Electrokinetic Chromatographic Separation Research Articles

Hydrophobic deep eutectic solvents as pseudo-stationary phases in capillary electrokinetic chromatography: An explorative study

Research into the application of deep eutectic solvents (DESs) in capillary electrophoresis (CE) is still in its infancy. Even so, a controversy has already arisen over the “true role” of DESs in CE. This is because the DESs employed previously were all hydrophilic, and it is therefore reasonable to speculate that they may be virtually completely dissociated into its initial components (hydrogen-bond acceptor (HBA) and hydrogen-bond donor (HBD)) in aqueous CE buffers. Since there seems to be no essential difference in CE performance between adding a DES and adding its two constituents separately (HBA and HBD) in a running buffer, the significance of using DESs in these CE systems has not been clearly established. In this context, this study established the first DES-based CE system in which a DES really worked as a single entity. To achieve this goal a hydrophobic DES (HDES), (−)-menthol:octanoic acid, was employed to act as a pseudo-stationary phase (PSP) in electrokinetic chromatography (EKC) mode. We found that the HDES is capable of forming stable aggregates (micro-droplets) which can act as a PSP in the running buffer. The novel HDES-type PSP exhibited impressive selectivities toward several groups of analogues, homologues, and isomers via hydrophobic mechanism. It can be used either solely for direct EKC separation, or in combination with traditional PSPs (e.g., cyclodextrins). As a proof-of-concept study, we have demonstrated that the synergistic effect generated in an “HDES + Hydroxypropyl-γ-cyclodextrin” dual-PSP system allowed an excellent separation of a simulated complex mixture consisting of 13 aromatic acids.

Ionic liquids in electrokinetic chromatography

There is an interest in the application of ionic liquids as additives into the separation media to improve achiral and chiral separations in electrokinetic chromatography (EKC). This review will critically discuss the developments on the use of ionic liquids in the different modes of EKC during the last five years (2015-mid 2020). A healthy number of 48 research articles searched through Scopus were categorised into two: ionic liquids as sole pseudophase (micelles, microemulsions, ligand exchange pseudophase or molecular pseudophase) and ionic liquids with pseudophase (achiral or chiral). More than half of the papers dealt with chiral separations that were mostly facilitated by another additive or pseudophase. The role of ionic liquids for improvement of separations were analysed, and we provided some recommendations for further investigations. Finally, the use of ionic liquids in different on-line sample concentration or stacking methods (i.e., field enhancement and sweeping) was briefly discussed.

Extraction and preconcentration of compounds from the l-tyrosine metabolic pathway prior to their micellar electrokinetic chromatography separation

The prominent biological effects of adrenaline (A), noradrenaline (NA) and dopamine (DA) as well as the clinical importance of their metabolites (such as dihydroxyphenylacetic acid (DOPAC), methoxy‑4-hydroxyphenyl glycol (MHPG), dihydroxyphenylglycol (DHPG), metanephrine (M), normetanephrine (NM), vanillylmandelic acid (VMA), homovanillic acid (HVA)) have forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. For this reason, the three most popular extraction techniques (dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME)) were tested. Micellar electrokinetic chromatography (MEKC) – a mode of capillary electrophoresis – with a diode array detector (DAD) was applied to assess the extraction efficiency. Next, the enrichment factor (EF) of each applied method was calculated in respect to standard mixtures of the analytes at the same concentration levels. The EF results of seven selected metabolites of biogenic amines (BAs) from urine after sample preparation procedures based on twenty-five different protocols (one DLLME, thirteen SPE and eleven SPME) were calculated and compared using hierarchical cluster analysis (HCA). The SPE as well as SPME procedures were proved to be the most effective approaches for the simultaneous extraction of the chosen compounds. Moreover, an ionic liquid (IL) – 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide – added to methanol in SPME additionally could successfully improve the extraction efficiency. It was also confirmed that the HCA approach could be considered a supportive tool in the selection of a suitable sample preparation procedure for that group of endogenous substances.

Chiral micellar electrokinetic chromatographic separation for determination of L- and D-primary amines released from murine islets of Langerhans.

D-amino acids have been located in various tissues including the endocrine portion of the pancreas, the islets of Langerhans. D-Serine (D-Ser), is of particular interest since it is an agonist for the ionotropic N-methyl-D-aspartate receptors. To examine the potential release of D-Ser and other D-amino acids from islets, a chiral micellar electrokinetic chromatography method was developed by derivatizing primary amines with 2,3-naphthalenedicarboxaldehyde and to achieve resolution of the enantiomers, two surfactants were used in the separation, sodium dodecyl sulfate and sodium deoxycholate. With the optimized conditions, 7 of 13 enantiomeric pairs that were tested had greater than baseline resolution, while the resolution of numerous other L-amino acids and small molecules were maintained. For the 17 compounds that were fully resolved, limits of detection were less than 10 nM. The resulting optimized separation method produced high efficiency peaks, with an average of 300,000 theoretical plates per peak and a peak capacity of 120. The method was used to examine the release of small molecules from groups of 50 murine islets of Langerhans. A peak was detected from islets incubated with 20 mM glucose that co-migrated with a D-Ser standard, although its level was below the quantifiable limit.

Study of aldehyde oxidase by micellar electrokinetic chromatography separation of O6 -benzylguanine and 8-oxo-O6 -benzylguanine.

A separation method for O6 -benzylguanine (O6 -BG) and 8-oxo-O6 -benzylguanine (8-oxo-O6 -BG) is developed by using MEKC. This study includes the optimization of separation and incubation parameters for both off-line and on-line procedures. The BGE consisted of 25 mM sodium phosphate buffer-methanol (70:30, v/v), apparent pH 7.4, in which SDS and methyl-β-cyclodextrin were dissolved yielding final concentrations of 50 and 15 mM, respectively. Separations were performed at 15 kV using an untreated fused-silica capillary (40 cm length, effective length is 30 cm) with the detection wavelength at 195 nm. The capillary was kept at 15°C. Good performances were demonstrated for the repeatability and linearity. The LOQ was determined to be 14 μM for 8-oxo-O6 -BG (S/N = 10). The accuracy values showed a bias of +7.9% for 50 μM and -7.0% for 100 μM. Premix and transverse diffusion of laminar flow profiles (TDLFP) methods were used for on-line mixing and reaction of the substrate O6 -BG with aldehyde oxidase. Both procedures were successful in mixing as well as subsequent separation of the substrate and the metabolite, while the repeatability of TDLFP (14.7%(n= 3)) was much better than the premix technique.

Evaluation of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) cationic polymer capillary coating for capillary electrophoresis and electrokinetic chromatography separations.

Capillary electrophoresis and electrokinetic chromatography are typically carried out in unmodified fused-silica capillaries under conditions that result in a strong negative zeta potential at the capillary wall and a robust cathodic electroosmotic flow. Modification of the capillary wall to reverse the zeta potential and mask silanol sites can improve separation performance by reducing or eliminating analyte adsorption, and is essential when conducting electrokinetic chromatography separations with cationic latex nanoparticle pseudo-stationary phases. Semipermanent modification of the capillary walls by coating with cationic polymers has proven to be facile and effective. In this study, poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymers were synthesized by reversible addition-fragmentation chain transfer polymerization and used as physically adsorbed semipermanent coatings for capillary electrophoresis and electrokinetic chromatography separations. An initial synthesis of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer coating produced strong and stable anodic electroosmotic flow of -5.7 to -5.4 × 10-4 cm2 /V⋅s over the pH range of 4-7. Significant differences in the magnitude of the electroosmotic flow and effectiveness were observed between synthetic batches, however. For electrokinetic chromatography separations, the best performing batches of poly([2-(acryloyloxy)ethyl]trimethylammonium chloride) polymer performed as well as the commercially available cationic polymer polyethyleneimine, whereas polydiallylammonium chloride and hexadimethrine bromide did not perform well.

Determination of selected synthetic cannabinoids and their metabolites by micellar electrokinetic chromatography – mass spectrometry employing perfluoroheptanoic acid-based micellar phase

Perfluoroheptanoic acid was employed as a volatile micellar phase in background electrolyte for micellar electrokinetic chromatography–tandem mass spectrometry separation and determination of 15 selected naphthoyl- and phenylacetylindole- synthetic cannabinoids and main metabolites derived from JWH-018, JWH-019, JWH-073, JWH-200 and JWH-250. The influence of concentration of perfluoroheptanoic acid in background electrolytes on the separation was studied as well as the influence of perfluoroheptanoic acid on mass spectrometry detection. The background electrolyte consisted of 75mM perfluoroheptanoic acid, 150mM ammonium hydroxide pH 9.2 with 10% (v/v) propane-2-ol allowed micellar electrokinetic chromatography separation together with mass spectrometry identification of the studied parent synthetic cannabinoids and their metabolites. The limits of detection of studied synthetic cannabinoids and metabolites were in the range from 0.9ng/mL for JWH-073 to 3.0ng/mL for JWH-200 employing liquid–liquid extraction. The developed method was applied on the separation and identification of studied analytes after liquid–liquid extraction of spiked urine and serum samples to demonstrate the potential of the method applicability for forensic and toxicological purposes.

Amperometric detection in the presence of carbon nanotubes dispersed in background electrolyte: Evaluating its suitability for capillary electrokinetic chromatography separations of polyphenolic compounds.

This work reports on the positive effects observed upon both the separation and analytical signals in electrophoretic separations of selected phenolic compounds when using aqueous BGE containing carbon nanotubes (CNTs) in connection to electrochemical detection (ECD). The influence of the presence of surfactant-coated CNTs in the BGE upon the amperometric response of probe compounds was evaluated under hydrodynamic regime in capillary flow injection experiments as well as electrophoretic separations. Among the surfactants employed to disperse CNTs within BGE, SDS shows the best results in terms of dispersion stability and degree of dispersion of the CNTs. ECD allows working with BGEs containing CNTs concentrations of, at least, 24.0 mg/L without increasing of baseline noise, on the opposite to that reported when using UV-visible detection, and the presence of CNTs in the BGE improves the electrochemical response of some of the tested compounds. These benefits were reflected in higher sensibility in the electrochemical signal and additional improved resolution in the electrophoretic separation of (±)-catechin and sinapic acid when using these BGE containing CNTs.

Investigation of the enantiomerization barriers of the phthalimidone derivatives EM12 and lenalidomide by dynamic electrokinetic chromatography.

The phthalimidone derivatives EM12 and lenalidomide, which are both structurally related to thalidomide, are highly interesting drugs and very recently lenalidomide attracted great attention as an antitumor and immune-modulating drug in the therapy for multiple myeloma. EM12 and lenalidomide are chiral, and the stereogenic carbon C-3 in the piperidine-2,6-dione moiety of these phthalimidone derivatives is prone to interconversion due to keto-enol tautomerization. The knowledge of the enantiomerization barrier is mandatory for pharmacokinetic studies and to develop a tailored therapy using the enantiopure or racemic drug. Here, we used dynamic EKC in combination with direct-calculation methods to determine the enantiomerization barriers of EM12 and lenalidomide. The separations of the enantiomers of EM12 and lenalidomide were performed in 50 mM aqueous disodium hydrogen phosphate buffer at pH 8 and 50 mM aqueous sodium tetraborate buffer at pH 9.3, respectively, using 20 mg/mL heptakis-(2,3-diacetyl-6-sulfato)-β-CD as a chiral additive. Enantiomerization of the compounds during the electrokinetic chromatographic separation resulted in pronounced plateau formation between the well-separated enantiomers. Peak form analysis of the experimentally obtained interconversion profiles yielded the enantiomerization rate constants k1 of EM12 and lenalidomide as well as the kinetic activation parameters ΔG(‡), ΔH(‡‡), and ΔS(‡) of enantiomerization by the evaluation of temperature-dependent measurements. The enantiomerization barrier ΔG(‡) was determined to be 98.3 ± 1.0 kJ/mol; the activation parameters ΔH(‡) = 46.1 ± 2.4 kJ/mol and ΔS(‡) = -170 ± 61 J/(K·mol) for EM12 and ΔG(‡) = 91.5 ± 1.0 kJ/mol, ΔH(‡) = 62.4 ± 5.4 kJ/mol, and ΔS(‡) = -98 ± 7 J/(K·mol) for lenalidomide. These findings were corroborated by density functional theory calculations at the B3LYP/3-21G level of theory of the ground state and intermediates considering an enantiomerization pathway via a keto-enol tautomerism.

Microemulsion Electrokinetic Chromatographic Separation and Determination of Three Benzoic Acid Compounds

Microemulsion Electrokinetic Chromatographic Separation and Determination of Three Benzoic Acid Compounds

Novel cation selective exhaustive injection-sweeping procedure for 5-nitroimidazole determination in waters by micellar electrokinetic chromatography using dispersive liquid–liquid microextraction

A novel method consisting of cation-selective exhaustive injection and sweeping (CSEI-sweeping) as on-line preconcentration followed by a micellar electrokinetic chromatography (MEKC) separation has been developed for the determination of 5-nitroimidazoles (5-NDZ) in environmental waters. Moreover, dispersive liquid–liquid microextraction (DLLME) has been proposed for first time as sample treatment technique prior to CSEI-sweeping-MEKC. DLLME was applied to 5mL of sample. Dibromomethane (1156μL) and 2-butanol (1363μL) were employed as extractant and dispersive solvents, respectively. Salting-out effect was achieved by the addition of 16% (w/v) NaCl to the samples. After DLLME and organic solvent evaporation, the residue was redissolved in a low conductivity solvent (5mM phosphoric acid with 5% of methanol) and electrokinetically injected at 9.8kV for 632s in a bare fused-silica capillary (57.2cm, 50μm I.D.). Prior to the injection, the capillary was rinsed with 50mM phosphate buffer pH 2.5, followed by a plug of a higher conductivity buffer (100mM phosphate pH 2.5, 50mbar, 264s) and a plug of water (50mbar, 2s). Separation was carried out applying −30kV at 20°C in 44mM phosphate buffer pH 2.5, containing 8% tetrahydrofuran and 123mM sodium dodecyl sulfate. Analytical signals were monitored at 276nm. Validation was performed in river and well waters, obtaining satisfactory results in terms of linearity, precision (% RSD generally lower than 10%) and trueness (recoveries higher than 70% in almost all cases). LODs ranged from 0.61 to 2.44ng/mL. The combination of this microextraction technique with the proposed capillary electrophoresis methodology supposes a simple, sensitive and cheap alternative for 5-NDZ analyses, in accordance with the aims of green chemistry.

Microwave-Plasma Dry-Etch for Fabrication of Conducting Polymer Microelectrodes

An inexpensive dry etch technology based on a low-pressure microwave plasma generated in a countertop microwave oven is characterized for the patterning of a conductive polymer microelectrode. The etch process is described, and the microwave-generated plasma is characterized by emission spectroscopy. The plasma is generated with an atmospheric mixture of mostly nitrogen and oxygen. A 10 μm wide band microelectrode composed of PEDOT:Tosylate, an optically transparent conductive polymer, is fabricated on a plastic substrate. Conductive polymer etch rates are approximately 280-300 nm/minute. A patterned microelectrode is characterized by atomic force microscopy. The horizontal distance of a 10-90% height of a plasma-etched 150 nm thick electrode was measured to be 360 ± 200 nm (n = 5). Electrodes are further characterized using steady-state cyclic voltammetry, and they have an electroactive area congruent with their geometric area. Finally, a complete device is assembled and used as a separation platform for biogenic amines. A microwave-etched 250 μm PEDOT:PSS electrode is employed for end-channel electrochemical detection on this microchip, where an electrophoretic separation of dopamine and catechol and a micellar electrokinetic chromatography separation of dopamine and serotonin are performed. Both mass and concentration LODs are comparable to other electrochemical detectors in an end-channel configuration. With the added advantages of easy processing, robustness, optical transparency, and low cost, we expect microwave-etched polymer films to be a viable alternative to traditional electrodes.

A Molecular Dynamics Simulation Study of Two Dipeptide Based Molecular Micelles: Effect of Amino Acid Order

Molecular dynamics (MD) simulations were used to compare the structures of the chiral molecular micelles (MM) poly-(sodium undecyl-(L,L)-leucine-valine) (poly(SULV)) and poly-(sodium undecyl-(L,L)-valine-leucine) (poly (SUVL)). Both MM contained polymerized surfactant monomers tenninated by chiral dipeptide headgroups. The study was undertaken to investigate why poly(SULV) is generally a better chiral selector compared to poly(SUVL) in electrokinetic chromatography separations. When comparing poly(SULV) to poly(SUVL), poly(SULV) had the more conformational flexible dipeptide headgroup and hydrogen bond analyses revealed that the poly(SULV) headgroup conformation allowed a larger number of intramolecular hydrogen bonds to form between monomer chains. In addition, a larger number of water molecules surrounded the chiral centers of the poly(SULV) molecular micelle. Poly(SULV) was also found to have a larger solvent accessible surface area (SASA) than poly(SUVL) and fluctuations in the poly(SULV) SASA during the MD simulation allowed dynamic monomer chain motions expected to be important in chiral recognition to be identified. Finally, approximately 50% of the Na+ counterions were found in the first three solvation shells surrounding both MM, with the remainder located in the bulk. Overall the MD simulations point to both greater headgroup flexibility and solvent and analyte access to the chiral centers of the dipeptide headgroup as factors contributing to the enhanced chiral selectivity observed with poly(SULV).

Polymeric micelle as the pseudostationary phase in electrokinetic chromatography

A simple, green, and novel approach to prepare polymeric micelle with amphiphilic random copolymer P (MMA-co-MAA) via neutralization in aqueous medium has been developed, and the polymeric micelle was firstly applied as a pseudostationary phase (PSP) in electrokinetic chromatography (EKC) in the present work. Three structurally similar corticosteroids namely hydrocortisone, prednisolone, and prednisone were separated with EKC using polymeric micelle as PSP to assess the separation performance. The effects of polymeric concentration and pH on micellar microstructure including size, morphology, surface charge density and EKC performances have been investigated. TEM showed that amphiphilic random copolymers were self-assembled via neutralization to form micelles with well-defined size and shape. The size and shape of the micelle depended on the P (MMA-co-MAA) concentration and pH. At the concentration of 0.048mM and pH 9.2, the polymeric micelles were of monodispersity and perfect spheres. DLS showed the size of micelle was almost equal as polymer concentration in the range of 0.0096–0.048mM, and then enlarged sharply at the concentration larger than 0.048mM. However, the zeta potentials of micelle were nearly unchanged. The polymer concentration is also the key parameter for EKC separation. Under the optimum conditions, three analytes could be baseline separated within 7.4min. Compared with typical MEKC, MEEKC, and MEKC modified with IL ([Bmim]BF4), the developed method was more rapid, efficient, and higher selective. The separation mechanism using polymeric micelle as PSP was reverse-phase interaction. The actual cosmetic samples were analyzed with recoveries between 97.3% and 113%.

STEROID FOCUSING BY MICELLE COLLAPSE IN MICELLAR ELECTROKINETIC CHROMATOGRAPHY

Preliminary studies on the separation of neutral steroids through analyte focusing by micelle collapse (AFMC) are presented to investigate its efficiency, sensitivity and limit of detection (LOD). The focusing mechanism of AFMC is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. The sample solution of the neutral analytes (S) is prepared using sodium dodecyl sulphate (SDS) at a concentration above the critical micelle concentration (cmc) with higher conductivity than the running buffer. Normal mode–micellar electrokinetic chromatography (NM–MEKC) separation was initially performed on 100 mg/L of six neutral steroids in methanol using 20 mM SDS, 10% (v/v) methanol and sodium borate buffer (pH 9.0) with positive applied voltage of 25 kV and pressure injection of 50 mbar for 1 sec at 25°C. The same buffer condition has been applied to AFMC–MEKC, whereby the mixture of six neutral steroids was dissolved in 2 mM SDS and 250 mM sodium borate buffer which gave a conductivity ratio of 0.49. Results showed a good separation of prednisolone, prednisone, betamethasone, testosterone, 17–α–methyltestosterone, and 4–androstene–3,17–dione at 240 nm with sensitivity enhancement factors of 2.08, 1.17, 0.92, 16.9, 0.8, and 1.21 respectively. AFMC–MEKC allowed several folds improvement in sensitivity compared to NM–MEKC.

Micellar electrokinetic chromatographic separation and quantitative analysis of thorium, uranium, gold, and mercury from environmental ore samples

Summary A rapid and reliable analytical procedure has been developed for the separation and analysis of dioxouranium, thorium, gold, and mercury by micellar electrokinetic chromatography (MEKC) using bis(acetylacetone)ethylenediamine (H2AA2en) as complexing reagent. Under the optimized conditions (64 mm borate buffer containing 12.25 mm SDS and 13% acetonitrile, pH 8.0, 25 kV, 311 nm as detection wavelength) the ions were separated within 5.0 min. Linear dynamic ranges were 1–5, 8–42, 10–50, and 2–40 μg mL−1, respectively, for gold, mercury, thorium, and uranium and the respective detection limits were 0.66, 3.33, 1.6, and 3.3 μg mL−1. The applicability of method has been evaluated by application to uranium and thorium from ore samples.

Micellar electrokinetic chromatographic separation and analysis of thorium, uranium, gold, and mercury in environmental ore samples

Micellar electrokinetic chromatographic separation and analysis of thorium, uranium, gold, and mercury in environmental ore samples

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5 mM Na 2B 4O 7, 7 mM Na 2HPO 4 and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 μl serum or plasma and reconstitution of the dried extract into 100 μl of a buffer composed of 0.5 mM Na 2B 4O 7 and 0.7 mM Na 2HPO 4 (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 μg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1–6 μg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.

Selectivity patterns in micellar electrokinetic chromatography: Characterization of fluorinated and aliphatic alcohol modifiers by micellar selectivity triangle

The usefulness of the micellar selectivity triangle (MST) for prediction and interpretation of separation patterns in micellar electrokinetic chromatography (MEKC) separations is presented. In addition, we demonstrate the capability of controlling selectivity properties of micelles through addition of organic modifiers with known solvation properties as predicted by MST. The examples are modification of the hydrogen bond donor (HBD) micelle of lithium perfluorooctanesulfonate, the hydrogen bond acceptor (HBA) micelle of tetradecyltrimethylammonium bromide, and the sodium dodecyl sulfate micelles with intermediate hydrogen bonding properties with two hydrophobic organic modifiers. One is an aliphatic alcohol, n-pentanol that can act as both a HBA and a HBD; by contrast, the other organic modifier is a fluorinated alcohol, hexafluoroisopropanol that is a strong HBD modifier and would enhance the hydrogen bond donor strength of micelles. A test sample composed of 20 small organic solutes representing HBA, HBD, and non-hydrogen bond aromatic compounds was carefully selected. The trends in retention behavior of these compounds in different micelles are consistent with the selectivity patterns predicted by the MST scheme. To the best of our knowledge, this is the first report on the unique selectivity of fluorinated alcohols as modifiers in MEKC. These results demonstrate the usefulness of the MST scheme for identifying pseudo-phases with highly similar or different selectivities and can serve as a guide for judicious selection of modifiers to create pseudo-phases with desired selectivity behavior on a rational basis.

Sodium cholate aggregation and chiral recognition of the probe molecule (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate (BNDHP) observed by 1H and 31P NMR spectroscopy.

Bile salt micelles can be employed as a pseudostationary phase in micellar electrokinetic capillary chromatography (MEKC) separations of chiral analytes. To improve MEKC separations of chiral analytes, a molecular level understanding of micelle aggregation in the presence of analyte is needed. Here, aggregation of sodium cholate has been observed by exploiting the presence of a model analyte molecule. The 31P and 1H nuclear magnetic resonance spectroscopy (NMR) chemical shifts of (R,S)-1,1'-binaphthyl-2,2'-diylhydrogenphosphate ((R,S)-BNDHP), a model analyte in chiral MEKC separations, are demonstrated to be very sensitive to the aggregation state of the bile salt sodium cholate. In addition to probing micellar aggregation, the NMR spectral resolution of enantiomeric species is also stronglycorrelated with chiral separations in MEKC. In this work, the aggregation of sodium cholate in basic solutions (pH 12) has been observed over the concentration range 0-100 mM. The primary critical micelle concentration (cmc) was found to be 14 +/- 1 mM for basic solutions of sodium cholate. In addition, a primitive aggregate is clearly observed to form at 7 +/- 1 mM sodium cholate. The data also show pseudo-cmc behavior for secondary aggregation observed in the regime of 50-60 mM cholate. Finally, the H5-H7 edge of BNDHP is shown to be sensitive to chirally selective interactions with primary cholate micelles.