Advantages of polar organic solvent chromatography for enantioseparation of chiral liquid crystals.

Three sets of fluorinated chiral liquid crystals were used to explore the polar organic solvent chromatography mode for their enantioseparation. The materials include a set of newly synthesized compounds with chiral center derived from 2-hexanol and two sets of compounds with chiral center derived from 2-/3-octanol. Baseline enantioseparation of all materials was achieved using binary mobile phases without additives. For some of the compounds exceedingly high values of enantioresolution (> 20) and enantioselectivity (> 4) were found. The chromatographic behavior of the sample set was studied on three different polysaccharide-based chiral columns - Chiralpak IA-U, IG-U and IB-U. Comparison of results from Chiralpak IA-U and IB-U shows the effect of amylose vs. cellulose polysaccharide backbone while comparison of Chiralpak IA-U and IG-U reveals the effect of 3,5-dimethylphenylcarbamate vs. 3­chloro-5-methylphenylcarbamate substituent. The mobile phases tested included whole range of acetonitrile/methanol mixtures to demonstrate that acetonitrile-rich and alcohol-rich mobile phases offer different enantiorecognition mechanisms and can provide complementarity to some extent. The effect of temperature on enantioseparation was investigated on Chiralpak IA-U by constructing van't Hoff plots for selected liquid crystals in pure acetonitrile and pure methanol as mobile phases.

Comparison of Chemical and Electrochemical Approaches to Abacavir Oxidative Stability Testing.

A novel electrochemical approach using two different electrode materials, platinum and boron-doped diamond (BDD), was employed to study the oxidative stability of the drug abacavir. Abacavir samples were subjected to oxidation and subsequently analysed using chromatography with mass detection. The type and amount of degradation products were evaluated, and results were compared with traditional chemical oxidation using 3% hydrogen peroxide. The effect of pH on the rate of degradation and the formation of degradation products were also investigated. In general, both approaches led to the same two degradation products, identified using mass spectrometry, and characterised by 319.20 and m/z 247.19. Similar results were obtained on a large-surface platinum electrode at a potential of +1.15 V and a BDD disc electrode at +4.0 V. Degradation of 20% of abacavir, the rate required for pharmaceutical stability studies, took only a few minutes compared to hours required for oxidation with hydrogen peroxide. Measurements further showed that electrochemical oxidation in ammonium acetate on both types of electrodes is strongly pHdependent. The fastest oxidation was achieved at pH 9. The pH also affects the composition of the products, which are formed in different proportions depending on the pH of the electrolyte.

Enantioseparation of liquid crystals and their utilization as enantiodiscrimination materials.

Liquid crystals can partake in enantioseparations in several different ways. Enantiomeric purity of chiral liquid crystals as analytes is often determined using enantioselective liquid or supercritical fluid chromatography. At the same time, chiral liquid crystalline materials can be applied as a chiral selector or they can function as an auxiliary material for a different chiral selector enabling enantioseparation of various solutes in miscellaneous separation techniques. They can also be used in fabrication of matrices or surfaces sensitive towards enantiomers or employed as chiral solvents for visualizing enantiomers in NMR. In this review, all of these aspects of relationship between liquid crystals and enantiodiscrimination are discussed in an effort to encompass their versatility and with emphasis on enantioseparations.

Alternative method for canagliflozin oxidation analysis using an electrochemical flow cell - Comparative study.

This paper highlights the potential of electrochemical flow cells for oxidative-stress testing of active pharmaceutical ingredients using canagliflozin as a model substance. Based on design of experiments, we developed our method through a reduced combinatorial design, optimizing the following independent variables: cell size, electrolyte flow rate, electrolyte concentration, and electrolyte pH. Using ammonium phosphate buffer with methanol in a 50/50 vol ratio as a working electrolyte, we electrochemically oxidized samples and analyzed them by high-performance liquid chromatography, considering the following dependent variables: peak area of each impurity, peak area of canagliflozin, and the percentage of the corresponding peak areas. Our results showed that the most significant independent variables were electrolyte pH and flow rate. By data optimization, we determined the most suitable conditions for electrochemical oxidation of canagliflozin, namely 50 µm cell size, 300 mM electrolyte concentration, 0.1 mL/h electrolyte flow rate, and electrolyte pH = 4. The repeatability of the method, expressed as the relative standard deviation of the canagliflozin peak area, measured in ten separately oxidized samples, was 1.64%. For comparison purposes, we performed a degradation experiment using hydrogen peroxide, identifying five identical impurities in both cases, as confirmed by mass spectrometry. The degradation products formed when using the chemical method after 1, 3, and 7 days totaled 0.09%, 0.75%, and 3.75%, respectively, and the degradation products formed when using the electrochemical method after 3 h totaled 3.11%. Oxidation with hydrogen peroxide required 7 days, whereas electrochemical oxidation was completed in 3 h. Overall, the electrochemical method significantly saves time and reduces the consumption of active ingredients and solvents thanks to the miniaturized size of the electrochemical cell, thereby minimizing the costs of forced degradation studies.

Comparison of static and dynamic mode in the electrochemical oxidation of fesoterodine with the use of experimental design approach.

Electrochemical conversion of fesoterodine to one of its oxidation products was evaluated with the application of the wall-jet flow cell. A traditional, "static" mode of electrolysis was compared with the "dynamic" mode of cell performance. For statistical assessment of the data, experiments were planned and performed with the application of design of experiments approach, namely Taguchi L18 design. After screening phase, the experimental settings were broadened or adjusted according to the results and optimization was performed. All of the samples were electrolysed with the use of chronoamperometric method in a three electrode system. The electrolysed samples were analysed using UHPLC-PDA-QDA method. The chromatographic run was performed in gradient elution with the application of C8 column. The response was expressed as % area of the main peak found with the PDA detection method whereas QDA detector was used in positive SIM mode for structural confirmation. All data obtained for both screening and optimization were treated together and linear models were adjusted. The use of large-surface glassy carbon electrode along with pH~7 were found to be the most significant factors influencing electrochemical oxidation of fesoterodine in both modes. The major differences were identified in terms of voltage applied to the electrodes which yielded the highest amounts of oxidation product. Evolution of electrochemical methods may serve as complementary technique in stress degradation studies in pharmaceutical industry.

Mobilization of electroosmotic flow markers in capillary zone electrophoresis.

UV-absorbing neutral substances are commonly used as markers of mean electroosmotic flow in capillary electrophoresis for their zero electrophoretic mobility in an electric field. However, some of these markers can interact with background electrolyte components and migrate at a different velocity than the electroosmotic flow. Thus, we tested 11 markers primarily varying in their degree of methylation and type of central atom in combination with five background electrolyte cations differing in their ionic radii and surface charge density, measuring the relative electrophoretic mobility using thiourea as a reference marker. Our results from this set of experiments showed some general trends in the mobilization of the markers based on the effects of marker structure and type of background electrolyte cation on the relative electrophoretic mobility. As an example, the effects of an inadequate choice of marker on analyte identification were illustrated in the electrophoretic separation of glucosinolates. Therefore, our findings may help electrophoretists appropriately select electroosmotic flow markers for various electrophoretic systems.

How mobile phase composition and column temperature affect enantiomer elution order of liquid crystals on amylose tris(3-chloro-5-methylphenylcarbamate) as chiral selector.

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed.

Enantiorecognition ability of different chiral selectors for separation of liquid crystals in supercritical fluid chromatography; critical evaluation.

Comprehensive study of enantioselective potential of eight different chiral stationary phases for chiral liquid crystal-forming molecules was conducted. The tested columns were: (i) polysaccharide-based Trefoil AMY1, CEL1 and CEL2 and (ii) superficially porous particles packed TeicoShell, VancoShell, TagShell, DMP-MaltoShell, and NicoShell. To test their enantioselective potential for these separations, twenty racemic mixtures of rod-like liquid crystalline materials comprising three different types of chiral centres and various other structural differences were used. Mobile phases consisting of supercritical carbon dioxide and alcohol as cosolvent were used; selected alcohols were methanol, ethanol and propan-2-ol. Effect of acidic and/or basic additives on enantioselectivity was also evaluated. Chiral stationary phases based on polysaccharides were found to have the greatest enantioselective potential for rod-like molecules that form liquid crystals, followed by TeicoShell, which proved suitable for enantioseparation of non-halogenated liquid crystals with lactic acid-based chiral centre.

Ultra-performance supercritical fluid chromatography: A powerful tool for the enantioseparation of thermotropic fluorinated liquid crystals.

A fast and simple supercritical fluid chromatography method for the enantioseparation of twenty newly synthesized orthoconic antiferroelectric liquid crystals is reported for the first time. The effects of alkoxy spacer length and fluorine atom presence and position in the phenyl ring on chromatographic behavior were investigated. Baseline enantioseparation of all compounds was achieved using simple mobile phases consisting of carbon dioxide and alcohol as cosolvent on (3,5-dimethylphenylcarbamate) derivative of amylose as chiral stationary phase. The analysis times ranged from 2 to 4 and from 4 to 7 min for most samples when using methanol and propane-2-ol, respectively. The significant effect of cosolvent type on the enantioseparation of these compounds was assessed and partial complementarity of methanol and propane-2-ol was observed.

Sample pretreatment for the capillary electrophoretic determination of organic acids in chromium(III) plating baths.

This work deals with the development and optimization of the sample pretreatment and consequent electrophoretic analysis of two modern plating baths containing chromium(III) and either citric acid or oxalic acid. Some model mixtures containing known amounts of components of industrial baths have been prepared to simulate simplified bath matrices. Prior to analysis, a sample pretreatment consisting of the addition of some agents that could release acid from the stable chromium complex was tested. Determination of organic anions was accomplished by indirect UV detection. The best results were achieved by precipitation of chromium(III) hydroxide. The content of oxalate and citrate in real samples was calculated as 96.5% (SD 2.3%) and 97.3% (SD 0.8%), respectively, of the declared amount. Very good robustness of the method and satisfactory repeatability of migration time and peak area were obtained. This simple inexpensive method is suitable for routine determination of citric and oxalic acid in chromium(III)-based plating baths.

Electrophoretic mobilities of neutral analytes and electroosmotic flow markers in aqueous solutions of Hofmeister salts.

Small neutral organic compounds have traditionally the role of EOF markers in electrophoresis, as they are expected to have zero electrophoretic mobility in external electric fields. The BGE contains, however, ions that have unequal affinities to the neutral molecules, which in turn results in their mobilization. In this study we focused on two EOF markers-thiourea and DMSO, as well as on N-methyl acetamide (NMA) as a model of the peptide bond. By means of CE and all atom molecular dynamics simulations we explored mobilization of these neutral compounds in large set of Hofmeister salts. Employing a statistical mechanics approach, we were able to reproduce by simulations the experimental electrophoretic mobility coefficients. We also established the role of the chemical composition of marker and the BGE on the measured electrophoretic mobility coefficient. For NMA, we interpreted the results in terms of the relative affinities of cations versus anions to the peptide bond.

PiB-Conjugated, Metal-Based Imaging Probes: Multimodal Approaches for the Visualization of ß-Amyloid Plaques.

In an effort toward the visualization of ß-amyloid plaques by in vivo imaging techniques, we have conjugated an optimized derivative of the Pittsburgh compound B (PiB), a well-established marker of Aß plaques, to DO3A-monoamide that is capable of forming stable, noncharged complexes with different trivalent metal ions including Gd(3+) for MRI and (111)In(3+) for SPECT applications. Proton relaxivity measurements evidenced binding of Gd(DO3A-PiB) to the amyloid peptide Aß1-40 and to human serum albumin, resulting in a two- and four-fold relaxivity increase, respectively. Ex vivo immunohistochemical studies showed that the DO3A-PiB complexes selectively target Aß plaques on Alzheimer's disease human brain tissue. Ex vivo biodistribution data obtained for the (111)In-analogue pointed to a moderate blood-brain barrier (BBB) penetration in adult male Swiss mice (without amyloid deposits) with 0.36% ID/g in the cortex at 2 min postinjection.

Overcharging in biological systems: reversal of electrophoretic mobility of aqueous polyaspartate by multivalent cations.

Charge reversal as an extreme case of charge compensation is directly observed by capillary electrophoresis for a negatively charged peptide in aqueous solutions of trivalent cations. Atomistic and coarse-grained simulations provide molecular interpretation of this effect showing that it is largely of electrostatic origin with a minor contribution of chemical specificity of the salt ions.

Counterion condensation in short cationic peptides: limiting mobilities beyond the Onsager-Fuoss theory.

We investigated the effect of the background electrolyte (BGE) anions on the electrophoretic mobilities of the cationic amino acids arginine and lysine and the polycationic peptides tetraarginine, tetralysine, nonaarginine, and nonalysine. BGEs composed of sodium chloride, sodium propane-1,3-disulfonate, and sodium sulfate were used. For the amino acids, determination of the limiting mobility by extrapolation, using the Onsager-Fuoss (OF) theory expression, yielded consistent estimates. For the peptides, however, the estimates of the limiting mobilities were found to spuriously depend on the BGE salt. This paradox was resolved using molecular modeling. Simulations, on all-atom as well as coarse-grained levels, show that significant counterion condensation, an effect not accounted for in OF theory, occurs for the tetra- and nonapeptides, even for low BGE concentrations. Including this effect in the quantitative estimation of the BGE effect on mobility removed the discrepancy between the estimated limiting mobilities in different salts. The counterion condensation was found to be mainly due to electrostatic interactions, with specific ion effects playing a secondary role. Therefore, the conclusions are likely to be generalizable to other analytes with a similar density of charged groups and OF theory is expected to fail in a predictable way for such analytes.

Microscale separation methods for enzyme kinetics assays.

Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.

UV-VIS detection of amino acids in liquid chromatography: online post-column solid-state derivatization with Cu(II) ions.

In this work, we describe the introduction of a post-column solid-state reactor in the HPLC system used for the analyses of amino acids. The reactor used was filled with copper(II) oxide. Passage of the analytes through the reactor leads to the formation of Cu(II) complexes. Unlike free amino acids, the Cu-complexes show significant absorbance in the UV region and accordingly sensitivity of UV-VIS detection is increased by two to three orders of magnitude. As a result of this improvement in sensitivity, we have obtained LOD values in micromolar range and good linearity over the studied concentration range (5.0×10(-5) to 2.0×10(-3) mol/L). The method exhibits advantages typical of solid-state reactors, such as negligible loss of efficiency due to the derivatization, simplicity of realization and a long-term durability. The presented system brings an easy and versatile solution for UV-VIS detection of coordinating compounds, which do not normally absorb well in the UV-VIS region.

Effect of association with sulfate on the electrophoretic mobility of polyarginine and polylysine.

Domains rich in cationic amino acids are ubiquitous in peptides with the ability to cross cell membranes, which is likely related to the binding of such polypeptides to anionic groups on the membrane surface. To shed more light on these interactions, we investigated specific interactions between basic amino acids and oligopeptides thereof and anions by means of electrophoretic experiments and molecular dynamics simulations. To this end, we measured the electrophoretic mobilities of arginine, lysine, tetraarginine, and tetralysine in sodium chloride and sodium sulfate electrolytes as a function of ionic strength. The mobility was found to be consistently lower in sodium sulfate than in sodium chloride at the same ionic strength. The decrease in mobility in sodium sulfate was greater for tetraarginine than for tetralysine and was larger for tetrapeptides compared to the corresponding free amino acids. On the basis of molecular dynamics simulations and Bjerrum theory, we rationalize these results in terms of enhanced association between the amino acid side chains and sulfate. Simulations also predict a greater affinity of sulfate to the guanidinium side chain groups of arginine than to the ammonium groups of lysine, as the planar guanidinium geometry allows simultaneous strong hydrogen bonding to two sulfate oxygens. We show that the sulfate binding to arginine, but not to lysine, is cooperative. These results are consistent with the greater decrease in the mobility of arginine compared to that of lysine upon addition of sulfate salt. The nonspecific mobility retardation by sulfate is ascribed to its electrostatic interaction with the cationic amino acid side chain groups.