Determination Of Acid Dissociation Constants Research Articles

Multi-wavelength spectrophotometric determination of acid dissociation constants: a validation study

The p K a values of 25 structurally diverse drug compounds including mono-, di- and tri-protic molecules were determined by a recently developed multi-wavelength spectrophotometric technique, called dip-probe absorption spectroscopy (D-PAS), and the traditional spectrophotometric titration. All experiments were performed at 25.0±0.5°C in an ionic strength of 0.15 M KCl in two laboratories (Semmelweis and Sirius), to evaluate the reproducibility and reliability of the spectrophotometric techniques for p K a determination. It was found that the p K a values determined by the D-PAS technique are in excellent agreement with values measured by the traditional spectrophotometric method (average deviation=0.08 units) and are consistent with the literature data where available. We have demonstrated that the D-PAS approach is a simple, precise and easy-to-use method for p K a determination of drug molecules with ionisable group(s) in proximity to chromophore(s). The advantages and limitations of spectrophotometric p K a determination are discussed

Determination of Acid Dissociation Constants of Neamine by Potentiometric and Electrospray Mass Spectral Techniques

The acid dissociation constants of neamine have been measured using potentiometric titrations. The pKa1, pKa2, and pKa3 values of neamine are 6.35 ± 0.2, 7.73 ± 0.15, and 8.62 ± 0.08, respectively. Neamine is readily characterized using positive-ion electrospray ionization–mass spectrometry (ESI–MS). Various protonated species and their solvated ions are mainly observed. Nevertheless, the abundances of the observed speciation over the pH range 5.0–9.8 do not reflect the variation in the bulk solution. Hence, reaction quotients determined from the ESI–MS investigations vary significantly from the pKa's determined from potentiometric titrations, thereby illustrating that care must be taken in determining thermodynamic properties using the former technique.

Equilibrium Analysis of Acid Dissociation Reactions of Phenolphthalein by Using Mobility Change in Capillary Zone Electrophoresis

Abstract An equilibrium analysis method by capillary zone electrophoresis was utilized for the determination of acid dissociation constants of phenolphthalein, which is unstable in an alkaline aqueous solution. Apparent electrophoretic mobility of phenolphthalein increased with increasing pH of a migrating solution. Acid dissociation constants (pKa) of phenolphthalein were determined as 8.84 and 9.40 by applying a non-linear least-squares analysis to the mobility change.

Multiwavelength spectrophotometric determination of acid dissociation constants: Part V: microconstants and tautomeric ratios of diprotic amphoteric drugs

The acid-base equilibria of several diprotic amphoteric drugs, namely, niflumic acid, norfloxacin, piroxicam, pyridoxine and 2-methyl-4-oxo-3H-quinazoline-3-acetic acid have been characterized in terms of microconstants and tautomeric ratios. A multiwavelength spectrophotometric (WApH) titration method for determination of acid dissociation constants (p K a values) of ionizable compounds developed previously was applied for this purpose. Microspeciation was investigated by three approaches: (1) selective monitoring of ionizable group by spectrophotometry, (2) deductive method and (3) k z method for determination of tautomeric ratio from co-solvent mixtures. The formulation for (3) has been derived and found to invoke fewer assumptions than a reported procedure (K. Takács-Novák, A. Avdeef, K.J Box, B. Podányi, G. Szász, J. Pharm. Biomed. Anal., 12 (1994) 1369–1377). It has been shown that the WApH technique, for such types of ampholytes, is able to deduce the microconstants and tautomeric ratios which are in good agreement with literature data.

Multiwavelength spectrophotometric determination of acid dissociation constants: Part IV. Water-insoluble pyridine derivatives

A multiwavelength spectrophotometric (WApH) titration method for determination of acid dissociation constants (p K a values) of ionizable compounds developed previously was applied in the case of pyridine derivatives of pharmaceutical interest. Specifically, UV absorption spectra of the drug solution are acquired in the course of a pH-metric titration using an optical device based on a fibre optics dip probe, a light source and a diode array detector. Target factor analysis was applied to deduce the p K a values from the spectral data recorded at different pH. Using this technique, the p K a values of six pyridine derivatives were determined successfully. It was demonstrated that the WApH technique in this case outperforms conventional pH-metric methods with respect to the measurement of p K a values of the sparingly water soluble samples reported in this study.

Multiwavelength spectrophotometric determination of acid dissociation constants: Part III. Resolution of multi-protic ionization systems

A multiwavelength spectrophotometric (WApH) titration method was applied to study several multi-protic histamine H 2-receptor antagonists which involved four acid dissociation constants (p K a values) over the pH range of 2–10. Specifically, UV absorption spectra of the drug solution were acquired in the course of a pH-metric titration using an optical device based on a fibre optics dip probe, a light source and a diode array detector. Target factor analysis was utilized to deduce the p K a values from the spectral data recorded at different pH. It was noted that some of the p K a values were within mid pH range which were difficult to obtain because of insufficient absorption spectra acquired in the un-buffered region of the titration curve. With the aid of the WApH technique coupled with an optically transparent buffer, all p K a values have been successfully determined and were in excellent agreement with those measured using a conventional pH-metric method.

Multiwavelength spectrophotometric determination of acid dissociation constants: Part II. First derivative vs. target factor analysis.

Acid dissociation constants (pKa values) denote the extent of ionization of drug molecules at different pH values, which is important in understanding their penetration through biological membranes and their interaction with the receptors. However, many drug molecules are sparingly soluble in water or contain ionization centres with overlapping pKa values, making precise pKa determination difficult using conventional spectrophotometric titration. In this work, we investigate a multiwavelength spectrophotometric titration (WApH) method for the determination of pKa values. Spectral changes which arise during pH-metric titrations of substances with concentration of about 10(-5) M were captured by means of an optical system developed in this study. All experiments were carried out in 0.15 M KCI solution at 25 +/- 0.5 degrees C. Mathematical treatments based on the first derivative spectrophotometry procedure and the target factor analysis method were applied to calculate the pKa values from the multiwavelength absorption titration data. pKa values were determined by the WApH technique for six ionizable substances, namely, benzoic acid, phenol, phthalic acid, nicotinic acid, p-aminosalicylic acid and phenolphthalein. The pKa values measured using the WApH technique are in excellent agreement with those measured pH-metrically. We have demonstrated that the first derivative spectrophometry procedure provides a relatively simple way to visualize the pKa values which are consistent with those determined using the target factor analysis method. However, for ionization systems with insufficient spectral data obtained around the sought pKa values or with closely overlapping pKa values, the target factor analysis method outperforms the first derivative procedure in terms of obtaining the results. Using the target factor analysis method, it has been shown that the two-step ionization of phenolphthalein involves a colorless anion intermediate and a red colored di-anion.

Theoretical calculations of acid-dissociation constants of proteins.

The purpose of this review is to introduce several computational procedures for the determination of acid-dissociation constants (pKa) of titratable groups in proteins. Several concepts, such as continuum electrostatics and the exact meaning of intrinsic and apparent pKas, will be explained in some detail. Each of the methods will be judged on its merits, and some comparisons between the methods will be presented. While the emphasis of this review will be on theoretical formulations, the experimental determination by means of nuclear magnetic resonance will be briefly explained. The determination of individual pKa values by nuclear magnetic resonance in combination with computationally determined pKas can provide unique information about the pH-dependent properties of proteins and their complexes with peptides, DNA, and ligands.

Determination of acid dissociation constants of peptide side-chain functional groups by two-dimensional NMR.

An NMR method is described for determining residue-specific acid dissociation constants for peptides which contain more than one residue of the same acidic or basic amino acid. The method is based on using the differences in C alpha H proton chemical shifts which result from peptide sequence nearest-neighbor and possibly secondary structure effects to resolve resonances for carbon-bonded reporter protons adjacent to each side-chain acidic group in two-dimensional total correlation spectroscopy (TOC-SY) spectra. Acid dissociation constants were determined for each of the four lysine side-chain ammonium groups of the peptide Lys-Asn-Asn-Gln-Lys-Ser-Glu-Pro-Leu-Ile-Gly-Arg-Lys-Lys-Thr-NH2. Resonances for the C epsilon H2 protons adjacent to the four side-chain ammonium groups, which overlap in the one-dimensional spectrum, were resolved using the C alpha H-C epsilon H2 cross peaks in the TOCSY spectrum. Chemical shift-pH titration data were obtained for each lysine side-chain ammonium group from one-dimensional subspectra taken from two-dimensional TOCSY spectra measured as a function of pH. The pKAs of the Lys1, Lys5, Lys13, and Lys14 side-chain ammonium groups were determined to be 11.14 +/- 0.01, 10.95 +/- 0.01, 10.96 +/- 0.02, and 11.09 +/- 0.02, respectively. The chemical shift-pH titration data were also analyzed by a pH-independent procedure to obtain relative acid dissociation constants: KA(Lys1)/KA(Lys5) = 0.663 +/- 0.009, KA(Lys1)/KA(Lys13) = 0.703 +/- 0.014, and KA(Lys1)/KA(Lys14) = 0.910 +/- 0.009, which correspond to relative acidities for the side-chain ammonium groups of Lys1, Lys5, Lys13, and Lys14 of 1:1.508:1.422:1.099. To further demonstrate the utility of this method, acid dissociation constants were determined for the six acidic groups of the peptide Glu-Ala-Cys-Asn-Pro-Ala-Ala-Gly-Arg-His-Tyr-Ser-Cys-NH2. Chemical shift-pH titration curves were obtained for the C beta H2 protons adjacent to the two cysteine thiol groups using one-dimensional subspectra taken from TOCSY spectra measured as a function of pH. The pKAs of the CyS3 and Cys13 thiol groups were determined to be 9.21 +/- 0.07 and 8.60 +/- 0.06, respectively. The relative acid dissociation constants (KA(Cys3)/ (KA(Cys13)) were found to be 0.21 +/- 0.06 by the pH-independent calculation procedure.

Determination of acid dissociation constants of anomers of amino sugars by 1H NMR spectroscopy

Acid dissociation constants of α- and β- d-glucos-, mannos-, and galactos-ammonium ions have been determined from 1H NMR chemical shifts of the individual anomers in D 2O. Values of p K a (D) for the α- and β-ammonium ions are, respectively: glucosamine, 8.12 and 7.87, mannosamine, 7.78 and 8.50, galactosamine, 8.49 and 8.02. The differences are ascribed largely to differences in the hydration requirements of ammonium and amino groups in the axial and equatorial positions and hydration at upper and lower faces of the sugars. Acid dissociation constants of the 1-hydroxyl group of nonionic d-glucosamine and d-glucose are higher for the β than the α anomer.

A New Liquid Chromatographic Method for the Determination of Acid Dissociation Constants

A new liquid chromatographic method for the determination of acid dissociation constants is presented on the basis of theoretical equations regarding the effect of background mobile phase ions on the retention of ionic analytes on nonionic polymer packings. The dissociation constants determined by the present method using Bio-Gel P-2 and Toyopearl HW- 40S as the column packings and water as the mobile phase solvent were in fairly good agreement with the values obtained by potentiometry. This method has an important advantage of permitting the simultaneous determination of thedissociation constants and the charges of analyte compounds. One can then readily assign a dissociation constant obtained for a polybasic acid to the corresponding step of the proton dissociation.

An improved method for determination of acid dissociation constants of peptides.

The method described here enables the proton dissociation constants of several amino acid residues of a peptide to be determined simultaneously in aqueous solution without prior knowledge of the exact concentration of the peptide. The method used here employs a non-linear fitting program, the BEST program, or a linear least-squares method in combination with the BEST program. These methods are discussed in detail with an emphasis on the quality of the potentiometric titration data that are obtained. Two representative peptides, one with two proton dissociation constants (Ka1, Ka2) and the other with four proton dissociation constants (Ka1-Ka4) were used to illustrate the advantages and the limitations of these two complementary methods. The pKa values of TVL, a schizophrenia-related tripeptide, were found to be 3.62 (+/- 0.02) and 7.17 (+/- 0.05); the pKa values of ELTLQE, a hexapeptide, were found to be 2.32, 3.77, 4.58 and 7.74. The methods reported here are generally applicable to a variety of peptides. The possibility of integrating these procedures into a preparative chromatographic system for the "on-line" assessment of the pKa values of peptides during the purification stage is an attractive and novel feature of this method.

Voltammetric determination of acid dissociation constants of pyrroloquinoline quinone and its reduced form under acidic conditions

The voltammetric properties of the coenzyme pyrroloquinoline quinone (PQQ ox) have been investigated in the pH range from 0 to 8. PQQ ox gives a quasi-reversible cyclic voltammogram at a platinum electrode, which is ascribed to a two-electron redox reaction of PQQ ox to pyrroloquinoline quinol (PQQ red). The standard redox potential ( E o′) of the PQQ ox/PQQ red couple has been determined using cyclic voltammetry. From the analysis of the E o′ vs. pH relationship, the four macroscopic acid dissociation constants ( K a) of both PQQ ox and PQQ red have been determined. The p K as have been assigned to the pyridyl nitrogen and three carboxyl groups. The E o′ of the PQQ ox/PQQ red couple at pH 7.0 has been determined as −0.175 V vs. SCE (a saturated calomel electrode) with a semiquinone (PQQ sem) formation constant of 0.02, where PQQ ox, PQQ red, and PQQ sem are present as trianions with the three carboxylate anion substituents. The pH dependence of the absorption spectra of PQQ ox has been interpreted in terms of the p K a values.

Voltammetric determination of acid dissociation constants of pyrroloquinoline quinone and its reduced form under acidic conditions

The voltammetric properties of the coenzyme pyrroloquinoline quinone (PQQ ox) have been investigated in the pH range from 0 to 8. PQQ ox gives a quasi-reversible cyclic voltammogram at a platinum electrode, which is ascribed to a two-electron redox reaction of PQQ ox to pyrroloquinoline quinol (PQQ red). The standard redox potential ( E o′) of the PQQ ox/PQQ red couple has been determined using cyclic voltammetry. From the analysis of the E o′ vs. pH relationship, the four macroscopic acid dissociation constants ( K a) of both PQQ ox and PQQ red have been determined. The p K as have been assigned to the pyridyl nitrogen and three carboxyl groups. The E o′ of the PQQ ox/PQQ red couple at pH 7.0 has been determined as −0.175 V vs. SCE (a saturated calomel electrode) with a semiquinone (PQQ sem) formation constant of 0.02, where PQQ ox, PQQ red, and PQQ sem are present as trianions with the three carboxylate anion substituents. The pH dependence of the absorption spectra of PQQ ox, has been interpreted in terms of the p K a values.

Measurement of acid dissociation constants of weak acids by cation exchange and conductometry

A simple strategy is presented for the determination of acid dissociation constants based on the measurement of conductance of a known concentration of the acid and/or the conductance of a solution of its fully or partially neutralized alkali-metal salts. For an n-protic acid, 2n conductance measurements are minimally necessary. In the simplest case of a typical monoprotic acid, the conductance of its alkali salt solution is measured before and after passage through an H{sup +}-form exchanger. From these data both the pK{sub a} of the acid and the equivalent conductance of the anion can be computed. The underlying equations are rigorously solved for monoprotic acids and some diprotic acid systems. For other diprotic and multiprotic acid systems, initial estimates are obtained by making approximations; the complete data set is then subjected to multiparametric fitting. The method does not require pH measurements; conductance can generally be measured accurately at low enough ionic strengths to obviate the need for major activity correction. Several experimental measurements are presented and excellent agreement with literature pK{sub a} values is observed. The reliability of the equivalent conductance values computed in this fashion is limited, however.

Determination of acid dissociation constants by high-performance liquid chromatography.

Values for the acid dissociation constant Ka of various acidic and basic compounds were determined from the pH-dependent capacity factor k' by reversed-phase high-performance liquid chromatography (HPLC) with octadecyl silane as the stationary phase. Both the neutral and ionic forms of acids and bases were retained on the stationary phase, and k' of the ionic form markedly influenced the calculation of Ka value. Values of Ka determined by HPLC agreed well with those determined by the conventional potentiometric method, indication the availability of this method for the determination of Ka values. The hydrophobicity of the ionized forms of these compounds is discussed in terms of the partition coefficient of the neutral form between octanol and water.

Determination of acid dissociation constants of histidine‐containing peptides by proton magnetic resonance spectroscopy

AbstractThe acid dissociation constant of the imidazolium ring of the decapeptide luliberin has been determined by 1H NMR‐followed titration in D2O. The normal procedure for the analysis of the titration curve, i.e. direct use of the Henderson‐Haselbalch equation, is still applicable in this case, but for more complex peptides a modified calculation procedure is proposed. Results obtained when both methods were applied to luliberin are compared. The influence of D2O when used as the solvent in this type of determination has been studied using Nα‐acetyl‐L‐histidine methyl ester as a model compound. The difference between the acid dissociation constant of this molecule determined in H2O and in D2O implies that a correction of −.25 unit is needed for those pKa values calculated by plotting the chemical shifts in D2O vs the apparent pH meter readings. The pKa found for Nα‐acetyl‐L‐histidine methyl ester, 6.30 ± 0.04, can be taken as a standard value for histidine‐containing peptides.

Simultaneous determination of acid dissociation constants and true partition coefficients by analysis of the apparent partition coefficients. III. The application of the Hammett acid function.

Equations for the simultaneous determination of acid dissociation constants (pKa) and true partition coefficients (P) of a very weak base or acid have been derived by applying the Hammett acid functions Ho and H- and by analyzing the acid function dependence of the apparent partition coefficient (Pa). The equations show that Pa-1 is linearly related to ho (ho=10-Ho) and h^ (h-=10-H-) for a base and an acid, respectively. Some experimental systems for bases have been successfully measured using octanol as an organic solvent phase and suitably diluted H2SO4 solution as an aqueous phase, the Ho value being used. The results obtained were reasonably consistent with the literature values. The application of this technique makes it possible to determine log P and pKa simultaneously, the latter values being smaller than 0 for bases and larger than 14 for acids.

Simultaneous determination of acid dissociation constants and true partition coefficients by analysis of the apparent partition coefficients. II. Dibasic acid and diacidic base.

The simultaneous determination of acid dissociation constants (pKa) and true partition coefficients (P) of a dibasic acid and a diacidic base was achieved by analyzing the pH dependence of the apparent partition coefficient (Pa). Equations derived theoretically show a parabolic relation of Pa to (H+)-1 and (H+) for a dibasic acid and a diacidic base, respectively, (H+) being the proton concentration. Theoretical considerations indicate

Simultaneous determination of acid dissociation constants and true partition coefficients by analyses of the apparent partition coefficients. I.

Since the partition coefficient P and the dissociation constant pKa are important in studies of structure-activity relationships, the pH dependence of the apparent partition coefficient Pa was quantitatively studied for the cases of acids and bases. Linear relations for mono-acids and mono-bases, but parabolic relations for the dissociation of two functional groups in the pH region studied were derived between Pa and proton concentration. The equations, however, are different for acids and bases. These equations make it possible to determine pKa and the log P simultaneously. Some experimental systems have been measured and compared with the results obtained by other techniques ; the agreement was good. A consideration of the dissociation in the organic phase showed that the correction factor due to ionic dissociation in the organic phase for an octanolbuffer system is negligible in the pH region near the pKa value, but is not negligible in the general equation connecting the Pa and P values.