Abdul Nabi

Bioluminescent assays with immobilized firefly luciferase based on flow injection analysis

Abstract A flow-injection manifold incorporating immobilized firefly luciferase is described. The detector design and reaction conditions are discussed and results are presented for the determination of adenosine-5′-triphosphate over the range 1 × 10−12−1 × 10−5 M. Modifications for creatine phoshokinase (10–400 U l−1) and creatine phosphate (10−t–10−1 M), both determined indirectly via ATP, are also described.

Flow‐injection determination of cysteine in pharmaceuticals based on luminol–persulphate chemiluminescence detection

A flow injection (FI) method is reported for the determination of l-cysteine, based on its enhancement on chemiluminescence (CL) emission of luminol oxidized by sodium persulphate in alkaline solution. The calibration graph was linear over the range 1.0 x 10(-9)-5.0 x 10(-7) mol/L (r(2) = 0.9992), with relative standard deviations (RSDs) in the range 1.1-2.3% (n = 4). The limit of detection (3 sigma blank) was 5.0 x 10(-10) mol/L with a sample throughput of 120/h. The method was applied to pharmaceuticals and the results obtained were in reasonable agreement with the amount labelled. The proposed method was also applied to cysteine in synthetic amino acid mixtures. Calibration graphs of N-acetylcysteine and glutathione over the range 1.0-50 x 10(-8) and 0.5-7.5 x 10(-7) mol/L were also established (r(2) = 0.998 and 0.9986) with RSDs in the range 1.0-2.0% (n = 4), and the limits of detection (3 sigma blank) were 5.0 x 10(-9) and 1.0 x 10(-8) mol/L, respectively.

Investigation of iron(III) reduction and trace metal interferences in the determination of dissolved iron in seawater using flow injection with luminol chemiluminescence detection

A detailed investigation into the performance of two flow injection-chemiluminescence (FI-CL) manifolds (with and without a preconcentration column) for the determination of sub-nanomolar dissolved iron (Fe(II)+Fe(III)), following the reduction of Fe(III) by sulphite, in seawater is described. Kinetic experiments were conducted to examine the efficiency of reduction of inorganic Fe(III) with sulphite under different conditions and a rigorous study of the potential interference caused by other transition metals present in seawater was conducted. Using 100microM concentrations of sulphite a reduction time of 4h was sufficient to quantitatively reduce Fe(III) in seawater. Under optimal conditions, cobalt(II) and vanadium(IV)/(III) were the major positive interferences and strategies for their removal are reported. Specifically, cobalt(II) was masked by the addition of dimethylglyoxime to the luminol solution and vanadium(IV) was removed by passing the sample through an 8-hydroxyquinoline column in a low pH carrier stream. Manganese(II) also interfered by suppression of the CL response but this was not significant at typical open ocean concentrations.

Determination of thiram in natural waters using flow-injection with cerium(IV)-quinine chemiluminescence system.

A simple and rapid flow-injection chemiluminescence method has been developed for the determination of dithiocarbamate fungicide thiram based on the chemiluminescence reaction of thiram with ceric sulfate and quinine in aqueous sulfuric acid. The present method allowed the determination of thiram in the concentration range of 7.5-2500 ng/mL and the detection limit (signal-to-noise ratio = 3) was 7.5 ng/mL with sample throughput of 120/h. The relative standard deviation was 2.5% for 10 replicate analyses of 500 ng/mL thiram. The effects of foreign species including various anions and cations present in water at environmentally relevant concentrations and some pesticides were also investigated. The proposed method was applied to determine thiram in spiked natural waters using octadecyl bonded phase silica (C(18)) cartridges for solid-phase extraction. The recoveries were in the range 99 +/- 1 to 104 +/- 1%.

Determination of nitrate and nitrite in freshwaters using flow-injection with luminol chemiluminescence detection

A simple and sensitive flow-injection (FI) method for the determination of nitrate and nitrite in natural waters, based on luminol chemiluminescence (CL) detection, is reported. Nitrate was reduced online to nitrite via a copperized cadmium (Cu-Cd) column and then reacted with acidic hydrogen peroxide to form peroxynitrous acid. CL emission was observed from the oxidation of luminol in an alkaline medium in the presence of the peroxynitrite anion. The limits of detection (S:N = 3) were 0.02 and 0.01 µg N/L, with sample throughputs of 40 and 90 /h for nitrate and nitrite, respectively. Calibration graphs were linear over the range 0.02-50 and 0.01-50 µg N/L [R2  = 0.9984 (n = 8) and R2  = 0.9965 (n = 7)] for nitrate and nitrite, respectively, with relative standard deviations (RSDs; n = 3) in the range 1.8-4.6%. The key chemical and physical variables (reagent concentrations, buffer pH, flow rates, sample volume, Cu-Cd reductor column length) were optimized and potential interferences investigated. The effect of cations [Ca(II), Mg(II), Co(II), Fe(II) and Cu(II)] was masked online with EDTA. Common anions (PO4(3-) , SO4(2-) and HCO3-) did not interfere at their maximum admissible concentrations in freshwaters. The effect of salinity on the luminol CL reaction with and without nitrate and nitrite (2 and 0.5 µg N/L, respectively) was also investigated. The method was successfully applied to freshwaters and the results obtained were in good agreement with those obtained by an automated segmented flow analyser reference method.

Bioluminescence assays with immobilised bacterial luciferase using flow injection analysis

A flow injection manifold incorporating bacterial luciferase and oxidoreductase (from Vibrio harveyi) co-immobilised on cyanogen bromide-activated Sepharose 4B is described. The protein recovery was 86 and 96% for luciferase and oxidoreductase, respectively, and the activity recovery was 14% for luciferase and 33% for oxidoreductase. The activity was retained for several days at room temperature. Calibration data for NADH, NADPH, FMN and FMNH2 over the range 1.0 × 10–9–1.0 × 10–4M is given for the optimised system.

Flow injection determination of thyroxine in pharmaceutical preparations using tris(2,2'-bipyridyl)ruthenium(III)-NADH chemiluminescence detection.

A flow injection (FI) method is reported for the determination of thyroxine based on its enhancement of chemiluminescence (CL) from the Ru(bpy)(3)(3+)-NADH system. The calibration graph was linear over the range 2.0-10x10(-8)mol L(-1) (r(2)=0.9989) with relative standard deviations (R.S.D.) in the range 2.0-4.5% (n=4). The limit of detection (3sigma blank) was 1.0x10(-9)mol L(-1) with sample throughput of 120h(-1). The effect of some organic compounds, anions and cations were studied for l-thyroxine determination. The method was applied to pharmaceutical preparations and the results obtained were in reasonable agreement with the amount labeled. The method was statistically compared with the results obtained by RIA; no significant disagreement at 95% confidence limit was observed. A calibration graph of NADH over the range 1.3x10(-8)-1.3x10(-6)mol L(-1) was also established (r(2)=0.9992) with R.S.D. in the range1.0-3.5% (n=4). The limit of detection (3sigma) was 1.0x10(-10)mol L(-1) NADH.

Dynamic surface tension analysis of dodecyl sulfate association complexes

First, a novel calibration method is used to expand the current understanding of spherical drop growth and elongation that occurs during on-line measurements of surface pressure using the dynamic surface tension detector (DSTD). Using a novel surface tension calibration method, the drop radius is calculated as a function of time from experimental drop pressure data and compared to the theoretical drop radius calculated from volumetric flow rate. From this comparison, the drop volume at which the drop shape starts to deviate ( approximately 4 mul) from a spherical shape is readily observed and deviates more significantly by approximately 6 mul drop volume (5% deviation in the ideal spherical drop radius) for the capillary sensing tip employed in the DSTD. From this assessment of drop shape, an experimental method for precise drop detachment referred to as pneumatic drop detachment is employed at a drop volume of 2 mul (two second drops at 60 mul/min) in order to provide rapid dynamic surface tension measurements via the novel on-line calibration methodology. Second, the DSTD is used to observe and study kinetic information for surface-active molecules and association complexes adsorbing to an air-liquid drop interface. Dynamic surface tension measurements are made for sodium dodecyl sulfate (SDS) in the absence and presence of either tetra butyl ammonium (TBA) or chromium (III). Sensitive, indirect detection of chromium and other multiply charged metals at low concentrations is also investigated. The DSTD is utilized in examining the dynamic nature of SDS: cation association at the air-liquid interface of a growing drop. Either TBA or Cr(III) were found to substantially enhance the surface tension lowering of dodecyl sulfate (DS), but the surface tension lowering is accompanied by a considerable kinetic dependence. Essentially, the surface tension lowering of these DS: cation complexes is found to be a fairly slow process in the context of the two second DSTD measurement. The limit of detection for both SDS and chromium (III) is in the 300-400 part-per-billion (by mass) range.

The bioluminescent determination of adenosine triphosphate with a flow-injection system

Abstract A procedure is described, for the bioluminescent determination of adenosine- 5′-triphosphate (ATP), based on firefly luciferin and luciferase and a purpose-built flow-through detector. The limit of detection is 10−14 mol and the sample throughput is 200 h−1. The useful response range for a 30-μl sample was 10−6–10−4 M ATP and the correlation coefficient (r) for a log-log plot over this range was 0.9991.

Determination of carbaryl by flow injection with luminol chemiluminescence inhibition detection

A flow-injection procedure is described for the determination of carbaryl based on its inhibition effect on luminol-cobalt(II) chemiluminescence reaction in alkaline medium in the presence of hydrogen peroxide. The calibration data over the range 5.0 × 10−7 to 20 × 10−6 M give a correlation coefficient (r 2) of 0.9972 with relative standard deviations (RSD; n = 4) in the range of 1.0–2.1% with a limit of detection (3 × blank noise) of 2.37 × 10−7 M for carbaryl. The sample throughput was 120 h−1. The effects of some carbamates, anions, and cations were studied on luminol CL system for carbaryl determination. The proposed method has been applied to determine carbaryl in natural waters.