory (DFT) using the Becke-half&half and B3LYP functionals on optimized molecular geometries have been carried out to investigate the energetics of gas-phase protonation. The electrostatic contribution to the solvation energies of protonated and deprotonated compounds were calculated by solving the Poisson equation using atomic charges generated by fitting the electrostatic potential derived from the molecular wave functions in vacuum. The combination of gas-phase and electrostatic solvation energies by means of the thermodynamic cycle enabled us to compute pKa values for the 26 compounds, which cover six distinct chemical groups (carboxylic acids, benzoic acids, phenols, imides, pyridines and imidazoles). The computational procedure for determining pKa values is accurate and transferable with a root-mean-square deviation of 0.53 and 0.57 pKa units and a maximum error of 1.0 pKa and 1.3 pKa units for Becke-half&half and B3LYP DFT functionals, respectively.     

Computational determination of aqueous pKa values of protonated benzimidazoles (part 1)

Benzimidazoles are the organic compounds investigated in this work. The experimental determination of the pKa values of protonated benzimidazoles in water is a challenge because of their low solubility. In addition, some derivatives are involved in tautomeric equilibria which increase the complexity of the theoretical pKa determinations. In the present study, different approaches are considered to develop a methodology for the accurate prediction of aqueous pKa values of protonated benzimidazoles at 298.15 K. We have considered different reaction schemes for approximating the acid dissociation equilibrium; two distinct equations are used for the calculation of pKa values, and a number of levels of theory and empirical corrections are applied in the process of working toward this aim. The best correlations between the experimental and calculated data are obtained at the B3LYP/6-31+G(d,p)-PCM(opt) level of theory. The predictive capabilities of the methodologies attempted are tested with two compounds that were not included in the set of benzimidazoles initially investigated. The direct calculations differ significantly from the expected values, but the pKa values calculated using the correlation equations are very similar and in reasonable agreement with the expected pKa values.     

Use of two different coating temperatures for a cold fiber headspace solid‐phase microextraction system to determine the volatile profile of Brazilian medicinal herbs

In this study, the experimental extraction conditions on applying headspace solid‐phase microextraction and cold fiber headspace solid‐phase microextraction (CF‐HS‐SPME) procedures to samples of six medicinal herbs commonly found in southern Brazil were optimized. The optimized conditions for headspace solid‐phase microextraction were found to be an extraction temperature of 60°C and extraction time of 40 min. For CF‐HS‐SPME, the corresponding values were 60°C and 15 min. In the case of the coating temperature for the CF‐HS‐SPME system, two approaches were investigated: (i) Temperature of 5°C applied during the whole extraction procedure; and (ii) the use of two fiber temperatures in the same extraction procedure with the aim of extracting the volatile and semivolatile compounds, the ideal condition being 60°C for the first 7.5 min and 5°C for the final 7.5 min. The three extraction procedures were compared. The CF‐HS‐SPME procedure had good performance only for the more volatile compounds whereas the strategy using two coating temperatures in the same procedure showed good performance for all compounds studied. It was also possible to determine the profile for the volatile fraction of each herb studied applying this technique followed by GC‐MS.