Analysis of fission products using capillary electrophoresis with on-line radioactivity detection

Capillary electrophoresis has been used to separate metal ions characteristically associated with nuclear fission. Indirect UV absorbance and on-line radioactivity detection were used simultaneously to monitor the analytes. The radioactivity detector consists of conical plastic scintillating material with the capillary passing through the center to provide a 4π detection geometry. The wide end of the cone is optically coupled to a photomultiplier tube. Transient isotachophoretic techniques were employed to stack large volumes of samples which had low specific activities. Radioactivity detection of (152)Eu and (137)Cs was achieved at the nanocurie level for 80-100 nL injections. The detector is approximately 80% efficient, enabling samples resident in the detector window for 0.1 min to be reliably assayed. The separation of (137)Cs and (137m)Ba isotopes, which are in secular equilibrium, was modeled to demonstrate the effects of the rapid decay of (137m)Ba.     

Speciation of mercury by hydrostatically modified electroosmotic flow capillary electrophoresis coupled with volatile species generation atomic fluorescence spectrometry

A novel method for speciation analysis of mercury was developed by on-line hyphenating capillary electrophoresis (CE) with atomic fluorescence spectrometry (AFS). The four mercury species of inorganic mercury Hg(II), methymercury MeHg(I), ethylmercury EtHg(I), and phenylmercury PhHg(I) were separated as mercury-cysteine complexes by CE in a 50-cm x 100-microm-i.d. fused-silica capillary at 15 kV and using a mixture of 100 mmol L(-1) of boric acid and 12% v/v methanol (pH 9.1) as electrolyte. A novel technique, hydrostatically modified electroosmotic flow (HSMEOF) in which the electroosmotic flow (EOF) was modified by applying hydrostatical pressure opposite to the direction of EOF was used to improve resolution. A volatile species generation technique was used to convert the mercury species into their respective volatile species. A newly developed CE-AFS interface was employed to provide an electrical connection for stable electrophoretic separations and to allow on-line volatile species formation. The generated volatile species were on-line detected with AFS. The precisions (RSD, n = 5) were in the range of 1.9-2.5% for migration time, 1.8-6.3% for peak area response, and 2.3-6.1% for peak height response for the four mercury species. The detection limits ranged from 6.8 to 16.5 microg L(-1) (as Hg). The recoveries of the four mercury species in the water samples were in the range of 86.6-111%. The developed technique was successfully applied to speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).     

Direct on-line injection in capillary electrophoresis

A direct method of sample introduction for capillary electrophoretic techniques is described using a cross configuration and high-voltage shunting. No physical disturbance of the separation capillary inlet is required, and the feasibility of direct on-line injection is demonstrated. Both full- and pinched-mode injections are evaluated, with pinched-mode injections showing superior performance. In the pinched mode, only a portion of the cross volume is introduced onto the separation capillary, as a result, a lower volume is injected, and wall effects within the cross are minimized. Preliminary studies indicate a peak height reproducibility for replicate injections of better than 4.1%, with area reproducibilities of less than 3.1% for nonoverlapping solutes. Utilizing this direct on-line injection method, many rigid or restricted capillary geometries can be accommodated, and extension to the wide range of capillary electrophoretic techniques is feasible.     

Microchip capillary electrophoresis with electrochemical detection

A novel microchip capillary electrophoresis system with electrochemical detection, using the replaceable microelectrode, is first reported. This kind of electrode can be fabricated in general laboratories and can be replaced quickly with electrodes of different materials according to the requirements of experiments. The end-column electrochemical detection on microchip CE was achieved by fixing the working electrode (such as carbon fiber, Pt, or Au, etc.) through a guide tube on the end of the separation channel. The experiment results indicate that the alignment of the electrode with the channel outlet can be carried out accurately and reproducibly, and therefore, the detection device has low noise and good reproducibility. The detection limit of dopamine is 2.4 x 10(-7) M, which is the lowest result reported so far. The separation and detection of dopamine, 5-hydroxytryptamine and epinephrine using carbon fiber and Pt microdisk electrodes within 50 s was successfully performed.     

DNA sequencing by capillary electrophoresis with four-decay fluorescence detection

A scheme for multiplex detection of dye-labeled DNA fragments in DNA sequencing is described in which on-the-fly, frequency-domain fluorescence lifetime detection is used to discriminate among the dye-labeled fragments of the four terminal bases in a single-lane CE separation. Two four-dye systems were evaluated, one excited at 488 nm and the other, at 514 nm. The 488 nm system proved successful for four-decay detection. Base calling was achieved either directly from on-the-fly lifetimes or from lifetime-resolved electropherograms recovered for each base from the electropherogram of the mixture of sequencing reaction products. The latter method was found to be more accurate (99% for two bases and 98.5% for three bases) and could achieve longer read lengths, but it was unsuccessful for sequencing of all four bases. The first method gave a base-calling accuracy of 96% for four-base sequencing over the fragment length range of 41-220 bases.     

Selective measurement of ultratrace methylmercury in fish by flow injection on-line microcolumn displacement sorption preconcentration and separation coupled with electrothermal atomic absorption spectrometry

A novel nonchromatographic speciation technique for ultratrace methylmercury in biological materials was developed by flow injection microcolumn displacement sorption preconcentration and separation coupled on-line with electrothermal atomic absorption spectrometry (ETAAS). In the developed technique, Cu(II) was first on-line complexed with diethyldithiocarbamate (DDTC), and the resultant Cu-DDTC was presorbed onto a microcolumn packed with the sorbent from a cigarette filter. Selective preconcentration of methylmercury (MeHg) in the presence of Hg(II), ethylmercury (EtHg), and phenylmercury (PhHg) was achieved at pH 6.8 through loading the sample solution onto the microcolumn due to a displacement reaction between MeHg and the presorbed Cu-DDTC. The retained MeHg was subsequently eluted with 50 microL of ethanol and on-line determined by ETAAS. Interferences from coexisting heavy metal ions with lower stability of their DDTC complexes relative to Cu-DDTC were minimized without the need of any masking reagents. No interferences from 5.5 mg L(-1) Cu(II), 4.5 mg L(-1) Cd(II), 2.5 mg L(-1) Cr(III), 3 mg L(-1) Fe(III), 10 mg L(-1) Ni(II), 10 mg L(-1) Pb(II), and at least 25 mg L(-1) Zn(II) were observed for the determination of MeHg at the 50 ng L(-1) level (as Hg). With the consumption of only 3.4 mL of sample solution, an enhancement factor of 75, a detection limit of 6.8 ng L(-1) (as Hg) in the digest (corresponding to 3.4 ng g(-1) in original solid sample for a final 50 mL of digest of 0.1 g of solid material), and a precision (RSD, n = 13) of 2.3% for the determination of methylmercury at the 50 ng L(-1) (as Hg) level were achieved at a sample throughput of 30 samples h(-1). The recoveries of methylmercury spike in real fish samples ranged from 97 to 108%. The developed technique was validated by determination of methylmercury in a certified reference material (DORM-2, dogfish muscle), and was shown to be useful for the determination of methylmercury in real fish samples.     

Hadamard transform capillary electrophoresis combined with absorption spectrometry

A novel injection device for applying absorption spectrometry to Hadamard transform (HT) capillary electrophoresis is described. A small hole, at the center part of the capillary, functions as an inlet port for the sample. The hole is immersed in a sample solution and the end of the capillary that is usually employed for sample introduction is immersed in a buffer solution. An ultraviolet absorption detector is placed between the sample injection port and the other end of the capillary filled with a buffer solution. A high potential is continuously applied between the injection port and the end of the capillary, which allows the sample solution to be introduced into the separation capillary. By application of a higher potential modulated according to a Hadamard code between both ends of the capillary, the buffer solution is injected into the separation capillary. In some preliminary experiments, this injection device was utilized to introduce a single sample segment into a capillary. As expected, a single peak was observed in the electropherogram for a sample containing a single component. This device was then employed for multiple sample injection in HT capillary electrophoresis. An 8-fold improvement in the S/N ratio was observed when the HT technique was used, in which a 255-order of a Hadamard matrix was used, as expected from theory. The present approach was also utilized for the sensitive detection of a sample comprised of multiple components.     

Automated sequential injection-microcolumn approach with on-line flame atomic absorption spectrometric detection for implementing metal fractionation schemes of homogeneous and nonhomogeneous solid samples of environmental interest

An automated sequential injection (SI) system incorporating a dual-conical microcolumn is proposed as a versatile approach for the accommodation of both single and sequential extraction schemes for metal fractionation of solid samples of environmental concern. Coupled to flame atomic absorption spectrometric detection and used for the determination of Cu as a model analyte, the potentials of this novel hyphenated approach are demonstrated by the ability of handling up to a 300 mg sample of a nonhomogeneous sewage amended soil (viz., CRM 483). The three steps of the endorsed Standards, Measurements, and Testing sequential extraction method have been also performed in a dynamic fashion and critically compared with the conventional batchwise protocols. The ecotoxicological relevance of the data provided by both methods with different operationally defined conditions is thoroughly discussed. As compared to traditional batch systems, the developed SI assembly offers minimal risks of sample contamination, the absence of metal re-distribution/readsorption, and dramatic saving of operational times (from 16 h to 40-80 min per partitioning step). It readily facilitates the accurate manipulation of the extracting reagents into the flow network and the minute, well-defined injection of the desired leachate volume into the detector. Moreover, highly time-resolved information on the ongoing extraction is given, which is particularly relevant for monitoring fast leaching kinetics, such as those involving strong chelating agents. On-line and off-line (for Cu, Pb, and Zn) single extraction schemes are also proven to constitute attractive alternatives for fast screening of metal pollution in solid samples and for predicting the current, rather than the potential, element bioavailability by the assessment of the readily mobilizable metal forms.     

Mobility moment analysis of molecular interactions by capillary electrophoresis

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.     

Electrothermal atomic absorption spectrometric diagnosis of familial hypercholesterolemia

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies.     

An absorption detection approach for multiplexed capillary electrophoresis using a linear photodiode array

A novel absorption detection method for highly multiplexed capillary electrophoresis is presented for zone electrophoresis and for micellar electrokinetic chromatography. The approach involves the use of a linear photodiode array on which a capillary array is imaged by a camera lens. Either a tungsten lamp or a mercury lamp can be used as the light source such that all common wavelengths for absorption detection are accessible by simply interchanging narrow-band filters. Each capillary spans several diodes in the photodiode array for absorption measurements. Over 100 densely packed capillaries can be monitored by a single photodiode array element with 1024 diodes. The detection limit for rhodamine 6G for each capillary in the multiplexed array is ～1.8 × 10(-)(8) M injected (S/N = 2). The cross-talk between adjacent capillaries is less than 0.2%. Simultaneous analysis of 96 samples is demonstrated.     

Characterization of surfactant coatings in capillary electrophoresis by atomic force microscopy

This paper describes the adsorption mechanisms and aggregation properties of cetyltrimethylammonium bromide (CTAB) and didodecyldimethylammonium bromide (DDAB) surfactants that are used for dynamic coatings in capillary electrophoresis (CE). Atomic force microscopy is used to directly visualize surfactant adsorption on fused silica. It was found that the single-chained surfactant CTAB forms spherical aggregates on silica while the double-chained surfactant DDAB forms a bilayer. Aggregation at the surface occurs at approximately the same surfactant concentration in which EOF reversal is observed in CE. The nearest-neighbor distance between CTAB aggregates varies inversely with buffer pH and becomes constant at the point when the silanol groups are fully ionized. DDAB forms a flat, uniform coating independent of pH. Increasing the buffer ionic strength changes the morphology of the CTAB aggregates from spherical to cylindrical. The change in morphology can alter the surface coverage, which is related to the "normalized" EOF measured in identical buffers. The morphology of a surfactant coating is also shown to affect its ability to inhibit protein adsorption to the capillary wall. Specifically, the full surface coverage provided by DDAB proved superior in a head-to-head comparison with CTAB.     

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.     

Elemental speciation analysis by multicapillary gas chromatography with microwave-induced plasma atomic spectrometric detection

Multicapillary column gas chromatography (MC-GC)/microwave-induced plasma atomic emission spectrometry (MIP AES) was developed for fast speciation analysis of organotin compounds in the environment. Ethylated butyltin compounds could be separated isothermally within less than 30 s (instead of ～5-10 min) without sacrificing either the resolution or the sample capacity of conventional capillary GC with oven temperature gradient programming. Careful optimization of the pressure and temperature GC program allowed a comprehensive organotin speciation analysis including phenyltin compounds within less than 2.5 min, increasing the sample throughput 6-fold. Compatibility of MC-GC with an MIP atomic emission detector (MIP-AED) was discussed. MC-GC/MIP-AES was validated for the analysis of sediment (PACS-1 and BCR 462) and biological (NIES11) certified reference materials.     

Contactless conductivity detection for capillary electrophoresis

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.     

Passive conductivity detection for capillary electrophoresis

A passive electrochemical detection principle that can be applied to capillary electrophoresis is presented. The separation electrical field is used to generate a potential difference between two electrodes located along the channel. For constant-current electrophoresis, the generated signal is proportional to the resistance of the solution passing between the two electrodes. Contrary to conductivity detectors that are ac driven and need to be decoupled from the separation field, the passive detection directly takes advantage of the separation field. The signal is simply measured by a high-impedance voltmeter. The detection concept has been validated by numerical simulations showing how the magnitude of the signal is related to the ratio between the electrode distance and the length of the sample plug. As a proof of the principle, this detection concept has been demonstrated by the electrophoretic separation of three alkali ions on a polymer microchip. Based on preliminary results, a detection limit of 20 microM and a dynamic range of up to 3 orders of magnitude have been achieved.     

Separation of serotonin from catechols by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis with electrochemical detection is demonstrated with columns having only 9-microns inner diameter. Amperometric detection limits of 0.7 amol are reported for serotonin. The difficult problem of resolving serotonin and dopamine--two neurotransmitters of interest having similar electrophoretic mobilities--is addressed by chemical means to improve selectivity. These include buffer modification with 2-propanol and a system employing borate complexation of the catechol in combination with sodium dodecyl sulfate micelles.