Violet light emitting diode-induced fluorescence detection combined with on-line sample concentration techniques for use in capillary electrophoresis

The first application of a violet light-emitting diode (LED) for fluorescence detection in capillary electrophoresis (CE) is described. The utility of violet LED (peak emission wavelength at 410 nm, approximately 2 mW) for fluorescence detection is demonstrated by examining reserpine and dopamine-labeled NDA (naphthalene-2,3-dicarboxaldehyde), respectively. The detection limit for reserpine was determined to be 2.5 x 10(-6) M by normal micellar electrokinetic capillary chromatography (MEKC) and this was improved to 2.0 x 10(-9) M and 2.0 x 10(-10) M when sweeping-MEKC and cation-selective exhaustive injection (CSEI)-sweep-MEKC techniques were applied, respectively. In addition, the detection limit of NDA-labeled dopamine was determined to be 6.3 x 10(-6) M by means of normal MEKC and this was improved to 3.0 x 10(-8) M when the sweeping-MEKC mode was applied.     

Large-volume sample sweeping with a high theoretical plate number using a coupled-capillary in capillary electrophoresis.

A large-volume sample injection (> 5 microL) with an extremely high theoretical plate number (N > 10(7)) was achieved when the sweeping-MEKC mode and a coupled-capillary (100 - 50 microm i.d.) were simultaneously used in a capillary electrophoresis (CE) separation. A low-cost and compact violet-LED ( approximately 2 mW) was used as the fluorescence excitation source. As a result, the theoretical plate numbers of the detected peaks (two model compounds: naphthalene-2,3-dicarboxaldehyde derivatized-dopamine and -norepinephrine) were 1.0 x 10(7) and 7.4 x 10(6), respectively. The limits of detection (at S/N = 3) of these were determined to be 2.8 x 10(-10) M (92 ppt) and 2.3 x 10(-10) M (83 ppt), respectively.     

Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries

Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.     

Micellar electrokinetic chromatography at low pH with polyelectrolyte-coated capillaries

The performance of capillaries coated with a poly(diallyldimethylammonium) (PDADMA) monolayer or poly(diallyldimethylammonium)-poly(styrenesulfonate) bilayer was investigated and compared under micellar electrokinetic chromatographic (MEKC) conditions. Both monolayer (positively charged) and bilayer (negatively charged) coatings with micellar (sodium dodecyl sulfate) electrolyte generated very stable and pH-independent cathodal electroosmotic flow (EOF). From the results obtained, it can be concluded that in a doubly coated capillary the second poly(styrenesulfonate) layer is replaced by sodium dodecyl sulfate micelles during flushing with micellar electrolyte. Consequently, in order to obtain a stable and pH-independent cathodal electroosmotic flow for the MEKC separations, the capillary coating with the second polyanion layer is not necessary. The importance of the PDADMA coating was illustrated by comparing MEKC separations of the common developing agents (hydroquinone, phenidone, pyrocatechol, pyrogallol and quinone) on a bare uncoated capillary with the coated capillary. The coating provides reproducible MEKC separations at low pH (pH 3.0) with relative standard deviation (R.S.D.) values for migration times and peak areas lower than 0.45 and 3.3%, respectively. Good linearities in the range from 5 x 10(-5) to 2 x 10(-3) mol l(-1) were obtained for all five compounds, with correlation coefficients higher than 0.998. The detection limits were in the range from 5 x 10(-6) mol l(-1) for pyrocatechol to 2 x 10(-5) mol l(-1) for quinone. The proposed MEKC system was applied to the determination of hydroquinone and phenidone in X-ray photographic developer solutions.     

Metal speciation using capillary electrophoresis--inductively coupled plasma atomic emission spectrometry and polytetrafluoroethylene capillaries

A capillary electrophoresis--inductively coupled plasma atomic-emission spectrometry (CE-ICP-AES) system using a polytetrafluoroethylene (PTFE) capillary has been developed. The CE-ICP interface was a modified concentric nebulizer. The PTFE capillary (50 microm internal diameter) was used as the central capillary of the nebulizer. Using the PTFE capillaries, the solution flow rate induced by the carrier gas flow was smaller than that of glass capillary. Solution flow was mainly induced by the CE electric field. Baseline separation of Ba2+/Mg2+ ion pair using simple buffer solution of 0.014 M sodium acetate was reported. Separation and correlation of metal species in metallothioneins (MT-1 and MT-2 in MT) of rabbit liver using the CE-ICP system were also discussed.     

Optimization of the separation and on-line sample concentration of phenethylamine designer drugs with capillary electrophoresis-fluorescence detection

Five 2C-series of phenethylamine designer drugs, including 2,5-dimethoxy-4-ethylthio-phenethylamine (2C-T-2), 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7), 4-chloro-2,5-dimethoxyphenethylamine (2C-C), 4-bromo-2,5-dimethoxy-phenethylamine (2C-B), 2,5-dimethoxy-4-iodo-phenethylamine (2C-I), were synthesized and standard GC/MS and fluorescence spectra are reported for them. A mixture of the five drugs was separated and detected by means of capillary electrophoresis (CE) with native fluorescence and light emitting diode (LED)-induced fluorescence (LIF) detection, respectively, for comparison. In the former case, exciting at a wavelength of 300 nm from a Xe lamp was used. The detection limits were found to be only in the range of approximately 10(-4) M by the micellar electrokinetic chromatography (MEKC) mode but were improved to approximately 10(-7) M when the sweeping-MEKC mode was used. For a highly sensitive analysis, LED-induced fluorescence detection was examined by derivatizing the compounds with a fluorescent dye, fluorescein isothiocyanate isomer I (FITC). A blue-LED (approximately 2 mW) was used as the fluorescence excitation source. The detection limits were improved to approximately 10(-7) and approximately 10(-8) M, respectively, when the MEKC and stacking-MEKC modes were applied. A mimic urine sample was obtained by spiking urine from a volunteer with the five standards, and after liquid-liquid extraction, the sample was examined by means of the MEKC-LIF mode. The extraction procedures used for the urine sample and the CE conditions for the separation were optimized.     

An interface chip connection between capillary electrophoresis and thermal lens microscope

A thermal lens microscope (TLM) detection of capillary electrophoresis (CE) utilizing microchip technology was developed. Fused-silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel in a microchip. The detection limit by TLM was estimated as 2.8 x 10(-7) absorbance by measuring pure water. The detection limit of derivatized amino acids determined by CE-TLM was estimated as 2.4 x 10(-8) M, which was 100 times lower than that of conventional absorbance detection.     

A chemometric study of active parameters and their interaction effects in a nebulized sheath-liquid electrospray interface for capillary electrophoresis-mass spectrometry

A chemometrics approach has been used for evaluating the effect of four experimental parameters when coupling capillary electrophoresis (CE) to electrospray ionization-mass spectrometry (ESI-MS). Electrospray voltage, sheath-liquid flow rate, nebulizing gas flow rate, and spray needle position in respect to the MS orifice were varied according to a full factorial design. In addition to main effects, two interaction effects could be identified as significant when measuring the peak intensity of the analytes, from a sample mixture containing peptides and pharmaceuticals. The first interaction effects, between the nebulizing gas flow rate and the sheath-liquid flow rate, and the second interaction effect, between the nebulizing gas flow rate and the spray position, could further explain the impact that these variables have on the spray performance. The number of theoretical plates and the baseline noise were also measured. The sheath-liquid flow was found to significantly affect the separation efficiency, while the noise level mainly was controlled by the nebulizing gas flow. The same factorial design was also used for a CE capillary with lower internal diameter (ID) and the effects of the same variables were compared on those capillaries using equal injection volume for both capillaries. Similar trends were obtained in both capillaries but capillary ID was shown to be a significant variable when evaluating both capillaries in a single model. It was found that a capillary with 25 microm ID provided improved CE-MS performance over than corresponding 50 microm ID capillary. Enhanced sensitivity was obtained using the narrow-bore capillary, and at lower sheath-liquid flow rate the 25 microm ID capillary also gave rise to more efficient peaks.     

Direct automatic determination of biogenic amines in wine by flow injection-capillary electrophoresis-mass spectrometry

A capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) method for the separation and determination of nine biogenic amines is proposed. Operational variables, such as the voltage, temperature, sheath liquid composition, flow-rate, and MS parameters, were optimized. Samples are injected in the hydrodynamic mode into a 75 cm x 50 microm ID coated capillary and separated by using 25 mM citric acid at pH 2.0. Heptylamine is used as internal standard. The experimental setup includes a flow manifold coupled to the CE system for automatic insertion of samples into the CE vials. The proposed method allows amines to be determined with limits of detection from 0.018 to 0.09 microg x mL(-1) and relative standard deviation (RSD) values from 2.4% to 5.0% (except 6.8% for histamine). The method was successfully used to determine biogenic amines in red and white wines.     

Iron protoporphyrin-modified monolithic support for on-line preconcentration of angiotensin prior to CE analysis

An on-line preconcentration method using a polymeric monolithic support is proposed for the retention of the decapeptide angiotensin I and its subsequent analysis by CZE. Monolithic capillary columns were prepared in fused-silica (FS) capillaries of 150 microm id by ionizing radiation-initiated in situ polymerization and cross-linking of diethylene glycol dimethacrylate and glycidyl methacrylate, and chemically modified with iron protoporphyrin IX (Fe-ProP). Monolithic microcolumns (8 mm long) were coupled on-line to the inlet of the separation capillary (FS capillary, 75 microm id x10 cm from the inlet to the microcolumn and 27 cm from the microcolumn to the detector). Angiotensin I was released from the sorbent by a 50 mM sodium phosphate, pH 2.5/ACN, 75:25 v/v solution and then analyzed by CZE with UV absorption detection at 214 nm. The concentration LOQ (CLOQ) was 0.5 ng/mL. The Fe-ProP-derivatized monolithic microcolumn coupled to the separation capillary exhibited a high retention capacity for peptide angiotensin I, and showed as much as 10,000-fold improvement in concentration sensitivity.     

Alcohols and wide-bore capillaries in nonaqueous capillary electrophoresis.

The feasibility of using C1-C5 alcohols as electrolyte solutions in nonaqueous capillary zone electrophoresis was investigated. The separation of basic narcotic analgesics and acidic diuretics was modified by changing the alcohol in an electrolyte solution containing alcohol-acetonitrile-acetic acid (50:49:1, v/v) and 20 mM ammonium acetate while other experimental conditions were kept constant. The alcohols studied were methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and 1-pentanol. The results indicate that even longer-chain alcohols can be used in nonaqueous capillary zone electrophoresis and, because of the lower currents they allow, they are especially advantageous in wider capillaries. Basic analytes were separated in 200 microm and 320 microm ID capillaries with 1-butanol-acetonitrile-acetic acid (50:49:1, v/v) containing 20 mM ammonium acetate as electrolyte solution. Problems related to the use of wide-bore capillaries are discussed.     

Protein separation and surfactant control of electroosmotic flow in poly(dimethylsiloxane)-coated capillaries and microchips

A thermally pyrolyzed poly(dimethylsiloxane) (PDMS) coating intended to prevent surface adsorption during capillary electrophoretic (CE) [Science 222 (1983) 266] separation of proteins, and to provide a substrate for surfactant adsorption for electroosmotic mobility control was prepared and evaluated. Coating fused-silica capillaries or glass microchip CE devices with a 1% solution of 100 cSt silicone oil in CH2Cl2, followed by forced N2 drying and thermal curing at 400 degrees C for 30 min produced a cross-linked PDMS layer. Addition of 0.01 to 0.02% Brij 35 to a 0.020 M phosphate buffer gave separations of lysozyme, cytochrome c, RNase, and fluorescein-labeled goat anti-human IgG Fab fragment. Respective plates/m typically obtained at 20 kV (740 V cm(-1)) were 2, 1.5, 1.25, and 9.4-10(5). In 50 mM ionic strength phosphate, 0.01% Brij 35 running buffer, the electroosmotic flow observed was about 25% of that in a bare capillary, and showed no pH dependence between pH 6.3-8.2. Addition of sodium dodecylsulfate (SDS) or cetyltrimethylammonium bromide (CTAB) to this running buffer allowed ready control of electroosmotic mobility, mu(eo). Concentrations of SDS between 0.005 to 0.1% resulted in mu(eo) ranging from 3 to 5 x 10(-4) cm2 V(-1) s(-1). Addition of 1 to 2.3 x 10(-4)% (2.7-6.3 microM) CTAB caused flow reversal. CTAB concentrations between 3.5 x 10(-4) and 0.05% (0.0014-1.37 mM) allowed control of mu(eo) between -1 x 10(-4) and -5.0 x 10(-4) cm2 V(-1) s(-1). For both surfactants the added presence of 0.01% Brij 35 provided slowly varying changes in mu(eo) with charged surfactant concentration.     

Low-temperature bath/coupled-capillary/sweeping-micellar electrokinetic capillary chromatography for the separation of naphthalene-2,3-dicarboxaldehyde-derivatized dopamine and norepinephrine

The use of a low-temperature (0 degrees C) bath-assisted coupled capillary for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized dopamine and norepinephrine using the sweeping-micellar electrokinetic capillary chromatography (MEKC) mode is described. In this technique, a capillary consisting of two portions with different inside diameters is used. Therefore, the field strength inside the capillary is different. Hence, the electrophoretic migration velocities of the analytes and the electro-osmotic flow (EOF) also are different. Furthermore, when a portion of the capillary (wide portion, used for sweeping) is immersed in a low-temperature bath, the viscosity of the buffer and the retention factor of the analytes inside are increased. Thus, not only are the interactions between the SDS micelles and the analytes increased, but the SDS-analytes also move more slowly. As a result, a more complete separation can be achieved, even when the sample injection volume is large, up to approximately 2 microL. In general, when the volume of an injected sample is larger, the effects of sweeping and separation would become insufficient, especially when the retention values (k) of the analytes are quite different. However, this limitation can be improved when the low-temperature bath/coupled capillary/sweeping-MEKC mode is used.     

Studies on the long-term thermal stability of particulate and monolithic poly(styrene-divinylbenzene) capillaries in reversed-phase liquid chromatography

In the present study, the long-term high-temperature (>80 degrees C) and temperature programming stability of fused silica capillaries packed with 5 microm PLRP-S 300 A and monolithic PS-DVB capillaries (both 180 microm id x 6 cm) under reversed-phase conditions has been examined. In isothermal mode, the columns were defined as temperature-stable when a less than 10% change in apparent retention factors (k) and a less than 20% change in "retention time/peak width"-factors (n) of the probe solutes (proteins) were observed after passing 7,500 void volumes of effluent through the columns (about 100 h operation). According to these criteria, the PLRP-S and monolithic capillaries were defined temperature-stable at 100 and 130 degrees C, respectively. Furthermore, when continuously running temperature programs between 50 degrees C and the upper temperature limit determined in isothermal mode, virtually no change in k or n were observed on neither of the columns after running more than 35,000 void volumes or 1,600 temperature programs. Additionally, temperature-programmed reversed-phase separations of proteins on both types of capillaries are demonstrated and discussed.     

Analysis of organic components of smokeless gunpowders: high-performance liquid chromatogaphy vs. micellar electrokinetic capillary chromatography

The aim of the present study was to verify the analytical performances of high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MEKC) for the separation and qualitative determination of a selected group of organic components of smokeless gunpowders. The HPLC method was based on a gradient reversed-phase elution with a mobile phase composed of 0.17 M H(3)PO(4)/methanol; detection was performed by UV absorption at the wavelengths of 220, 254, and 270 nm. The MEKC experiments were carried out by using uncoated fused-silica capillaries (50 microm inside diameter, 50 cm effective length) and a running buffer composed of 10 mM sodium tetraborate at pH 9.24 added with 25 mM sodium dodecyl sulfate (SDS); the applied voltage was 25 kV; detection was either at a fixed wavelength UV of 214 nm or with a diode-array detector operating in the wavelength range from 190 to 350 nm. Both reversed-phase HPLC and MEKC techniques succeeded in resolving the tested standard mixtures of organic components of smokeless powders. Although the sequence of elution of the different analytes was slightly different between HPLC and MEKC, a statistical analysis based on the Spearman's rank correlation test showed that the two separation patterns were highly correlated. HPLC and MEKC were comparable in terms of elution/migration time precision, whereas MEKC showed higher reproducibility of peak areas. The interfacing of capillary electrophoresis with diode array UV detection provided distinct UV spectra of the individual analytes, thus improving, on the detection side, the analytical selectivity and identification power of capillary electrophoresis.     

Streaming current measurements in zirconia-coated capillaries

The electroosmotic flow created in zirconia-modified capillaries has been previously investigated. In this paper, we compared the electroosmotic data set with streaming current measurements and we related all these data through zeta-potential. Streaming current measurements give an excellent indication on the direction and the value of the electroosmotic mobility of an electrolyte/capillary system for a large set of experimental conditions: 2 < pH < 12, 0 < ACN < 80 %, 10(-4) M < [SO(2- )4 ] < 4 x 10(-2) M. A good correlation between zeta-potential from streaming current measurements and zeta-potential from electroosmotic mobility measurements was observed (r2 = 0.95). However, the values obtained from streaming current were always slightly lower than the one calculated from electroosmotic mobility (slope = 0.86, sigma = 0.06). In zirconia-coated capillaries the zeta-potential can be tuned from -50 to +100 mV depending on the composition of the electrolyte.     

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.     

Use of vancomycin silica stationary phase in packed capillary electrochromatography: III. enantiomeric separation of basic compounds with the polar organic mobile phase

The separation of basic compounds into their enantiomers was achieved using capillary electrochromatography in 50 or 75 microm inner diameter (ID) fused-silica capillaries packed with silica a stationary phase derivatized with vancomycin and mobile phases composed of mixtures of polar organic solvents containing 13 mM ammonium acetate. Enantiomer resolution, electroosmotic flow, and the number of theoretical plates were strongly influenced by the type and concentration of the organic solvent. Mobile phases composed of 13 mM ammonium acetate dissolved in mixtures of acetonitrile/methanol, ethanol, n-propanol, or isopropanol were tested and the highest enantioresolutions were achieved using the first mobile phase, allowing the separation of almost all investigated enantiomers (9 from 11 basic compounds). The use of capillaries with different ID (50 and 75 microm ID) packed with the same chiral stationary phase revealed that a higher number of theoretical plates and higher enantioresolution was achieved with the tube with lowest ID.     

Toward million-fold sensitivity enhancement by sweeping in capillary electrophoresis combined with thermal lens microscopic detection using an interface chip

This paper reports a thermal lens microscope (TLM) detection coupled with capillary electrophoresis (CE) by using an interface chip (IFChip) to achieve highly sensitive detection with high reproducibility. Fused silica capillaries with an inner diameter of 50 microm were directly connected to a microchannel on the IFChip. In comparison with an on-capillary detection method in CE-TLM, ca. 10-fold improvements in the reproducibility for peak height were obtained by using IFChips. The detection limit of an azo dye was estimated to be 3.6 x 10(-7)M (100 ppb), which was above 100-times lower than that of conventional absorbance detection. Toward further improvement of the detectability for nonfluorescent compounds, on-line sample preconcentration by sweeping was applied to the CE-TLM using the IFChip. Due to the sweeping effect, 3900000-fold increase in the sensitivity was successfully achieved.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.