High-sensitivity detection of biological amines using fast Hadamard transform CE coupled with photolytic optical gating

Here, we report the first utilization of Hadamard transform CE (HTCE), a high-sensitivity, multiplexed CE technique, with photolytic optical gating sample injection of caged fluorescent labels for the detection of biologically important amines. Previous implementations of HTCE have relied upon photobleaching optical gating sample injection of fluorescent dyes. Photolysis of caged fluorescent labels reduces the fluorescence background, providing marked enhancements in sensitivity compared to photobleaching. Application of fast Hadamard transform CE (fHTCE) for fluorescein-based dyes yields a ten-fold higher sensitivity for photolytic injections compared to photobleaching injections, due primarily to the reduced fluorescent background provided by caged fluorescent dyes. Detection limits as low as 5 pM (ca. 18 molecules per injection event) were obtained with on-column LIF detection using fHTCE in less than 25 s, with the capacity for continuous, online separations. Detection limits for glutamate and aspartate below 150 pM (1-2 amol/injection event) were obtained using photolytic sample injection, with separation efficiencies exceeding 1 x 10(6) plates/m and total multiplexed separation times as low as 8 s. These results strongly support the feasibility of this approach for high-sensitivity dynamic chemical monitoring applications.     

DNA separations in microfabricated devices with automated capillary sample introduction

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.     

Parallel separations of oligonucleotides with optically gated sample introduction on multichannel microchips

With the release of the human genome sequence, there has been increasing attention given to other genetic analyses, including the detection of genetic variations and fast sequencing of multiple samples for pharmacogenomics studies. Rapid injections of samples in multiplexed separation channels by optically gated sample introduction are shown here for DNA separation. Serial separations of four amino acids are shown in less than four seconds on a microchip with four multiplexed channels. Five short oligonucleotides have also been rapidly separated in 2% LPA with four channels using this technique. In addition, multiple unique samples have been simultaneously separated and five-base resolution has been demonstrated.     

Characterization of electrokinetic gating valve in microfluidic channels

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.     

Investigation of the mechanism of pH-mediated stacking of anions for the analysis of physiological samples by capillary electrophoresis

Capillary electrophoresis has been widely used for the analysis of physiological samples such as plasma and microdialysate. However, sample destacking can occur during the analysis of these high-ionic strength samples, resulting in poor separation efficiency and reduced sensitivity. A technique termed pH-mediated stacking of anions (base stacking) has previously been developed to analyze microdialysate samples and achieve on-line preconcentration of analytes by following sample injection with an injection of sodium hydroxide. In this work, the mechanism of base stacking was investigated. Peak efficiency was shown to be a function of background electrolyte and sample ionic strength. Analytes representing several classes of compounds with a wide range of mobilities were used to study the effects of multiple parameters on sample stacking. The length of hydroxide injection required for stacking was shown to be dependent on analyte mobility and the type of amine background electrolyte used. Combinations of electrokinetic and hydrodynamic injections of sample and hydroxide were examined and it was concluded that although stacking could be achieved with several injection modes, electrokinetic injection of both sample and hydroxide was most effective for sample stacking. The mechanism of pH-mediated stacking for each of these modes is presented.     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

Robustness of a method based on the serial coupling of reversed‐phase and zwitterionic hydrophilic interaction LC–MS for the analysis of phenols

We have recently proved that the serial coupling of RP and zwitterionic hydrophilic interaction LC with mass spectrometric detection is a versatile and reliable technique to simultaneously separate polar and apolar compounds in complex samples, for example, phenols in wine. In order to evaluate the system suitability for long‐term usage, the robustness of a method based on the serial coupling of RP and zwitterionic hydrophilic interaction LC was evaluated after one year of analyses comprising >1100 injections. The retention time and peak shape of phenol standards and phenols in a red wine were compared to the values previously published. Phenol retention times were shifted <30 s. However, the peak widths were increased, partially due to the deterioration of the stationary phases. Nevertheless, the method was still highly reliable for the analysis of phenols in wine.     

The use of anion-exchange disks in an optrode coupled to a multi-syringe flow-injection system for the determination and speciation analysis of iron in natural water samples

A combination of multi-syringe flow-injection analysis (MSFIA) technique with an optical fibre reflectance sensor for the determination of iron in water samples has been developed in this work. Anion-exchange solid phase extraction (SPE) disks have been used as solid phase. Ammonium thiocyanate has been chosen as chromogenic reagent for Fe(III). The complex Fe[SCN]₆³⁻ is retained onto the SPE disk and spectrophotometrically detected at 480 nm. The complex is eluted with 0.25 mol l⁻¹ hydrochloric acid in 75% ethanol. Total iron can be determined by oxidising Fe(II) to Fe(III) with hydrogen peroxide.A mass calibration was run within the range of 0.4-37.5 ng. The detection limit (3s_b/S) was 0.4 ng. The repeatability (RSD), calculated from 9 replicates using 0.5 ml injections of a 25 μg l⁻¹ concentration, was 3.6%. The repeatability between five anion-exchange disks was 5.4%. An injection throughput of 7 injections per hour for a sampling volume of 1 ml has been achieved.The applicability of the proposed methodology in natural water samples has been proved.The properties of anion-exchange and chelating SPE disks have been studied and compared.     

Flow Injection Analysis of Mixtures of Dopamine,Adrenaline and Ephedrine in Human Biofluids Using Stabilized after Storage in Air Lipid Membranes with a Novel Incorporated Resorcin[4]arene Receptor

This work describes a technique for the rapid, selective and sensitive electrochemical flow injection analysis of mixtures of the stimulating compounds adrenaline, dopamine, and ephedrine using stabilized after storage in air bilayer lipid membranes (BLMs) with incorporated resorcin[4]arene receptor. Injections of the stimulating compounds were made into flowing streams of a carrier electrolyte solution and a transient current signal, with duration of seconds, reproducibly appeared in less than two min after exposure of the lipid membranes to the compounds. The magnitude of this signal was linearly related to the concentration of the compound, which could be determined at micromolar levels. Repetitive cycles of injection of stimulating compounds have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each stimulating compound and increased in the order of adrenaline, dopamine and ephedrine. The difference in time of response has allowed selective detection and analysis of these compounds in mixtures. The investigation of the effect of potent interferences included a wide range of compounds usually found in human biofluids, as well as proteins and lipids. The results showed that only proteins (most common in lipid film based biosensors) pose a problem that can be eliminated by modulation of the carrier solution to flow rates that do not allow adsorption of these compounds in the lipid films. The technique was applied in human urine samples.     

Increased throughput in quantitative bioanalysis using parallel-column liquid chromatography with mass spectrometric detection

The feasibility of quantitative bioanalysis by parallel-column liquid chromatography in conjunction with a conventional single-source electrospray mass spectrometer has been investigated using plasma samples containing a drug and its three metabolites. Within a single chromatographic run time, sample injections were made alternately onto each of two analytical columns in parallel at specified intervals, with a mass spectrometer data file opened at every injection. Thus, the mass spectrometer collected data from two sample injections into separate data files within a single chromatographic run time. Therefore, without sacrificing the chromatographic separation or the selected reaction monitoring (SRM) dwell time, the sample throughput was increased by a factor of two. Comparing the method validation results obtained using the two-column system with those obtained using the corresponding conventional single-column approach, the methods on the two systems were found to be equivalent in terms of accuracy and precision. The parallel-column system is simple and can be implemented using existing laboratory equipment with no additional capital outlays. A parallel-column system configured in this manner can be used not only for the within-a-run analysis of two samples containing two different sets of chemical entities, but also for the within-a-run analysis of two samples containing the same set of chemical entities.     

Flow injection analysis for calcium in serum,water and waste waters by spectrophotometry and by ion-selective electrode

The Flow Injection technique is shown to provide fast, reliable and sensitive methods for the determination of calcium in various aqueous as well as serum samples; spectrophotometric or potentiometric detection can be used. At sampling rates of 100–110 samples per hour, with 30-μl sample injections, high reproducibility of measurement and low reagent consumption are achieved in both methods. In the spectrophotometric method, the analytical readout is available within 12 s after sample injection at a total reagent consumption of 0.75 ml per analysis. The potentiometric measurement of the calcium activity in serum is placed on a reliable basis by alternating measurements of serum samples and aqueous standards without incurring any non-reproducible changes in potential between aqueous and serum solutions. This permits the simultaneous determination of pH and pCa, the analytical readout being available within XXX s of sample injection. The good agreement between the results obtained with the Flow Injection method and those attained by atomic absorption and EDTA titrations as well as pCa stat-measurements show that the new methods are potentially suitable for routine analysis.     

An automated capillary electrophoresis system for high-speed separation of DNA fragments based on a short capillary

A high-speed DNA fragment separation system was developed based on a short capillary and a slotted-vial array automated sample introduction system. The injection process of DNA sample in a short capillary was investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca. 20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of ΦX174-Hae III digest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in <100 s with plate heights ranging from 0.21 to 0.74 μm (corresponding to plate numbers from 4.86 × 10(6) to 1.36 × 10(6)/m). The repeatabilities for the migration time of the eleven fragments were between 0.4 and 1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.     

Enhanced pH-mediated stacking of anions for CE incorporating a dynamic pH junction

A technique has been developed to enhance analyte focusing for CE for the analysis of physiological samples. High-ionic-strength samples are titrated to low-ionic-strength on-line using pH-mediated sample stacking in conjunction with a dynamic pH junction. This method concentrates analytes by reducing their electrophoretic mobility during field-amplification. Parameters responsible for enhanced focusing were investigated, and an enhanced pH-mediated stacking method was optimized for anionic nucleosides. The process results in ultra-narrow peak widths, for example, 0.28 s for thymidine with a 10 min analysis time. Peak width and resolution with the enhanced stacking method were also compared to normal base stacking and electrokinetic injection. With this technique, mass-loading capacity can be increased without degradation in peak shape and resolution is dramatically improved.     

Capillary electrophoretic separation of dsDNA under nonuniform electric fields

Improved sensitivity for the analysis of DNA by capillary electrophoresis has been achieved, based on simultaneous increases in optical path length and injection volume. To increase the optical path length, bubble cells with diameters ranging from 150 to 450 microm have been fabricated and tested. In terms of resolution and sensitivity, a bubble cell of 300 microm diameter is appropriate when using 75-microm capillaries. To allow greater injection volumes, we performed on-line concentration of DNA in the presence of electroosmotic flow (EOF) using 2.0% poly(ethylene oxide) (PEO). With a 300-microm bubble cell, a 170-fold improvement in the sensitivity for the 89-bp fragment has been accomplished when injecting about 0.33 microL DNA. In the presence of the bubble cell, the resolution for the large fragments improves while that for the small ones (<124 base pair) decreases. The effect of bubble cells was further investigated by conducting DNA separation in the absence of EOF, showing that improvements in resolution are mainly due to increased migration differences when DNA migrated at low electric field strengths in the bubble region. We have suggested that such an effect is more profound using shorter capillaries, leading to complete separation of phiX 174 RF DNA-Hae III digest in 2 min.     

Headspace analysis with cryogenic focusing: A procedure for increasing the sensitivity of automated capillary headspace analysis

Summary A convenient procedure has been described for enhancing the sensitivity of equilibrium headspace gas chromatography. The technique involves the cryogenic focusing of headspace vapors at the head of a 0.32 mm fusedsilica capillary column which has been coupled to a packed column injection port. Cryofocusing is accomplished automatically by cooling the GC oven and results in enhanced sensitivity with improved resolution. With this simple focusing technique no sample splitting is required which gives at least a 10-fold increase in sensitivity. A Multiple Headspace Injection (MHI) technique has been described which allows several rapid headspace injections to be made at the start of a single chromatographic run. Cryofocusing causes the injections to be superimposed, thus increasing the sensitivity even further. These procedures have been demonstrated for flavor and aroma analyses of toothpaste and soft drink samples. Presented at the Symposium on Headspace GC, National American Chemical Society Meeting, Chicago, Ill., September 8–13, 1985     

pH-mediated field-amplified sample stacking of pharmaceutical cations in high-ionic strength samples

Capillary electrophoretic separation of samples of physiological origin typically have both poor resolution and efficiency due to destacking. We have previously reported a stacking method for concentration of catecholamines in artificial dialysate, or Ringer's solution. However, pH-mediated sample stacking of other cations has not been investigated. In this report, pH-mediated stacking has been extended to eletripan, dofetilide, doxazosin, sildenafil, UK-103,320, UK-202,581, and CP-122,288. These compounds were chosen without prior structural screening except that they were cationic at the pH of our background electrolyte (BGE). Capillary electrophoretic behavior of samples in BGE is compared with those of samples in Ringer's solution with and without pH-mediated acid stacking. Results indicate that the peak heights and efficiencies for acid-stacked samples are increased compared to the unstacked samples in Ringer's solution or BGE. For example, the peak efficiencies for 5 s injections of eletriptan in BGE and Ringer's solution are 138,000 and 72,000 plates, respectively. In contrast, a 10 s injection of eletriptan followed by acid injection for 16 s produces a peak with 246,000 plates. Evaluation of the stacking effect was performed by comparison of the peak height at similar peak efficiencies for samples in Ringer's solution with and without stacking. Using this method, pH-mediated acid stacking provides a 10- to 27-fold sensitivity enhancement for the seven cations.     

On-line liquid chromatography – gas chromatography for automated clean-up and analysis of polycyclic aromatic hydrocarbons

A fully automated system, comprising a liquid chromatograph (LC) coupled on-line to a gas chromatograph (GC) by means of a loop interface, has been constructed for clean-up and analysis of polycyclic aromatic hydrocarbons (PAH). An autosampler was utilized for sample injection into the LC. By the use of a back-flush technique in conjunction with an ordinary analytical aminopropylsilica column, PAH could be isolated by LC: a concurrent solvent evaporation injection technique was then used for on-line transfer of the PAH fraction to the GC, where the PAH analysis was completed. Compared with ordinary off-line LC clean-up followed by GC analysis, the sensitivity has been increased by a factor of 50–100, yielding a detection limit for individual PAH of a few nanograms per sample when using flame ionization detection. Further, irreproducible losses of low molecular weight PAH as a result of solvent evaporation steps in off-line clean-up procedures have been eliminated. Reproducibility of retention times and relative peak areas is high, facilitating automatic peak identification and calculation of concentrations, and the system can thus be used for automatic sample evaluation. The total time required for clean-up and analysis is only 1.5 hours, and the demand on personnel time for the analysis of PAH has been drastically reduced. The technique has been demonstrated with samples of urban air and of used automobile engine lubricating oil.     

Modified Hadamard transform microchip electrophoresis

Sensitivity is a crucial point in the development applications for medicine or environmental samples in which the analytes are present in the nanomolar range. Besides further technical development of detection systems, the multiplex sample injection technique can be applied for enhancing the signal-to-noise ratio. Hadamard transform is easily applied to microchip electrophoresis due to the fact that sample injection is generally achieved through cross, double-tee, or tee injector structures. This paper reports the first demonstration of a modified Hadamard transform electrophoresis on a microchip by using an amperometric detector. Contrary to the previous Hadamard applications, the resolution (number of points per unit of time) of electropherograms obtained is independent of the number of injections.     

Performance of programmed temperature vaporizer, pulsed splitless and on-column injection techniques in analysis of pesticide residues in plant matrices.

A programmed temperature vaporizer (PTV) injection technique has been recently implemented in our laboratory. In present paper its performance is compared with other GC injection techniques commonly used in trace analysis of organic contaminants. Twenty-six pesticides representing different chemical classes were selected for the study. This group comprised compounds typically subjected to discrimination in the injection port of the gas chromatograph, e.g., polar organophosphorus pesticides and thermolabile carbamates. In the first set of experiments standards in pure solvent were injected into GC systems employing different types of injection, i.e., (i) on-column, (ii) pulsed splitless, (iii) PTV solvent split, (iv) PTV splitless, and the responses of analytes were compared. Discrimination of troublesome compounds was significantly decreased with the application of PTV solvent split injection. In the second set of experiments repetitive injections of purified wheat samples were performed, with aims to evaluate the long-term stability of responses, as well as matrix effects in different stages of system contamination for each injection technique. The tolerance of the GC system to co-injected matrix components was increased in the order: on-column相似文献