Microemulsion electrokinetic chromatography with on-line atmospheric pressure photoionization mass spectrometric detection

In the present paper we report, for the first time, the successful on-line coupling of microemulsion electrokinetic chromatography (MEEKC) with mass spectrometric (MS) detection using an atmospheric pressure photoionization interface. Microemulsions (MEs) including mostly volatile ingredients and classical MEs based on nonvolatile buffer components and sodium dodecyl sulfate (SDS) as surfactant were compared with respect to their compatibility with MS detection. The investigations performed revealed that MEs with up to 3% SDS and buffers containing sodium borate can be employed without significant suppression of the MS signals. A test mixture of nine substances could be separated by MEEKC using a ME consisting of 0.8% octane, 2% SDS, 6.6% butanol, and 90.6% of 20-mmol ammonium hydrogencarbonate buffer (pH 9.5). Operating the MS instrument in the MS(2) mode provided improved signal/noise ratios for analytes leading to characteristic MS-MS transitions. Thereby, limits of detection ranging between 0.5 (carbamazepine) and 5 microg mL(-1) (phenacetin) could be obtained.     

Quenched phosphorescence as a detection method in capillary electrophoretic chiral separations. Monitoring the stereoselective biodegradation of camphorquinone by yeast

Quenched phosphorescence detection of camphorquinone in cyclodextrin-based electrokinetic chromatography provides very favorable detection limits, i.e., 7 x 10(-)(7) M, 3 orders of magnitude lower than conventional UV absorption detection at 200 nm. The detection is based on the dynamic quenching by the analyte of the strong phosphorescence emission of brominated naphthalenesulfonate, under deoxygenated buffer solution conditions. This approach has been used to detect (1S)-(+)- and (1R)-(-)-camphorquinone after enantiomeric separation by CE. Although the use of the negatively charged carboxymethyl beta-cyclodextrin (CM-beta-CD) alone was not successful, the addition of a second, neutral cyclodextrin, alpha-CD, provided an adequate enantiomeric separation of camphorquinone. Using 25 mM borate buffer (pH 8.5) with 10 mM CM-beta-CD and 20 mM alpha-CD (applied voltage 20 kV, ambient temperature), the enantiomeric separation was performed in approximately 14 min. The chiral method was applied to monitor the stereoselectivity of the biotransformation of a racemic mixture of camphorquinone by yeast. It was found that the enantiomeric ratio calculated from the peak areas in the electropherogram (RSD = 5%) after 24 h of incubation decreased from 0.92 for the control solution (culture medium without yeast) to 0.24 for the culture medium; a similar ratio of 0.25 was observed for cell extract solutions. Therefore, racemic camphorquinone is enantioselectively degraded by yeast, the biodegradation of (1S)-(+)-camphorquinone being faster than that of the (1R)-(-)-enantiomer.     

Micelle stacking in micellar electrokinetic chromatography

In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.     

Sensitized enantioselective laser-induced phosphorescence detection in chiral capillary electrophoresis

The sensitivity of enantioselective cyclodextrin-induced room-temperature phosphorescence detection of camphorquinone (CQ) is enhanced using sensitization via a donor with a high extinction coefficient. The enantiomeric distinction is based on the different phosphorescence lifetimes of (+)-CQ and (-)-CQ after their complexation with α-cyclodextrin (α-CD). The collisional Dexter energy transfer from the selected donor 2,6-naphthalenedisulfonic acid (2,6-NS) to the acceptor CQ is still very efficient despite the inclusion of the acceptor into CD. For coupling to the chiral separation of (±)-CQ in cyclodextrin-based electrokinetic chromatography, the donor was added to the deoxygenated background electrolyte that consisted of 20 mM α-CD, 10 mM carboxymethyl-β-CD, and 25 mM borate buffer at pH 9.0. Time-resolved batch studies on sensitized phosphorescence show a significant enantioselectivity for (+)- and (-)-CQ in the presence of both α-CD and CM-β-CD although the lifetime difference is somewhat reduced with respect to direct excitation. The enantiomers were distinguished after their separation using an online time-resolved detection system. Excitation was performed at 266 nm with a pulsed, small-sized, quadrupled Nd:YAG laser. With 1 × 10(-5) M 2,6-NS, limits of detection of 4.1 × 10(-8) M and 5.2 × 10(-8) M were found for (+)-CQ and (-)-CQ, respectively. The online measured lifetimes were 238 ± 8 μs for (+)-CQ and 126 ± 10 μs for (-)-CQ. The method was used to determine the concentration of (±)-CQ leaching from a cured dental resin into water. The extracts contained 4.7 ± 0.1 × 10(-7) M of both (+)-CQ and (-)-CQ.     

Separation and detection of explosives on a microchip using micellar electrokinetic chromatography and indirect laser-induced fluorescence

A new approach for sensitive detection on a microfabricated chip is presented. Indirect laser-induced-fluorescence (IDLIF) was used to detect explosive compounds after separation by micellar electrokinetic chromatography (MEKC). The detection setup was used in an epifluorescence configuration with excitation provided by a near-IR diode laser operating at 750 nm. To achieve indirect detection, a low concentration of a dye (5 microM Cy7) was added to the running buffer as a visualizing agent. Using this methodology, a sample containing 14 explosives (EPA 8330 mixture) was examined. Concentrations of 1 ppm of trinitrobenzene (TNB), trinitrotoluene (TNT), dinitrobenzene (DNB), tetryl, and 2,4-dinitrotoluene (2,4-DNT) could be detected with S/N ratios between 3 and 10. Analyses showing 10 peaks, with plate numbers on the order of 60000, were completed within 60 s using a 65 mm long separation channel. The three isomers of nitrotoluene (2-, 3-, and 4-nitrotoluene) were not resolved. Additionally, the two nitramines (HMX and RDX) could only be detected at much higher concentrations, likely due to the low fluorescence quenching efficiencies of these compounds. The analysis method was also used to separate and detect nitroaromatic compounds in extracts from spiked soil samples. The presence of 1 ppm (1 microg of analyte/1 g of soil) of TNB, DNB, TNT, tetryl, 2,4-DNT, 2,6-DNT, 2-NH2-4,6-DNT, and 4-NH2-2,6-DNT could readily be detected. In the interest of increasing the sensitivity of the analysis, various on-chip injection schemes were evaluated. It was found that a 250 microm double-T injector gave a 35% increase in peak signal compared to a straight-cross injector, which is less than expected based on injected volume.     

Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Individual nuclei isolated from the human leukemia CCRF-CEM and CEM-C2 cells treated with doxorubicin (DOX) were in-column lysed with a sodium dodecyl sulfate (SDS) containing buffer, their contents were then separated by micellar electrokinetic capillary chromatography using the same lysing buffer, and the DOX content was detected by laser-induced fluorescence. Use of a microscope for the selection of one nucleus from the nuclear preparation decreases the possibility of introduction of other subcellular components that are commonly found as impurities in subcellular fractions. The presence of SDS in the running buffer made negligible the DNA's quenching effect on DOX fluorescence, which often compromises quantification of DOX by direct imaging, making it possible to carry out the first direct measurement of the doxorubicin content of isolated nuclei. On average, nuclei from CCRF-CEM and CEM/C2 cell lines contained 85 +/-64 (n = 6) and 91 +/- 51 (n =7) amol of DOX, respectively. These values correspond to 74 and 65% of the average total cellular content as determined by single-cell analysis of the corresponding cell types. It is envisioned that this approach could become an important bioanalytical tool to investigate the effect of treatments with fluorescent drugs targeting the nucleus.     

Quantification of etoposide and etoposide phosphate in human plasma by micellar electrokinetic chromatography and near-field thermal lens detection

A method employing micellar electrokinetic chromatography in combination with near-field thermal lens detection (NF-TLD) was developed for the rapid simultaneous determination of etoposide phosphate and etoposide in human blood plasma, taking teniposide as internal standard. The method developed allows the baseline separation of the solutes of interest from each other and from potential interfering matrix constituents within 4 min. The NF-TLD device employed permits detection of solutes absorbing electromagnetic radiation at lambda = 257 nm in fused-silica capillaries with 75-microm i.d. via the near-field thermal lens effect with LODs of 100 microg L(-1) for etoposide phosphate and 170 microg L(-1) for etoposide. Comparison of the performance of this detector to the performance of a commercial absorption spectrometric detector working at lambda = 257 nm showed a substantial improvement in detection limits (up to 60-fold improvement) for the near-field thermal lens detector.     

Simultaneous enantioseparation and tandem UV-MS detection of eight beta-blockers in micellar electrokinetic chromatography using a chiral molecular micelle

The feasibility of using a new and more versatile polymeric chiral surfactant, i.e., poly(sodium N-undecenoxy carbonyl-L-leucinate (poly-L-SUCL) is investigated for simultaneous enantioseparation and detection of eight structurally similar beta-blockers with tandem UV and MS detection. Three optimization approaches, i.e., direct infusion-MS, capillary zone electrophoresis-MS, and chiral micellar electrokinetic chromatography-mass spectrometry (CMEKC-MS), were investigated to optimize sheath liquid parameters, spray chamber parameters, and CMEKC separation parameters for maximum sensitivity and chiral resolution. Compared to unpolymerized micelle of L-SUCL, the use of micelle polymer (i.e., poly-L-SUCL) provided significantly higher separation efficiency, lower separation current, and higher detection sensitivity for CMEKC-ESI-MS of beta-blockers. It was also observed that, unlike monomeric L-SUCL, polymeric L-SUCL provided enantioseparation of all beta-blockers even at the lowest surfactant concentration (i.e., 5 mM poly-L-SUCL). Under optimum CMEKC and ESI-MS conditions (15 mM poly-L-SUCL, 25 mM each of NH4OAc and TEA (pH 8.0); 80% (v/v) methanol sheath liquid containing 40 mM NH4OAc (pH 8.0); sheath liquid flow rate, 5.0 microL/min; drying gas flow rate, 5 L/min; drying gas temperature, 200 degrees C; nebulizing pressure, 6 psi (0.414 bar); capillary voltage, +2.5 kV; fragmentor voltage, 85 V), baseline enantioseparation of eight beta-blockers was achieved by tandem UV (in approximately 30 min) and MS (in approximately 60 min) detection. Calibration curves for all beta-blockers were linear in the range of 0.01-0.6 mM for both CMEKC-UV and CMEKC-MS methods, but the later method provided better concentration limit of detection with similar RSD for migration time and peak areas. The CMEKC-ESI-MS method appears suitable for use as a routine procedure for high-throughput separation of beta-blockers with high sensitivity.     

Sweeping of neutral analytes in electrokinetic chromatography with high-salt-containing matrixes

The concept of sweeping neutral analytes using a high-conductivity matrix or under a reduced electric field in micellar electrokinetic chromatography (MEKC) using anionic micelles and in the presence of electroosmotic flow is presented. Three important processes are identified. First, stacking of the micelles at the cathodic interface between the sample solution (S) and background solution (BGS) zones is identified. This is then followed by the sweeping of analyte molecules by the stacked micelles that enter the S zone. Finally, the destacking of the stacked micelles at the anodic interface between the S and BGS zones occurs. The stacking of the micelles improves the focusing effect of sweeping by a factor approximately equal to the ratio of conductivities between the S and BGS zones (ratio = enhancement factor = gamma'). However, the destacking of the stacked micelles broadens the swept zones by a factor approximately equal to 1/gamma'. In effect, the focusing effect of sweeping using a matrix with equal or higher conductivity compared to the BGS will be roughly the same. The micelle stacking and destacking mechanisms are verified experimentally. This paper also provides comments on the mechanism of neutral analyte focusing under similar conditions proposed by another group.     

Analysis of illicit drugs in human urine by micellar electrokinetic capillary chromatography with on-column fast scanning polychrome absorption detection.

Using micellar electrokinetic capillary chromatography (MECC) with a borate/phosphate buffer containing 75 mM SDS (pH 9.1), common drugs of abuse and/or their metabolites, including opioids, benzoylecgonine, amphetamines, and methaqualone, can easily be analyzed. After solid-phase extraction of 5 mL of urine, drug concentrations down to about 100 ng/mL can be unambiguously monitored with on-column multiwavelength detection. Peak assignment is achieved through comparison of retention times and absorption spectra of eluting peaks with those of computer-stored model runs. The effectiveness of the approach is demonstrated with data obtained from different patient urines which tested positively for one or several drugs using nonisotopic immunoassays. Results suggest that MECC of illicit drugs is a highly specific and sensitive instrumental approach suitable for confirmation testing following a positive response of a toxicological screening procedure.     

Migration behavior of cationic solutes in micellar electrokinetic capillary chromatography.

A phenomenological approach is presented to describe the migration of cationic solutes in micellar electrokinetic capillary chromatography (MECC). The migration behavior of an organic base is complicated by the presence of an acid-base equilibrium, the ion-pairing formation between the conjugated acid of the base and the monomer surfactants, and the interactions of both the base and its conjugated acid with the micellar pseudophase. An equation was derived that allows the calculation of the migration factor of a cationic solute in MECC with anionic micelles. Two limiting cases were considered: first the cationic solute completely associates with the anionic surfactant (ion-pair formation constant, KIP, approaches infinity), and therefore there is no free charged species in the solution; second, the KIP = 0 and the free conjugated acid, BH+ migrates in the aqueous bulk solvent at its own electrophoretic velocity. An estimate for the ion-pair formation constant between cationic solutes and free surfactant can be obtained by using the model.     

Identification of PCR products using PNA amphiphiles in micellar electrokinetic chromatography

We present a method to identify single-stranded PCR products of varying lengths by hybridization of n-alkylated peptide nucleic acids (PNA amphiphiles) to the products, followed by separation with micellar electrokinetic chromatography (MEKC). These end-attached PNA amphiphiles (PNAA) partition to nonionic micelles in the running buffer (Triton X-100), linking the tagged DNA to the micellar drag-tag. This linkage shifts the electrophoretic mobility of a tagged component away from both untagged DNA and tagged DNA of different lengths. The mobility of the tagged DNA is established by its extent of partitioning to the micelle phase as well as its size relative to the attached micelle. A model is presented that can be used to determine the length of an unknown oligomer given an experimentally obtained mobility. We find that the collective action of micelles that transiently attach to the tagged DNA impart about the same hydrodynamic drag as covalently bound "drag-tags" of a similar size. With the use of the PNAA-MEKC method, PCR products of 88, 134, 216, and 447 bases are clearly resolved in less than 5 min. To our knowledge, this work represents the first use of surfactant micelles as drag-tags to separate DNA in capillary electrophoresis. Furthermore, the PNAA tag only attaches to DNA containing a target sequence, helping ensure that only the desired PCR products are analyzed.     

Sensitized and quenched phosphorescence as a detection mode in capillary electrophoresis

The potential of sensitized and quenched phosphorescence of biacetyl as an optical detection mode in capillary electrophoresis (CE), complementary to absorption and fluorescence detection, was explored. From 24 naphthalenesulfonates (NS) that were studied in batch experiments, 5 NS were used as test compounds in CE. The technique is based on the intense phosphorescence emission of biacetyl (present as a constituent of the CE buffer) at room temperature in deoxygenated liquid solutions. A simple device, based on purging with nitrogen gas, was developed to meet this deoxygenation requirement in CE. A standard liquid chromatography luminescence detector, provided with a pulsed xenon light source, was used for detection. In view of the phosphoresence lifetime of biacetyl (70 μs under present solution conditions), the background caused by scattered excitation light could be readily suppressed by using a delay time for detection. Both phosphorescence modes can be applied at a 0.02 M biacetyl concentration, though in the quenched mode a biacetyl concentration of 0.05 M yields better results. From the five test analytes considered, three show sensitized phosphorescence and two dynamically quenched phosphorescence. Though various experimental parameters still have to be optimized further, the results are quite encouraging: under stacking conditions (pt = 768 mbar·s), detection limits ranged from 5 × 10(-)(8) to 4 × 10(-)(7) M.     

Congeneric behavior in estimations of octanol-water partition coefficients by micellar electrokinetic chromatography

Linear Solvation Energy Relationships (LSERs) are used to explain the congeneric behavior observed when using Micellar Electrokinetic Chromatography (MEKC) to estimate the octanol-water partition coefficient scale of solute hydrophobicity. Such studies provide useful insights about the nature of solute interactions that are responsible for the sources of congeneric relationships between MEKC retention and log Po/w. It was determined that solute dipolarity/polarizability and hydrogen-bonding character play the most important roles in the congeneric behavior observed for many surfactant systems. The individual dipolarity/polarizability and hydrogen-bonding contributions to the free energy of transfer were also investigated.     

On-line concentration of acidic compounds by anion-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

An easy, simple, and highly efficient on-line preconcentration method for acidic compounds in capillary electrophoresis was investigated. It combined two on-line concentration techniques, field-amplified sample injection (FASI) and sweeping. A low-pH (2.5) background electrolyte was used to suppress the electroosmotic flow (EOF), obviating the need of a coated capillary, as well as to neutralize the weakly acidic analytes. After injection of a plug of water inside the separation capillary, negative voltage was applied to initialize FASI for a much longer time than usual. The anions experienced a high electric field and moved quickly to the boundary of the water and the low-pH nonmicellar electrolyte. When the anions encountered the low-pH electrolyte, they were neutralized and a focused sample zone was formed. Then both inlet and outlet vials were changed to those containing the low-pH micellar background electrolyte. As negative voltage was applied, the anionic micelles moved into the capillary, and sweeping and separation began. The novelty in the present procedure is that a low-pH buffer is used to suppress the EOF and also the ionization of the analytes, without need of any other additives or use of a coated capillary. This method afforded 100,000-fold improvement in peak heights for some phenoxy acidic herbicides. The detection limits for these compounds could be low as 100 pg/mL     

Determination of biological toxins using capillary electrokinetic chromatography with multiphoton-excited fluorescence

We report a highly sensitive and rapid strategy for characterizing biological toxins based on capillary electrokinetic chromatography with multiphoton-excited fluorescence. In this approach, aflatoxins B1, B2, and G1 and the cholera toxin A-subunit are fractionated in approximately 80 s in a narrow-bore electrophoretic channel using the negatively charged pseudostationary phase, carboxymethyl-beta-cyclodextrin. The aflatoxins--highly mutagenic multiple-ringed heterocycles produced by Aspergillus fungi--are excited at the capillary outlet through the simultaneous absorption of two to three 750-nm photons to yield characteristic blue fluorescence; cholera toxin A-subunit, the catalytic domain of the bacterial protein toxin from Vibrio cholera, is excited through an unidentified multiphoton pathway that apparently includes photochemical transformation of an aromatic residue in the polypeptide. The anionic carboxymethyl-beta-cyclodextrin, used to chromatographically resolve the uncharged aflatoxins, enhances emission from these compounds without contributing substantially to the background. Detection limits for these toxins separated in 2.1-micron-i.d. capillaries range from 4.4 zmol (approximately 2700 molecules) for aflatoxin B2 to 3.4 amol for the cholera toxin A-subunit. Larger (16-micron-i.d.) separation capillaries provide concentration detection limits for aflatoxins in the 0.2-0.4 nM range, severalfold lower than achieved in 2.1-micron capillaries. These results represent an improvement of > 10(4) in mass detectability compared to previously published capillary separations of aflatoxins and demonstrate new possibilities for the analysis of proteins and peptides.     

Hydrophobicity of cationic solutes measured by electrokinetic chromatography with cationic microemulsions

The effect of the structure of employed surfactants on the selectivity of microemulsion electrokinetic chromatography (MEEKC) was investigated. Both their ionic groups and their hydrocarbon chain lengths did not affect the selectivity for neutral solutes. However, for cationic solutes, ionic interactions between catonic solutes and anionic surfactants both in the aqueous phase and in the microemulsion phase were observed. Based on these results, determination of the hydrophobicity of cationic solutes was done using MEEKC with cationic microemulsions. The obtained migration index (MI), which had been proposed as a novel hydrophobicity scale measured by MEEKC (Ishihama, Y.; et al. Anal. Chem. 1995, 67, 1588), correlated with conventional hydrophobicity scales for a water/1-octanol system, a liposome system, and an HPLC system using a phospholipid-immobilized artificial membrane (IAM) column. Quantitative structure-activity relationship studies on a protein binding affinity using MI as well as the hydrophobicity scales using the liposome and the IAM column provided better results than those using the water/1-octanol system.