Fast high-throughput method for the determination of acidity constants by capillary electrophoresis. II. Acidic internal standards

A fast method for the determination of acidity constants by CZE has been recently developed. This method is based on the use of an internal standard of pK(a) similar to that of the analyte. In this paper we establish the reference pK(a) values of a set of 24 monoprotic neutral acids of varied structure that we propose as internal standards. These compounds cover the most usual working pH range in CZE and facilitate the selection of adequate internal standards for a given determination. The reference pK(a) values of the acids have been established by the own internal standard method, i.e. from the mobility differences between different acids of similar pK(a) in the same pH buffers. The determined pK(a) values have been contrasted to the literature pK(a) values and confirmed by determination of the pK(a) values of some acids of the set by the classical CE method. Some systematic deviations of mobilities have been observed in NaOH buffer in reference to the other used buffers, overcoming the use of NaOH in the classical CE method. However, the deviations affect in a similar degree to the test compounds and internal standards allowing thus, the use of NaOH buffer in the internal standard method. This fact demonstrates the better performance of the internal standard method over the classical method to correct mobility deviations, which together with its fastness makes it an interesting method for the routine determination of accurate pK(a) values of new pharmaceutical drugs and drug precursors.     

Determination of dissociation constants of compounds with potential cognition enhancing activity by capillary zone electrophoresis

Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. In this work we have used CZE for determination of pK(a) values of new compounds prepared from intermediates, 2, 3 and 4-(2-chloro-acetylamino)-phenoxyacetic acids, by substituting chloride for 2-oxo-pyrrolidine, 2-oxo-piperidine or 2-oxo-azepane. These substances are expected to have a cognition enhancing activity and free radicals scavenging effect. Measurements were performed in a polyacrylamide-coated fused-silica capillary of 0.075 mm ID using direct UV detection at 254 nm. Three electrolyte systems were used for measurements to eliminate effects of potential interactions between tested compounds and components of the BGE. In the pH range 2.7-5.4, chloride, formate, acetate and phosphate were used as BGE co-ions, and sodium, beta-alanine and epsilon-aminocaproate as counterions. Mobility standards were measured simultaneously with the tested compounds for calculations of correct electrophoretic mobilities. Several approaches for the calculation of the pK(a) values were used. The values of pK(a) were determined by standard point-to-point calculation using Henderson-Hasselbach equation. Mobility and pH data were also evaluated by using nonlinear regression. Three parameter sigmoidal function fitted the experimental data with correlation coefficients higher than 0.99. Results from CZE measurements were compared with spectrophotometric measurements performed in sodium formate buffer solutions and evaluated at wavelength where the highest absorbance difference for varying pH was recorded. The experimental pK(a) values were compared with corresponding values calculated by the SPARC online calculator. Results of all three used methods were in good correlation.     

Determination of acid-base dissociation constants of azahelicenes by capillary zone electrophoresis

CZE was employed to determine acid-base dissociation constants (pK(a)) of ionogenic groups of azahelicenes in methanol (MeOH). Azahelicenes are unique 3-D aromatic systems, which consist of ortho-fused benzene/pyridine units and exhibit helical chirality. The pK(a) values of pyridinium groups of the studied azahelicenes were determined from the dependence of their effective electrophoretic mobility on pH by a nonlinear regression analysis. The effective mobilities of azahelicenes were determined by CZE at pH range between 2.1 and 10.5. Thermodynamic pK(a) values of monobasic 1-aza[6]helicene and 2-aza[6]helicene in MeOH were determined to be 4.94 +/- 0.05 and 5.68 +/- 0.05, respectively, and pK(a) values of dibasic 1,14-diaza[5]helicene were found to be equal to 7.56 +/- 0.38 and 8.85 +/- 0.26. From these values, the aqueous pK(a) of these compounds was estimated.     

Ampholytes as background electrolytes in capillary zone electrophoresis: sense or nonsense? Histidine as a model ampholyte

A lot of phenomena, occuring in capillary zone electrophoresis (CZE), are linked with the ionic concentration of the background electrolyte (BGE). If weak bases and acids are used as BGEs in CZE, at a pH where they are scarcely ionized, the ionic concentration of the BGE is very low and this brings a strong peak broadening, limited sample stacking and low sample load. Because the electromigration dispersion increases extremely, moreover, the existence of low-conductivity BGEs in CZE is a contradiction in terms. The behavior of ampholytes as BGE in CZE is examined, by means of histidine as a model ampholyte. For BGEs consisting of histidine, important parameters, including the ionic concentrations, buffer capacity, transfer ratio, and the indicator for electromigration dispersion E(1)m(1)/E(2)m(2), are calculated at various pH. Although the transfer ratio is fairly constant over the whole pH traject, the ionic concentration and buffer capacity decrease whereas the electromigration dispersion strongly increases near the pI of histidine. I.e., that ampholytes can be applied as BGEs in CZE, however, just not at pH near their pI value, except as the difference between the pK values of the basic and acidic group, the deltapK value, is very small. For ampholytes with a low deltapK value or at high concentrations, all the before-mentioned effects are less fatal, but in that case we can not speak of a real low-conductivity BGE. If ampholytes are used at pH near their pK values, the use of ampholytes as BGE is not advantageously compared with simple weak bases and acids. This has been confirmed by calculations and experiments.     

Determination of acid-base dissociation constants of very weak zwitterionic heterocyclic bases by capillary zone electrophoresis

Thermodynamic acid-base dissociation (ionization) constants (pK(a)) of seven zwitterionic heterocyclic bases, first representatives of new heterocyclic family (2,3,5,7,8,9-hexahydro-1H-diimidazo[1,2-c:2',1'-f][1,3,2]diazaphosphinin-4-ium-5-olate 5-oxides), originally designed as chiral Lewis base catalysts for enantioselective reactions, were determined by capillary zone electrophoresis (CZE). The pK(a) values of the above very weak zwitterionic bases were determined from the dependence of their effective electrophoretic mobility on pH in strongly acidic background electrolytes (pH 0.85-2.80). Prior to pK(a) calculation by non-linear regression analysis, the CZE measured effective mobilities were corrected to reference temperature, 25°C, and constant ionic strength, 25 mM. Thermodynamic pK(a) values of the analyzed zwitterionic heterocyclic bases were found to be particularly low, in the range 0.04-0.32. Moreover, from the pH dependence of effective mobility of the bases, some other relevant characteristics, such as actual and absolute ionic mobilities and hydrodynamic radii of the acidic cationic forms of the bases were determined.     

Electrochemical Behavior of Cu Electrode and Its Application in CE Determination of Polyols

The electrochemical behavior of a copper (Cu) electrode and its application in capillary electrophoresis determination of polyols was investigated in order to understand the redox property of Cu and achieve better separation efficiency. Electrochemical measurements were performed using a Cu electrode (fresh or oxidized) in buffer solutions having different pH values (7.8–13.0) by cyclic voltammetry. The Cu electrode showed higher electroactivity under stronger alkaline conditions. Further, the Cu electrode was found suitable for detecting weak oxidizing or reducing polyhydroxy compounds because of the redox reactions among Cu, Cu(I), and Cu(II) species. Thus, the Cu electrode was used in capillary zone electrophoresis (CZE) for separation and determination of propanediol and glycerol, weak electroactive polyols, using different separation (pH 8.7) and detection (pH 11.04) buffers. Separation and detection buffers with different pH values in CZE technology could offer efficient separation efficiency and detection limits at the same time.     

Separation and migration behavior of structurally related phenothiazines in cyclodextrin-modified capillary zone electrophoresis

The influences of buffer pH and the concentration of beta-cyclodextrins (beta-CDs) on the separation and migration behavior of 13 structurally related phenothiazines in CD-modified capillary zone electrophoresis (CD-CZE) using a phosphate background electrolyte at low pH were investigated. We focused on the separation of these phenothiazines, including the enantiomers of chiral analytes, with the use of beta-CD and hydroxypropyl-beta-CD (HP-beta-CD) as electrolyte modifiers or chiral selectors at concentrations less than 8 mM. The results indicate that the interactions of phenothiazines with beta-CDs are very strong and that effective separations of 13 analytes can be achieved with addition of 0.3 mM beta-CD or 0.5 mM HP-beta-CD in a phosphate buffer at pH 3.0. Binding constants of phenothiazines to beta-CDs were evaluated for a better understanding of the interactions of phenothiazines with beta-CDs.     

Separation and Migration Behavior of Dichlorophenols in β‐Cyclodextrin‐Modified Capillary Zone Electrophoresis

The separation and migration behavior of six isomeric dichlorophenols (DCPs) in cyclodextrin‐modified capillary zone electrophoresis (CD‐CZE) using a phosphate‐borate buffer at alkaline pH with β‐CD and hydroxypropyl‐β‐CD (HP‐β‐CD) as electrolyte modifiers were investigated. The influence of buffer pH and the concentration of β‐cyclodextrins were examined. The results indicate that baseline separation of six isomeric DCPs can be achieved with addition of β‐CD concentration in the range of 2.0‐10 mM or HP‐β‐CD concentration in the range of 4.0‐10 mM at pH 10.0. Binding constants of DCPs to β‐CDs were evaluated for a better understanding of the interaction of DCPs with β‐CDs.     

毛细管区带电泳法测定卷烟中的生物碱

在磷酸缓冲体系中采用毛细管区带电泳法测定卷烟中的生物碱时,检测灵敏度低,分离度差。考察了卷烟中生物碱的 提取条件,分离缓冲溶液的类型、pH值和浓度,卷烟中生物碱测定方法的线性范围、检出限、重现性和回收率。结果发 现,当采用410 mmol/L的酒石酸溶液（pH 2.8）为缓冲体系时,卷烟中生物碱的检测灵敏度和分离度均有明显改善,烟碱 的线性范围为0.06~0.80 mg/L（其他生物碱为0.006~0.10 mg/L）,检出限为0.002~0.01 mg/L,相对标准偏差为2.2%~10%,回收率为87.6%~102%。     

Enantioseparation of phenothiazines in cyclodextrin-modified capillary zone electrophoresis using sulfated cyclodextrins as chiral selectors

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in CD-modified CZE using dual CD systems consisting of randomly sulfate-substituted CD (MI-S-beta-CD) and a neutral CD as chiral selectors in a citrate buffer (100 mM) at pH 3.0 were investigated. The results indicate that MI-S-beta-CD is an excellent chiral selector for enantioseparation of ethopropazine. The enantiomers of promethazine can also be baseline-resolved with MI-S-beta-CD at concentrations in the range of 0.5-1.0% w/v. On the other hand, thioridazine and trimeprazine interact strongly with neutral CDs. As a result, the enantioselectivity of these two phenothiazines is remarkably and synergistically enhanced with increasing the concentration of neutral CDs in the presence of MI-S-beta-CD and simultaneous enantioseparations of these phenothiazines, except for methotrimeprazine, could favorably be achieved with the use of dual CD systems. Moreover, by varying the concentration of beta-CD or gamma-CD at a fixed concentration of MI-S-beta-CD (0.75% w/v) reversal of the enantiomer migration order of promethazine occurred. This may be attributable to the opposite effects of charged and neutral CDs on the mobility of the enantiomers of promethazine.     

A simple method for the titration of multicomponent acid-base mixtures

A titration method has been developed to analyze acid mixtures, in which the pK values differed by 0.5 to 1 pH units. Instead of the use of equivalence points a buffer capacity curve is numerically derived from the titration curve resulting in a presentation similar to chromatograms and spectra. The consumption of the NaOH titrant is determined within pH intervals of 0.2-0.6 units around the pK values of the components. The buffer capacity measured is expressed as the consumption of NaOH over the pH interval. The amounts of compounds are found from these results using simple models of chemometrics. The method has been applied to a mixture of formic and acetic acids with concentrations of 0-3 mmol/20 ml with an error of 0-0.05 mmol. Furthermore a curve-fitting method based on a theoretical equation of buffer capacity can be applied with an error slightly larger than with the previous method. In this case, the pK values were also computed. For titrations beyond the pH range 4-10, the buffer capacity of water has to be subtracted from the titration results. The whole procedure requires including the computations about 3-6 min.     

苯胺及其衍生物的毛细管电泳行为研究

采用紫外吸收检测，毛细管电泳分离，研究了九种苯胺及其衍生物在毛细管区带电泳(CZE)体系和胶束电动毛细管色谱(MECC)体系中的行为特征。讨论了缓冲溶液的浓度与pH、胶束浓度及混合胶束等在不同体系中对分离组分的影响，发现在CZE体系中，控制分离的主要因素是pKb值；在MECC体系中，控制分离的主要因素是溶质分子中碳原子数。建立了一种分离测定九种苯胺及其衍生物的高效毛细管电泳方法。     

Separation mechanism and determination of flavanones with capillary electrophoresis and high-performance liquid chromatography

To probe separation mechanism and determination with capillary zone electrophoresis (CZE) and liquid chromatography (LC), nine compounds with identical flavanone skeleton were studied. Optimum separation of LC was attained with gradient of acetonitrile and 5mM phosphate buffer (pH 6.9). For CE, electrolyte buffer was 4.5mM SDS in 32mM sodium tetraborate buffer (pH 9.2). The distinguishing feature in this work was successful separation of monohydroxyl stereoisomers by CZE. Polarity is generally increased with hydroxyl groups. In a separation mechanism study, polarity would be reduced by intramolecular hydrogen bond between hydroxyl of C5 and carbonyl group of C4. Comparison of the retention results among monohydroxyl flavanones shows polarity with hydroxyl at C6 the least, and that at C4' and C7 nearly equal. Also, elution order of flavones and flavanones would be adverse due to the hydroxyl at C3 in LC. From the numerical value pK(a) of flavanone, the C7-OH is the smallest, and two hydroxyl groups in an adjacent position is always less than the unique one caused by forming a stable 5-membered ring. Investigation of separation mechanism yield only the effect of constituent but also reasonable explanation for contradictory results between Wulf and our laboratory, this due to the hydroxyl at C3.     

Influence of buffer substances and urea on the beta-cyclodextrin-mediated chiral separation of dipeptides in CE

The influence of buffering substances and urea on the beta-CD-mediated chiral separations of the dipeptides Ala-Phe and Ala-Tyr was studied in the pH range of 2.5-3.8. Only minor effects of the buffer substances on the chiral separation selectivity alpha were observed at a beta-CD concentration of 15 mg/mL. In contrast, the selectivity improved at pH 2.5 but decreased at pH 3.8 upon the addition of 2 M urea. Complexation by beta-CD resulted in a shift of the pK(a) values toward higher values which was more pronounced for the DD-enantiomers of both dipeptides than for the LL-enantiomers. Addition of urea further increased the pK(a) shift. The consequence of this pK(a) shift is an increase of the fraction of the protonated, positively charged form of the peptides which explained the improved chiral separation at pH 2.5 and the reduced selectivity at pH 3.8. A pK(a) shift by the addition of urea was also observed for N-tert-butyloxycarbonyl phenylalanine (BOC-Phe) as a model compound that is strongly complexed by beta-CD. This effect was not stereospecific. Addition of urea resulted in a decrease of the apparent complexation constants between beta-CD and the BOC-Phe enantiomers to the same extent but this did not affect the separation selectivity alpha. For chiral separations that display strong pH dependence such as peptide enantioseparations close to the pK(a) values of the compounds, urea may not solely be regarded as a solubility enhancer for beta-CD but may also influence the separation.     

Experimental design for the estimation of photophysical parameters of the two-state excited-state proton-exchange reaction in the presence of pH buffer

The excited-state proton-exchange reaction of commonly used fluorescent pH probes at physiological pH becomes reversible upon addition of pH buffer. Using computer-generated fluorescence decay surfaces, we investigated under which experimental conditions (pH, buffer concentration, and excitation and emission wavelengths) the rate constants describing the excited-state processes (k(ij)) and the spectral parameters related to excitation ((~)b(1)) and emission ((~)c(1)) can be accurately and precisely estimated by global compartmental curve fitting. It was found that a minimum of three fluorescence decay traces should be collected for the pH probe in the presence of buffer. These three decays should be characterized by at least two different pH values and at least two different buffer concentrations. In addition to these three traces, a minimum of one trace corresponding to the pH probe without buffer has to be recorded. Furthermore, for the accurate estimation of k(ij), (~)b(1), and (~)c(1), at least two of these traces should be collected at the same pH and excitation and emission wavelengths. The experimental conditions should be chosen in such a way that decays with unambiguous biexponential character are obtained. For fluorescent pH probes with pK(a) approximately equal to 7 that are responsive in the near-neutral pH range, it is advisable to use buffers with pK(B)(a) values comparable to or higher than the pK(a) of the probe. Because the changes in the decay times are already apparent with a small quantity of buffer, there is no need to use excessively high buffer concentrations. From a practical point of view, the best experimental design is attained when one combines in a single fluorescence decay surface traces originating from samples characterized by different pH values at the same buffer concentration with traces characterized by different buffer concentrations at the same pH and decays of samples without buffer measured at several pH values.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

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.     

Electrochemical Oxidation of Phenothiazine Derivatives at Glassy Carbon Electrodes and Their Differential Pulse and Square‐wave Voltammetric Determination in Pharmaceuticals

Abstract

Three 2,10‐disubstituted phenothiazines—chlorpromazine hydrochloride (CPM), thioridazine hydrochloride (TR) and propericiazine (PRC)—were electrochemically studied in various buffer systems at different pH values, using a glassy carbon electrode. The substances were electrochemically oxidized at potential range 0.55–0.75 V. The oxidation was reversible and exhibited diffusion‐controlled process. The mechanism of the oxidation process is discussed. According to the linear relation between peak current and concentration, differential pulse voltammetry (DPV) and square wave voltammetry (SWV) methods for quantitative determination of chlorpromazine and propericiazine in 0.1 M HClO₄, and thioridazine in pH 2 phosphate buffer, was applied. Both the repeatability and reproducibility of the methods were also determined for all studied substances. The developed procedures were successfully applied to the determination of chlorpromazine and thioridazine in pharmaceutical dosage forms.     

A reversed-phase thin-layer chromotographic method for the determination of relative partition coefficients of very lipophilic compounds.

A reversed-phase thin-layer chromatographic method has been developed for the determination of partition coefficients. A support phase has been chosen, following investigation of the lack of adsorptive properties, which has a minimal effect on the pH of the buffer system. A stationary phase has been chosen to give deltaRm values of the same magnitude as Hansch pi values for a series of phenothiazines. The method can be applied to molecules of a wide range of lipophilicity following preliminary investigations of suitable phase-volume ratios and of the pH and composition of the binary mobile phase, providing adsorption on the support phase is excluded.     

Interaction of Chlorpromazine and Trifluoperazine with Anionic Sodium Dodecyl Sulfate (SDS) Micelles: Electronic Absorption and Fluorescence Studies

The characteristics of binding of two phenothiazine antipsychothic drugs, chlorpromazine (CPZ) and trifluoperazine (TFP), to anionic sodium dodecyl sulfate (SDS) monomers and/or micelles were investigated using electronic absorption and fluorescence spectroscopies. Binding constants K(b) and pK(a) values for the drugs in SDS micelles were estimated using the red shifts of the maximum absorption and changes in absorption upon alkalization or in the presence of surfactant. The pK(a) shift of CPZ due to its interaction with SDS micelles is about 0.7 unit to higher values, as compared to the reported value of pK(a) obtained in buffer around 9.3. For TFP the pK(a) shift is 0.4 unit to higher values compared to that in buffer, reported as 4.0. The electronic absorption spectroscopic data suggest a biphasic interaction as a function of detergent concentration which is quite dependent of the protonation states of the drugs. In the case of TFP a very strong binding takes place when the drug is fully protonated (pH 2.0) and a distinct binding takes place at stoichiometric (low) surfactant concentrations (interaction via surfactant monomers) and at higher concentrations (in the presence of micelles). Static fluorescence probe analysis using pyrene was used to study the nature of the phenothiazine-surfactant premicellar and self-aggregates. The I(3)/I(1) and I(475)/I(1) ratios associated to pyrene fluorescence vibronic bands and excimer intensities ratios, respectively, were monitored for several ratios [SDS]/[drug] and significant changes, dependent of the drug presence and its protonation state, have been observed revealing a hydrophobic microenvironment provided by TFP-SDS aggregates in comparison with CPZ both at pH 7.0 and 4.0. Static anisotropy was also used to monitor the changes of the self-aggregates and micellar packing in the presence of the phenothiazine drugs. In aqueous solutions the anisotropy of the fluorescent probe dipyridamole (DIP) is quite low, being around 0.005 at pH 7.0 and 0.025 at pH 4.0, and the addition of detergent leads to an increase in the values of anisotropy to 0.030 at pH 7.0 and 0.070 at pH 4.0. In the presence of the phenothiazine drugs, and in the premicellar detergent concentration range, the anisotropy of DIP increases to 0.134 and 0.111 (dependent on drug concentration) for CPZ and TFP, respectively, at pH 4.0. These results suggest that the presence of both phenotiazine drugs makes the premicellar aggregates more rigid by decreasing the probe mobility, and are consistent with a more polar localization of the CPZ in the micelles as compared with TFP. At pH 7.0 the anisotropy changes are smaller, suggesting a slight decrease in CMC induced by the phenothiazines. Copyright 2000 Academic Press.