Open-tubular capillary electrochromatography/electrospray ionization-mass spectrometry using polymeric surfactant as a stationary phase coating

We describe the use of the polymeric surfactant poly(sodium undecylenic sulfate) (poly-SUS) as a stationary phase coating in open-tubular capillary electrochromatography (OT-CEC) coupled with electrospray ionization-mass spectrometry (ESI-MS) for the analysis of beta-blocker and benzodiazepine analytes. The production of a polymeric surfactant coating on the capillary inner wall involves (i) adsorption of the cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) to the inner surface of capillary, and (ii) adsorption of the negatively charged poly-SUS onto the cationic polymer layer via strong physical interaction of the two polymer layers. As compared with micellar electrokinetic chromatography (MEKC) coupled with ESI-MS, the main advantage of this proposed method is minimization of introduction of the monomeric or polymeric surfactant into the mass spectrometer, thus avoiding the interference of the nonvolatile micelle in ESI-MS. The effects of buffer pH and applied voltage on the separation of the analytes are also discussed. Under optimum conditions, four of the five beta-blockers and four benzodiazepines are separated.     

Open-tubular capillary electrochromatography using a capillary coated with octadecylamine-capped gold nanoparticles

Octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and characterized by using UV-Vis adsorption spectrum, transmission electron chromatography (TEM), SEM, and FT-IR. A simple but robust hydrophobic coating was easily developed by flushing a capillary with a solution of ODA-Au-NPs, because the positive charges were carried by the nanoparticles which strongly adsorb to the negatively charged inner surface of a fused-silica capillary via electrostatic and hydrophobic interactions. The chromatographic characteristics of the coated capillary was investigated by varying the experimental parameters such as buffer pH, buffer concentration, and percentage of organic modifier in the mobile phase. The results show that (i) resolution between thiourea and naphthalene is almost the same when comparing the electrochromatograms obtained using pH 7 buffer as mobile phase after and before the capillary column was operated using pH 11 and 3 mobile phase; (ii) no significant changes in retention time and deterioration in peak efficiency were found after 60 runs of test aromatic mixtures; and (iii) column efficiency up to 189 000 theoretical plates/meter for testosterone was obtained. All of the results indicated that the coating could act as a stable stationary phase for open tubular CEC as well as for bioanalysis.     

Open-tubular electrochromatography of organic phosphates on a sapphyrin-modified capillary.

Sapphyrin coating of the inner wall of the capillary results in a distinct interaction of the phosphate residue-possessing compounds as proven by a seven-membered model mixture of nucleoside mono- and diphosphates and ATP. Modification of the inner surface of the capillary not only alters the endoosmotic flow (as would have been expected) but brings about an electrochromatographic effect based on the interaction of tested phosphate moiety-bearing solutes with the immobilized sapphyrin layer. Elution of the sample can be achieved by using either 25 mM borate-acetate buffer in which monophosphates are not only separated from each other, but also selectively separated from di- and triphosphates (ATP). With the other two buffer systems tested, i.e. borate-phosphate and Tris-HCl, better selectivity (though smaller interaction with the capillary coating) was observed. The coating is relatively stable (can be used for 20 subsequent runs at least), simple to materialize, and in spite of a strong UV absorbancy of sapphyrin at the wavelength used (254 nm), decreases the limit of detection by no more than one order of magnitude as compared to the untreated capillary. Resolution factors (calculated to the preceding peak) are in most cases better in the electrochromatographic separation mode as compared to the separation in the untreated capillary, which reflects both the decrease in the electroosmotic flow and the interaction with the capillary wall coating.     

Pressurized-flow anion-exchange capillary electrochromatography using a polymeric ion-exchange stationary phase

The feasibility of using capillary columns equipped with silica frits and packed with a polymer-based anion exchanger (Dionex AS9-HC) for CEC separations of inorganic anions has been investigated. Experiments using a conventional 25 cm packed bed, and mobile phase flow that is a combination of hydrodynamic and electroosmotic flow were used to demonstrate that by varying the applied voltage (electrophoresis component) or the concentration of the competing ion in the mobile phase (ion-exchange component), considerable changes in the separation selectivity could be obtained. Using an artificial neural network, this separation system was modelled and the results obtained used to determine the optimum conditions (9 mM perchlorate and −10 kV) for the separation of eight inorganic anions. When a short (8 cm) packed bed was used, with detection immediately following the packed section, the separation of eight test analytes in under 2.2 min was possible using pressure-driven flow and a simple step voltage gradient. A more rapid separation of these analytes was obtained by only applying high voltage (−30 kV), where many of the same analytes were separated in less than 20 s and with a different separation selectivity to that obtained in conventional ion-exchange or capillary electrophoresis separations.     

Open-tubular capillary electrochromatography of small polar molecules using etched, chemically modified capillaries

The migration characteristics of small polar molecules are evaluated on etched, chemically modified capillaries with four different moieties (C5, C18, diol and cholesterol) bonded onto a silica hydride surface. The effects of pH on migration are used to determine the possible contributions of eletrophoretic mobility, electroosmotic flow (EOF) and analyte/bonded phase interactions. The EOF on etched capillaries is more complicated than on ordinary fused capillaries because it changes from anodic to cathodic as the pH is raised. A mixture of neurotransmitters and related compounds is used to further evaluate the effects of the bonded moiety on the separation properties of this particular electrophoretic format.     

Open-tubular capillary electrochromatography of bovine beta-lactoglobulin variants A and B using an aptamer stationary phase.

DNA aptamers that form a G-quartet conformation were covalently attached to a capillary surface for open-tubular capillary electrochromatographic separation of bovine beta-lactoglobulin variants A and B, which vary by 2 of their 162 amino acid residues. Separation was achieved using a 4-plane, G-quartet aptamer stationary phase with tris(hydroxymethyl)aminomethane (Tris) or phosphate buffer as the mobile phase. In control experiments, separation did not occur using either an oligonucleotide of similar base composition but which does not form a G-quartet structure, or using capillary zone electrophoresis on a bare capillary under similar experimental conditions. Separation was achieved using a capillary coated only with the covalent linker molecule. In phosphate buffer, the separations were similar for aptamer-coated and linker-only stationary phases, while in Tris buffer, retention times were almost doubled for the linker-only capillary. When Tris buffer is the mobile phase, there appears to be weaker interactions between the proteins and the stationary phase that may result in a gentler, less denaturing separation than is commonly achieved using hydrocarbon-based stationary phases.     

Polydopamine-based permanent coating capillary electrochromatography for auxin determination

A novel, simple, and economical method for the preparation of open-tubular capillary column using polydopamine coating was reported for the first time. After the capillary was filled with dopamine solution for 20h, polydopamine was formed and deposited on the inner wall of capillary as permanent coating via the oxidation of dopamine by the oxygen dissolved in the solution. Moreover, the electroosmotic flow of the coated capillaries was measured to be dependent on the repetitive coating times. The performance of the polydopamine-coated capillary electrochromatography was validated by the analysis of four auxins, indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), indole-3-acetic acid (IAA), and phenoxyacetic acid (PAA). The precisions (RSD, n=5) were in the range of 1.6-2.4% for migration time, 4.0-6.5% for peak area response, and 3.6-4.7% for peak height response for the four auxins at 1microgmL(-1) level. The detection limits were 0.185, 0.172, 0.177, and 0.259microg/mL for IBA, dCPAA, IAA, and PAA, respectively. The method was successfully used to the determination of IAA in the culture media of IAA-producing bacteria.     

Open-tubular capillary electrochromatography using capillary columns chemically bonded with the new host molecules calix[6]crown,calix[6]arene

Two host molecules, p-tert-butylcalix[6]-1,4-crown-4 and p-tert-butylcalix[6]arene were prepared and attached onto the inner surfaces of capillaries for open-tubular electrochromatography with the aid of gamma-glycidoxypropyl-trimethoxysilane (KH-560) as bridging agent. The successful bonding was confirmed by infrared (IR) results and greatly decreased electroosmotic flow (EOF). Parameters affecting separation, such as buffer pH and organic modifier were studied. The two novel stationary phases were evaluated by the separation of isomeric toluidines, a mixture of pyridine and isomeric picolines, and isomeric dihydroxybenzenes; comparisons between capillaries coated with the two stationary phases and bare capillary were investigated. The special selectivity of these two novel stationary phases showed a certain extent of supramolecular interactions between stationary phases and solutes.     

Open-tubular capillary columns with a porous layer of monolithic polymer for highly efficient and fast separations in electrochromatography

Open-tubular columns for CEC separations having inner-wall coated with a thin layer of porous monolithic polymer have been studied. A two-step process including (i) UV-initiated polymerization leading to a layer of porous poly(butyl methacrylate-co-ethylene dimethacrylate), and (ii) UV-initiated grafting of ionizable monomers appear to be well suited for the preparation of these columns. The thickness of the porous polymer layer is controlled by the percentage of monomers in the polymerization mixture and/or length of the irradiation time. The layer thickness significantly affects retention, efficiency, and resolution in open-tubular CEC. Under optimized conditions, column efficiencies up to 400,000 plates/m can be achieved. Use of higher temperature and application of pressure enables a significant acceleration of the open-tubular CEC separations.     

Microchip-based capillary electrochromatography using packed beds

Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.     

Acrylic-based high internal phase emulsion polymeric monolith for capillary electrochromatography

The use of high internal phase emulsion polymers (polyHIPEs) for CEC applications has remained relatively unexplored. A few reports exist in the literature for the preparation of similar structures. In this study, polyHIPEs having high porosity, and interconnected open-cell structure, were introduced and evaluated as stationary phase for CEC. The polyHIPE monolithic columns were prepared by the in situ polymerization of isodecylacrylate (IDA) and divinylbenzene (DVB) in the continuous phase of a high internal phase emulsion (HIPE). Due to its well-defined polyHIPE structure with interconnected micron size spherical voids, the columns synthesized with different initiator concentrations were successfully used for the separation of alkylbenzenes. Furthermore, the columns indicated a strong electroosmotic flow (EOF) without any additional EOF generating monomer probably due to the presence of ionizable sulfate groups coming from the water-soluble initiator used in the preparation of polyHIPE matrix. The best chromatographic performance in the separation of alkylbenzenes was achieved by using 70% ACN in the mobile phase with high column efficiency (up to 200 000 plates/m).     

Separation of nucleosides using capillary electrochromatography

The analysis of nucleosides and nucleotides have in most cases been performed by HPLC using either reversed-phase HPLC with gradient elution or using reversed-phase ion-pair chromatography. In this paper we have explored the possibility of using capillary electrochromatography (CEC) in order to avoid the use of gradients or ion-pairing reagents. CEC is in many ways comparable to HPLC, but CEC is theoretically able to provide better separations due to the higher efficiency caused by the flowfront being more plug-like as also is the case in CE, which is to be compared to the more parabolic flow observed in HPLC. The separation of six nucleosides (adenosine, cytidine, guanosine, inosine, thymidine and uridine) was investigated with respect to concentration of buffers, pH, amount of acetonitrile, temperature and voltage in order to optimise the separation. Baseline separation was achieved for the six nucleosides in less than 13 min using a background electrolyte consisting of (5 mM acetic acid, 3 mM triethylamine, pH 5.0)-acetonitrile (92:8, v/v).     

Separation of benzodiazepines using capillary electrochromatography

Benzodiazepines are often used for the treatment of epilepsy, convulsions, and many psychiatric disorders. The widespread use of this class of drugs has occasionally raised concern about recreational benzodiazepine abuse and has led to the erroneous impression that benzodiazepines have a relatively high abuse liability among recreational drug users. Therefore, the separation and identification of these compounds is of great interest. In general, the separation of benzodiazepines is performed using high-performance liquid chromatography (HPLC). Recently, capillary electrochromatography, which combines the high efficiency of capillary zone electrophoresis and the high selectivity of HPLC, has gained much attention. The focus of the work reported here is the use of a 40-cm packed bed of Reliasil 3- microm C(18) stationary phase to separate seven benzodiazepines. Optimal conditions are established by varying the mobile phase, amount of organic modifier, buffer concentration, applied voltage, and column temperature. A mobile phase composition of Tris-HCl (pH 8)-acetonitrile (60:40), an electrolyte concentration of 30mM, and a temperature of 15 degrees C with an applied voltage of 20 kV proves to be optimum. In addition, the method developed here is applied to the characterization of oxazepam in a standard urine sample.     

Affinity electrochromatography of acidic drugs using a liposome-modified capillary

Liposomes can be effectively deposited on the inner surface of a capillary wall by flushing the electrophoretic system with a liposome suspension followed by air-drying of the capillary and removal of the excess of loosely bound liposomes by a 0.1 M NaOH wash. It was demonstrated that capillaries prepared in this way could be used for studies of analyte (drug)–liposome binding. The results were expressed as free binding energy changes [Δ(ΔG⁰)] relatively to an arbitrarily selected standard (acetylsalicylic acid). The results were compared to [Δ(ΔG⁰)] changes obtained from binding studies effected by capillary electrophoresis using a stable liposome plug in a capillary with minimized endoosmotic flow. Good agreement of data reported in the literature (without correction for the residual endoosmotic flow), our previous data obtained in a similar way (however, after the correction for the residual endoosmotic flow) and data obtained by the immobilized liposome affinity electrochromatography reported in this communication was achieved.     

Open-tubular gas chromatography using capillary coated with octadecylamine-capped gold nanoparticles

The octadecylamine-capped gold nanoparticles (ODA-Au-NPs) were prepared and directly used to coat the capillary wall. The hydrophobic coating acted as the stationary phase for open-tubular gas chromatography (OTGC). The ODA-Au-NPs can be adsorbed tightly onto the inner surface of fused silica capillary column via electrostatic interaction and enhanced interaction of van der Waals between gold nanoparticles and the capillary wall. Thus, the modification of the inner surface of capillary column by ODA-Au-NPs can be achieved simply by flushing the capillary with a solution of ODA-Au-NPs and the resulted ODA-Au-NPs coating is very stable. No perceptible degradation in the ODA-Au-NPs-based separation was observed after ∼1900 sample runs. This type of columns also provided excellent chromatographic performances: high number of theoretical plates, outstanding run-to-run and column-to-column reproducibility, and high selectivity for a wide range of test mixtures. An efficiency of 2474 theoretical plates per meter for chlorobenzene was obtained on an ODA-Au-NPs-modified 1.6 m × 100 μm i.d. fused silica capillary column.     

Miniaturization of capillary electrochromatography using an ultrashort capillary column

The instrumentation for miniaturization of capillary electrochromatography was devised and an injection method for this apparatus was proposed. By using an ultra short capillary column (15 mm packed length, 36 mm total length, 75 microm inner diameter, packed with cation exchange supports), the separation of five biochemical compounds was performed within 1 min. The high separation efficiency of 9780 plates was achieved by using an ultrashort capillary column. The miniaturized instrumentation for capillary electrochromatography might be utilized as one of the possible methods in microfabricated analysis or in an alternative approach to it.     

Polydopamine as an adhesive coating for open tubular capillary electrochromatography

Polydopamine (PolyD) coating was used as an adhesive layer in the preparation of biological stationary phases for open tubular capillary electrochromatography (OT-CEC). The influence of coating solution freshness, coating time, temperature and dopamine hydrochloride concentration on the PolyD layer formation was studied. The performance of the polyD coating was monitored by measuring the electro-osmotic flow in coated capillaries. Following polyD coating of the capillary, secondary layer material (e.g. cell membrane solutions, phospholipid mixtures or mitochondria) was inserted into the capillary for at least 1?h. The performance of these double-coated capillaries (a polyD layer+a biological material layer) was compared with capillaries containing the respective biological material directly attached to the capillary wall. The study reveals that the presence of polyD layer in fused silica capillaries improves the performance of lipid and membrane fragment coatings in capillaries. At the same time, the thickness of the polyD layer does not have marked impact on the secondary coatings. Analysis with test analytes demonstrated that double-coated capillaries can be applied to study membrane-drug interactions.     

Development and application of polymeric monolithic stationary phases for capillary electrochromatography

Monolithic columns for capillary electrochromatography are receiving quite remarkable attention. This review summarizes results excerpted from numerous papers concerning this rapidly growing area with a focus on monoliths prepared from synthetic polymers. Both the simplicity of the in situ preparation and the large number of readily available chemistries make the monolithic separation media a vital alternative to capillary columns packed with particulate materials. Therefore, they are now a well-established stationary phase format in the field of capillary electrochromatography. A wide variety of synthetic approaches as well as materials used for the preparation of the monolithic stationary phases are presented in detail. The analytical potential of these columns is demonstrated with separations involving various families of compounds and different chromatographic modes.     

Packed-column capillary electrochromatography and capillary electrochromatography-mass spectrometry using a lithocholic acid stationary phase

The preparation and characterization of a novel lithocholic acid (LCA)-based liquid crystalline (LC) stationary phase (SP) suitable for application in packed-column CEC and CEC coupled to MS is described. The extent of bonding reactions of LCA-SP was assessed using 1H-NMR, 13C-NMR and elemental analysis. This characterization is followed by application of the LCA-SP for separation of beta-blockers, phenylethylamines (PEAs), polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Using the optimum mobile phase operating conditions (pH 3.0-4.5, 10 mM ammonium acetate, 85% v/v ACN), a comparison of the chromatographic ability of the aminopropyl silica phase vs. the LCA-bonded phase was conducted. The results showed improved selectivity for all test analytes using the latter phase. For example, the CEC-MS of beta-blockers demonstrated that the LCA-bonded phase provides separation of six out of seven beta-blockers, whereas the amino silica phase provides four peaks of several co-eluting beta-blockers. For the CEC-MS analysis of PEAs, the LCA-bonded phase showed improved resolution and different selectivity as compared to the aminopropyl phase. An evaluation of the retention trends for PEAs on both phases suggested that the PEAs were retained based on varying degree of hydroxyl substitution on the aromatic ring. In addition, the MS characterization shows several PEAs fragment in the electrospray either by loss of an alkyl group and/or by loss of H2O. Finally, the LCA-bonded phase displayed significantly higher separation selectivity for PAHs and PCBs as compared to the amino silica phase.     

Enantiomeric impurity determination of levetiracetam using capillary electrochromatography

CEC was used to develop a method for the enantiomeric excess determination of levetiracetam, an antiepileptic drug. Different types of calibration curve were evaluated for use in the range between 0.01 and 1 mg/mL when aniracetam was used as an internal standard. The method gave comparable results when only the areas of the impurity were used in the calibration curve. The predicted detection and quantification limits from the S/N were 1.1 and 3.6 microg/mL, respectively. However, experimental results showed that LOD and LOQ were underestimated. Repeatability of injection was demonstrated by the RSD values obtained for retention time, resolution, ratios of the areas impurity/internal standard, and areas of impurity and internal standard individually, which were below or equal to 9.30%. The between-days variability experiments indicated that it is better to make a calibration curve daily. The finally selected calibration curves were used to test the accuracy of the developed method on bulk samples and Keppra tablets containing 250 mg levetiracetam. Both selected calibration curves performed similarly. The one using the internal standard information gave overall recoveries between 88 and 118%, while the one using areas gave results between 84 and 118%.