Mixed-mode electrokinetic chromatography of aromatic bases with two pseudo-stationary phases and pH control

The electrokinetic chromatographic (EKC) separation of a series of aromatic bases was achieved utilising an electrolyte system comprising an anionic soluble polymer (polyvinylsulfonic acid, PVS) and a neutral beta-cyclodextrin (beta-CD) as pseudo-stationary phases. The separation mechanism was based on a combination of electrophoresis, ion-exchange interactions with PVS, and hydrophobic interactions with beta-CD. The extent of each chromatographic interaction was independently variable, allowing for control of the separation selectivity of the system. The ion-exchange and the hydrophobic interactions could be varied by changing the PVS and the beta-CD concentrations, respectively. Additionally, mobilities of the bases could be controlled by varying pH, due to their large range of pKa values. The separation system was very robust with reproducibility of migration times being <2% RSD. The two-dimensional parameter space defined by the two variables, [beta-CD] and %PVS, was modelled using a physical model derived from first principles. This model gave very good correlation between predicted and observed mobilities (r2=0.999) for the 13 aromatic bases and parameters derived from the model agreed with the expected ion-exchange and hydrophobic character of each analyte. The complexity of the mathematical model was increased to include pH and this three-dimensional system was modelled successfully using an artificial neural network (ANN). Optimisation of both the two-dimensional and three-dimensional systems was achieved using the normalised resolution product and minimum resolution criteria. An example of using the ANN to predict conditions needed to obtain a separation with a desired migration order between two of the analytes is also shown.     

Separation of opiate alkaloids by electrokinetic chromatography with sulfated-cyclodextrin as a pseudo-stationary phase

The separation of six related opiate alkaloids (morphine, thebaine, 10-hydroxythebaine, codeine, oripavine and laudanine) was studied using sulfated-cyclodextrin (s-CD) as a cation-exchange pseudo-stationary phase. Cation-exchange interactions between the cationic analytes and the anionic s-CD (7-11 mol of sulfate groups per mole CD) were found to bethe predominant mechanism, allowing the separations to be performed at low pH where the opiates are protonated and exhibit very similar mobilities. The concentrations of the s-CD and the competing ion (Na+ or Mg2+) in the electrolyte were used to govern the extent of the ion-exchange interactions. Interactions with the sulfated-cyclodextrin differed for each analyte, with oripavine exhibiting the strongest interaction and 10-thebaine and laudanine showing the weakest interactions. Despite the very similar structures of the analytes, these differences resulted in significant changes in separation selectivity. The separation was modelled using a migration equation derived from first principles and based on ion-exchange interactions between the s-CD and the opiates. Constants within the model were obtained by non-linear regression using a small subset of experimentally determined migration times. These constants related to the ion-exchange affinities of the s-CD for the various opiates. When the model was used to predict migration times under other experimental conditions, a very good correlation was obtained between observed and predicted mobilities (r2=0.996). Optimisation of the system was performed using the normalised resolution product and minimum resolution criteria and this process provided two optimised separations, each exhibiting a different separation selectivity.     

Optimisation of selectivity in the separation of aromatic amino acid enantiomers using sulfated beta-cyclodextrin and dextran sulfate as pseudostationary phases

Control of selectivity in the enantiomeric separation of three aromatic amino acids (phenylalanine, tyrosine and tryptophan) was demonstrated utilising two separate electrolyte additives. Sulfated-beta-cyclodextrin (s-beta-CD) was chosen as the chiral selector while the addition of dextran sulfate provided a means with which to predictably fine-tune separation selectivity. The two additives were found to interact independently with the amino acids, with the s-CD providing chiral interactions while the dextran sulfate provided ion-exchange (IE) interactions. The system was also very robust with reproducibility of migration times being < 2.0% RSD between runs and < 2.6% on using a new capillary. A physical model derived from first principles was also successfully used to describe the two additive system. The model accurately described the observed separations over the range of 0-20 mM s-beta-CD and 0-1% dextran sulfate with a correlation coefficient of 0.998 between predicted and observed mobilities. The physical model also provided useful information about the system including association constants between the analytes and the pseudostationary phases, together with the mobilities of the associated complexes (analyte-cyclodextrin and analyte-dextran sulfate). Selectivity optimisation was achieved using the normalised resolution product and minimum resolution criteria. The physical model also allowed a desired separation selectivity to be obtained, such that experimental conditions could be predicted to lead to a particular migration order.     

Enantioselective separation of azole compounds by EKC. Reversal of migration order of enantiomers with CD concentration

The enantioselective separation of a group of six weak base azole compounds was achieved in this work using EKC with three neutral beta-CDs as chiral selectors. The native beta-CD and two other beta-CD derivatives with different types and positions of the substituents on the CD rim ((2-hydroxy)propyl-beta-CD (HP-beta-CD) and heptakis-2,3,6-tri-O-methyl-beta-CD (TM-beta-CD)) were employed. Apparent binding constants for each pair compound-CD were determined in order to study analyte-CD interactions. The best enantiomeric resolutions for miconazole, econazole, and sulconazole were observed with HP-beta-CD whereas for the separation of the enantiomers of ketoconazole, terconazole, and bifonazole, TM-beta-CD was the best chiral selector. The enantioseparations obtained were discussed on the basis of the structure of the compounds taking into account that inclusion into the hydrophobic CD cavity occurred through the phenyl ring closer to the azole group. In addition, a change in the migration order for the enantiomers of two of the compounds studied (ketoconazole and terconazole) with the concentration of HP-beta-CD was observed for the first time.     

Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

A highly water-soluble new cyclodextrin (CD) derivative 2-O-acetonyl-2-O-hydroxypropyl-beta-CD (2-AHP-beta-CD) was synthesized and tested as an effective chiral selector for the capillary zone electrophoretic resolution (Rs) of several basic and acidic analytes. The primary purpose of the research was to explore the capability of the 2-AHP-beta-CD as chiral selectors on comparison with the neutral CDs such as beta-CD, DM-beta-CD and HP-beta-CD. Substitution with 2-O-acetonyl-2-O-hydroxypropyl group at the secondary hydroxyl sites of the CD is aimed at influencing the magnitude and selectivity of analyte-CD interactions. The chiral resolution was strongly influenced by the concentration of the CDs and buffer pH. 2-AHP-beta-CD showed the best enantiomer resolution properties among the tested compounds, while the other CDs showed inferior or no performances at all.     

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

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

Microemulsion electrokinetic chromatography of corticosteroids. Effect of surfactants and cyclodextrins on the separation selectivity

The separation of neutral hydrophobic corticosteroids (cortisone, cortisone acetate, hydrocortisone, hydrocortisone acetate, prednisolone and prednisolone acetate) by microemulsion electrokinetic chromatography (MEEKC) was studied. In the preparation of microemulsion, heptane was the solvent, n-butanol the co-surfactant and, as anionic surfactants, sodium dodecyl sulfate (SDS) or taurodeoxycholic acid sodium salt (STDC) were employed. Using an acidic running buffer, (phosphate pH 2.5) a strong suppression of the electroosmotic flow (EOF) was observed; this resulted in a fast anodic migration of the analytes partitioned into the negatively charged microemulsion droplets. Under these conditions, STDC showed better separation of corticosteroids than the conventional SDS; however, the use of a single anionic surfactant did not provide the required selectivity. The addition of the neutral surfactant polyoxyethylene glycol octadecyl ether (Brij 76) significantly altered the migration of each analytes allowing a better tuning of separation; however, in order to obtain adequate resolution between couples of adjacent critical peaks, the addition of neutral cyclodextrins (CDs) was found to be essential. This apparently complex system (CD-MEEKC), was optimized by studying the effect of the most important parameters affecting separation: STDC concentration, Brij 76 concentration, nature and concentration of cyclodextrins. Following a rational step-by-step approach, the optimised conditions providing the complete separation of the analytes were found to be: 4.0% STDC, 2.5% Brij 76, 6.6% n-butanol, 1.36% heptane and 85.54% of a solution 5 mM beta-CD in 50 mM phosphate buffer (pH 2.5). The optimized system was preliminary applied to the detection of corticosteroids related substances at impurity level and it could be considered a useful orthogonal alternative to HPLC methods.     

Separation of basic drug enantiomers by capillary zone electrophoresis using glucuronyl glucosyl beta-cyclodextrin as a chiral selector.

Separations of basic drug enantiomers have been investigated using glucuronyl glucosyl beta-cyclodextrin (GUG beta-CD) as a chiral selector in the background electrolyte by capillary zone electrophoresis. The effects of GUG beta-CD concentration and running buffer pH on the migration times and resolution of 16 basic drug enantiomers were precisely examined using a linear polyacrylamide-coated capillary. High resolution of 16 basic drug enantiomers was generally attained with a running buffer pH 2.5 or 3.5 containing 10 mM GUG beta-CD. Next, we compared the chiral resolution abilities of GUG beta-CD with those of beta-CD and maltosyl beta-CD (G2 beta-CD). GUG beta-CD showed higher resolution for basic drug enantiomers tested than beta-CD and G2 beta-CD. This could be due to that hydrogen bonding or ionic interactions of uncharged and charged glucuronyl glucosyl groups of GUG beta-CD with an analyte could stabilize the inclusion complex.     

Modelling and optimization of the electrokinetic chromatographic separation of mixtures of organic anions and cations using poly(diallydimethyl- ammonium chloride) and hexanesulfonate as mixed pseudostationary phases

The separation of a series of aromatic carboxylic acids, sulfonates and opiates using electrokinetic chromatography employing a mixture of the soluble cationic polymer poly(diallydimethylammonium chloride) (PDDAC) and the amphiphilic anion hexanesulfonate as pseudostationary phases is described. In this system, the PDDAC pseudostationary phase interacts with the anionic analytes, whereas the hexanesulfonate pseudostationary phase interacts with the cationic analytes. A migration model has been derived which takes into account the ion-exchange (IE) interactions between the anions and the cationic PDDAC as well as the ion-pair (IP) interactions between the opiates and the hexanesulfonate. A further interaction between the combined PDDAC-hexanesulfonate complex and the more hydrophobic analytes is also evident and is accounted for in the model. Constants obtained by applying the model agreed well with the expected trends in IE affinities of the anions for PDDAC and also corresponded with the hydrophobic natures of the analytes. Optimization of the PDDAC and hexanesulfonate concentrations was performed using the normalized resolution product and minimum resolution product criteria. The minimum resolution product criterion proved to be most successful. An advantage of the described system is the improvement in peak shapes obtained after addition of hexanesulfonate to the electrolyte, resulting in increased plate numbers and better resolution. The system was very robust with mobilities varying by less than 2% over a period of days and on using different capillaries.     

Migration order of dipeptide and tripeptide enantiomers in the presence of single isomer and randomly sulfated cyclodextrins as a function of pH

The present study was conducted in order to evaluate the cyclodextrin (CD)-mediated chiral separation of peptide enantiomers as uncharged analytes at pH 5.3 using randomly sulfated beta-cyclodextrin, heptakis-6-sulfato-beta-CD and heptakis-(2,3-diacetyl-6-sulfato)-beta-CD as chiral selectors. Although less effective compared to stronger acidic conditions, the CDs proved to be suitable chiral selectors for the present set of peptides at pH 5.3. The carrier ability of the negatively charged CDs upon reversal of the applied voltage may also be exploited leading to a reversal of the migration order. In addition, reversal of the enantiomer migration order upon increasing the buffer pH from 2.5 to 5.3 was also observed for Ala-Tyr in the presence of randomly sulfated beta-CD, for Ala-Phe, Ala-Tyr, Phe-Phe, Asp-PheNH(2) and Gly-Ala-Phe in the presence of heptakis-6-sulfato-beta-CD, and for Phe-Phe and Ala-Leu in the presence of heptakis-(2,3-diacetyl-6-sulfato)-beta-CD. The migration behavior could be explained on the basis of the complexation constants and the mobilities of the peptide-CD complexes. While a change in the affinity pattern of the CDs upon increasing the pH was observed for some peptides, complex mobility was the primary factor for other peptide-CD combinations affecting the enantiomer migration order at the two pH values studied.     

Separation of neutral and basic enantiomers by cyclodextrin electrokinetic chromatography using anionic cyclodextrin derivatives as chiral pseudo-stationary phases

Separations of neutral and basic racemates were performed using five different anionic cyclodextrin (CD) derivatives as chiral selectors, viz. carboxymethylated β-CD, β-CD phosphate sodium salt, sulfobutyl ether β-CD sodium salt, carboxymethylated γ-CD, and γ-CD phosphate sodium salt. For the separation of neutral racemates, an untreated fused silica capillary was employed and various neutral racemates were successfully separated. Since the pH of the buffer affected the electroosmotic flow (EOF), the resolution was improved by changing the buffer pH. A polyacrylamide coated capillary was employed for the separation of basic racemates to suppress EOF and to prevent adsorption of cationic analyte on the capillary surface. By choosing an appropriate type and concentration of anionic CD, about 40 basic racemates were successfully separated. Some rough binding constants of basic analytes with an anionic β-CD were measured to discuss the optimum concentration of the CD. The migration direction was dependent on the binding constants and the concentration of the CD. The analyte strongly bound to the anionic CD migrated towards the anode but the weakly bound one moved towards the cathode. Anionic γ-CDs were also very useful for the separation of basic enantiomers. Five neutral CDs were employed as chiral selectors to compare selectivity between charged and neutral CDs, and eleven racemates could only be resolved using anionic CDs. The separation of some basic racemates in human plasma was also described. The direct injection of plasma samples was possible for some enantiomers that did not interact strongly with plasma proteins.     

Synthesis of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose, a single-isomer, monosubstituted, permanently dicationic beta-CD and its use for enantiomer separations by CE

The dichloride salt of mono-6-deoxy-6-N,N,N',N',N'-pentamethylethylenediammonio-cyclomaltoheptaose (PEMEDA-BCD), the first single-isomer, monosubstituted, permanently dicationic beta-CD has been synthesized, analytically characterized, and used for the capillary electrophoretic separation of the enantiomers of a group of analytes in acidic and basic BGEs. When the concentration of PEMEDA-BCD was changed in the BGEs, the resulting effective mobilities of the analytes and the respective separation selectivities followed the predictions of the ionic strength-corrected charged resolving agent migration model. Good separation selectivities and favorable normalized EOF mobilities allowed for the rapid, efficient separation of the enantiomers of anionic, weak acid and nonionic analytes in the low- and/or high-pH BGEs.     

Enantiomer separation of drugs by capillary electrophoresis using mixtures of beta-cyclodextrin sulfate and neutral cyclodextrins

Direct separation of enantiomers of drugs was investigated by capillary electrophoresis employing mixtures of charged cyclodextrin derivatives (CDs) and electrically neutral CDs (i.e., dual CD system). Among various charged CDs employed, it was found that beta-CD sulfate showed relatively wide enantioselectivity for a wide variety of drugs under acidic conditions. Then separation of enantiomers was performed by employing beta-CD sulfate and the effect of the addition of electrically neutral CDs to the buffers containing beta-CD sulfate was investigated. Through the addition of electrically neutral CDs to the buffers containing the charged CD, resolution of most of the enantiomers was improved, compared with those with the charged CD alone. It was also found that the ring size (alpha, beta, gamma,), the substitution groups and the concentration of the additional electrically neutral CDs affected the enantioselectivity. For example, alpha-CD addition was effective for the separation of enantiomers of chlorpheniramine and hydroxypropyl-beta-CD was effective for the enantiomer separation of trimetoquinol isomer. The application of the method in optical purity testing is also briefly mentioned.     

Enantioseparation of chiral N-imidazole derivatives by electrokinetic chromatography using highly sulfated cyclodextrins: mechanism of enantioselective recognition

Baseline separation of ten new substituted [1-(imidazo-1-yl)-1-phenylmethyl)] benzothiazolinone and benzoxazolinone derivatives, with one chiral center, was achieved by CD-EKC using highly sulfated CDs (alpha, beta, gamma highly S-CDs) as chiral selectors. The influence of the type and concentration of the chiral selectors on the enantioseparations was investigated. The highly S-CDs exhibit a very high enantioselectivity power since they allow excellent enantiomeric resolutions compared to those obtained with the neutral CDs. The enantiomers were resolved with analysis times inferior to 2.5 min and resolution factors R(s) of 3.73, 3.90, 1.40, and 4.35 for compounds 1, 2, 3, and 5, respectively, using 25 mM phosphate buffer at pH 2.5 containing either highly S-alpha-CD, highly S-beta-CD, and highly S-gamma-CD (3 or 4% w/v) at 298 K, with an applied field of 0.30 kV/cm. The determination of the enantiomer migration order for the various analytes and the study of the analyte structure-enantioseparation relationships display the high contribution of the interactions between the analytes phenyl ring and the CDs to the enantiorecognition process. The thermodynamic study of the analyte-CD affinities permits us to improve our knowledge about the enantioseparation mechanism.     

Separation of monomethyl-benz[a]anthracene isomers using cyclodextrin-modified electrokinetic chromatography

Cyclodextrin-modified electrokinetic chromatography (CD-EKC) was investigated for the separation of 12 monomethylbenz[a]anthracene (MBA) isomers. Combined use of a polymeric surfactant, poly(sodium 10-undecenyl sulfate) (poly-SUS), with various types of neutral cyclodextrins (CDs) [beta-CD, gamma-CD, dimethyl-beta-CD (DM-beta-CD), trimethyl-beta-CD (TM-beta-CD) and hydroxypropyl-beta-CD (HP-beta-CD)] were successful in CD-EKC separation of the MBA isomers. Baseline resolution of 10 of the 12 isomers, except for 9-MBA and 2-MBA, was achieved with gamma-CD at pH 9.75. The beta-CD, gamma-CD, and beta-CD derivatives (DM-beta-CD, TM-beta-CD, HP-beta-CD) were found to have different resolution and selectivity. Additionally, the tR/t0 values of isomers were found to be dependent on the type and concentration of the CD additives. In general, tR/t0 values of MBA isomers decrease with an increase in the concentration of beta-CD derivatives, whereas the reversed was true when the concentrations of native beta-CD and gamma-CD were varied. The combination of 5 mM gamma-CD, 0.5% (w/v) poly-SUS, 35% (v/v) acetonitrile at a pH of 9.75 provided the best selectivity and resolution of the MBA isomers with a separation time of 110 min. However, the use of 30 mM DM-beta-CD under similar EKC conditions resulted in much faster separation (ca. 16 min) of 10 MBA isomers.     

Use of multivariate analysis for optimization of separation parameters and prediction of migration time, resolution, and resolution per unit time in micellar electrokinetic chromatography

The optimization of separation parameters in chromatography for better separation and resolution of analytes continues to be a labor intensive procedure usually performed by a trial and error method. A multivariate analysis in the form of multilinear regression (MLR) is used to optimize separation parameters and predict the migration behavior, resolution, and resolution per unit time of achiral (4-chlorophenol, pentachlorophenol, clonazepam, and diazepam) and chiral (1,1'-binaphthyl 2,2'-dihydrogen phosphate (BNP), and 1,1'-bi-2-naphthol (BOH)) compounds in MEKC. Separations of achiral and chiral analytes were performed using an achiral (poly(sodium N-undecylenic sulfate)) molecular micelle and chiral (poly(sodium N-undecanoyl-L-leucylvalinate) or poly(sodium N-undecanoyl-L-isoleucylvalinate)) molecular micelle, respectively, at various operating temperatures, applied voltages, pH values, and molecular micelle concentrations in the BGE. The separation parameters were subsequently used as input variables for MLR models. The models were validated with independent samples. The root-mean-square percent relative error (RMS%RE) is used as a figure of merit for characterizing the performance of the migration time, resolution, and resolution per unit time models. The RMS%RE obtained for predicted migrated times, resolutions, and resolution per unit time of 4-chlorophenol, pentachlorophenol, clonazepam, diazepam, BNP, and BOH ranged between 8 and 19%. The same experimental procedure was used to optimize the separation parameters of six other chiral analytes of different compound class. The predicted migration times, resolutions, and resolution per unit time of the chiral as well as the achiral analytes compare favorably with the experimental migration times and resolutions, indicating versatility and wide applicability of the technique in MEKC.     

Synthesis, analytical characterization and initial capillary electrophoretic use in an acidic background electrolyte of a new, single-isomer chiral resolving agent: heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)-β-cyclodextrin

The sodium salt of heptakis(2-O-sulfo-3-O-methyl-6-O-acetyl)cyclomaltoheptaose (HAMS), the first single-isomer sulfated β-CD that carries the sulfo group exclusively at the C2 position, has been synthesized. The purity of each synthetic intermediate and of the final product was determined by hydrophilic interaction (HILIC) and reversed-phase HPLC. The structural identity of each intermediate and of the final product was verified by 1-D and 2-D NMR spectroscopy and MALDI-TOF MS. HAMS was used for the capillary electrophoretic separation of the enantiomers of a set of non-ionic and weak base analytes in pH 2.5 background electrolytes. Rapid separations with satisfactory peak resolution values were obtained for most enantiomers using low concentrations of HAMS. The effective mobilities and separation selectivities were dependent on the concentration of HAMS according to the predictions of the charged resolving agent migration model. The separation selectivities observed with HAMS, heptakis(2-O-methyl-3-O-acetyl-6-O-sulfo)cyclomaltoheptaose and heptakis(2-O-methyl-3,6-di-O-sulfo)cyclomaltoheptaose were different for some of the analytes studied in detail.     

Modeling and optimization of the chiral selectivity of basic analytes in chiral capillary electrophoresis with negatively charged cyclodextrins using electrochemical detection

A mathematical model concerning the separation selectivity of basic analytes in chiral capillary electrophoresis (CE) modified with negatively charged cyclodextrins (CDs) has been presented to describe the dependence of chiral selectivity on the buffer pH and the chiral selector concentration. The electrophoretic method to determine the parameters of the model has also been developed. The model has been tested with racemic epinephrine and isoproterenol as target analytes and sulfonated beta-CD as chiral selector. The agreements have been found between the calculated and the measured values. Some significant conclusions to optimize chiral CE separation have been derived from the model and proven by the experiments. Electrochemical detection was used to meet the requirement of the low introduced concentration of analytes.     

Cyclodextrin‐mediated capillary electrophoresis enantioseparation of dansylated β‐amino acids with bicyclo[2.2.2]octane,bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures

The present study investigated the separation of bicyclic β‐amino acids with bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane and cyclopenta[d][1,2]oxazole core structures by capillary electrophoresis using native cyclodextrins as well as neutral and charged derivatives as chiral selectors. The amino acids were derivatized with dansyl chloride to provide a UV chromophore. Separations were carried out at 20°C in a 48.5/40 cm, 50 µm fused‐silica capillary at an applied voltage of 20 kV. Fifty millimolar sodium phosphate background electrolytes pH 2.5 and 7.2 containing either 5 or 30 mg/mL of the CDs were used. For the majority of the investigated CDs, enantioseparations could only be achieved at pH 2.5 when the analytes are positively charged. Successful enantioseparations as negatively charged analytes at pH 7.2 were only observed for few compounds. In the case of methyl‐γ‐cyclodextrin, opposite enantiomer migration order was observed in pH 2.5 or 7.2 background electrolytes. Dependence of the enantiomer migration order on the size of the cavity of the cyclodextrins was also found. Furthermore, the degree of methylation of β‐cyclodextrin derivatives affected the migration order of several analyte enantiomers.     

Separation selectivity patterns of fully charged achiral compounds in capillary electrophoresis with a neutral cyclodextrin

Based on the separation selectivity equation, related to the dimensionless parameters for fully charged achiral analytes using a neutral CD, the separation selectivity can be classified into seven patterns. With respect to CZE without CD, the presence of CD in the buffer may improve, or reduce, the separation selectivity with this effect being accompanied by the same or reversed electrophoretic mobility order for charged analytes. This can depend on the separation selectivity of the two analytes in free solution, the binding selectivity, the separation selectivity of analyte–CD complexes and the ratio of electrophoretic mobility of the analytes in free, and complexed forms. Using positional isomers of benzoic acids and phenoxy acids as test analytes and α‐CD as a selector, the observed separation selectivity shapes were found to be in excellent agreement with the predicted separation selectivities.