The First Online Capillary Electrophoresis-Microscale Thermophoresis (CE-MST) Method for the Analysis of Dynamic Equilibria-The Determination of the Acidity Constant of Fluorescein Isothiocyanate.

This article presents the first successful application of a capillary electrophoresis-microscale thermophoresis tandem technique (CE-MST) for determining the values of equilibrium constant, realized by connecting online the CE and MST instruments using a fused-silica capillary. The acid-base dissociation of fluorescein isothiocyanate, expressed by the acidity constant value (pKa), was used as a model. The measurement procedure consisted of introducing a mixture containing the analyte and a deliberately added interferent into the CE capillary, electrophoretic separation of the analyte from the interferent, the detection of the analyte with a CE-integrated detector, detection with a MST detector, and then stopping the flow temporarily by turning off the voltage source to conduct the thermophoretic measurement. The analysis of migration times, peak areas and MST responses obtained concurrently for the same sample allowed us to determine the pKa value using three independent methods integrated within one instrumentation. The analyte was effectively separated from the interferent, and the acidity values turned out to be consistent with each other. An attempt was also made to replace the standard commercial CE instrument with a home-made portable CE setup. As a result, the similar pKa value was obtained, at the same time proving the possibility of increasing cost efficiency and reducing energy consumption. Overall, the CE-MST technique has a number of limitations, but its unique analytical capabilities may be beneficial for some applications, especially when sample separation is needed prior to the thermophoretic measurement.

A sustainable approach for the stability study of psychotropic substances using vitreous humor and liver as alternative matrices.

The stability of psychotropic substances representing various drug groups important from the perspective of forensic chemistry, including benzodiazepines, antidepressants, carbamazepine, cocaine, and their selected metabolites, was investigated for 1 month in two alternative biological matrices, vitreous humor and liver homogenate. Three different thermal storage conditions (-20, 4, and 20 °C) were tested. Liquid chromatography-mass spectrometry (LC-MS) analysis was preceded by an effective solid-phase microextraction (SPME) procedure. The results were statistically analyzed using one-way ANOVA to find significant concentration variations over time. The results obtained allowed for dividing the analytes into four groups: stable under all tested conditions, only at -20 and 4 °C, only at 20 °C, and overall unstable. Nordiazepam, venlafaxine, and cocaine and its metabolites turned out to be the most unstable substances, while fluoxetine showed the highest storage stability in both matrices. The SPME/LC-MS method was comprehensively evaluated according to the principles of white analytical chemistry (WAC), which reconcile the greenness and functionality of the method. A close to 100% whiteness score proves its sustainability and suitability for the intended application.

The Acid-Base/Deprotonation Equilibrium Can Be Studied with a MicroScale Thermophoresis (MST).

MicroScale thermophoresis (MST) is a rapidly developing bioanalytical technique used routinely for the examination of ligand-target affinity. It has never been used so far for the analysis of acid-base dissociation and the determination of pKa constant. This work is the-proof-of-concept of this new idea. It demonstrates that the pKa values obtained from the thermophoretic data are consistent with the reference methods. As a result, the analytical potential and utility of the MST technology can become even greater, especially if the new detection system of thermophoretic movement will be developed in the future. Even now, taking into account the necessity to use fluorescence, the proposed method may be useful in many respects.

A Perspective of the Comprehensive and Objective Assessment of Analytical Methods Including the Greenness and Functionality Criteria: Application to the Determination of Zinc in Aqueous Samples.

The recently proposed concept of White Analytical Chemistry (WAC), referring to the Red-Green-Blue color model, combines ecological aspects (green) with functionality (red and blue criteria), presenting the complete method as "white". However, it is not easy to carry out an overall quantitative evaluation of the analytical method in line with the WAC idea in an objective manner. This paper outlines the perspective of the future development of such a possibility by attempting to answer selected questions about the evaluation process. Based on the study consisting in the evaluation of selected model methods by a group of 12 independent analysts, it was shown how well individual criteria are assessed, whether the variability of assessments by different people is comparable for each criterion, how large it is, and whether averaging the scores from different researchers can help to choose the best method more objectively.

Comprehensive Assessment of Flow and Other Analytical Methods Dedicated to the Determination of Zinc in Water.

An original strategy to evaluate analytical procedures is proposed and applied to verify if the flow-based methods, generally favorable in terms of green chemistry, are competitive when their evaluation also relies on other criteria. To this end, eight methods for the determination of zinc in waters, including four flow-based ones, were compared and the Red-Green-Blue (RGB) model was exploited. This model takes into account several features related to the general quality of an analytical method, namely, its analytical efficiency, compliance with the green analytical chemistry, as well as practical and economic usefulness. Amongst the investigated methods, the best was the flow-based spectrofluorimetric one, and a negative example was that one involving a flow module, ICP ionization and MS detection, which was very good in analytical terms, but worse in relation to other aspects, which significantly limits its overall potential. Good assessments were also noted for non-flow electrochemical methods, which attract attention with a high degree of balance of features and, therefore, high versatility. The original attempt to confront several worldwide accepted analytical strategies, although to some extent subjective and with limitations, provides interesting information and indications, establishing a novel direction towards the development and evaluation of analytical methods.

Differentiation of isomeric metabolites of carbamazepine based on acid-base properties; Experimental vs theoretical approach.

Metabolism of carbamazepine is complex and leads to the three isomeric derivatives whose occurrence is dependent on the type of sample material. Their unambiguous differentiation is overall important. In this work, the qualitative analysis of 2-hydroxycarbamazepine, 3-hydroxycrbamazepine and carbamazepine-10,11-epoxide was attempted for the first time using capillary zone electrophoresis, based on the models linking electrophoretic mobility with pKa value determining the acidity of the hydroxyl groups. For this purpose, pKa values ​​were determined using electrophoretic and theoretical methods, and then the compliance of the obtained mobility models with the measured values ​​was analyzed. Despite the slight difference in acidity ​​(0.3-0.4 pH unit), the obtained results prove that the correct identification of the metabolites under consideration, and reliable prediction of the selectivity of their separation, are possible on the basis of experimentally determined pKa values, even with highly simplified methods assuming the lack of certain data. However, it is important to choose the optimal pH value, which should be close to pKa. On the other hand, worse results were obtained for the theoretically determined mobilities, which differed significantly from the experimental values. An attempt was also made to explain the acidity of hydroxycarbamazepines and the associated thermodynamic parameters - deprotonation enthalpy and entropy, with respect to their structure. The lack of intramolecular hydrogen bonds and the significant contribution of entropic effects stabilizing the protonated form seems to be significant. The higher pKa value for CBZ-2-OH probably results from the stronger effect of the energetically unfavorable organization of solvent dipoles due to ionization.

Simultaneous quantification of food colorants and preservatives in sports drinks by the high performance liquid chromatography and capillary electrophoresis methods evaluated using the red-green-blue model.

Consumption of sports drinks is generally associated with a healthy lifestyle, despites a common addition of colorants and preservatives to these products. This work presents the novel methods based on high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), aimed at the simultaneous quantification of both these groups of additives within a single separation. The results obtained show that colorants are generally added in low and rather safe amounts, whereas preservatives (benzoate and sorbate) are much more abundant, and likely pose a potential risk, especially for children. The overall potential of the HPLC and CE methods was critically assessed and compared using the RGB additive color model, a user-friendly tool that allows one to point out and encode particular capabilities and general potential of a method using colors, and to express its holistic effectiveness with one integrated quantitative parameter from 0 to 100%. As a result, the potential predispositions of both methods were delineated.

Acidity constant of pH indicators in the supramolecular systems studied by two CE-based methods compared using the RGB additive color model.

Acid-base properties of methyl orange, bromocresol green, bromophenol blue, and bromothymol blue were thoroughly investigated in the past due to their application as colorimetric pH indicators. However, it is still unknown how these properties change upon the supramolecular host-guest interactions. Owing to the growing interest in using supramolecular host-guest interactions to reach expected modification of various physicochemical properties of guests, we decided to address this question in the present article. We estimated the shifts of pKa values induced by diverse hosts (cyclodextrins, cucurbiturils, calixarenes, micelles, and serum albumin) and performed a thermodynamic analysis of the selected systems. To make a deeper insight, we confronted the aforementioned dyes with the other kinds of molecules studied by us in the past. In overall, the results obtained demonstrate a large multiplicity of possible pKa behaviors, their poor predictability, and the existence of subtle structure-acidity relationships. In addition, we observed three thermodynamically different mechanisms of pKa alteration. Therefore, more studies are needed to bring closer the promising perspective of a programmable acidity's tuning. Our methodology was based on capillary electrophoresis (CE) applied in two parallel variants: a classical method based on the fitting of a nonlinear function, and an alternative two-value method (TVM), which requires over twice less measurements to estimate pKa. To identify the optimal approach for further studies, both methods were comprehensively compared and discussed based on the RGB additive color model, a user-friendly scale that integrates three primary aspects of an analytical method: analytical performance, green chemistry, and practicality.

Cyclodextrin-induced acidity modification of substituted cathinones studied by capillary electrophoresis supported by density functional theory calculations.

This paper shows that the acidity of substituted cathinones can change in a diversified and poorly predictable manner upon supramolecular interaction with cyclodextrins used as buffer additives in capillary electrophoresis. The direction and range of pKa shifts may be noticeably different for the particular cyclodextrins and cathinones, suggesting a strict correlation with structure. The most interesting results were observed for 2-hydroxyethyl-ß-cyclodextrin, which is capable for inducing the large negative and enantioselective apparent pKa shifts for α-pyrrolidinovalerophenone and methylenedioxypyrovalerone, even much above -1.0 pH unit. A thermodynamic analysis was performed, to identify the role of enthalpy and entropy in the formation and deprotonation of the respective diastereomeric complexes. The former process turned out to be driven by an energetically favorable increase in entropy, related probably to a hydrophobic effect. Deprotonation enthalpy in the complexed state, in turn, occurred to be more favorable than in the free molecule state, entailing the large drop in pKa after complexation. The DFT calculations allowed us to identify some structural effects that most likely contribute to these phenomena. At last, we have demonstrated that at low cyclodextrin concentration and pH ensuring partial ionization, pKa shifts contribute to chiral separation of the abovementioned cathinones. This analytically important effect may be helpful in anticipating the most efficient chiral separation mechanism of other systems.

Simultaneous enantioseparation of methcathinone and two isomeric methylmethcathinones using capillary electrophoresis assisted by 2-hydroxyethyl-ß-cyclodextrin.

Methcathinone (ephedrone), 4-methylmethcathinone (mephedrone), and 3-methylmethcathinone (metaphedrone) are toxicologically-important cathinone derivatives used commonly as designer drugs. In this work we show the first method allowing to separate simultaneously all these molecules in a chiral medium, ensuring good resolution between all enantiomers. Eight cyclodextrins have been tested as potential chiral selectors, the best results were obtained with 2-hydroxyethyl-ß-cyclodextrin, unreported so far for efficient separation of cathinones. After optimization, the method was calibrated and validated with and without the use of internal standard. The addition of standard improved an overall repeatability and precision, the use of electrophoretic mobility ratio was especially favorable (RSD < 1%). It was demonstrated that the method may be easily extended by introducing the additional cathinone-related drugs to the sample, maintaining satisfactory separation efficiency.

Flow variation as a factor determining repeatability of the internal standard-based qualitative and quantitative analyses by capillary electrophoresis.

The use of migration times and peak areas referred to another sample component - internal standard, brings many benefits in improving reliability of capillary electrophoresis. However, it is quite commonly overlooked that despite relative migration time and peak area ratio are more stable than the absolute values upon alteration in the flow rate, some shift should always be expected. The present work offers a new look at this analytically-important issue. We have derived a simple model allowing to estimate the magnitude of error for the selected pair of molecules of known mobilities upon the given flow alteration. Then, we have confronted the theoretical predictions with the experimental results obtained for the model sample separated in various flow conditions reached by the external pressure manipulation, including several internal standards of different mobilities. A good agreement has been obtained, pointing out that the magnitude of error may be large even for the seemingly "good" internal standards. Several potentially useful means have been tested to address this issue: the use of electrophoretic mobilities and electrophoretic mobility ratios instead migration times in the qualitative analysis, and performing time-correction of peak area ratios, or alternatively, transformation of electropherograms from the time-related scale into the electrophoretic mobility-related scale in the quantitative analysis. We have also considered some additional factors. The results may be of interest for all users dealing with the development and optimization of analytical methods using capillary electrophoresis.

Thermodynamics of acid-base dissociation of several cathinones and 1-phenylethylamine, studied by an accurate capillary electrophoresis method free from the Joule heating impact.

Capillary electrophoresis is often used to the determination of the acid-base dissociation/deprotonation constant (pKa), and the more advanced thermodynamic quantities describing this process (ΔH°, -TΔS°). Remarkably, it is commonly overlooked that due to insufficient dissipation of Joule heating the accuracy of parameters determined using a standard approach may be questionable. In this work we show an effective method allowing to enhance reliability of these parameters, and to estimate the magnitude of errors. It relies on finding a relationship between electrophoretic mobility and actual temperature, and performing pKa determination with the corrected mobility values. It has been employed to accurately examine the thermodynamics of acid-base dissociation of several amine compounds - known for their strong dependency of pKa on temperature: six cathinones (2-methylmethcathinone, 3-methylmethcathinone, 4-methylmethcathinone, α-pyrrolidinovalerophenone, methylenedioxypyrovalerone, and ephedrone); and structurally similar 1-phenylethylamine. The average pKa error caused by Joule heating noted at 25 °C was relatively small - 0.04-0.05 pH unit, however, a more significant inaccuracy was observed in the enthalpic and, in particular, entropic terms. An alternative correction method has also been proposed, simpler and faster, but not such effective in correcting ΔH°/-TΔS° terms. The corrected thermodynamic data have been interpreted with the aid of theoretical calculations, on a ground of the enthalpy-entropy relationships and the most probable structural effects accounting for them. Finally, we have demonstrated that the thermal dependencies of electrophoretic mobility, modelled during the correction procedure, may be directly used to find optimal temperature providing a maximal separation efficiency.

Acidity of substituted cathinones studied by capillary electrophoresis using the standard and fast alternative approaches.

Cathinone derivatives are notorious but still weakly characterized molecules, known mainly as components of the designer and illicit drugs. The knowledge on their acidity is scarce and incomplete, therefore, we decided to determine the pKa values for six of them: 2-methylmethcathinone, 3-methylmethcathinone, 4-methylmethcathinone, α-pyrrolidinovalerophenone, methylenedioxypyrovalerone and ephedrone. For that purpose we employed capillary electrophoresis, which is known for its accurateness in comparison to other analytical techniques. We used and compared two methodologically different approaches. The standard method relied on measuring electrophoretic mobility across the broad pH range and fitting the sigmoidal function. The obtained pKa values were in the range 8.59-9.10, thus these molecules remain mostly protonated and positively ionized in the physiological conditions. The alternative two-values (TVM) and one-value methods (OVM), proposed by us previously, have been used herein for the first time to the cationic molecules. TVM enables estimation of pKa using only two electrophoretic mobility values, referring to the total and partial ionization. OVM requires only a single measurement because mobility of ion is predicted theoretically. Both TVM and OVM yielded only a small deviation of pKa from the standard approach, averagely 0.07-0.09pH unit. Two important issues have also been addressed: the potential of a maximally fast calculation method using no repetition at the given pH, and the accuracy of method with regard to pH attributed to partial ionization. As a whole, the analytical potential of the TVM/OVM approach seems to be huge and invaluable for fast pKa screening/estimation.

Improving repeatability of capillary electrophoresis-a critical comparison of ten different capillary inner surfaces and three criteria of peak identification.

A poor repeatability of migration times caused by the fluctuations of electroosmotic flow (EOF) is an inherent weakness of capillary electrophoresis. Most researchers endeavor to prevent this problem using relative migration times or various capillary coatings which are expensive and not easy in comparison. Herein, we present an original approach to this problem: we apply a model sample designed to induce significant EOF instability, in order to critically compare ten capillary types with different physicochemical characteristics. Moreover, we accompany capillary modification with the evaluation of various criteria of peak identification: migration time, migration times ratio, and electrophoretic mobility. Our results show a great effectiveness of a dynamic coating in the stabilization of migration times, with the average RSD(%) value reduced from 3.5% (bare silica capillary) down to 0.5%. The good outcomes were also obtained for the surfactant-modified silica and amine capillaries. For the capillaries exhibiting significant instability of EOF, electrophoretic mobility turned out to be a more universal and reliable criterion of peak identification than relative migration time. It can be explained by an intrinsic dependency of migration times ratio on EOF change, which should always be considered during the selection of an internal standard.

Origin of Remarkably Different Acidity of Hydroxycoumarins-Joint Experimental and Theoretical Studies.

In the present work the origin of highly varied acidity of hydroxycoumarins (pKa values) has been for the first time investigated by joint experimental and computational studies. The structurally simple regio-isomers differing in the location of hydroxyl group, 3-hydroxycoumarin (3-HC), 4-hydroxycoumarin (4-HC), 6-hydroxycoumarin (6-HC), 7-hydroxycoumarin (7-HC), as well as 4,7-dihydroxycoumarin (4,7-HC) and the larger 4-hydroxycoumarin-based derivatives: warfarin (WAR), 7-hydroxywarfarin (W7), coumatetralyl (CT), and 10-hydroxywarfarin (W10), have been compared in terms of enthalpy-entropy relationships accounting for the observed pKa values. We have revealed that in the case of large molecules the acidic proton is stabilized by the following noncovalent interactions OH···O (WAR and W7), OH···π (CT), and OH···OH···O (W10), this effect leads to a compensatory enthalpy-entropy relation and yields a moderate pKa increase. On the other hand, different location of the hydroxyl group in the regio-isomers (3-HC, 4-HC, 6-HC, and 7-HC) leads to the massive changes in acidity due to a lack of enthalpy-entropy compensation. Our results suggest that the solvent-solute interactions and electron delocalization degree in anions contribute to the observed behaviors. Such knowledge can be useful in the future to design novel systems exhibiting desired acid-base properties, and to elucidate enthalpy-entropy compensation phenomena.

Enhancing effectiveness of capillary electrophoresis as an analytical tool in the supramolecular acidity modification.

A strategic modification of acidity (pK a values) by the non-covalent host-guest interactions is one of the most promising concepts in current supramolecular chemistry. This work is aimed at enhancing the effectiveness of capillary electrophoresis (CE) in determination of pK a shifts caused by such interactions and their thermal dependencies crucial in a deep thermodynamic description. We show how to (i) minimize the systematic errors related to Joule heating, (ii) minimize the influence of a voltage ramp time, (iii) speed up pK a shift identification and estimation, (iv) interpret thermal effects related to two overlapped dynamic equilibria, and (v) determine pK a shifts by an alternative spectrophotometric method (CE-DAD). The proposed solutions were implemented to examine the supramolecular pK a shifts of several coumarin derivatives, caused by a variety of structurally different cyclodextrins. It was revealed that a specific host substitution pattern determines the magnitude of apparent pK a shifts. Accordingly, heptakis(2,6-di-O-methyl)-ß-cyclodextrin induces the much stronger shifts than both non-methylated-ß-cyclodextrin and heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin applied at the same concentration. We also show that insofar as the complexation of 4-hydroxycoumarin and its derivative (coumatetralyl) are similarly exothermic, the thermal effects accompanying the deprotonation process are remarkably different for both molecules. The pK a shift induced by complexation with calixarene was also for the first time determined by a CE method. These observations throw a new light on the background of acidity modification and confirm the applicability of CE as an analytical tool.

Minimizing the impact of Joule heating as a prerequisite for the reliable analysis of metal-protein complexes by capillary electrophoresis.

Herein we report on a drastic release of metal ions from the Fe-bound transferrin, and Fe- or Mn-bound lactoferrin, observed upon the increase in the separation voltage during CE-based analysis. To verify whether this process is caused directly by electric field, we developed an Isothermal Voltage Increase approach (IVI), which is the extension of methods reported by Krylov et al. IVI ensures isothermal conditions while increasing separation voltage by a hydrodynamic pushing of the injected sample to the actively cooled capillary section, combined with a rationale choice of cooling temperature, dependent on the value of current. Interestingly, the application of IVI revealed that the previously observed effect was caused solely by the insufficient dissipation of Joule heating - the saturation of each protein remained unchanged despite a significant rise in the electric field. This outcome demonstrates how crucial is to ensure an effective temperature control for preventing systematic errors in the analysis of biomolecular complexes. IVI seems also to be a simple and useful tool for discovering new potential processes that may be stimulated directly by electric field.

Seven Approaches to Elimination of the Inherent Systematic Errors in Determination of Electrophoretic Mobility by Capillary Electrophoresis.

Electrophoretic mobility is a basic parameter that describes the electromigration of an ionized particle, which is used in many fields of analytical and physicochemical science. Its determination by capillary electrophoresis (CE), using a routine method, is intrinsically affected by the generation of Joule heating, entailing a drop in viscosity and possible alteration of the degree of ionization, and also by other commonly overlooked effects: axial electric field distortion and voltage ramping. The objective of this work was to provide the first theoretical overview and experimental comparison of all accessible methods that could be used to prevent these sources of inaccuracy. We have discussed seven independent approaches: (i) extrapolation of mobility to the zero power, (ii) initial buffer resistance-based correction, (iii) rational cooling adjustment, (iv) elimination of the nonthermostated capillary part, (v) inter/extrapolation to the nominal temperature, (vi) internal standard-based correction, and (vii) simple recalculation based on the temperature rise. Two methodologies (v and vi) have been proposed for the first time. Furthermore, we have shown how some approaches can be further developed, obtaining several novel and more sophisticated methods, which are also included in the comparison. Our investigation will help researchers to choose the optimal approach. We have also demonstrated for the first time how to measure the independent impact of four different effects. The outcomes reveal the compensatory character of some phenomena and explain the highly diverse and unpredictable magnitude of the total errors. The use of a correction method seems crucial for ensuring the high reliability of CE-based analyses.

Capillary coating as an important factor in optimization of the off-line and on-line MEKC assays of the highly hydrophobic enzyme chlorophyllase.

The choice between bare and coated capillaries is a key decision in the development and use of any methods based on capillary electrophoresis. In this work several permanently and dynamically coated capillaries were successfully implemented in a previously developed micellar electrokinetic chromatography (MEKC) assay of the plant membrane enzyme chlorophyllase. The results obtained demonstrate the rationale behind the use of capillary coating, which is crucial for successful optimization of both the off-line mode and the on-line/electrophoretically mediated microanalysis assay mode. The application of an amine permanently coated capillary (eCAP) is a simple way to significantly increase the repeatability of migration times and peak areas, and to ensure a strong electroosmotic flow that considerably decreases the overall analysis time. A dynamic coating (CEofix) allows one to apply an on-line incubation to control the reaction progress inside the capillary, and to increase the signal-to-noise ratio and peak efficiency. The dynamic coating is possible with use of both the normally applied uncoated silica capillary and the precoated amine capillary, which ensures more repeatable migration times. The strong points of the uncoated silica capillary are its attractive price and wide range of pH that can be applied. The characteristics presented may simplify the choice of capillary modification, especially in the case of hydrophobic analytes, MEKC-based separations, and other enzymatic assays.

Cyclodextrin-assisted enantioseparation of warfarin and 10-hydroxywarfarin by capillary electrophoresis studied from the analytical and thermodynamic points of view.

In this work cyclodextrin-assisted enantioseparation of warfarin and 10-hydroxywarfarin by CE has been studied from the analytical and thermodynamic points of view. The role of cyclodextrin concentration and temperature has been analyzed in reference to three different analytical parameters, corresponding to selectivity, resolution and resolution/analysis time ratio. The optimal conditions for enantioseparation have been found, they have been selected on the basis of critical difference in electrophoretic mobility and possibly short analysis time. The values of complexation percentage have also been calculated, to provide a link between the state of complexation equilibrium and the effectiveness of enantioseparation. In the optimal conditions the difference in complexation degree between enantiomers reaches 2.5% and 7.3% for warfarin and 10-hydroxywarfarin, respectively. At each temperature the highest enantioresolution is observed when the average complexation degree is close to 50%. In each case complexation is exothermic and driven by some enthalpically favorable process. 10-hydroxywarfarin exerts the stronger affinity to cyclodextrin and the stronger stereoselective effect. The presented results may be helpful in optimization and understanding of chiral separations by CE.