Signal enhancement by on-column fluorimetric detection in gradient elution of dansyl amino acids in liquid chromatography

Summary Fluorimetric detection in the presence of a stationary phase has been applied to gradient elution of dansyl amino acids in liquid chromatography. A 1.5 mm ID quartz tube packed with the same materials as the separation column was employed for the flow cell. Conventional-size columns were employed. The peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase, leading to improvements in sensitivity, which was pronounced for analytes eluting late. The lower the gradient, the larger the improvement in sensitivity achieved. Detection limits were improved by a factor of up to 5.1 by fluorimetric detection using the packed flow cell, compared with those achieved using a common empty flow cell.     

Signal enhancement by on-column fluorimetric detection in gradient elution of dansyl amino acids in liquid chromatography

Summary Fluorimetric detection in the presence of a stationary phase has been applied to gradient elution of dansyl amino acids in liquid chromatography. A 1.5 mm ID quartz tube packed with the same materials as the separation column was employed for the flow cell. Conventional-size columns were employed. The peak height of analytes increased with increasing retention owing to focusing and environmental effects of the stationary phase, leading to improvements in sensitivity, which was pronounced for analytes eluting late. The lower the gradient, the larger the improvement in sensitivity achieved. Detection limits were improved by a factor of up to 5.1 by fluorimetric detection using the packed flow cell, compared with those achieved using a common empty flow cell.     

Microchip reversed-phase liquid chromatography with packed column and electrochemical flow cell using polystyrene/poly(dimethylsiloxane)

A microchip pressure-driven liquid chromatography (LC) with a packed column and an electrochemical flow cell has been developed by using polystyrene (PS) and poly(dimethylsiloxane) (PDMS). The cylindrical separation column with packed octadecyl silica particles was fabricated in the PS substrate. The three electrode system (working, reference, and counter electrode) for amperometric detection was fabricated onto the PS substrate, using the Au deposition, photolithography, and chemical etching. The detector flow cell was formed by sealing the electrode system with a PDMS chip containing a channel. In this flow cell, the effect of working electrode width (in the direction of flow) on chromatographic parameters, such as peak width and peak resolution were studied in electrode width ranging 50-5,000 microm. The effect of electrode width on sensitivity (current intensity, current density, and S/N ratio) was also examined. The sensitivity was discussed by simulating the concentration profile generated around the working electrode. The effects of the column packing size and the column size on the separation efficiency were examined. In this study, a good separation of three catechins was successfully achieved and the detection limits for (+)-catechin, epicatechin, and epigallocatechin gallate were 350, 450, and 160 nM, respectively.     

New approaches on gas phase molecular absorption spectrometry detection in gas chromatography

A hyphenated technique for coupling a spectrophotometric detector to a gas chromatograph with a packed column is described. The packed column was connected directly to the flow cell (path length 1 cm) which was placed in a UV-visible molecular absorption spectrophotometer with diode array detector. This detection system was employed for separating and determining benzene, toluene, 1,4-xylene, 1,2-xylene and mesitylene. Molecular absorption spectra of all the separated compounds have also been obtained. A new possibility for coupling capillary gas chromatography and gas phase molecular absorption spectrometry is currently being tested. In this new case, two fibre optics are used and the measurement is performed in the capillary column.     

New approaches on gas phase molecular absorption spectrometry detection in gas chromatography

A hyphenated technique for coupling a spectrophotometric detector to a gas chromatograph with a packed column is described. The packed column was connected directly to the flow cell (path length 1 cm) which was placed in a UV-visible molecular absorption spectrophotometer with diode array detector. This detection system was employed for separating and determining benzene, toluene, 1,4-xylene, 1,2-xylene and mesitylene. Molecular absorption spectra of all the separated compounds have also been obtained. A new possibility for coupling capillary gas chromatography and gas phase molecular absorption spectrometry is currently being tested. In this new case, two fibre optics are used and the measurement is performed in the capillary column.     

A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter min-1. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.     

Flow injection electrochemical hydride generation of hydrogen selenide on lead cathode: critical study of the influence of experimental parameters.

A flow injection system for the determination of selenium by electrochemical hydride generation and quartz tube atomic absorption spectrometry is described. The generator consists of an electrolytic flow-through cell with a concentric arrangement and a packed cathode made of particulated lead. The influences of sample flow rate, carrier gas flow rate and electrolysis current on the hydrogen selenide generation have been critically studied. Both sample flow rate and electrolysis current play important roles in the efficiency of the hydride generation process. A characteristic mass of 2.4 ng and a concentration detection limit of 17 microg l(-1) were obtained for a sample volume of 420 microl.     

Radiochemical detection for packed capillary liquid chromatography-mass spectrometry

For the identification of trace level organic molecules, such as drug or pesticide metabolites, there is need of a practical method to do packed capillary liquid chromatography-mass spectrometry (LC-MS) with radiochemical detection. This problem has been successfully solved by use of a post column flow-splitter, with coaxially transported makeup flow that increases the split flow rate to a flow compatible with commercially available radiochemical flow cells. To test the device, 14C-labeled azoxystrobin, a commercial fungicide, was analyzed by liquid chromatography-radiochemical activity monitor-mass spectrometry (LC-RAM-MS) using a 0.32 mm i.d. packed capillary column. Azoxystrobin could be detected at 500 pCi with good signal/noise. The method is general and can be used with capillary LC columns of smaller diameters. Column efficiency of about 20,000 theoretical plates/m was achieved using either radiochemical or mass spectrometric data, thus demonstrating the lack of band broadening using the described method for radiochemical detection. The simple hardware described allows the routine use of packed capillary LC with radiochemical detection.     

On-line SFC/FT-IR analysis of agriculturally-related compounds

Several agricultural compounds which are used as herbicide precursors have been analyzed by SFC and are detected on-line via a flow cell FT-IR interface. A mixture of ureas is separated on a microbore packed column; while a mixture of benzamides and anilides and a mixture of sulfonamides are separated on capillary columns. The use of 100% CO₂ as the mobile phase enables an infrared spectrum of each eluting analyte to be obtained on-line with good signal to noise ratio. The urea mixture is also separated with a conventional cyanopropyl bonded phase analytical scale packed column using a methanol-modified CO₂ mobile phase and UV detection. The separations achieved with and without modifier are compared.     

Scintillator-fiber flow-cell radioactivity detector for liquid chromatography

Summary A new type of flow-cell radiation detector for use in liquid chromatography which is packed with aligned scintillator fibers is described. A primary advantage of the fiber packed cell is that light generated by the scintillator is absorbed to a much less extent by the fibers than by the powder scintillator used in conventional flow cells. A detection efficiency of 55% has been obtained for carbon-14 using 0.1-mm diameter hand-pulled glass fibers. Computer modeling has shown that even better results can be obtained by using smaller and more uniform diameter fibers which will allow better packing. The fiber cell also demonstrates back pressures which are a factor of 50 less than the conventional cell and much less susceptibility to absorption of compounds because of its lower surface area. Research sponsored by the Office of Energy Research, U.S. Department of Energy, under contract DE-AC05-840R21400 with Martin Marietta Energy Systems, Inc.     

Comparison of post-column and on-column fluorescence detection of UO22+ in water

Summary Automatic equipment for the treatment and subsequent injection of a liquid sample connected to a lab-made fluorescence cell is described. Adsorption and desorption of the selected solute were observed for the system of the flow cell following a packed column (post-column detection) and compared with a packed flow cell where the sorption material is localized directly in the fluorescence cell (on-column detection). Applicability of the system was demonstrated using the example of the determination of UO ₂ ²⁺ cation in surface water. The analysis of 272 l of the mixture of a real sample (10 l^–1 uranium) and buffer takes 14.5 min with RSD=±4.2% with enrichment of the solute on the pre-column and subsequent desorption. The system of on-column detection decreases the minimal detectable concentration eight times.     

Minicolumn field sampling--preconcentration of trace zinc from seawater and its laboratory detection by flow injection flame atomic absorption spectrometry.

A simple field sampling-preconcentration method for zinc determination in seawater is described. Seawater was collected in situ by pumping it through a minicolumn packed with a chelating resin (Chelite P) connected to a field flow preconcentration system (FFPS). These packed minicolumns retain the dissolved zinc, and once are loaded with the analyte, they are returned to the laboratory where they are sequentially inserted into a flow injection system for on-line zinc elution with diluted hydrochloric acid and flame atomic absorption spectrometric detection. A factorial design has been used to optimize the FFPS and the flow injection elution process. The proposed method has a linear calibration range from 0.07 to at least 9.4 microg L(-1) of zinc, with a detection limit of 0.02 microg L(-1) and a throughput of 26 samples h(-1). Validation was carried out against certified reference water samples. This procedure has been successfully applied to the determination of Zn in seawater samples from Galicia (Spain).     

A windowless flow cell-based miniaturized fluorescence detector for capillary flow systems

A miniaturized fluorescence detector utilizing a three-dimensional windowless flow cell has been constructed and evaluated. The inlet and outlet liquid channels are collinear and are located in the same plane as the excitation paths, while the optical fiber used to collect the emission light is perpendicular to this plane. The straightforward arrangement of the flow path minimizes band dispersion and eliminates bubble formation or accumulation inside the cell. The use of high-brightness light-emitting diodes (LEDs) as the excitation source and a miniaturized metal package photomultiplier tube (PMT) results in a compact and sensitive fluorescence detector. The detection limit obtained from the system for fluorescein isothiocyanate (FITC) in flow injection mode is 2.6 nmol/L. The analysis of riboflavin and FITC by packed capillary liquid chromatography is demonstrated.      

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.     

Solid-phase UV spectroscopic multisensor for the simultaneous determination of caffeine, dimenhydrinate and acetaminophen by using partial least squares multicalibration

A straightforward flow-through multisensor was developed for the fast simultaneous determination of caffeine (CF), dimenhydrinate (DMH) and acetaminophen (AAP) based on the integration of their retention and UV detection. A diode array spectrophotometer was used to monitor the inherent UV full-spectra in the range 245-310 nm of the analytes retained on C(18) bonded phase beads packed in a flow cell, without requiring additional reagents or derivatization processes. The extensively overlapped spectra of the analytes retained on the solid support could be resolved by partial least squares (PLS) regression. After collecting the response of the multisensor, its active microzone was regenerated by using methanol as the eluting agent, leaving it ready for the next determination. The proposed multisensor has been satisfactorily applied for the analysis of synthetic and real samples with different nominal contents of these active principles.     

Pressurized-flow anion-exchange capillary electrochromatography using a polymeric ion-exchange stationary phase

The feasibility of using capillary columns equipped with silica frits and packed with a polymer-based anion exchanger (Dionex AS9-HC) for CEC separations of inorganic anions has been investigated. Experiments using a conventional 25 cm packed bed, and mobile phase flow that is a combination of hydrodynamic and electroosmotic flow were used to demonstrate that by varying the applied voltage (electrophoresis component) or the concentration of the competing ion in the mobile phase (ion-exchange component), considerable changes in the separation selectivity could be obtained. Using an artificial neural network, this separation system was modelled and the results obtained used to determine the optimum conditions (9 mM perchlorate and −10 kV) for the separation of eight inorganic anions. When a short (8 cm) packed bed was used, with detection immediately following the packed section, the separation of eight test analytes in under 2.2 min was possible using pressure-driven flow and a simple step voltage gradient. A more rapid separation of these analytes was obtained by only applying high voltage (−30 kV), where many of the same analytes were separated in less than 20 s and with a different separation selectivity to that obtained in conventional ion-exchange or capillary electrophoresis separations.     

Development of solid-phase chemiluminescence immunoassays for digoxin comparing flow injection and sequential injection techniques.

The development of a competitive solid-phase immunoassay for digoxin making use of the acridinium chemiluminescence system is described. Two different instrumental approaches are compared. One is based on a continuous flow system using a peristaltic flow injection analysis pump; the other uses a new sequential injection technique. In both systems a flow cell, consisting of transparent PTFE tubing packed with immobilized antibodies, acts as an immunoreactor. The entire assay, including both the immunoreaction and the chemiluminescence reaction, takes place in this immunoreactor cell. Compared with the flow injection technique, the sequential injection mode showed higher precision, ranging from 2.16 to 5.5% RSD depending on concentration. The total assay time, including regeneration, is less than 8 min with the sequential injection technique. The detection limit for both techniques is in the low femtomole range.     

降低二硫化碳萃取液中痕量苯检出下限的气相色谱法

摘要：提出一个直接进样口（岛津WBI-17）和SE-30填充柱测定海水中微量苯的二硫化碳萃取气相色谱分析方法。方法的检测下限低、操作简单、样品用量少、重复性好。     

Simultaneous determination of trace metals by solid phase absorptiometry: application to flow analysis of some rare earths

Solid phase absorptiometry was applied to the simultaneous flow analysis of trace metals in combination with a micro black flow-through cell packed with ion exchanger beads and a multi-channel photodetector connected with optical fibers. A 4.0 cm³ sample solution containing five rare earth metal ions (Dy, Ho, Tm, Er and Nd) was introduced into a flow system and these metal ions were concentrated on a cation exchanger (Muromac 50W-X4) in a flow-through cell. The absorbance increases originating from the f-f or d-f transition bands of these metal ions were directly and continuously measured at 910 nm for Dy, at 530 nm for Ho, at 683 nm for Tm, 522 nm for Er and at 790 nm for Nd, respectively. A multi-variable analysis method was combined with the flow analysis because the absorption spectra of the five rare earth metal ions partly overlapped one another. Although no coloring reagents were used, the proposed method was about ¶200 times more sensitive than the corresponding solution method. The reproducibility of this method was less than ± 5%. The detection limits were 0.03, 0.40, 0.30, 0.35 and 0.23 mg dm^–3 for Dy, Ho, Tm, Er and Nd, respectively. Five rare earth metal ions could be precisely determined in practical samples such as yttrium concentrate.     

Low-level mercury determination with thiamine by fluorescence optosensing

A sensitive fluorescence optosensing method for the determination of Hg(II) in water samples is described. The method, using a flow injection technique, is based on the immobilization on a non-ionic-exchanger solid support (packed in a flow cell placed in a conventional fluorimeter) of the thiochrome formed by the oxidation of thiamine with Hg(II) in a continuous flow carrier at pH 8.1. Experimental parameters such as the solid support, the carrier pH, the thiamine concentration and the flow-rate were investigated to select the optimum operating conditions. The proposed optosensor showed a relative standard deviation of + 3.0% for ten replicates analysis of 100 ng ml(-1) of mercury(II). A detection limit of 3 ng ml(-1) for mercury(II) was achieved for 4-ml sample injections. A detailed study of interferences (possible elements present in natural waters) demonstrated that this optosensing method is virtually specific for this metal, because it allows the determination of mercury in the presence of relatively large amounts of other heavy metals and compounds present in natural waters, such as Mg(II) or Ca(II). The method was successfully applied to the determination of Hg(II) in spiked samples of mineral, tap and sea water.