CE-ICP-MS for studying interactions between metals and biomolecules

Metal-biomolecule interactions comprise an important research area in metallomics, and are significant for biology, medicine, pharmacy, nutrition, metabolism, and environmental science. Hybrid techniques are preferred for studying interactions between metals and biomolecules. Of all the separation techniques, capillary electrophoresis (CE) exhibits high resolution, minimal sample and reagent consumption, and rapid and efficient separations with minor disturbance of the existing equilibrium between the metal species and their biomolecular complexes. Inductively coupled plasma mass spectrometry (ICP-MS) presents high sensitivity to most of elements and offers multi-element detection.This article provides an overview of CE-ICP-MS for the study of metal-biomolecule interactions. We discuss applications of CE-ICP-MS to the study of interactions between metals or metalloids and natural ligands, such as humic substances or fulvic acids, and the interchange of metal complexes with metal species in metalloproteins.     

Protein-aptamer binding studies using microchip affinity capillary electrophoresis

The use of traditional CE to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombin-binding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with an LOD for thrombin of 5 nM at a three-fold S/N.     

Binding energies of water to doubly hydrated cationized glutamine and structural analogues in the gas phase

The modes of metal-ion and water binding in doubly hydrated complexes of lithiated and sodiated glutamine (Gln) are probed using blackbody infrared radiative dissociation experiments and density functional theory calculations. Threshold dissociation energies, E0, for loss of a water molecule from these complexes are obtained from master-equation modeling of these data. The values of E0 are 36 +/- 1 and 38 +/- 2 kJ/mol for the lithiated and sodiated glutamine complexes, respectively, and are consistent with calculated water binding energies for the nonzwitterionic form of these complexes. Calculated water binding energies for the zwitterionic forms of these complexes are significantly higher. In contrast, calculations indicate that the zwitterionic form of Gln in these complexes is more stable than the nonzwitterionic form by 8 and 15 kJ/mol when lithiated and sodiated, respectively. Doubly hydrated lithiated and sodiated complexes of asparagine methyl ester (AsnOMe), asparagine ethyl ester (AsnOEt), and glutamine methyl ester (GlnOMe) were also studied for comparison to Gln. Although these clusters lack the acidic group of Gln and therefore have different water coordination behavior, these results further support the conclusion that Gln is nonzwitterionic in these clusters. Surprisingly, the complexes containing sodium are more stable than those containing lithium, a result that is attributed to subtle differences in how these two metal ions bind to the amino acid esters in these complexes.     

Capillary electrophoresis for diastereomers of (R,S)-tetrahydroisoquinoline-3-carboxylic acid derivatized with (R)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole: effect of molecular geometries

Diastereomers derived from (R,S)-tetrahydroisoquinoline-3-carboxylic acid (Tic), a potential neurotoxin with a chiral fluorescence tagging reagent, (R)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy), are well resolved by capillary electrophoresis (CE). For a better understanding of the separation mechanism, a semiempirical computational method (i.e., AM1 method) is used to study the molecular geometry, relative energy, and size of the derivatives. The molecular sizes are estimated to be 216.3 and 240.6 cm3/mol for (R)-NBD-APy-(R)-Tic and (R)-NBD-APy-(S)-Tic, respectively. The CE elution order of the diastereomeric derivatives confirms the AM1 computational results: (R)-NBD-APy-(R)-Tic elutes before (R)-NBD-APy-(S)-Tic. The effects of running buffer pH and the addition of a chiral selector, beta-cyclodextrin (beta-CD), on the separation are studied. In the presence of beta-CD, the migration behavior of the diastereomers is changed because of the formation of CD inclusion complexes. Study of the space-filling models for optimized conformations of the diastereomeric derivatives and beta-CD suggests that the geometries of the diastereomers decides that the diastereomers are incorporated into the CD cavity to form CD inclusion complexes with different volumes. Experimental results from CE separations conclude the same.     

A novel specific heparin-binding activity of bovine folate-binding protein characterized by capillary electrophoresis

Folate-binding proteins (FBPs) are ubiquitous, soluble and membrane-bound high-affinity receptors for folate, an essential nutrient involved in nucleic and amino acid metabolism. In the course of optimizing CE separation conditions for FBP purified from cow's milk we discovered a novel specific heparin-binding activity of FBP by affinity CE. Heparin is a highly sulfated glycosaminoglycan and thus prone to induce anodic migration shifts of complexing analytes. Prior complexation of FBP with folate abolished heparin binding, and thus folate competes with heparin for binding to FBP. It was estimated that heparin bound several orders of magnitude less strongly than folate with an average dissociation constant in the 1-10 microM range. In contrast to the mobility shifts induced by heparin, free and folate-bound FBP were not separated by CE. However, binding of folate induced a distinct increase in FBP-peak symmetry, and using heparin as an affinity displacer, the free FBP in equilibrium with folate-FBP complexes could readily be separated from the complexes. While the folate-FBP interaction was too strong to be characterized quantitatively because of inadequate detection limits of a UV-based detection system, it was possible to estimate the folate-FBP binding stoichiometry using this approach. The heparin interaction fractionated FBP into distinct subfractions, and the CE approach thus promises to be useful for unraveling the complex oligomerization behavior of FBP isoforms as well as for evaluating the FBP affinity for various species and analogs of glycosaminoglycans and folate.     

Protolytic dissociation mechanisms and comparative acid stabilities of palladium(II), zinc(II), copper(II), and nickel(II) complexes of alkylated dipyrrins

Complexes of Pd(II), Cu(II), Ni(II), and Zn(II) with alkylated dipyrrins (Hdpm) were synthesized and characterized by physicochemical and spectroscopic methods. Protolytic dissociation kinetics of these complexes in benzene in the presence of acetic and trichloroacetic acid was studied. A protonated dipyrrin is the reaction product of protolytic dissociation of the complexes in acid solutions. The observed and true dissociation rate constants, as well as activation reaction parameters, were calculated. Kinetic models of the processes are proposed, and the patterns of influence of the ligand nature on dissociation kinetics were determined. The Pd(II) complexes proved to be much more stable than other those of the other metals, according to the results of the kinetic studies. The lability of the complexes strongly depends on the length and position of the alkyl substituent of the ligand. The dissociation of the Ni(II) complex gives a heteroligand complex at low concentrations of acid, but the complex undergoes full protolytic dissociation at higher concentrations of acid. The dissociation of the complex of Cu(II) is an equilibrium process, involving formation of the protonated form of the ligand.     

Isoelectric focusing of small non-covalent metal species from plants

IEF is known as a powerful electrophoretic separation technique for amphoteric molecules, in particular for proteins. The objective of the present work is to prove the suitability of IEF also for the separation of small, non-covalent metal species. Investigations are performed with copper-glutathione complexes, with the synthetic ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) and respective metal complexes (Fe, Ga, Al, Ni, Zn), and with the phytosiderophore 2'-deoxymugineic acid (DMA) and its ferric complex. It is shown that ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid and DMA species are stable during preparative scale IEF, whereas copper-glutathione dissociates considerably. It is also shown that preparative scale IEF can be applied successfully to isolate ferric DMA from real plant samples, and that multidimensional separations are possible by combining preparative scale IEF with subsequent HPLC-MS analysis. Focusing of free ligands and respective metal complexes with di- and trivalent metals results in different pIs, but CIEF is usually needed for a reliable estimation of pI values. Limitations of the proposed methods (preparative IEF and CIEF) and consequences of the results with respect to metal speciation in plants are discussed.     

Determination of Dynamic Metal Complexes and their Diffusion Coefficients in the Presence of Different Humic Substances by Combining Two Analytical Techniques

The combined use of a competing ligand exchange (CLE) method and a diffusive gradient in thin films (DGT) technique in a quasi-labile system provides a better understanding of dynamic metal (Cu and Ni) complexes in the presence of humic substances of different origins. The CLE and DGT techniques provide total labile (dynamic) metal complexes (Cu and Ni) and their dissociation rate constants in environmental systems. DGT was found to estimate lower concentrations of labile metal complexes than CLE. These discrepancies were caused by diffusion controlled metal flux (towards the binding resin gel) in the diffusive gel of DGT. The interactions of Cu and Ni with humic acids are stronger than their interactions with fulvic acid and natural organic matter. Changes in the lability of Ni and Cu complexes (complexed with humic substances of different origins) with the changing analytical detection window indicate that the complexes of these metals were formed with different binding sites with diverse binding energies in the humic substances. The combination of these two techniques was found to be very useful in determining diffusion coefficients of labile metal-humate complexes in quasi-labile systems. The values of diffusion coefficients of labile Ni and Cu complexes determined in this study are in good agreement with limited results from the literature. This finding is novel and can be very useful in further improving our understanding of the metal-humate interactions in natural environments.     

Effect of buffer,electric field,and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis

The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.     

High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts

High-efficiency capillary isoelectric focusing (cIEF) separations of protein complexes obtained from soluble protein fractions are demonstrated. Size-exclusion chromatography was used as a first dimension separation to fractionate putative protein complexes with apparent molecular masses of up to 1,500,000 from an Escherichia coli cytosolic fraction. Non-denaturing cIEF separations using highly hydrophilic polymer-coated capillaries constituted the second dimension. The conditions developed produced reproducible and high-efficiency separations, corresponding to approximately 2 x 10(6) theoretical plates and peak capacities of approximately 10(3) for pH 3-10 cIEF separations in 65 cm long capillaries. Combination of the two non-denaturing separation dimensions permitted isolation and analysis of individual protein complexes from complicated biological samples. Studies indicated that many E. coli complexes were stable on the time scale of the cIEF separations, but were degraded upon more extended periods of storage on ice, necessitating rapid sample processing and fast analysis techniques.     

Online analysis of europium and gadolinium species complexed or uncomplexed with humic acid by capillary electrophoresis–inductively coupled plasma mass spectrometry

Detailed information on the geochemical behavior of radioactive and toxic metal ions under environmental conditions (in geological matrices and aquifer systems) is needed in order to assess the long-term safety of waste repositories. This includes knowledge of the mechanisms of relevant geochemical reactions, as well as associated thermodynamic and kinetic data. Several previous studies have shown that humic acid can play an important role in the immobilization or mobilization of metal ions due to complexation and colloid formation. In our project we investigate the complexation behavior of (purified Aldrich) humic acid and its influence on the migration of the lanthanides europium and gadolinium (homologs of the actinides americium and curium) in the ternary system consisting of these heavy metals, humic acid and kaolinite (KGa-1b) under almost natural conditions. Capillary electrophoresis (CE, Beckman Coulter P/ACE MDQ), with its excellent separation performance, was hyphenated with a homemade interface to inductively coupled plasma mass spectrometry (ICP–MS, VG Elemental PlasmaQuad 3) giving a system that is highly sensitive to the rare-earth element species of europium and gadolinium with humic acid. The humic acid used was also halogenated with iodine, which acted as an ICP–MS marker. To couple CE to ICP–MS, a fused silica CE capillary was flexibly fitted into a MicroMist 50 μl nebulizer with a Cinnabar cyclonic spray chamber in the external homemade interface. The chamber was chilled to a temperature of 4 °C to optimize the sensitivity. 200 ppb of cesium were added to the CE separation buffer so that the capillary flow could be observed. A make-up fluid including 4 ppb Ho as an internal standard was combined with the flow from the capillary within the interface in order to get a fluid throughput high enough to maintain continuous nebulization. Very low detection limits were achieved: 125 ppt for ¹⁵³Eu and 250 ppt for ¹⁵⁸Gd. Using this optimized CE–ICP–MS coupling system it was possible to quantify metal concentrations from the detection limit up to approximately 1 ppm (the linear range). This set-up was used to separate metal/humic acid-species in a 100 mM acetic acid/10 mM acetate buffer system. Using humic acid as the complexing ligand, uncomplexed metal ion species could be separated from metal–humate complexes on a time-resolved scale.      

Interaction of molecular hydrogen with open transition metal centers for enhanced binding in metal-organic frameworks: a computational study

Molecular hydrogen is known to form stable, "nonclassical" sigma complexes with transition metal centers that are stabilized by donor-acceptor interactions and electrostatics. In this computational study, we establish that strong H2 sorption sites can be obtained in metal-organic frameworks by incorporating open transition metal sites on the organic linkers. Using density functional theory and energy decomposition analysis, we investigate the nature and characteristics of the H2 interaction with models of exposed open metal binding sites {half-sandwich piano-stool shaped complexes of the form (Arene)ML(3- n)(H2)n [M=Cr, Mo, V(-), Mn(+); Arene = C6H5X (X=H, F, Cl, OCH3, NH2, CH3, CF3) or C6H3Y2X (Y=COOH, X=CF3, Cl; L=CO; n=1-3]}. The metal-H2 bond dissociation energy of the studied complexes is calculated to be between 48 and 84 kJ/mol, based on the introduction of arene substituents, changes to the metal core, and of charge-balancing ligands. Thus, design of the binding site controls the H2 binding affinity and could be potentially used to control the magnitude of the H2 interaction energy to achieve reversible sorption characteristics at ambient conditions. Energy decomposition analysis illuminates both the possibilities and present challenges associated with rational materials design.     

Dynamic unfolding of a regulatory subunit of cAMP-dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability

Characterization of the unfolding dynamics of a recombinant type IA regulatory subunit (RIalpha) of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (cAPK) was examined by CE with UV detection. Electrophoretic separation of RIalpha by CE in a buffer devoid of cAMP resulted in rapid dissociation of the complex from the original sample due to the high negative mobility of the ligand relative to receptor. This process enabled in-capillary generation of cAMP-stripped RIalpha, which was used to estimate the apparent dissociation constant (Kd) of 0.6 +/- 0.2 microM. A comparison of RIalpha dynamic unfolding processes with urea denaturation was performed by CE with (i.e., RIalpha-cAMP) and without (i.e., cAMP-stripped RIalpha) excess cAMP in the buffer during electromigration. The presence of cAMP in the buffer confirmed greater stabilization of the protein, as reflected by a higher standard free energy change (DeltaG(U) degrees) of 10.1 +/- 0.5 kcal x mol(+1) and greater cooperativity in unfolding (m) of -2.30 +/- 0.11 kcal x mol(-1) M(-1). CE offers a rapid, yet versatile platform for probing the thermodynamics of cAPK and other types of receptor-ligand complexes in free solution.     

Free and bound phenolic compounds in barley (Hordeum vulgare L.) flours. evaluation of the extraction capability of different solvent mixtures and pressurized liquid methods by micellar electrokinetic chromatography and spectrophotometry

The solution speciation of metals is a critical parameter controlling the bioavailability, solution-solid phase distribution and transport of metals in soils. The natural metal-complexing ligands that exist in soil solution include inorganic anions, inorganic colloids, organic humic substances, amino acids (notably phytosiderophores and bacterial siderophores) and low-molecular mass organic acids. The latter two groups are of particular significance in the soil surrounding plant roots (the rhizosphere). A number of analytical methodologies, encompassing computational, spectroscopic, physico-chemical and separation techniques, have been applied to the measurement of the solution speciation of metals in the environment. However, perhaps with the exception of the determination of the free metal cation, the majority of these techniques rarely provide species specific information. High-performance liquid chromatography (HPLC) coupled to a sensitive detection system, such as inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionisation mass spectrometry (ESI-MS), offers the possibility of separating and detecting metal-organic acid complexes at the very low concentrations normally found in the soil environment. This review, therefore, critically examines the literature reporting the HPLC separation of metal-organic acid complexes with reference to thermodynamic equilibrium and kinetic considerations. The limitations of HPLC techniques (and the use of thermodynamic equilibrium calculations to validate analytical results) are discussed and the metal complex characteristics necessary for chromatographic separation are described.     

沉淀聚合法制备三聚氰胺分子印迹聚合物微球

以三聚氰胺为模板分子,以甲基丙烯酸为功能单体,乙二醇二甲基丙烯酸酯为交联剂,在乙腈-乙二醇(20∶1,V/V)混合溶剂中沉淀聚合制备了分子印迹聚合物微球.利用1H-NMR和紫外光谱方法研究了模板与功能单体相互作用情况.结果表明,三聚氰胺与甲基丙烯酸(MAA)分子通过协同氢键作用形成1∶2型氢键配合物.利用扫描电镜和红外光谱对聚合物微球的结构进行了表征.结果表明,印迹聚合物近似圆球形,粒径约为400～500 nm,且大于非印迹聚合物微球,表面存在大量的结合位点.通过静态平衡吸附实验研究了聚合物微球对模板分子的结合能力,印迹聚合物微球在4 h后逐渐达到吸附平衡,Scatchard分析表明,印迹聚合物微球主要存在两类不同的结合位点,最大表观结合量(Qmax)和平衡离解常数(Kd)分别为Qmax1=22.97μmol/g,Kd1=0.14×10-3 mol/L;Qmax2=157.65μmol/g,Kd2=2.55×10-3 mol/L,计算得出表观印迹效率和有效印迹效率分别为68%和58%.此方法合成的印迹聚合物微球对三聚氰胺有较好的结合性能,可应用于三聚氰胺的分离检测.     

Cation‐enhanced capillary electrophoresis separation of atropoisomer anions

CE was used to study the separation of the atropoisomers of four phosphoric acids and two sulfonic acids and the enantiomers of two phosphoric acids. All solutes are in their anionic forms in aqueous electrolytes. The chiral additives were two hydroxypropyl cyclodextrins (CDs) and cyclofructan 6 (CF6). The CDs were able to separate four solutes and the CF6 additive could separate only one: 1,1′‐binaphthyl‐2,2′‐diyl hydrogenphosphate (BHP). Since CF6 is able to bind with cations, nitrate of alkaline metals, Ba²⁺, and Pb²⁺ were added, greatly improving the BHP separation at the expense of longer migration times. There seems to be a link between CF6–cation‐binding constants and BHP resolution factors. Cation additions were also performed with CD selectors that are less prone to form complexes with cations. Significant improvements of enantiomer or atropoisomer separations were observed also associated with longer migration times. It is speculated that the anionic solutes associate with the added cations forming larger entities better differentiated by CDs.     

Cross-linked poly(vinyl alcohol) as permanent hydrophilic column coating for capillary electrophoresis.

A fast method for the generation of permanent hydrophilic capillary coatings for capillary electrophoresis (CE) is presented. Such interior coating is effected by treating the surface to be coated with a solution of glutaraldehyde as cross-linking agent followed by a solution of poly(vinyl alcohol) (PVA), which results in an immobilization of the polymer on the capillary surface. Applied for capillary zone electrophoresis (CZE) such capillaries coated with cross-linked PVA exhibit excellent separation performance of adsorptive analytes like basic proteins due to the reduction of analyte-wall interactions. The long-term stability of cross-linked PVA coatings could be proved in very long series of CZE separations. More than 1000 repetitive CE separations of basic proteins were performed with stable absolute migration times relative standard deviation (RSD > 1.2%) and without loss of separation efficiency. Cross-linked PVA coatings exhibit a suppressed electroosmotic flow and excellent stability over a wide pH range.     

Metal Ion Chelation Chromatography on Complexon Sorbed Stannic Silicate

Abstract

Stannic silicate has been sorbed with complexones like xylenol orange, eriochrome black T and 1,10-phenanthroline for use as a chelate ion exchanger. The sorption capacity for different metals has been worked out. Kd values have been determined. Xylenol orange was used for the separation of Th(IV) from Cd(II) and Zn(II) and Cu(II) from Cd(II) and Zn(II), 1,10 phenanthroline for the separation of Fe(II) from Fe(III). These separations are based on the stabilities of the various complexes formed by the interaction of metal inns with complexones. By elution of metal ions which forms less stable complexes with the complexones no evidence of complexing agent in the eluate was found.     

Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye: noncovalent labeling and nonequilibrium measurement of association constants

In response to a growing interest in the use of smaller, faster microchip (mu-chip) methods for the separation of proteins, advancements are proposed that employ the asymmetric squarylium dye Red-1c as a noncovalent label in mu-chip CE separations. This work compares on-column and precolumn labeling methods for the proteins BSA, beta-lactoglobulin B (beta-LB), and alpha-lactalbumin (alpha-LA). Nonequilibrium CE of equilibrium mixtures (NECEEM) represents an efficient method to determine equilibrium parameters associated with the formation of intermolecular complexes, such as those formed between the dye and proteins in this work, and it allows for the use of weak affinity probes in protein quantitation. In particular, nonequilibrium methods employing both mu-chip and conventional CE systems were implemented to determine association constants governing the formation of noncovalent complexes of the red luminescent squarylium dye Red-1c with BSA and beta-LB. By our mu-chip NECEEM method, the association constants K(assoc) for beta-LB and BSA complexes with Red-1c were found to be 3.53 x 10(3) and 1.65 x 10(5) M(-1), respectively, whereas association constants found by our conventional CE-LIF NECEEM method for these same protein-dye systems were some ten times higher. Despite discrepancies between the two methods, both confirmed the preferential interaction of Red-1c with BSA. In addition, the effect of protein concentration on measured association constant was assessed by conventional CE methods. Although a small decrease in K(assoc) was observed with the increase in protein concentration, our studies indicate that absolute protein concentration may affect the equilibrium determination less than the relative concentration of protein-to-dye.     

Separation of positional isomers by cucurbit[7]uril-mediated capillary electrophoresis

A novel macrocyclic molecule, cucurbit[7]uril (CB[7]) was for the first time employed as an additive in capillary electrophoresis (CE). In similarity to other macrocyclic molecules, such as crown ethers, cyclodextrins (CDs) and calixarenes, CB[7] can form inclusion complexes with a variety of guest molecules due to its inner cavity. Thus, it can be used like other macrocyclic molecules to manipulate selectivities in CE. During the running process, CB[7] bears a positive charge under the studied pH range (pH 2.5-7) and can be adsorbed onto the inner wall of a fused-silica capillary, leading to a reversal of the electroosmotic flow (EOF). Electrophoretic behaviors of nitrotoluene, nitrophenol, nitroaniline, and methylaniline isomers were studied under various conditions. The electrophoretic separations of the isomers can be accomplished with a buffer containing CB[7]. Furthermore, a probable separation mechanism in the presence of CB[7] was also proposed.