Strasbourg: Council of Europe; British Pharmacopoeia. London: Her Majesty's Stationary Office; Landers JP. Handbook of capillary and microchip electrophoresis and associated microtechniques. Overview of capillary electrophoresis and capillary electrochromatography.
J Chromatogr A. A brief introduction to capillary electrophoresis. Forensic Sci Int. Development and optimisation of a generic micellar electrokinetic capillary chromatography method to support analysis of a wide range of pharmaceuticals and excipients.
J Pharm Biomed Anal. Micelles as pseudo-stationary phases in micellar electrokinetic chromatography. Fundamentals of micellar electrokinetic chromatography. Eur J Chem. Advances in capillary electrophoresis. Selectivity in capillary electrophoresis in the presence of micelles, chiral selectors and non-aqueous media. Ahuja S, Jimidar MI. Capillary electrophoresis methods for pharmaceutical analysis. London: Academic Press; Deva: Editura Intelcredo; Enhanced pharmaceutical analysis by ce using dynamic surface coating system.
Otsuka K, Terabe S. Enantiomer separation of drugs by micellar electrokinetic chromatography using chiral surfactants. Fanali S. Enantioselective determination by capillary electrophoresis with cyclodextrins as chiral selectors. Gubitz G, Schmid MG. Chiral separation principles in capillary electrophoresis. Current applications in the analysis of pharmaceuticals by capillary electrophoresisI.
Trend Anal Chem. Current applications in the analysis of pharmaceuticals by capillary electrophoresisII. Development of a screening method for analytical control of antibiotic residues by micellar electrokinetic capillary chromatography. Anal Chim Acta. Analysis of different beta-lactams antibiotics in pharmaceutical preparations using micellar electrokinetic capillary chromatography.
Pajchel G, Tyski S. Adaptation of capillary electrophoresis to the determination of selected cephalosporins for injection. Micellar electrokinetic capillary chromatography of macrolide antibioticsSeparation of tylosin, erythromycin and their related substances. Wienen F, Holzgrabe U. A new micellar electrokinetic capillary chromatography method for separation of the components of the aminoglycoside antibiotics. Migration behavior and separation of tetracycline antibiotics by micellar electrokinetic chromatography.
Rapid determination of sulfonamides in milk using micellar electrokinetic chromatography with fluorescence detection. Optimization and validation of the micellar electrokinetic capillary chromatographic method for simultaneous determination of sulfonamide and amphenicol-type drugs in poultry tissue.
Development of capillary electrophoresis methods for the analysis of fluoroquinolones and application to the study of the influence of humic substances on their photodegradation in aqueous phase. Simultaneous determination of triazole antifungal drugs in human plasma by sweeping-micellar electrokinetic chromatography. Anal Bioanal Chem. Application of micellar electrokinetic capillary chromatography to forensic analysis of barbiturates in biological fluids.
J Forensic Sci. Micellar electrokinetic capillary chromatography of benzodiazepines in human urine. Separation of 1,4-benzodiazepines by micellar elektrokinetic capillary chromatography. J Biochem Biophys Methods. Enantioseparation of phenothiazines in cyclodextrin-modified micellar electrokinetic chromatography.
Determination of tricyclic antidepressants in human plasma by micellar electrokinetic capillary chromatography. J Chromatogr.
Blanco M, Valverde I. Electrophoretic behaviour of pharmacologically active alkylxanthines. Chiral separation by capillary electromigration techniques. An integrated process for measuring the physicochemical properties of drug candidates in a preclinical discovery environment.
It covers the analysis, formation mechanisms, and bioavailability of tea polyphenols and discusses bioactivities of teas including anticancer, anti-inflammatory, anti-obesity, and anti diabetes. Increased awareness of the many health benefits of tea has fueled an increase in the market for ready to drink teas and tea products in general that will continue to grow.
This expanding market requires a resource that provides the evidence. The editors of this volume have more than research publications in tea, and experience in editing more than 50 books between them. Under their expertise and editorial guidance, the contributors present chapters that explore the science behind the health claims of teas.
With contributions from international experts, Chiral Separations by Capillary Electrophoresis provides a general overview of the principles of chiral separation by capillary electrophoresis and the different chiral selectors available. The book discusses the most important as well as several new chiral selectors used in capillary electrophoresis.
It reviews recent pharmaceutical and biomedical applications and explores novel techniques, such as capillary electrophoresis coupled to mass spectrometry and microchip technology. The book also examines the quantitative aspects of capillary electrophoresis, the possibilities of capillary electrochromatography, and the various chiral columns available.
Capillary electrophoresis has proven to be an effective tool for chiral separation. This book explains how this technique can be used in the separation of molecules, offering insight into both existing and emerging applications. It has changed from being an exploratory technique, mainly of academic interest, to one that is applied to solve "real" analytical problems. CE is easily adapted to its various modes of operation, often requiring little more than a change of the buffer solution, and is quickly becoming the preferred technique when analyzing minute amounts of available material.
Featuring new chapters on CE analysis of inorganic ions and carbohydrates, the new edition of Capillary Electrophoresis not only presents this method as an academic tool, but also provides applications for solving "real-world" analytical problems. This updated Second Edition reflects the increasing use of CE over the last 10 years, how it is being applied, and the basic theoretical aspects of the separation and detection methodology of CE.
Capillary Electrophoresis: Theory and Practice will appeal to students and professionals of analytical chemistry, physical chemistry, biochemistry, and biotechnology and includes suitable experiments designed to be attempted by university or college students, or anyone else wishing to familiarize themselves with CE.
However, the assumption that N is independent of k' is clearly a first-order approximation, since most researchers have predicted or reported a slight-to- moderate dependence. Theory A theory for the optimization of the retention of neutral solutes was reported in and is summarized below. A similar theory for charged solutes is discussed near the end of Section VI [Equations ].
The theory established the existence of an optimum k' range within which good resolution would be obtained. It also defined quantitatively the upper and lower bounds for this range in terms of resolution and resolution per unit time, i.
With regard to option 2 , although the main effect of an organic solvent is to lower the polarity of the aqueous phase and to decrease somewhat the electroosmotic flow, solvents can also modify the micelles, resulting in changes in selectivity as well as various micellar parameters cmc, aggregation number, etc. This may be less of a concern for polymerized micelles and other pseudophases [cyclodextrins, starburst dendrimers] whose physical state is relatively insensitive to organic solvents.
When the initial selectivity and elution range are satisfactory, changing the surfactant concentration option 1 is the preferred method for adjusting retention in MEKC. The derivation is generally quite similar to the approach used for other column chromatographic methods.
This interval widens as the elution window tmlU becomes larger and is consistent with the effect of tmJt o on the peak capacity Equation Alterna- tively, one can define an optimum interval ofretention factors as that interval k'iow to k'high over which the resolution is within an arbitrary percentage e.
In contrast to the similarities ofk'iow among the three intervals in Table 4. Control Suifactant. Equation 13 is an expression for the retention factor. The phase ratio Equation 12 can be written explicitly in terms of surfactant concentration [SURF], the critical micelle con- centration erne , and the partial molar volume V of the surfactant.
Since the parameters in Equation 27 are either experimentally measurable or physical constants, a priori prediction of an optimum surfactant concentration is possible. The lowest usable surfactant concentration will be determined by the need for reproducible concentrations of micelles in order to obtain reproducible retention times.
The upper limit of the surfactant concentration is likely to be governed by such adverse solution properties as high viscosity or high conductance Joule heating. From our own experience and that reported in the literature, the highest tolerable concentration for monovalent surfactants SDS, CTAB, etc. On the other hand, neither surfactant system without organic solvent was ideally suited for all the amino acids.
Organic Modifier. Solutes of moderate-to-high hydrophobicity are difficult to analyze by MEKC using totally aqueous buffer systems. These types of solute elute near or with the micelle at tme' One way to increase the affinity of the solute for the mobile phase is to add an organic modifier to the buffer system.
The use of an organic modifier will alter the retention, k', of the solutes. In RPLC the addition of an organic modifier alters the analyte's affinity for the stationary phase by changing: 1 the polarity of the mobile phase, and 2 the retention characteristics of the stationary phase, usually to a much lesser degree. There are four predominant interactions between molecules in the liquid phase: 1 dispersion or London forces, 2 induction forces, 3 hydrogen bonding, and 4 orientation forces.
The total interaction of a solute molecule with a solvent molecule is described by all four interactions in combination. Solvent strength is directly related to polarity, thus "polar" solvents have an affinity for "polar" analytes. Since an organic modifier is less polar than water, any combination of organic modifier and water will be less polar than pure water, hence the mobile phase will be stronger and retention will be reduced.
In MEKC the addition of organic modifier alters not only the partitioning coefficient Pwm but also the phase ratio P , the surfactant's cmc, the zeta potentials of the capillary wall and the micelles, and the solution viscosity.
The effect of organic solvent on micelle formation and solute migration reproducibility govern the upper limit of organic solvent that can be used in MEKC. A fixed surfactant concentration e. Adapted from Ahuja, E. Of the two approaches, the latter is probably more convenient. Along this line, Ahuja and Foley have shown that solute retention can be varied much more with dodecyl sulfate micelles in the presence of lithium counterions i. In pure aqueous butfers, the retention with equimolar concentrations of SDS and LiDS is nearly identical, but as acetonitrile is added, the retention of all four solutes and particularly the more highly retained ones decreases more rapidly note log scale on retention axis in the LiDS system.
For example, k'hcnzophcnol1c decreases from The elimination of micelles and hence the MEKC separation mechanism by the addition of organic solvent to the buffer does not always mean that separations will no longer be possible. In two studies, sufficient organic modifier was added to suppress micellization and cause ionic surfactant monomers and neutral solutes to form charged solvophobic complexes that could be separated electrophoretically. Theory A very significant limitation to the application of MEKC to complex samples is the low peak capacity due to the limited elution range Equation The problem of increasing the elution range can be solved in several ways.
As Ilcp,me approaches -Ileo' the micelles become "stationary" and the elution range becomes infinite. Mobile phases with high surfactant concentrations generate large currents which enhance the Joule heating effect and result in poor reproducibility. Control Kaneta et al. Several reports have also established that the elution range is a function of the surfactant counterion The electroosmotic flow can be reduced by decreasing the 1; zeta potential of the capillary wall.
One parameter that can be used to manipulate the 1; potential is pH. Surface modification of the capillary is another method that can be used to reduce the 1; potential. Then the absolute values of the reciprocal velocities, electroos- motic and micellar, are plotted against the concentration of Brij and least squares lines are fitted through the data. The point at which the two lines intersect is the concentration of Brij necessary to achieve an infinite elution for the given SDS concentration.
In this case, the peak capacity is increased significantly, albeit at the expense of analysis time and possibly efficiency. One of the easiest ways to increase tmJto somewhat by a factor of 1. It is a simple and effective way to significantly increase tmJto , although it usually results in reduced electroos- motic flow, thereby increasing the analysis time proportionally. A somewhat larger 2. Selectivity Selectivity in MEKC is defined as the ratio of analyte partition coefficients Pwm or retention factors k' , the same as in GC and HPLC, and it is essentially the ability of a micellar system to separate two or more compounds that differ by one or more chemical groups.
As a increases, there is a very dramatic drop in N needed for the separation. Also, as the elution range tmdto increases, fewer theoretical plates are needed. It is often convenient to distinguish between two types of selectivity, hydrophobic and polar group selectivity, depending on whether the functional groups that differ among the compounds being separated are nonpolar or polar. For quantitative purposes, hydrophobic selectivity can be defined as the methylene selectivity or the selectivity between adjacent homologs.
It is most easily measured from the ratio of retention factors of adjacent homologs or from the slope of a In k vs. Polar group selectivity in MEKC is the ability of a micellar system to separate two or more compounds that differ by a polar functional group, and for convenience it is measured as the ratio of the retention factor of a derivative of benzene and benzene itself, i.
In k,olutc, w ,urf. Another way to characterize micellar selectivity is through linear solvation energy rela- tionships. By performing multivariate linear regression on a relatively extensive retention data set, micelle characteristics a, b, s, and m can be measured and compared for various surfactant systems. Tables 4.
The results show that hydrophobic selectivity is similar to that achieved in reverse-phase liquid chromatography RPLC , typically 0. A more detailed discussion of a is beyond the scope of this chapter, but some general principles can be summarized. First, a is independent of applied voltage, assuming that Joule Table 4. From Little, E. Second, a is also independent of surfactant concentration, assuming there are no solute-specific interactions with the buffered micellar media.
This assumption is valid for virtually all neutral analytes and for some charged analytes. Finally, several chemical variables of the running buffer may be changed to alter selectivity and sometimes the analytes' Pwm : 1 adding or changing organic modifiers e. Background Many useful insights can be gained by viewing chromatographic processes from a molec- ular perspective.
While a group of molecules is displaced along the separation axis, the individual molecules are essentially engaging in random, erratic motion. It is very difficult to predict the behavior of a single molecule, but it is possible to use a statistical approach to describe the behavior of a group of molecules i.
The use of a random walk model in chromatography has been used by researchers to describe the various mech- anisms that contribute to zone spreading. Common Sources of Chromatographic Band Broadening There are three basic sources of intracolumn band broadening plate height that are common to most forms of chromatography: 1 eddy dispersion, 2 longitudinal diffusion; and 3 resistance to mass transfer.
The first, eddy dispersion, is characteristic of flow in a packed chromatographic bed. The irregularly shaped particles form irregularly shaped chan- nels between particles which cause a nonuniform flow velocity.
This source of plate height does not exist in unpacked open tubular columns and is not applicable to MEKC. The second band-broadening phenomenon, longitudinal diffusion, results from the ran- dom Brownian motion diffusion that all solutes undergo in all directions as they are displaced along the column.
Although diffusion in the radial direction is a desirable means of mass transport, solute diffusion along the separation axis, i. The third source of band broadening, resistance to mass transfer, is itself made up of several components.
The first is the finite rate of solute mass transfer from the mobile aqueous phase to the pseudostationary phase and vice versa. Since the adsorption-desorption process occurs randomly, the motion of solute molecules is quite erratic and can result in zone spreading if the kinetics are relatively slow.
A second component of resistance to mass transfer is the finite rate of solute mass transfer in the mobile phase. Given the almost perfectly flat flow profile provided by electroosmotic transport in MEKC except in the diffuse double layer near the capillary wall , this source of band broadening is always negligible for neutral analytes and usually negligible for charged analytes, except when excessive Joule heating results in radial temperature and viscosity gradients, the latter giving rise to radial electrophoretic velocity gradients for charged analytes.
We have purposely avoided exces- sive editorial comments in this section so as not to unduly influence the reader. There are at least two other sources of band broadening unique to MEKC. First, micelles have a characteristic aggregation number indicative of the average number of surfactant monomers that make up a micelle. These aggregation numbers are characteristic not only of the identity of the surfactant but also the chemical environment that the micelle is in. For SDS under proper conditions the aggregation number is It is important to keep in mind that there may be micelles that have more or fewer monomers than the aggregation number.
This results in a size distribution of micelles and is termed the polydispersity. Since our systems utilize ionic buffers, the micelles are charged and any variation in the number of monomers in the micelle will give rise to a different charge for the micelle and hence a different electrophoretic mobility f. In addition, the micelle shape may be affected, and this may also affect f. And because the heat is primarily dissipated at the capillary walls, a radial temperature gradient exists across the capillary.
In one approach to this problem of temperature-induced band broadening, the temperature dependence of the viscosity of the buffer in the capillary was considered to be the mechanism which determined the velocity profiles of the solute. It was subsequently shown that the temperature gradient, treated in this manner, was not a significant source of band broadening.
In another approach, it was postulated that a radial gradient in k' exists because the Pwm is a function of temperature and will vary over the cross-section of the capillary. This causes solute molecules to spend a greater fraction of time in the mobile phase at the capillary center than near the walls of the capillary, and this means that a solute will have a greater vavg at the center of the capillary, decreasing toward the capillary wall.
Davis has used a random walk approach to characterize this potential band-broadening mechanism and termed it transchannel mass transfer. Yet as the vel, was increased, other band- broadening mechanisms became apparent. There has been considerable debate on the contribution of intermicellar mass transfer to plate height.
Sepaniak et al. But other research- ers have presented evidence, both theoretical and experimental, that the observed effects under these conditions are due to overloading the micelles with solute. Another mass transfer mechanism is partitioning the solute into and out of the micelle, termed micellar mass transfer. But micellar mass transfer has also been determined to be virtually insignificant as a source of band broadening under the experimental conditions examined.
The use of organic solvents should be kept to a minimum. Sample solvent and running buffer should be matched as closely as possible. Inlet and outlet buffer reservoir levels should differ at most by 0. To minimize H inj , the length of the injection plug should not exceed 0. Injection times should also be relatively short. When choosing a buffer and surfactant system, select those that minimize the overall conductance or ionic strength.
For a given buffer system, keep the applied voltage at the upper limit of the linear portion of the "Ohm's law plot" a graph of current vs. Capillary cuts should be as square as possible to avoid peak tailing, and samples should be filtered. Interactive agents should be of the highest purity, in order to minimize Hpd' 9. When possible, use field amplified sample stacking. If the length of the detector-illuminated volume of the capillary can be varied, it should be made as short as possible e.
Although we have considered intracolumn sources of band broadening in some detail, there are of course extracolumn sources of band broadening due to injection and detection. Limited numbers of studies have been conducted on the effects of injection technique on plate height in MEKC. This results in a narrow sample plug being put at the beginning of the column and hence better efficiency. There have also been a few recent reports on the application to MEKC of zone sharpening, i. JO The contribution to cr 2T from the detector and related electronics has also been examined in detai1.
Based on a critical evaluation of the results of others as well as our own observations, we summarize in Table 4. Practical Strategies There have been several reports on optimization of resolution by various methods. Unfor- tunately the term optimization means different things to different researchers. One should be aware that approaches to the optimization of separations based 1 on fundamental theories e.
In order to truly optimize a separation, one approach should not be unduly favored over another. The approach of McNair et a1. Equation 20 : 1 increase N; 2 increase ex; 3 increase the retention term s. Since ex is very hard to predict and manipulate, their study focused on Nand f k'. At low values of veo and k' it was possible to get infinite R.
Their conclusion was that R. This approach appears to be good for the simultaneous optimization of two or perhaps three parameters. Castagnola et al. Some may be less suited for the discrete changes in solution variables pH, surfactant concentration, etc.
A systematic evaluation of optimization of Rs by manipulating surfactant concentration has been reported. The theory helps to predict the optimum surfactant concentration for a given pair of solutes.
As Equation 24 shows, the optimum retention factor for a given separation is related to tmjtw One potential difficulty is that as the surfactant concentration is increased, the tmjt" value may vary con- siderably, depending on the surfactant system used. As noted earlier, there were several advantages to using the mixed surfactant system, but for the present discussion the interesting point is the difference in the behavior of tmJt" for the two systems.
The optimization of Rs for systems that have variable tmjt" is less desirable because an iterative process is needed to converge to the optimum surfactant concentration. But only two iterations at most should be required for the proper convergence. A more detailed discussion of mixed micel1ar systems is presented in a later section.
When MEKC is used to separate inorganic ions, the surfactant employed is often oppositely charged in order to avoid electrostatic repulsion which would tend to inhibit ion-micel1e interactions. Electrostatic interactions, presumably at or near the surface of the micel1e, are often dominant due to the polar character hydrophilicity of most inorganic ions.
In one study, cetyltrimethylammonium chloride was used above and below its cmc to separate the fol1owing monovalent anions: bromide, nitrate, bromate, iodide, and iodate. Based on Equation 33, one might expect that a plot of lI!
Ion-exchange models have also been employed to describe MEKC separations of anions and transition metal ions, the latter using SDS and tartaric acid in the buffer.
Migration and Retention of Ionizable Species Khaledi et al. The blue arrows show the difference in the speed of the migration of enantiomers upon interaction with the CS. Identification Identification of of Individual Enantiomers Individual Enantiomers The The identification identification of of individual enantiomers is individual enantiomers is highly highly valuable valuablefor forthe thequantitation quantitation of of impurity enantiomers, and to follow up a stereoselective synthesis procedure or toor impurity enantiomers, and to follow up a stereoselective synthesis procedure de-to determine a stereoselective metabolite [].
The identification of individual termine a stereoselective metabolite []. The identification of individual enantiomers of enantiomers of a racemate a racemate after after enantiomericseparation enantiomeric separationcan candirectly directlybebeachieved achievedby byrunning runninga areference reference optically opticallypure puresingle singleenantiomer enantiomerunder underthethesame sameelectrophoretic electrophoreticconditions conditionsand andcomparing compar- the ing obtained the obtainedmigration migration time of of time thethe peak peakwith withthe themigration migrationtimes times ofof the two peaks the two peaksofof the the separated separated enantiomers enantiomers peak peak matching.
Alternatively, Alternatively,the thepeak peakspiking spikingapproach approachtoto the the racemate sampleisisalso racemate sample alsoa acommon common method. WhenWhenthe the individual individual enantiomers enantiomers are are un- unavailable, several alternative approaches are used with different available, several alternative approaches are used with different degrees of success and degrees of success and limitations.
The The polarimetry polarimetry or electronic or electronic circular circular dichroism dichroism can be can be used used to identify to identify the the individual enantiomers [,]. Enantiomers will have mirrored individual enantiomers [,]. Enantiomers will have mirrored circular dichroism circular dichroism spectra, spectra, which which would would be be equal equal but opposite.
Liu but opposite. Liuet etal. SevenSeven enantiomeric enantiomeric substances substances tryptophan, tyrosine, phenylalanine, Boc-valine, Boc-leucine, ibuprofen, tryptophan, tyrosine, phenylalanine, Boc-valine, Boc-leucine, ibuprofen, and naproxen and naproxen were used in the study.
The method was found to be more suitable were used in the study. The method was found to be more suitable for a nonracemic mix- for a nonracemic mixture ture withwith good good and and stable stable electronic electronic circular circular dichroism dichroism signals. Enantiomers of each compound have opposite signals. Reprinted with permission fromreference from reference[]. Computational Computational docking studies can docking studies can give give poor poor information information not notaccurate accurate whenwhenthe the structure structureof ofthethechiral chiralselector selectorisisavailable availabledepending depending ononthethe different differentstrengths strengths of complex- of com- ation discussed plexation below.
Figureillustrates the chiral 8 schematically discrimination process, illustrates the chiral assuming the discrimination influence process, of EOF assuming theand the migration influence of EOF and direction the mi-of the CDs.
Figure Schematicrepresentation 8. Schematic representationofofthe thechiral chiraldiscrimination discriminationprocess processbybyCDs. For For enantiomeric enantiomeric impurity impurity determination, determination, e. Accord- ing to ICH to According guidelines, the enantiomeric ICH guidelines, impurity the enantiomeric shouldshould impurity not exceed 0. The determination of impurity The determination enantiomer of impurity of 0.
Whilein determination also importantfrom biological in this samples regard, absolute concentration where sensitivitypair the enantiomeric plays oratheir moremetabolites crucial role are in determination present in very from lowbiological samples concentration.
It where is worth thenoting enantiomeric that most pair or their metabolites commercially available are present single in enantiomeric pure very low concentration. Design of Experiment 3. Design of Experiment In the last decades, pharmaceutical regulatory authorities and organizations have been In the last decades, demanding pharmaceutical implementation regulatory of a risk-based authorities approach during and organizations analytical methodhave de- been demanding implementation of a risk-based approach velopment to assure the quality of experimental designs adopted to reach the optimumduring analytical method de- velopment to assure the quality of experimental designs adopted analytical conditions prior to wet lab development, in addition to delimiting the Design to reach the optimum analytical conditions prior to wet lab development, in addition to delimiting the Design Space and robust areas of an analytical method before proceeding to its method valida- Space and robust areas of an analytical method before proceeding to its method valida- tion.
Notions like analytical quality by design AQbD were introduced to represent the tion. In CEKC, AQbD started replacing Quality by Testing in the last decade, not only to find optimum values of the Critical Method Parameters CMPs within the Design Space DS also known as Method Operability Design Region, Knowledge Space, Response Surface, Resolution Map, Contour Plot and Surface Plot , but also to enable a deep understanding of the interactions taking place between analytes enantiomers and the CSto reveal dynamics of the enantiorecognition mechanisms supported by computer modeling , enhance gained knowledge and risk management of the method that allows enlightened continuous method improvement.
Design of Experiments DoE has been adopted as the systematic approach to assure the quality of the experimental design. The most common designs during this screening phase are Fractional Factorial and Placket Burman designs, yet some reports overlook a multivariate screening and resort to the more time-consuming One-factor-at-a-time OFAT Table 2.
Settling on the most relevant cmPs is usually aided by Main Effect Plots and Pareto charts, however none of the methods reported in the interval January March invested effort in this phase where many used the OFAT approach, while others did not proceed via the statistical analysis step Table 1 , probably due to the established cmPs in CEKC, namely, voltage, temperature, nature and concentration of chiral modifier and buffer in the BGE.
In a study by Milan et al. All computation was indeed focused on the optimization phase, where designs like full factorial, face-centered central composite designs, D-optimal designs are adopted to produce the response surfaces and generate the multivariate or least-square regression models related the significant cmPs to cmAs in addition to Monte Carlo simulations that are used for accurate definition of the DS, that enables selection of the optimal working point, robust range, or sweet spot revealed by the overlay of multiple methods operable design region MODR.
Responses in multivariate optimization in CEKC are mostly selectivity or resolution, and analysis time, where their simultaneous optimization is sometimes performed via the desirability function [,]. Selection of the appropriate optimization method is critical to the accuracy of fit of the regression model obtained. Long expertise of the analyst sometimes allows him to skip this step where a selection of relevant factors to carry to the optimization design is an expert decision; sometimes, classic trends between cmPs and CQAs like the direct relation of run time with buffer concentration and its inverse relation with voltage can lead to skipping those two factors in a screening study.
In a study by Abdel-Megied et al. As such, the determination of a working point may be possible, yet the definition of the MODR is impossible without a proper fit of the regression model Table 2. Due to the importance of CEKC in the determination of the eudismic ratio and chiral purity of drugs, where the eutomer has a significantly higher effect of distomer or where the latter has undesirable side effects, many studies consider DoE a backbone of this type of studies, despite the flaws in reproducibility that seem to exist in column preparation, thus affecting the reliability of regression models [].
Harnisch et al. DoE tools were also used to test method robustness prior to validation [,]. Even though fractional factorial designs are more common in electrophoretic and chromatographic DoE based studies, yet the CNX tool nicely identified CEKC injection, detection, capillary factors, and BGE factors, such as type and concentration of buffer, type of organic modifier, type of CD, and its degree of substitution as Constant C.
The friendly PBD was used to test method robustness prior to validation with values around the working point by Harnisch et al. It is also possible to use this quick technique to validate the MODR per se, but here at extremes of the cmP ranges and to verify that the ATP is also fulfilled in these extreme points as reported by Pasquini et al.
Reports do not reason this selection of limited space face-centered CCD [,,], yet it is well known that experimental points out of the cube space are on many occasions not practically applicable, due to instrument limitations, here probably voltage and temperature which have recommended high and low settings, respectively. It is indeed of paramount importance to select an applicable experimental domain to avoid breaking an experimental design for practical or technical reasons. Molecules , 26, 18 of 31 Table 2.
Type of buffer. Design Rs; t; max analyses time 20 min. Type of CD: Quantify 0. Molecular Modeling Applied to Enantioseparations As mentioned in the previous sections, CE plays a crucial role in enantiomeric discrimina- tion.
In this sense, aiming to elucidate the enantioseparation process at the molecular level, as well as the EMO, molecular modeling has proven to be an excellent auxiliary tool for the understand- ing of molecular recognition mechanism between selector and selectand. Besides, theoretical investigations have also been considered as powerful allies in CE experiments [].
Association i. In special, several studies have unequivocally shown that the inclusion complex formation is not a prerequisite for chiral recognition by CDs, mainly when substituted-CDs are used, which can offer sites of interactions with selectand.
Thus, the most theoretical studies in the enantioseparation field are devoted to CD host-guest inclusion complexes to comprehend the complexation mechanism and to correlate the experimental results with the mode of inclusion of the guest molecule analyte into the CD host chiral selector.
Furthermore, molecular modeling has been used to explain not only selector-selectand interaction, but also the migration phenomena observed in CE [,]. Previous works presented the elucidation of chiral recognition mechanisms in separation sciences by various selectors. Scriba reviewed the main contributions in the literature about the enantiorecognition mechanisms of main chiral selectors, i. Peluso et al. In this context, this review section illustrates the combined approach using CE and molecular modeling for understanding the chiral recognition mechanism using CDs.
From a structural point of view, CD complexes differ from each other in terms of the spatial and geometric arrangement, due to the chemical nature, strength, and number of interactions established.
Table 3 contains an overview of theoretical works, along with experimental data, from the years January to March that deals with the study of chiral discrimination using CDs. As can be seen, several research groups have dedicated themselves to study the chiral discrimination mechanism by CDs, aiming to predict or confirm EMO.
It is important to mention that the selector—selectand interaction is only one part of the entire separation process in CE. Another part also important is associated with the dynamics mobilities involved in chiral discrimination. In this sense, MD simulations have been used in an attempt to explain, at the molecular level, the mechanisms of chiral separation and EMO. In , Salgado et al.
The binding energies be- tween each of the enantiomers and the evaluated CDs were calculated, in addition to the different contributions to total binding energy, divided into van der Waals, coulombic, des- olvation, and nonpolar energy.
In view of the numerical data, the authors showed that the energies computed via MD, were proportional to the experimental equilibrium bond constants. Molecules , 26, 21 of 31 Table 3. Molecules , 26, 23 of 31 Although most of the theoretical calculations are performed via MD simulations, some precaution is necessary.
One aspect that must be considered in MD simulations is related to the selector-selectand interactions. It is known that multiple long- and short-range interac- tions are involved in the chiral separations, but not all these forces are enantioselective. In this sense, MD simulations must also be used cautiously, especially in the studies aimed to explain EMO, which must also consider systems that have a reverse migration order, due to concentration, pH, or other variables [55,].
Moreover, all possible conditions must be inserted and considered in the MD simulations, for example, selector and selectand charges, force field, nature of the electrolytes, among others, along with experimental validation of studied CD complexes [,]. On the other hand, the use of quantum mechanical high-level calculations, such as DFT, to study the enantioseparation process has risen, especially in recent years. DFT is fundamentally a more precise theoretical methodology than those based on molecular mechanics, since it is more reliable and efficient in describing the energy and electronic structure of chemical systems and complexes.
In , Michalska et al. In , Cecilio et al. Along with the theoretical results, fractions of pure enantiomers were obtained and injected into the electrophoretic system, whose migration times by experimental studies corroborated with theoretical predictions.
In , Pires et al. After optimizing the experimental conditions that influence the separation, such as concentration and pH of BGE and concentration and type of CD, it was possible to obtain an electropherogram with optimized conditions. Notably, the theoretical study contribution to the experiment proved to be very relevant, since it was possible to determine the EMO. Figure 9 shows an electrophero- gram with optimized conditions, which are similar to those proposed by Hancu et al.
In this condition, R -amlodipine enantiomer presented a migration time shorter than the S -amlodipine enantiomer. Electropherogram related to conditions optimized for enantioseparation of amlodipine and diastereoisomeric Figure 9.
Experimental conditions: 5. Despite the rapid increase in the number of papers involving DFT calculations Despite the rapid increase in the number of papers involving DFT calculations in in the last years, semiempirical methods still attract a great deal of attention owing the last years, semiempirical methods still attract a great deal of attention owing to their to their lower computational demands. For example, semiempirical methods, such as lower PM3 and computational demands.
PM6, have been foundFor example, to give good semiempirical methods,properties estimates of molecular such as PM3thanand PM6, haveand ab initio been DFT found to give methods good lower at even estimates of molecular computational properties cost, making it than ab initio and an attractive DFT method for the description of inclusion complexes []. A systematic theoretical analysis, when used in combination potential of molecular modeling for understanding enantioselective recognition mecha- or not with CE experiments, can provide a good perspective on the enantioseparation process, serving as nisms.
A systematic theoretical analysis, when used in combination or not with CE exper- a valuable auxiliary method for studying chiral recognition mechanisms. Finally, efforts iments, can provide a good perspective on the enantioseparation process, serving as a in the computational chemistry field are continuously required—since the mechanistic valuable auxiliary method for studying chiral recognition mechanisms.
Finally, efforts in aspects of selector-selectand interactions, at the molecular level, are still, in many cases, not the fullycomputational understood. Author Contributions: Conceptualization, S. All authors S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available within the article or on request from the corresponding author. Conflicts of Interest: The authors have declared no conflict of interest.
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