Liquid-liquid-liquid microextraction for sample preparation of biological fluids prior to capillary electrophoresis.

Methamphetamine as a model compound was extracted from 2.5-mL aqueous samples adjusted to pH 13 (donor solution) through a thin phase of 1-octanol inside the pores of a polypropylene hollow fiber and finally into a 25-microL acidic acceptor solution inside the hollow fiber. Following this liquid-liquid-liquid microextraction (LLLME), the acceptor solutions were analyzed by capillary zone electrophoresis (CE). Extractions were performed in simple disposable devices each consisting of a conventional 4-mL sample vial, two needles for introduction and collection of the acceptor solution, and a 8-cm piece of a porous polypropylene hollow fiber. From 5 to 20 different samples were extracted in parallel for 45 min, providing a high sample capacity. Methamphetamine was preconcentrated by a factor of 75 from aqueous standard solutions, human urine, and human plasma utilizing 10(-1) M HCl as the acceptor phase and 10(-1) M NaOH in the donor solution. In addition to preconcentration, LLLME also served as a technique for sample cleanup since large molecules, acidic compounds, and neutral components were not extracted into the acceptor phase. Utilizing diphenhydramine hydrochloride as internal standard, repetitive extractions varied less than 5.2% RSD (n = 6), while the calibration curve for methamphetamine was linear within the range 20 ng/microL to 10 micrograms/mL (r = 0.9983). The detection limit of methamphetamine utilizing LLLME/CE was 5 ng/mL (S/N = 3) in both human urine and plasma.     

Liquid-phase microextraction combined with hollow fiber as a sample preparation technique prior to gas chromatography/mass spectrometry

Two modes of liquid-phase microextraction (LPME) combined with hollow fiber (HF) were developed for gas chromatography/mass spectrometry (GC/MS). Both methodologies, that is, static LPME with HF and dynamic LPME with HF, involved the use of a small volume of organic solvent impregnated in the hollow fiber, which was held by the needle of a conventional GC syringe. In static LPME/HF, the hollow fiber impregnated with solvent was immersed in the aqueous sample, and the extraction processed under stirring; in dynamic LPME/HF, the solvent was repeatedly withdrawn into and discharged from the hollow fiber by a syringe pump. This is believed to be the first reported instance of a semiautomated liquid microextraction procedure. The performance of the two techniques was demonstrated in the analysis of two PAH compounds in an aqueous sample. Static LPME/HF provided approximately 35-fold enrichment in 10 min and good reproducibility (approximately 4%). Dynamic LPME/HF could provide higher enrichment (approximately 75-fold) in 10 min and even better reproducibility (approximately 3%). Both methods allow the direct transfer of extracted analytes to a GC/MS system for analysis.     

Cloud point extraction as a preconcentration step prior to capillary electrophoresis

Cloud point extraction was applied as a preconcentration step prior to capillary electrophoresis. The behavior of a surfactant-rich micellar phase injected into a capillary electrophoresis system was studied using different separation modes: micellar electrokinetic capillary chromatography and capillary zone electrophoresis (CZE). A problem that appeared on introducing a surfactant-rich phase into a bare fused silica capillary was that the surfactant was adsorbed onto the wall of the capillary, leading to a marked loss of efficiency and reproducibility both in the migration times and in the areas of the electrophoretic peaks. The use of cetyltrimethylammonium bromide dynamically coated capillaries afforded reproducible results, although the half-life of the capillary was short. The most satisfactory results were obtained by using nonaqueous media in the CZE mode, thus avoiding surfactant adsorption. Other parameters related to the composition of the injection medium were also studied to optimize the electrophoretic behavior of the analytes and the sensitivity of the determination. The optimized procedure was applied to the determination of triazines in tap and river water samples.     

Liquid-phase microextraction as an on-line preconcentration method in capillary electrophoresis

A simple and efficient sample preconcentration method for capillary electrophoresis has been developed using liquid-phase microextraction (LPME). A thin layer of an organic liquid was used to separate a drop of the aqueous acceptor phase hanging at the inlet of a capillary from the bulk aqueous donor phase. The donor-phase pH was 1.0, and the acceptor phase pH was 9.5. This pH difference caused the preconcentration of the acidic compounds, fluorescein and fluorescein isothiocyanate, into the acceptor-phase drop. Enrichment factors of 3 orders of magnitude were obtained with 30-min LPME at 35 degrees C.     

Attenuated total internal reflectance infrared microspectroscopy as a detection technique for capillary electrophoresis

A novel detector for capillary electrophoresis (CE) using single-bounce attenuated total internal reflectance (ATR) Fourier transform infrared (FT-IR) microspectroscopy is presented. The terminus of the CE capillary is placed approximately 1 microm from the internal reflectance crystal at the focus of an ATR infrared microscope. Using pressure driven flow injection, concentration and volume detection limits have been determined for 25- and 10-microm-i.d. silica capillaries. Upon injection of 820 pL of succinylcholine chloride in a 10-microm capillary, a concentration detection limit of approximately 0.5 parts per thousand (ppt), or 410 pg, is found. The injection volume detection limit using a 108 ppt solution is 2.0 pL (216 pg). Sample separations using a programmed series of pressure, voltage, and again pressure on 25-, 50-, and 75-microm-i.d. capillaries are shown. CE separations of citrate and nitrate, as well as succinylcholine chloride with sodium salicylate using acetone as a neutral marker, are demonstrated. Several advantages of this CE-FT-IR technique include: (1) minimization of postcolumn broadening as a result of a small detector volume; (2) the ability to signal average spectra of the same aliquot, thereby improving the signal-to-noise in a stopped-flow environment; and (3) simplicity of design.     

Peak-shape correction to symmetry for pressure-driven sample injection in capillary electrophoresis

Pressure-driven sample injection in capillary electrophoresis results in asymmetric peaks due to difference in shapes between the front and the back boundaries of the sample plug. Uneven velocity profile of fluid flow across the capillary gives the front boundary a parabolic shape. The back side, on the other hand, has a flat interface with the electrophoresis run buffer. Here, we propose a simple means of correcting this asymmetry by pressure-driven "propagation" of the injected plug, with the parabolic sample-buffer interface established at the back. We prove experimentally that such a propagation procedure corrects peak asymmetry to the level comparable to injection through electroosmosis. Importantly, the propagation-based correction procedure also solves a problem of transferring the sample into the efficiently cooled zone of the capillary for capillary electrophoresis (CE) instruments with active cooling. The suggested peak correction procedure will find applications in all CE methods that rely on peak shape analysis, e.g., nonequilibrium capillary electrophoresis of equilibrium mixtures.     

On-column sample gating for high-speed capillary zone electrophoresis

High-speed zone electrophoresis in a fused-silica capillary is described. Elevated electric fields and short capillary lengths allow a mixture of fluorescein isothiocyanate (FITC) labeled amino acids to be separated in times as short as 1.5 s. Formation of the analyte zone at the head of the capillary is controlled by laser-induced photolysis of a tagging reagent. This gating procedure allows rapid and automated introduction of sample into the capillary. Ultimately, Joule heating of the buffer limits the speed and efficiency of the separation.     

Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits

A method based on solid-phase microextraction (SPME) and capillary electrophoresis/mass spectrometry (CE/ MS) is described for determining simultaneously five acidic pesticides (o-phenylphenol, ioxynil, haloxyfop, acifluorfen, picloram) in fruits. The CE device is coupled to an electrospray interface by a commercial sheath-flow adapter. Emphasis is placed on fulfillment of the speed and sensitivity requirements. The best separation is achieved using 32 mM ammonium formate/acid formic buffer at pH 3.1, with a working voltage of 25 kV. The MS detection of the five pesticides was performed in negative ionization mode. Full-scan spectra with base peaks corresponding to [M-H]- were obtained except for acifluorfen, which gives [M-H-CO2]- as most abundant ion. Compared with the conventional EC-UV, the limits of detection were lower for acifluorfen, haloxyfop, ioxynil, and picloram, by a factor of 20, 20, 50, and 2, respectively. Extraction involved fruit sample homogenization with an acetone-water solution (5:1), filtration, and acetone evaporation prior to fiber extraction. SPME conditions such as time, pH, ion strength, stationary phase of the fiber, sample matrix, and desorption solvents were examined. The recovery of the analytes ranged from 7 to 94%, and the relative standard deviation was between 3 and, 13%. The method was found to be linear between 0.02 and 500 mg kg(-1) with correlation coefficients ranging from 0.992 to 0.997. The limits of quantification were from 0.02 to 5 mg kg(-1). The optimized method was successfully applied to the analysis of acid pesticides in fruit samples.     

Narrow sample channel injectors for capillary electrophoresis on microchips

In microchip CE, sample injection is generally achieved through cross, double-tee, or tee injector structures. In these reported approaches, channel width and depth are uniform at the injection intersection. Here, we present cross and tee injectors having narrow sample channels. Using a cross injector with reduced sample channel width, resolution, column efficiency, and sensitivity are remarkably improved. Furthermore, no leakage control is required in both injection and separation phases, making the microchip CE system more user-friendly. Good resolution can also be obtained using tee injectors with narrow sample channels, which would otherwise be impossible using conventional tee injectors. Using the narrow sample channel tee injector instead of conventional cross and double-tee injectors, the number of reservoirs in multiplexed systems can be reduced to N + 2 (N, the number of paralleled CE systems), the real theoretical limit. The virtues of the novel injectors were demonstrated with poly(dimethylsiloxane)-glass chips incorporating eight parallel CE channels.     

Well-less capillary array electrophoresis chip using hydrophilic sample bridges

An innovative sample introduction method that facilitates extreme simplification of the layout of a capillary array electrophoresis (CAE) chip is described. Multiple samples were directly injected onto CAE channels from sample loaders using hydrophilic sample bridges, thus obviating auxiliary components of sample wells and sampling channels of a typical CAE chip. Hydrophilic sample bridges were spontaneously formed in hydrophobic surroundings to connect sample loaders to corresponding CAE channels, through which electrosample injections were effectively made. Sample dispersion was intrinsically avoided due to the "sticky" nature of the bridges. Utilizing hydrophilic interactions, target spots for formation of sample bridges can be expanded from the actual openings of CAE channels, which reduces the burden of precise positioning of samples toward CAE channels. In this work, simultaneous introduction of multiple samples onto well-less CAE channels arranged with 1-mm gaps was successfully demonstrated. Well-less CAE chips, feasible now due to the hydrophilic sample bridges, would bring unprecedented advantages in both production and operation because of their extreme simplicity.     

Microwave-assisted sample combustion: a technique for sample preparation in trace element determination

A novel digestion procedure based on sample combustion ignited by microwave radiation is proposed for organic samples. Certified samples of bovine liver, pig kidney, and skim milk were used as examples to demonstrate the performance of the proposed procedure. Cadmium and copper were determined in these samples by electrothermal atomic absorption spectrometry. Samples (between 50 and 250 mg) were wrapped with paper and placed on a homemade quartz holder that was positioned inside to quartz vessels used in a commercial microwave oven. Ammonium nitrate solution was added to the paper, and vessels were pressurized with oxygen to 15 bar. The rotor containing four vessels was placed inside the oven, and microwave radiation was applied for 20 s at 1400 W. Combustion was complete in few seconds, and an additional reflux step, which was optional, was applied. The agreement to the certified values was between 96 and 105% for both analytes. Only with the combustion step, the residual carbon (RC) was below 1.3%. The RC decreased to less than 0.4% when an additional reflux step with concentrated nitric acid was applied.     

Field amplified separation in capillary electrophoresis: a capillary electrophoresis mode

In field-amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electroosmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field-amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field-amplified zone, using what we have called field amplified separation in capillary electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near-zero eof at the selected separation pH. Using FAsCE, improvements of 20% for separation speed and 40% for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, a part of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient, and fast CE separation mode.     

Quantitative injection from a microloop. Reproducible volumetric sample introduction in capillary zone electrophoresis

A wire loop deployed at the tip of a capillary electrophoresis system has been investigated as a means of quantitative injection. A thin film of a liquid is formed on the loop, the loop is transferred to a sealed chamber, and then pneumatic pressure is applied to introduce the contents of the loop into the capillary. As long as the applied pressure is below a certain threshold, no air is introduced into the capillary, even after the loop contents have been fully introduced. Sample surface tension and viscosity do not have a significant effect on the injected volume. The small loop injection technique appears to be a robust and reproducible alternative to presently practiced approaches to sample injection in CE.     

On-line sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis

Previous reports describing sample stacking on microchip capillary electrophoresis (microCE) have regarded the microchip channels as a closed system and treated the bulk flow as in traditional capillary electrophoresis. This work demonstrates that the flows arising from the intersection should be investigated as an open system. It is shown that the pressure-driven flows into or from the branch channels due to bulk velocity mismatch in the main channel should not be neglected but can be used for liquid transportation in the channels. On the basis of these concepts, a sample preconcentration scheme was developed in a commercially available single-cross glass chip for microCE. Similar to field-amplified stacking injection in traditional CE, a low conductivity sample buffer plug was introduced into the separation channel immediately before the negatively charged analyte molecules were injected. The detection sensitivity was improved by 94-, 108-, and 160-fold for fluorescein-5-isothiocyanate, fluorescein disodium, and 5-carboxyfluorescein, respectively, relative to a traditional pinched injection. The calibration curves for fluorescein and 5-carboxyfluorescein demonstrated good linearity in the concentration range (1-60 nM) investigated with acceptable reproducibility of migration time and peak height and area ratios (4-5% RSD). This preconcentration scheme will be of particular significance to the practical use of microCE in the emerging miniaturized analytical instrumentation.     

Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique

A split-flow capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface is introduced, in which the electrical connection to the CE capillary outlet is achieved by diverting part of the CE buffer out of the capillary through an opening near the capillary outlet. The CE buffer exiting the opening contacts a sheath metal tube which acts as the CE outlet/ESI shared electrode. In cases in which the ESI source uses a metal needle, the voltage contact to the CE buffer is achieved by simply inserting the outlet of the CE capillary, which contains an opening, into the existing ESI needle (thereby greatly simplifying the CE to MS interfacing). As a result of the concentration-sensitive nature of ESI, splitting a small percentage of the CE flow has minimal effect on the sensitivity of detection. In addition, because the liquid is flowing through the opening and out of the capillary, there is no dead volume associated with this interface. Moreover, bubble formation due to redox reactions of water at the electrode does not effect CE/ESI-MS performance, because the actual metal/liquid contact occurs outside of the CE capillary. The sensitivity associated with a sheathless CE/MS interface, the ease of fabrication, universality, and lack of any dead volume make this design a superior CE/ESI-MS interface. The performance of this interface is demonstrated by analyses of a peptide standard and a protein digest using a variety of capillary dimensions.     

Interface for coupling capillary electrophoresis to inductively coupled plasma and on-column concentration technique

A new interface for coupling capillary electrophoresis (CE) to inductively coupled plasma (ICP) was developed. The interface was built outside and independent of the nebulizer and could be easily connected with a microconcentric nebulizer (MCN) as well as conventional pneumatic nebulizers. An on-column concentration technique was used to increase the sensitivity and to enhance the resolution of the system of capillary electrophoresis-inductively coupled plasma atomic emission spectrometry (CE-ICP-AES). By doing this, it was possible to analyze 1 μg/mL of total chromium (prepared with K(2)Cr(2)O(7) and CrCl(3)) and 1 μg/mL of copper consisting of Cu(2+) and Cu(EDTA)(2-) with good spectroscopic intensity and efficient separation. Detection limits of 18 elements for MCN-ICP-AES coupled with CE were assessed by continuous sample introduction without applying high voltage and were found to be 1-4 times higher than those typically obtained by using MCN-ICP-AES for elemental analysis (without connection to the CE interface). When the on-column concentration technique was used, the sensitivities and separations were further improved by increasing the amount of sample. A simple electrolyte (0.05 M HNO(3)) and a large inner diameter capillary (150 μm) could also be used to attain efficient separation.     

Numerical analysis and experimental estimation of a low-leakage injection technique for capillary electrophoresis

This paper presents an experimental and numerical investigation into the use of low-leakage injection techniques to deliver sample plugs within electrophoresis microchips. The study addresses the principal material transport mechanisms such as electrokinetic migration, fluid flow, and diffusion and gives detail analyses to the double-L injection technique, which employs electrokinetic manipulations to avoid sample leakage within the microchip. Electrical potential contour, velocity vector, and streamline distribution in the micro CE chip are successfully developed. Experimental and numerical testing results show the double-L injection technique is capable of reducing sample leakage within cross-form microfluidic chips. The current study confirms the double-L injection technique has an exciting potential for use in high-quality, high-throughput chemical analysis applications and in many other applications throughout the field of micro total analysis systems.     

Online sample preconcentration in capillary electrophoresis using analyte focusing by micelle collapse

A dimension for online sample preconcentration in capillary electrophoresis (CE) without modification of current CE commercial instrumentation is introduced. The focusing mechanism is based on the transport, release, and accumulation of molecules bound to micelle carriers that are made to collapse into a liquid phase zone. More than 2 orders of magnitude improvement in detection sensitivity for model steroidal compounds using sodium dodecyl sulfate micelles as carrier is demonstrated.     

A high-throughput continuous sample introduction interface for microfluidic chip-based capillary electrophoresis systems

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with <4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.     

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.