Indirect Laser-induced Fluorescence Research Articles

Optimisation of analytical conditions for simultaneous determination of phenol and chlorophenols by capillary electrophoresis with indirect laser-induced fluorescence detection

This work presented a capillary electrophoresis method with indirect laser-induced fluorescence detection for the simultaneous determination of phenol and four chlorophenol derivatives. The separation was obtained within 20 minutes with a background electrolyte composed of 5 mM borax and 1 mM fluorescein (pH=9.75), 17 kV of applied voltage, and 120 s of hydrostatic injection. At the optimal conditions, the limit of detections for phenolic compounds was in the range of 0.08-0.23 mg/l, and the RSD data of repeatability and reproducibility were less than 8.0% for both migration times and peak areas. This developed method was applied to analyse concentrations of phenolic compounds in surface water and wastewater samples, with the recoveries ranging from 59.4 to 102.5%.

Determination of alditols by capillary electrophoresis with indirect laser-induced fluorescence detection

A novel method was developed based on capillary electrophoresis with indirect laser-induced fluorescence detection for the determination of alditols including xylitol, sorbitol, arabinitol and mannitol. The baseline separation was achieved with a capillary with 30μm inner diameter and running buffer (pH 9.6) composed of 40mM sodium tetraborate, 5% (v/v) methanol, 10−5M sodium fluorescein as background fluorescence. Calibration curves (0.2–2.0mgmL−1) showed good linear correlations (r>0.999). The detection limits of the alditols were in the range of 19.0–24.4μgmL−1. The intra-day and inter-day RSDs for migration times and peak areas were less than 1.6% and 4.3%, respectively. The average recoveries were in the range of 99.6–105.5%. The developed method was successfully applied to determination of alditols in different real samples, and the results were compared with those of capillary electrophoresis with indirect ultraviolet detection method.

Determination of five quinolone antibiotic residues in foods by micellar electrokinetic capillary chromatography with quantum dot indirect laser-induced fluorescence

A new assay was developed for the determination of five quinolone antibiotic residues in foods, loxacin, enrofloxacin, ciprofloxacin, lomefloxacin, and norfloxacin, by micellar electrokinetic capillary chromatography with indirect laser-induced fluorescence, in which cadmium telluride quantum dots were used as a fluorescent background substance. Some factors that affected the peak height and the resolution were examined. The optimized running buffer was composed of 20 mM SDS, 7.2 mg/L quantum dots, and 10 mM borate at pH 8.8. The separation voltage was 20 kV. Under these conditions, five quinolone antibiotic residues were separated successfully within 8 min. The detection limits ranged from 0.003 to 0.008 mg/kg; the linear dynamic ranges were all 0.01 ∼ 10 mg/kg; and the average recoveries of the spiked samples were 81.4 ∼ 94.6 %. The assay can meet the requirement of maximum residue limits to these five quinolone antibiotics in the regulations of the European Union and Japan and has been applied for determining their residues in animal-derived food.

Determination of nicotinyl pesticide residues in vegetables by micellar electrokinetic capillary chromatography with quantum dot indirect laser‐induced fluorescence

A new assay was developed by use of micellar electrokinetic capillary chromatography with indirect LIF fluorescence for the determination of thiamethoxam, acetamiprid, and imidacloprid residues in vegetables, in which the cadmium telluride quantum dots (QDs) synthesized in aqueous phase were used as fluorescent background substance and their excitation and emission wavelengths matched with LIF detector by engineering their size. The factors that affected the peak height and the resolution were optimized. The running buffer was composed of 4.4 μM cadmium telluride QDs as fluorescent background substance, 40 mM borate and 60 mM SDS, and its pH was adjusted to 8.0. The separation voltage was 25 kV. Under the optimum conditions, the detection limits were 0.05, 0.01, and 0.009 mg/kg; the linear dynamic ranges were 0.5-30, 0.1-30, and 0.1-30 mg/L; and the average recoveries of spiked samples were 72.0-101.2, 74.0-106.7, and 77.8-105.1% for thiamethoxam, acetamiprid, and imidacloprid, respectively. The assay can meet the requirement of maximum residue limits to these three pesticides in the regulations of European Union and Japan, and has been applied for determining their residues in vegetables.

Rapid and simple determination of adenine and guanine in DNA extract by micellar electrokinetic chromatography with indirect laser-induced fluorescence detection

This paper describes a micellar electrokinetic chromatography with indirect laser-induced fluorescence detection method for the simultaneous determination of adenine and guanine in DNA extracts from fungus, maize and soybean. The baseline separation was achieved within 2.5 min with running buffer (pH 9.3) composed of 10 mM borate, 20 mM SDS, 3.0 × 10 −7 M fluorescein sodium as background reagent, applied voltage of 27.5 kV, cartridge temperature of 25.0 °C. Good linearity relationships (correlation coefficients > 0.9917) between the second-order derivative peak heights (RFU) and concentrations of the analytes (mg ml −1) were obtained. The detection limits in second-order derivative electrophoregrams were 1.16 × 10 −3 mg ml −1 for adenine and 0.29 × 10 −3 mg ml −1 for guanine, respectively. The RSD data of intra-day for migration times and second-order derivative peak heights were less than 0.59 and 4.09%, respectively. This developed method was applied to the analysis of the two purines in DNA extracts with recoveries in the range of 85.3–110.2%. In this work, although the detection sensitivity was lower than that of direct LIF, yet the method has the advantages of rapidness and simplicity. And it would also extend the application range of LIF detector.

Detection of nitrated benzodiazepines by indirect laser-induced fluorescence detection on a microfluidic device

Recently there has been concern regarding the use of flunitrazepam and other low-dose benzodiazepines in drug-facilitated sexual assault. These compounds are placed in drinks of unsuspecting victims and produce a sedative effect with anterorgrade amnesia. Chip-based microfluidic systems can provide a quick and disposable procedure for the detection of flunitrazepam and other nitrated benzodiazepines used in these crimes. This paper describes the application of indirect quenching of cyanine dye (Cy5) for detection of nitrated benzodiazepines. The separation is performed on a microfluidic device with a separation channel 8 cm long and 50 μm wide and utilizes indirect fluorescence detection with 635 nm laser excitation. The optimization of the separation using micellar electrokinetic chromatography with organic modifiers is described. A borate buffer containing 2.6 μM Cy5 dye, 15 mM sodium dodecyl sulphate (SDS) and 20% methanol is used. Complete separation of four target drugs occurs in under 2 min with limits of detection in the low μg/ml range. Overall the method provides a rapid and simple analysis for the presence of nitrated benzodiazepines in beverages and other similar preparations.

Method development for the determination of coumarin compounds by capillary electrophoresis with indirect laser-induced fluorescence detection

A capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence detection (ILIFD) method is described for the simultaneous determination of esculin, esculetin, isofraxidin, genistein, naringin and sophoricoside. The baseline separation was achieved within 5 min with running buffer (pH 9.4) composed of 5 mM borate, 20% methanol (v/v) as organic modifier, 10 −7 M fluorescein sodium as background fluorophore and 20 kV of applied voltage at 30 °C of cartridge temperature. Good linearity relationships (correlation coefficients >0.9900) between the second-order derivative peak-heights (RFU) and concentrations of the analytes (mol L −1) were obtained. The detection limits for all analytes in second-order derivative electrophoregrams were in the range of 3.8–15 μM. The RSD data of intra-day for migration times and second-order derivative peak-height were less than 0.95 and 5.02%, respectively. This developed method was applied to the analysis of the courmin compounds in herb plants with recoveries in the range of 94.7–102.1%. In this work, although the detection sensitivity was lower than that of direct LIF, yet the method would extend the application range of LIF detection.

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.

Determination of EOF of PMMA microfluidic chip by indirect laser-induced fluorescence detection

A simplified method for mobility determination of electroosmotic flow (EOF) of polymethyl methacrylate (PMMA) microfluidic chips is developed with indirect laser-induced fluorescence detection (IDLIFD). Based on the interval between the beginning of separation and the apex of converse peak, the present technique highlights in circumventing the great demand for sensitivity and baseline stability of routine methods. IDLIFD is performed by a home-made laser-induced fluorescence detector of confocal optical configuration, mainly composed of a 473 nm laser diode double-pumped solid state laser (LD-DPSSL) and a 520 nm band-pass interference filter. Amino acetate (NH 4Ac) solution containing Vitamin B 2 is used as the background fluorescent reagent (BFR) solution. The pre-treatment procedures of chip inter-surface and the voltage program of injection and separation are studied. Reasons for the low background fluorescent response during injection are also discussed. Even this phenomenon has a good effect on the accurate recording of the interval between the baseline starting of separation and the lowest point of the converse peak. The results demonstrate that the reciprocals of EOF mobility ( υ eo) of PMMA microfluidic chip are in line with the ion strength of background electrolyte (BGE) solution. Furthermore, the υ eo of PMMA material is related to the pH of BGE solution. Compared with those results obtained from the Current-Monitoring method, no apparent difference has been found.

Indirect Fluorescence of Amines in Capillary Electrophoresis, Using Cresyl Violet

This paper describes the analysis of amines under acidic conditions by capillary electrophoresis (CE) in conjunction with indirect laser‐induced fluorescence (ILIF), using cresyl violet as a probe. In the system, a small pinhole (0.2 mm) and an interference filter (589 nm) were used to confine the light and to minimize the plasma interference from a He–Ne laser, respectively, improving the baseline stability. Adding lithium ions to the background electrolytes (BGEs) is effective to achieve narrower peak profiles, leading to better resolution. The analysis of six amines by CE–ILIF using an aqueous solution, pH 3.5, containing 5.0% methanol, 0.1 mM sulfuric acid, 0.1 mM cresyl violet, and 0.3 mM lithium was complete in 5 min, with the limits of detection (LOD) on the level of µM. Negative peak profiles are for amines with greater electrophoretic mobility than that of lithium ions, but positive peaks for the slower ones. To further improve the sensitivity, on‐line concentration based on pH junction has been demonstrated. When injecting the sample prepared in a solution of 0.2 mM sulfuric acid, pH 3.3, at 15 kV for 60 s, and conducting the separation using the above‐mentioned condition, the sensitivity improvements are greater than 10‐fold compared to that injecting at 15 kV for 5 s. With the advantages of rapidity, sensitivity, and low cost, this method has proven potential for the analysis of trace amines in biological samples.

Indirect laser‐induced fluorescence detection for capillary electrophoresis using a frequency‐doubled diode laser

A blue (452 nm) frequency-doubled diode laser with a quasi-cw optical output power of 10 microW is used for indirect laser-induced fluorescence detection in combination with the capillary electrophoretic separation of inorganic anions. As fluorescing probe ion the anion of 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was selected having an absorption maximum of 454 nm in alkaline medium. Employing a capillary coated with linear acrylamide, baseline separation of eight inorganic anions was possible within 5 min. With a separation buffer containing 50 micromol.L(-1) HPTS and 10 mmol.L(-1) lysine the limits of detection for sulfate, nitrite, nitrate, azide, thiocyanate, and chlorate were between 0.9 and 4.7 micromol.L(-1). Separation of chloride and sulfate was achieved by adding 0.25 mmol.L(-1) calcium hydroxide to the separation buffer. Inorganic anions in several mineral and tap water samples have been determined with the technique developed and results are compared to data obtained by ion chromatography in combination with conductivity detection after conductivity suppression.

Capillary electrophoretic analysis of arsenic species with indirect laser induced fluorescence detection

The development of a method for determining arsenic species by capillary zone electrophoresis (CZE) with indirect laser-induced fluorescence (LIF) is described in this paper. The buffer pH, the concentration of fluorescein, the nature and the concentration of the background electrolytes (BGEs) were defined. When 2.0 mM NaHCO3 (pH 9.28) with 10–7 M fluorescein was used as the buffer, arsenite (As(III)), dimethylarsonic acid (DMA), monomethylarsonic acid (MMA), and arsenate (As(V)) were all separated from one another. The limits of detection for the four arsenic species were in the range of 0.12–0.54 mg/L. This method was used in the analysis of spiked arsenic species in tap and mineral water to demonstrate its usefulness. The results showed that both the recovery and the reproducibility of the developed method were acceptable.

Indirect fluorescence of aliphatic carboxylic acids in nonaqueous capillary electrophoresis using merocyanine 540.

A method for the analysis of aliphatic carboxylic acids (ACAs) in nonaqueous capillary electrophoresis (NACE) in conjunction with indirect laser-induced fluorescence (ILIF) using merocyanine 540 (MC 540) is described. Performing the analysis in organic solvent is advantageous when using MC 540, because of its greater quantum yield in aprotic solvent. To achieve a high dynamic reserve (DR) and optimize resolution, we have tested a number of aqueous mixtures containing alcohols and acetonitrile (ACN). The optimum buffer for the analysis of C2-C18 ACAs, in terms of sensitivity, resolution, and speed, is an aqueous mixture of 40% ACN, 30% ethanol, and 1 mM Tris at apparent pH 7.4 (adjusted with ascorbic acid). Under this condition, the DR is greater than 1000, thereby the limits of detection for acids are in the range of sub-microM to microM. Linear plots show that the dynamic ranges for the analysis of ACAs are at least two decades in concentration, with regression coefficients all greater than 0.98. The relative standard deviations of the migration times and peak heights for all ACAs are less than 2.0%. Furthermore, this simple and cost-effective method has been applied to the analysis of marine lipid concentrate, with the concentrations of 1.67+/-0.03 and 4.50+/-0.05 mM (n = 5) for C14 and C16 acids, respectively, in a tablet of marine lipid concentrate sample.

Bi-enzymatic and capillary electrophoretic analysis of non-fluorescent compounds in microfluidic devices: Determination of xanthine

This report describes general investigations for xanthine determination in microfluidic devices. Xanthine is chosen as a model analyte for the detection of compounds via bi-enzymatic systems on one hand, and via capillary electrophoresis (CE) of non-fluorescent analytes on the other. The bi-enzyme system comprises xanthine oxidase (XOD) and horseradish peroxidase (HRP) with XOD giving the specificity for the analyte and HRP allowing chemiluminescent (CL) detection. Studies include the use of enzyme solutions, as well as enzyme coated beads pumped continuously through the device or trapped in a 330 pL cavity. In a second approach, indirect laser-induced fluorescence (LIF) is used as a detection method for microchip-based CE separations. Although the detection methods are not optimised, this report demonstrates general new possibilities for a fast determination of such compounds, in particular xanthine, in microfluidic devices.

Indirect laser-induced fluorescence detection for capillary electrophoresis using a violet diode laser.

The violet (415 nm) diode laser is used for indirect laser-induced fluorescence detection in capillary electrophoretic separations of inorganic anions and chemical warfare agent degradation products. Inorganic anions were detected using 8-hydroxypyrene-1,3,6-trisulfonic acid as the indirect probe and achieved submicromolar (40-80 ppb) detection limits in a 2-min separation. The chemical warfare agent degradation products methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate were detected using the porphyrin tetrakis(4-sulfophenyl)porphine as the indirect probe and achieved detection limits of 0.1 microM (9 ppb), which are 1 order of magnitude better than that achieved using indirect UV detection. Baseline stability achieved with the violet diode laser was excellent, with dynamic reserve (DR) values of > 1000, which are 15 times better than that achieved using an unstabilized HeCd laser.

Indirect laser-induced fluorescence detection of explosive compounds using capillary electrochromatography and micellar electrokinetic chromatography

Mixtures of nitroaromatic and nitramine explosive compounds and their degradation products were analyzed using electrokinetically driven separations with both indirect laser-induced fluorescence (IDLIF) and UV absorption detection. Complete separations of the 14-component mixture (EPA 8330) were achieved using both capillary electrochromatography (CEC) and micellar electrokinetic chromatography (MEKC). IDLIF detection was performed using an epifluorescence system with excitation provided by a 635 nm diode laser and micromolar concentrations of the dye Cy-5 as the visualizing agent. While the sensitivity of the two detection methods was similar for the nitroaromatic compounds, the nitramines could only be detected using UV absorption due to their low fluorescence quenching efficiency of Cy-5. The detection sensitivity using IDLIF was limited by low frequency oscillations in the fluorescence background. The oscillations increased with higher electric field strength and were attributed to thermal fluctuations caused by Joule heating. Due to the more conductive running buffer and higher separation currents used in MEKC, sensitive IDLIF detection could only be achieved using low (approximately 100 V/cm) field strengths, resulting in long analysis times. CEC separations, which are typically run with low conductivity mobile phases to avoid bubble formation, are less sensitive to this effect. In CEC separations with IDLIF detection a stable fluorescence background using Cy-5 could be established using only a nonporous stationary phase. In capillaries packed with porous silica particles, anomalous migration behavior was observed with charged dye molecules and a stable fluorescence background could not be established under electrokinetic flow. This is the first demonstration of IDLIF in packed channel CEC.

Separation and detection of explosives on a microchip using micellar electrokinetic chromatography and indirect laser-induced fluorescence

A new approach for sensitive detection on a microfabricated chip is presented. Indirect laser-induced-fluorescence (IDLIF) was used to detect explosive compounds after separation by micellar electrokinetic chromatography (MEKC). The detection setup was used in an epifluorescence configuration with excitation provided by a near-IR diode laser operating at 750 nm. To achieve indirect detection, a low concentration of a dye (5 microM Cy7) was added to the running buffer as a visualizing agent. Using this methodology, a sample containing 14 explosives (EPA 8330 mixture) was examined. Concentrations of 1 ppm of trinitrobenzene (TNB), trinitrotoluene (TNT), dinitrobenzene (DNB), tetryl, and 2,4-dinitrotoluene (2,4-DNT) could be detected with S/N ratios between 3 and 10. Analyses showing 10 peaks, with plate numbers on the order of 60000, were completed within 60 s using a 65 mm long separation channel. The three isomers of nitrotoluene (2-, 3-, and 4-nitrotoluene) were not resolved. Additionally, the two nitramines (HMX and RDX) could only be detected at much higher concentrations, likely due to the low fluorescence quenching efficiencies of these compounds. The analysis method was also used to separate and detect nitroaromatic compounds in extracts from spiked soil samples. The presence of 1 ppm (1 microg of analyte/1 g of soil) of TNB, DNB, TNT, tetryl, 2,4-DNT, 2,6-DNT, 2-NH2-4,6-DNT, and 4-NH2-2,6-DNT could readily be detected. In the interest of increasing the sensitivity of the analysis, various on-chip injection schemes were evaluated. It was found that a 250 microm double-T injector gave a 35% increase in peak signal compared to a straight-cross injector, which is less than expected based on injected volume.

Analysis of glycosaminoglycan monosaccharides by capillary electrophoresis using indirect laser-induced fluorescence detection

Two methods for monosaccharide analysis by capillary electrophoresis (CE) using counterelectroosmotic and coelectroosmotic modes with indirect laser-induced fluorescence detection were optimised and compared. A mixture of seven glycosaminoglycan-derived hexoses was separated in alkaline fluorescein-based electrolytes and detected in both counterelectroosmotic and coelectroosmotic conditions. The fluorescein concentration and pH of the background electrolyte, and the influence of the reversal of electroosmotic flow by addition of hexadimethrine bromide on the separation were studied. Coelectroosmotic CE conditions provided better resolution and limits of detection. A 10 −6 M fluorescein solution at pH 12.25 containing 0.0005% (w/v) hexadimethrine bromide was used as background electrolyte. Quality parameters such as run-to-run, day-to-day precision and limits of detection were calculated, and better figures of merit were obtained for the coelectrooosmotic conditions than for the counterelectroosmotic mode. The coelectroosmotic method was applied to the quantitation of the hexosamine contents in glycosaminoglycans after acid hydrolysis. The method proved to be suitable for the determination of dermatan sulfate in heparin down to 2% (w/w).

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection.

Indirect detection of organic acids in non-aqueous capillary electrophoresis

Non-aqueous capillary electrophoresis (NACE) with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2–C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (pKa) values of C8–C18 FAs obtained from the slope of the linear plot −log [(μ0/μ)−1] vs. pH, using 20% isopropanol and 40% acetonitrile as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 (MC540) as fluorophores, has been performed to the analysis of AA and its stereoisomer, isoascorbic acid (IAA), and the limits of detection of AA and IAA are 0.30 μM and 0.17 μM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with IAA as the internal standard in less than 3 min and its concentration is 76.7±0.4 mM.