Planar chip technology for capillary electrophoresis

Miniaturization of separation columns implies equally reduced volumes of injectors, detectors and the connecting channels. Planar chip technology provides a powerful means for the fabrication of micron sized structures such as channels. This is demonstrated with three examples. An optical absorbance detector chip exhibits the expected behavior of a 1 mm optical pathlength cell despite its volume of 4 nL. A capillary electrophoresis device allows for integrated injections of 100 pL samples, for efficiencies of 70 000 to 160 000 theoretical plates in 10 to 20 seconds, and for external laser-induced fluorescence detection at any capillary length of choice between 5 and 50 mm. A system for synchronized cyclic capillary electrophoresis is also presented in which plate numbers per volt can be dramatically increased.     

High performance optical absorbance detectors based on low noise switched integrators

Optical absorption detection is the most common analytical measurement principle in liquid phase analysis. The current state-of-the-art of commercially available detectors exhibit peak-to-peak (p-p) noise levels in the range of 1 x 10(-5)-2 x 10(-5) absorbance units (10-20 microAU). Using circuitry based on newly available switched integrator integrated circuit (IC) packages, it is possible to construct inexpensive absorbance detectors with p-p noise levels as low as 3 microAU under actual use conditions. The necessary electronics are described and performance data are reported with light emitting diodes (LEDs) as light sources. Even in the capillary format with a rectangular capillary (50 x 1000 microm cross section) with a slitwidth <50 microm and with the 1000 microm dimension as the nominal pathlength, p-p noise levels of 10 microAU are observed, from which a concentration limit of detection (LOD) of 10 nM for bromothymol blue (BTB) can be estimated with a 660 nm light source.     

Performance of an in-plane detection cell with integrated waveguides for UV/Vis absorbance measurements on microfluidic separation devices

A microfluidic device with integrated waveguides and a long path length detection cell for UV/Vis absorbance detection is presented. The 750 microm U-cell detection geometry was evaluated in terms of its optical performance as well as its influence on efficiency for electrophoretic separations in the microdevice. Stray light was found to have a strong effect on both, the sensitivity of the detection and the available linear range. The long path length U-cell showed a 9 times higher sensitivity when compared to a conventional capillary electrophoresis (CE) system with a 75 microm inner diameter (ID) capillary, and a 22 times higher sensitivity than with a 50 microm ID capillary. The linear range was comparable to that achieved in a 75 microm ID capillary and more than twice as large as in a 50 microm ID capillary. The use of the 750 microm U-cell did not contribute significantly to band broadening; however, a clear quantification was made difficult by the convolution of several other band broadening sources.     

A multireflection cell for enhanced absorbance detection in microchip-based capillary electrophoresis devices

The design, fabrication and testing of a photolithographically fabricated, glass-based multireflection absorbance cell for microfluidic devices, in particular microchip-based capillary electrophoresis (CE) systems is described. A multireflection cell was fabricated lithographically using a three-mask process to pattern aluminum mirrors above and below a flow channel in a chip, with 30 microm diameter optical entrance or exit apertures (one in each mirror) positioned 200 microm apart. Source and detector were positioned on opposite sides, and the metal mirrors were made 1 cm square, to reduce stray light effects. Calibration curves using bromothymol blue (BTB) with a 633 nm source (He:Ne laser) were linear to at least 0.5 absorbance units, with typical r2 values of 0.9997, relative standard deviations in the slopes of +/- 1.3%, and intercepts of zero within experimental error. Effective optical pathlengths of 50-272 microm were achieved, compared to single-pass pathlengths of 10-30 microm, corresponding to sensitivity enhancements (i.e., optical path length increase) of 5 to 10-fold over single-pass devices. Baseline absorbance noise varied within a factor of two in almost all devices, depending only weakly on path length. This device can give much higher absorbance sensitivity, and should be much easier to manufacture than planar, glass-based devices previously reported.     

Analysis of sludge extracts by high resolution GC with selective detectors

Combination of various GC detectors by using a Varian effluent splitter with glass capillary columns has been found to be a rapid procedure for profiling organics extracted from sludges and river sediments. The selectivity and the increased sensitivity of the thermionic nitrogen-phosphorus detector (TSD), the electron capture detector (ECD), and the flame photometric detector (FPD) over the flame ionization detectors (FID) or mass spectrometers allow the detection of compounds present at trace levels without need for extensive sample cleanup. Furthermore, the combination of two selective detectors may supplement the information with regard to the chemical functionalities required for structure elucidation.     

Performance of a simple UV LED light source in the capillary electrophoresis of inorganic anions with indirect detection using a chromate background electrolyte

Light emitting diodes (LEDs) are known to be excellent light sources for detectors in liquid chromatography and capillary electromigration separation techniques, but to date only LEDs emitting in the visible range have been used. In this work, a UV LED was investigated as a simple alternative light source to standard mercury or deuterium lamps for use in indirect photometric detection of inorganic anions using capillary electrophoresis with a chromate background electrolyte (BGE). The UV LED used had an emission maximum at 379.5 nm, a wavelength at which chromate absorbs strongly and exhibits a 47% higher molar absorptivity than at 254 nm when using a standard mercury light source. The noise, sensitivity and linearity of the LED detector were evaluated and all exhibited superior performance to the mercury light source (up to 70% decrease in noise, up to 26.2% increase in sensitivity, and over 100% increase in linear range). Using the LED detector with a simple chromate-diethanolamine background electrolyte, limits of detection for the common inorganic anions, Cl-, NO3-, SO4(2-), F- and PO4(3-) ranged from 3 to 14 microg L(-1), using electrostatic injection at -5 kV for 5 s.     

Optimisation of probe concentration in indirect photometric detection in capillary electrophoresis using highly absorbing dyes

In indirect photometric detection in capillary electrophoresis, the concentration of the absorbing probe ion in the background electrolyte should be as high as possible in order to increase the dynamic range of the detection method. For relatively low absorptivity probes (epsilon < 2000 L mol(-1)cm(-1)) used under typical conditions (75 microm ID capillary) the maximum probe concentration is normally limited by the separation current. However, for medium (epsilon approximately/= 2000-15000 L mol(-1)cm(-1)) and especially for high (epsilon > 15000 L mol(-1)cm(-1)) absorptivity probes such as dyes, the maximum concentration may be limited by the background absorbance of the electrolyte which must fall within the linearity range of the detector. In this work, it is shown that another practical factor limiting the probe concentration is the adsorption of probe onto the capillary wall at higher concentrations, resulting in unstable baseline and increased noise. Use of a zwitterionic surfactant to suppress adsorption enabled the concentration of a model probe anion (tartrazine) to be increased by a factor of six times (to 3 mM). This resulted in significant improvements in peaks shapes, resolution between peaks, detection sensitivity and linear calibration range for the analyte anions. Baseline separation of a test mixture was maintained up to 7.5 mM total concentration of sample coions injected (13.7 nL) for the 3 mM electrolyte, with detection limits ranging from 0.63 to 0.94 microM. Peak height reproducibility (over 20 consecutive injections) was improved (values ranging from 1.1 to 1.9%) compared with electrolytes containing lower concentrations of the probe. Overall, the optimised, higher concentration probe electrolyte provided the sensitivity benefits of highly absorbing probes with the additional benefits of ruggedness and improved stacking, peak shapes and resolution.     

Evaluation of the performance of various universal and selective detectors for sulfur determination in natural gas

In the present contribution, the performance of a number of commercially available selective and universal detectors that can be used in the analysis of sulfur components in natural gas is evaluated in terms of sensitivity, selectivity, reproducibility, quenching effect, stability, and compound dependence of the sulfur response. Investigated detectors include the sulfur chemiluminescnce, the flame photometric, the electron capture, the mass spectrometric, the thermal conductivity, and the flame ionization detector. The sulfur chemiluminescence detector was found to have the best overall performance, e.g., low picogram amounts of sulfur can be detected accurately and the linear dynamic range is more than five orders of magnitude. After careful optimization, the sulfur response of this detector was found to be almost compound independent. All other detectors, including the flame photometric and the electron capture detector, have more or less compound dependent responses.     

Enhancement of detection sensitivity for indirect photometric detection of anions and cations in capillary electrophoresis

This review focuses on the indirect photometric detection of anions and cations by capillary electrophoresis. Special emphasis has been placed on the sensitivity of the technique and approaches taken to enhance detection limits. Theoretical considerations and requirements have been discussed, including buffering, detection sensitivity, separation of cations, and detector linearity. A series of tables detailing highly absorbing probes and the conditions of their use for indirect photometric detection are included.     

Indirect photometric detection in capillary zone electrophoresis

An indirect photometric detection method is described which is based on the use of an absorbing co-ion as the principal component of the background electrolyte. The zones of non-absorbing ionic species are revealed by changes in light absorption due to charge displacement of the absorbing co-ion. Theoretical considerations are given for selecting a suitable absorbing co-ion to achieve a high sensitivity of detection.The role of electromigration dispersion is illustrated by experiments and the effects of the differences in the effective mobilities of sample ions and that of the absorbing co-ion are discussed. The highest sensitivity can be achieved for sample ions having an effective mobility close to the mobility of the absorbing co-ion. In such a case, the concentration of the sample component in its migrating zone can be high while electromigration dispersion is still negligible. The useful dynamic range of the detection is then limited by the linearity and noise of the detector, the former parameter being given mostly by the shape of the on-column detection cell. The best sensitivities can be obtained in low-concentration background electrolytes containing a co-ion with high absorption at a given detection wavelength.It is shown that indirect photometric detection can be useful for detecting substances that have no optical absorption in the UV and/or visible region, provided that the composition of the background electrolyte is selected correctly.     

Electrophoretically mediated microanalysis with small molecules: the Jaffé method for creatinine carried out in a capillary tube

An eletrophoretically mediated microanalysis (EMMA) approach, used to perform online chemistry between two small molecules, has been characterized and optimized. The "plug-plug" type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of creatinine-containing samples and alkaline picrate (Jaffe reaction) within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure injecting a plug of picrate followed by a plug of the creatinine-containing sample. A potential was then applied to electrophoretically mix the two reactants, and an incubation time of up to 6 min allowed the reaction to proceed prior to the application of a 27 kV separation potential with absorbance detection at 485 nm. The use of a 50 microm inner diameter(ID) extended light path capillary (150 microm pathlength) was found to be adequate for determining elevated levels of creatinine in human blood sera, but could not be used to quantify normal levels. Quantification of both normal and elevated levels of creatinine in sera was possible with a 75 microm ID high-sensitivity cell (1200 microm pathlength). Calibration plots using the latter for creatinine in human blood sera spanned the expected clinical range and were linear between 40 microM and 1.2 mM (r2 = 0.996) with an estimated limit of detection of 17 microM (signal-to-noise ratio S/N = 3). A quantitative comparison of results obtained with the reported EMMA method and accepted clinical methodology correlated very well (slope = 1.001).     

Design and performance of a light-emitting diode detector compatible with a commercial capillary electrophoresis instrument

Indirect photometric detection in capillary electrophoresis (CE) has been predominantly performed in the UV region, in part due to a lack of suitable high-intensity and low-noise light sources in the visible spectral region. A new photometric detector based on light-emitting diodes (LEDs) as light sources and compatible with a commercially available CE instrument has been designed and constructed and its performance evaluated. The utility of this detector was successfully demonstrated by the indirect photometric detection of anions using a dye as probe and absorbance measured in the visible region. The detector exhibited very low baseline noise (around 0.03 mAU), stable output, and improved upper limit of detection linearity (502 mAU) compared with previously used LED detectors. The detector was tested for indirect detection of anions separated with an electrolyte containing 4 mM Orange G as the indirect detection probe, 10 mM histidine as an isoelectric buffer, and 0.05% hydroxypropylmethylcellulose to suppress the electroosmotic flow. Extremely low detection limits were obtained ranging from 0.16-0.36 microM (excluding chloride 0.56 microM), with separation efficiencies in the range of 154,000-274,000 theoretical plates.     

Improved dual photometric-contactless conductometric detector for capillary electrophoresis

A new design of a dual, UV photometric - contactless conductometric detector is described. The separation capillary with an optical window created is pressed onto two semitubular electrodes, 3 mm wide and 2 mm apart. The electrodes form the detection cell of the contactless conductometric detector. An optical fiber, placed in the gap between the conductometric electrodes, brings radiation from the source. The radiation that passes through the separation capillary is recorded by a large-area photodiode. The optical fiber and the photodiode operate the photometric cell which is between the conductometric electrodes. The detector thus permits simultaneous photometric and conductometric detection in the same place of the capillary, while exchanging of the separation capillary is easy and without effect on the detector geometry and performance.     

Miniaturization of separation techniques using planar chip technology

Miniaturization of separation columns implies equally reduced vol- umes of injectors, detectors, and the connecting channels. Planar chip technology provides a powerful means for the fabrication of micron-sized structures such as channels. This is demonstrated by two examples. An optical absorbance detector chip exhibits the expected behavior of a 1 mm optical path length cell despite its volume of 1 nL. A capillary electrophoresis device allows integrated injections of 100 pL samples, efficiencies of 70,000 to 160,000 theoretical plates in 10 to 20 seconds, and external laser-induced fluorescence detection at any capillary length of choice between 5 and 50 mm.     

Determination of triphenyltin hydroxide derivatives by capillary GC and tin-selective FPD

A new method for the determination of triphenyltin hydroxide using capillary column gas chromatography with a tin-selective flame photometric detector has been developed. Triphenyltin hydroxide and its potential metabolites are converted to methyl derivatives and separated on glass capillary columns coated with OV-101. Derivatization of triphenyltin hydroxide, triphenyltin chloride, diphenyltin dichloride, phenyltin trichloride, and bis-triphenyltin oxide is nearly quantitative with a minimum of redistribution products. The selectivity of the flame photometric detector is cearly demonstrated by the comparison of chromatographic profiles obtained from using both the flame photometric and flame ionization detectors. The use of this chromatographic system in the analysis of triphenyltin hydroxide in a fortified water sample demonstrates the potential use of this system in organotin residue chemistry.     

Indirect fluorometric detection of tryptic digests separated by capillary zone electrophoresis

This work demonstrates the analytical utility of indirect fluorescence detection with capillary zone electrophoresis (CZE) for the analysis of trace quantities of macromolecular mixtures. Detection is based upon charge displacement and is not based upon any absorption or emission property of the analyte. No derivatization step is required. Indirect fluorescence is therefore a general detector for electrophoresis. Subfemtomolar quantities of tryptic digest mixtures are separated within three minutes, and reproducible peaks are obtained from the mixtures. Mass limits of detection are 3000 times lower than those of commercial high-performance liquid chromatography (HPLC) UV absorbance detectors and 180 times lower than those of UV absorbance detectors in CZE systems. This separation and detection system should be well suited to analysis of trace quantities of mixtures of peptides.     

毛细管电泳光学检测器的进展

对毛细管电泳的光学检测器作了简要评述。根据所采用的检测原理。光源检测器可分为紫外检测器，激光诱导荧光检测器、化学肆光检测器、荷耦合器件检测器、折射指数检测器等许多种类，具有简单方便、使用广泛，信息量较大等特点，是一类有良好诉检测器。     

Characteristics and performance of gas chromatographic detectors with glass capillary columns

A comparison of the characteristics and Performance of the flame ionization, electron capture, and flame photometric detectors with capillary columns is described. Factors which may affect the limits of detection, linearity, chromatographic peak shape, and other detector performance characteristics are discussed and compared with the results of a model derived for the behavior of concentration and mass flow-rate dependent chromatographic detectors used with capillary GC systems. Examples are given of highly complex and labile mixtures such as pesticide residues and products from coal hydrogenation.     

Spectroelectrochemical detector for flow-injection systems and liquid chromatography

Flow-through spectroelectrochemical detectors for flow-injection systems and liquid chromatography are described. The detectors have a rectangular flow channel with a reticulated vitreous carbon working electrode followed by an open optical window. The dead volumes of the cells are 27 μl (liquid chromatography) and 80 μl (flow injection). In situ spectral monitoring of reaction products and intermediates for compounds that are both weakly and highly absorbing is demonstrated by using o-tolidine and N,N,N′,N′-tetramethyl-p-phenylenediamine. As a detector for flow-injection systems, components in two-component mixtures can be quantified. As a detector for liquid chromatography, simultaneous absorbance and electrochemical chromatograms allow more eluting compounds to be identified and quantified. Mixtures of nitro- and chloro-phenols are used to illustrate the simultaneous profiling of spectral and redox properties.     

Miniaturized movable contactless conductivity detection cell for capillary electrophoresis

A miniaturized capacitively coupled contactless conductivity detector (mini-C(4)D) cell has been designed which is small enough to allow it to slide along the effective capillary length inside the capillary cassette of an Agilent capiillary electrophoresis system (CE) (or other CE brand of similar construction), including the possibility of positioning it close to the point of optical detection (4 cm), or even putting two such detector cells in one cassette. The cell was tested and the performance characteristics (noise, sensitivity, and peak width) were compared with those obtained with the previously used large C(4)D cell. No significant differences were observed. The mini-C(4)D was used in simultaneous separations of common cations and anions where its advantage over a larger C(4)D cell is the ability to vary the point of detection with the mini-C(4)D cell continuously at any point along the capillary length, so that the optimum apparent selectivity can be chosen. Other applications include providing a convenient second point of detection in addition to photometric detection, such as to measure accurately the linear velocity of a zone, or to allow placement of two mini-C(4)D cells in one capillary cassette simultaneously.