Peroxyoxalate Chemiluminescence Reaction Research Articles

Sensitive peroxyoxalate chemiluminescence determination of psychotropic indole derivatives

The continuous-addition-of-reagent (CAR) technique was used for the determination of hallucinogenic alkaloids such as N-methyltryptamine, 5-methyltryptamine, α-methyltryptamine, bufotenine and psilocin by measuring the peroxyoxalate chemiluminescence produced on reaction with the bis(2,4,6-trichlorophenyl)oxalate–hydrogen peroxide system following derivatization with dansyl chloride. This is the first reported use of chemiluminescence for the determination of these compounds. The special ability of the CAR technique to suppress background emission in peroxyoxalate chemiluminescence reactions provides a highly sensitive means of determination (the response to indole alkaloids is linear over two orders of magnitude and detection limits are in the picomole range). The proposed method compares favourably with existing alternatives in terms of sensitivity and was validated in the determination of psilocin in mushroom samples.

Kinetic studies on the peroxyoxalate chemiluminescent reaction: imidazole as a nucleophilic catalyst

The peroxyoxalate system undergoes one of the most efficient chemiluminescence reactions and is the only one supposed to involve an intermolecular chemically induced electron exchange luminescence (CIEEL) mechanism, with proven high efficiency.In this work we report kinetic results on the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) with hydrogen peroxide, catalysed by imidazole (IMI), in the presence of chemiluminescence activators. The kinetics were followed by measurement of the intensity of light emission and the 2,4,6- trichlorophenol (TCP) release, observed as an absorption change. From the dependence of the observed rate constants on the concentrations of TCPO, imidazole, hydrogen peroxide and activator, we attribute rate constants to three elementary steps in the proposed simplified mechanistic scheme. The initial step consists of attack of the nucleophilic imidazole on TCPO. A bimolecular (k1(2)= 1.4 ± 0.1 dm3 mol–1 s–1) and a trimolecular [k1(3)=(9.78 ± 0.08) 102 dm6 mol–2 s–1] rate constant can be attributed to this step. The imidazolide subsequently suffers imidazole-catalysed peroxide attack, leading to a peracid derivative; trimolecular rate constants k2(3)=(1.86 ± 0.06)104 dm6 mol–2s–1 and k2(3)′=(8.7 ± 0.2) 103 dm6 mol–2 s–1 can be obtained for this step from the peroxide and the imidazole dependence, respectively. The cyclization of the peracid affords the reactive intermediate, which we believe to be 1,2-dioxetanedione; a rough estimate of k3∼ 0.2 s–1(at [IMI]= 1.0 mmol dm–3) is obtained for this step. Finally, the interaction of the activator with the reactive intermediate, probably involving the CIEEL sequence and leading ultimately to light emission, is extremely fast and cannot be observed kinetically.

Zero-Dead-Volume Peroxyoxalate Chemiluminescence Detection in Liquid Chromatography

A simple, flexible, and inexpensive peroxyoxalate chemiluminescence detection reactor for liquid chromatography is reported. This postcolumn device permits the efficient mixing of the mobile phase and reactants, bis(2,4,6-trichlorophenyl) oxalate and hydrogen peroxide, with a zero dead-volume and the following features : (a) highly flexible optimization of chemical variables affecting the peroxyoxalate chemiluminescence reaction, which can take place outside the chromatographic system ; (b) use of high oxidant/oxalate ester concentration ratios, which decrease background emission and increase the signal/noise ratio ; and (c) use of large reaction volumes in the detection cell without excessive additional band broadening (the chemiluminescence reaction is very rapid under the selected experimental conditions). The proposed detection system was tested in the high-performance liquid chromatographic determination of polycyclic aromatic hydrocarbons, where it exhibited higher sensitivity (limits of detection in the femtomole range) than existing alternatives.

A further study on the degradation mechanism of rhodamine 6G in the peroxyoxalate chemiluminescent reaction

In previous work, we have demonstrated that the degradation of rhodamine 6G (R6G) in the peroxyoxalate chemiluminescent reaction may be related to the formation of an intermediate and that the oxidation effect of H 2O 2 is insignificant. In this paper, the effect of 9,10-diphenylanthracene (DPA) on the degradation of R6G has been examined. The experimental results show that, as the concentration of DPA is increased, the degradation rate of R6G decreases. This can be interpreted in terms of the competitive interaction of DPA and R6G molecules with the intermediate. It was also found that N 2 bubbling of the reaction mixture leads to a decrease in the degradation rate of R6G. On the basis of these experimental results, a mechanism for R6G degradation in the peroxyoxalate chemiluminescent system, involving a reaction intermediate and dissolved oxygen, is suggested.

1,2-Diarylethylenediamines as sensitive pre-column derivatizing reagents for chemiluminescence detection of catecholamines in HPLC

Chemiluminescence detection in high-performance liquid chromatography for derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol was studied on the basis of the peroxyoxalate chemiluminescence reaction. The amines and isoproterenol, derivatized with 1,2-diarylethylenediamines, were separated on a reversed-phase HPLC column (TSK gel ODS-120T) with isocratic elution using a mixture of imidazole buffer (pH 5.8, 120 mM)-methanol-acetonitrile (6:2:9, v/v/v). The eluate was detected by a post-column chemiluminescence reaction system, using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate and hydrogen peroxide. Of the 14 1,2-diarylethylenediamines investigated, it was found that 1,2-bis(3-chlorophenyl)ethylenediamine, 1,2-bis(3,4-dichlorophenyl)-ethylenediamine and 1,2-bis(4-chlorophyenyl)ethylenediamine were the most sensitive derivatives for all catecholamines. The derivatization and peroxyoxalate chemiluminescence reaction conditions were optimized for 1,2-bis(3-chlorophenyl)-ethylenediamine. The chromatographic detection limits for catecholamines were approximately 40–120 amol for an injection volume of 100 μl (signal-to-noise ratio of 3).

High-Performance Liquid Chromatographic Determination of Hydrogen Peroxide with Peroxyoxalate Chemiluminescence Detection

Abstract A high performance liquid chromatographic method with peroxyoxalate chemiluminescence (PO-CL) detection was developed for the determination of hydrogen peroxide. Hydrogen peroxide separated by a reversed phase column was detected with the CL produced by the PO-CL reaction using a mixture of 2,4,6,8-tetrathiomorpholi-nopyrimido[5,4-d]pyrimidine and bis[2-(3,6,9-trioxade-cyloxycarbonyl)-4-nitrophenyl]oxalate or bis(2,4,6-trichlorophenyl)oxalate in acetonitrile as a post column reagent. Hydrogen peroxide could be determined over the range of 5–100 pmol on column and the detection limit was 188 fmol at a signal-to-noise ratio of 3. Relative standard deviation of 10-replicate measurements of peak heights for 50 pmol was 2.36%. The detection system applied to the assay of hydrogen peroxide in cola drinks. Trace amounts of hydrogen peroxide could be determined in six cola drinks. The levels were 68.4 ± 2.6–174.2 ± 8.4 nmol/dl. Hydrogen peroxide contaminated in commercially available hydroperoxides was also determined by the proposed method. The amounts obtained were 0.061% for t-butyl hydroper-oxide and 0.003% for cumene hydroperoxide.

A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection.

A fully automated in-line extraction reversed-phase high-performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N-Methyldopamine (N-MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N-MeDA, and in-line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT-CEX). This pre-extraction process was coupled with an HPLC system including reversed-phase mode separation on an analytical column (TSK gel ODS-80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis [4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated. The detection limit for all the CAs was about 1 fmol (signal-to-noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 microL of human plasma (n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within-day assay and 5.0, 3.8 and 4.0%, respectively, for the between-day assay. The method was applicable to the determination of CAs in 25-50 microL of human or rat plasma.

Screening of urinary coproporphyrin using cloud point extraction and chemiluminescence detection

A simple screening test was developed for the sensitive and selective measurement of urinary coproporphyrin. In this screening test, efficient and selective extraction/pre-concentration of coproporphyrin from the aqueous medium(urine) into a much smaller volume phase containing a common non-ionic surfactant (Triton X-100) and ethyl acetate was accomplished by the addition of a relatively large amount of a cloud point depressing electrolyte (K 3PO 4) into the sample solution to effect cloud point separation. Sensitive and selective detection of coproporphyrin in the mixed Triton X-100 and ethyl acetate phase was performed via chemical excitation using the peroxyoxalate chemiluminescence reaction. The effects of surfactant and cations (from the cloud point depressing electrolyte) on the chemiluminescence intensity of coproporphyrin were briefly investigated. Furthermore, the spectrum of urinary coproporphyrin obtained using the present chemiluminescence method was briefly compared with that obtained from fluorescence method.

Light emission from bilirubin using the peroxyoxalate chemiluminescence reaction

The observation of light emission from bilirubin in an organic solvent as a result of the peroxyoxalate chemiluminescence reaction is reported for the first time. The results presented herein indicate that bilirubin chemiluminescence can be detected in N,N′ dimethylformamide but not in chloroform, dimethylsulfoxide, or pyridine. To elucidate this phenomenon, the effects of solvent on the chemiluminescence intensity were examined as a function of solvent composition. Furthermore, analytical utilization of the present observation for the determination of bilirubin in biological materials was evaluated. Relative and absolute limits of detection were found to be 8.5 nmol 1 −1 and 5.0 ng, respectively, and the response was found to be linear at least over three orders of magnitude. Selectivity for the detection of bilirubin in the presence of a number of common interference species was also examined. Finally, the visible spectrum of bovine serum in the blue-green region was obtained using the present chemiluminescence method and was briefly compared with those obtained from absorption ad fluorescence methods.

Influence of metal ions on peroxyoxalate chemiluminescence

The influence of metal ions on the peroxyoxalate chemiluminescence reaction was investigated using batch experiments. A number of metal ions showed an improvement of the chemiluminescence reaction of bis(2-nitrophenyl) oxalate with hydrogen peroxide in the presence of 3-aminofluoranthene as the fluorophore and imidazole as the catalyst. Copper(II) gave the highest enhancement, of about one order of magnitude. The reason for the increase in the chemiluminescence intensity by metal ions is probably a change in the mechanism of the peroxyoxalate chemiluminescence reaction due to stabilization of the oxalate ester-imidazole complex by the metal ion, leading to a higher efficiency of the chemiluminescence reaction.

Photographic Detection of Sub-Picomole Fluorescers by Peroxyoxalate Chemiluminescence Reaction

Femtomole fluorescers such as perylene or 5, 12-bis(phenylethynyl)naphthacence (PEN) were detected photographicallythrough the peroxyoxalate chemiluminescence reaction (CL). With the use of X-ray film, the CL emissions from 9.6× 10-14mol fluorescers were recorded as black-and-white spots, while they are recorded as color spots on color films such as instant color films (ISO 80-100) or high speed 35mm roll films (ISO 400 and 3200) with the detection limit of 9.6× 10-13mol. This technique has been applied to the detection of albumin labeled with fluorescer. As little as 0.30μg of pyrene labeled bovine serum albumin or 2.4μg of tetramethylrhodamine labeled albumin was detected by this technique.

Determination of femtomole concentrations of catecholamines by high-performance liquid chromatography with peroxyoxalate chemiluminescence detection

A highly sensitive method for determination of the plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) is described. The method consists of the extraction of the catecholamines, using 3,4-dihydroxybenzylamine as internal standard, from plasma with alumina (5 mg), followed by a reversed-phase column separation, on-column fluorogenic derivatization with ethylenediamine (ED) and post-column peroxyoxalate chemiluminescent reaction detection utilizing bis[4-nitro-2-(3,6,9-trioxadecyl-oxycarbonyl)phenyl] oxalate (TDPO) and hydrogen peroxide. In order to optimize the reaction conditions for high-performance liquid chromatography to obtain highly sensitive detection, the effects of changing reagent compositions on the chemiluminescence yield were investigated. The following are the optimized conditions. Eluent, a mixture of 50 mmol l-1 potassium acetate (pH 3.20)-50 mmol l-1 potassium phosphate (pH 3.20)-acetonitrile (90.15 + 4.85 + 3 v/v/v) containing 1 mmol l-1 sodium hexanesulfonate (40 degrees C) and flow rate, 0.5 ml min-1. Fluorogenic reagent solution, 105 mmol l-1 ED and 175 mmol l-1 imidazole in acetonitrile-ethanol (90 + 10 v/v) and flow rate, 0.25 ml min-1. Reaction coil (15 m x 0.5 mm i.d.) heated at 80 degrees C. Chemiluminogenic reagent solution, 0.25 mmol l-1 TDPO, 150 mmol l-1 hydrogen peroxide and 110 mmol l-1 trifluoroacetic acid in dioxane-ethyl acetate (50:50 v/v) and flow rate, 1.4 ml min-1. The detection limits for all the catecholamines were 1 fmol (signal-to-noise ratio at 2). The standard deviations of the method for the determination of NE, E and DA added to rat plasma (2.5 nM) were 3, 3 and 4%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic study of background emission from peroxyoxalate chemiluminescence reaction and application to the improvement of detection limits in liquid chromatography

Kinetic study of background emission from peroxyoxalate chemiluminescence reaction and application to the improvement of detection limits in liquid chromatography

Peroxyoxalate Chemiluminescence Assay of Hydrogen Peroxide and Glucose Using 2,4,6,8-Tetrathiomorpholuiopyrimido[5,4-d]-pyrimidine as a Fluorescent Component

Peroxyoxalate chemiluminescence (CL) assay of hydrogen peroxide (H2O2) or glucose was developed by using 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine as a fluorescent component and bis(2,4,6-trichlorophenyl)oxalate (TCPO) as an oxalate. Linear relationships between CL intensity and final concentration of H2O2 from 10-8 to 10-4 M were obtained. The detection limit at the ratio of CL intensities for sample and blank (S/B) of 3 was 10 nM. The precision for five replicate measurements at 10-5 and 10-6 M of H2O2 were 17.6 and 15.7% of relative standard deviations, respectively. α-D-Glucose was transformed to β-D-glucose with mutarotase and converted to H2O2 and D-gluconic acid with glucose oxidase, which was detected by using peroxyoxalate CL reaction. A linear calibration graph was obtained up to 1.5×10-4 M of glucose solution. The method was applied to the assay of glucose in human serum. The recovery was 98.2% (N=4). The method correlated well with the conventional colorimetric method (r=0.968).

Flow-injection chemiluminescent method for an enzyme-labelled DNA probe

A flow-injection chemiluminescent (CL) method was investigated for the determination of hydrogen peroxide generated from an enzyme-labelled DNA probe. Biotinylated DNA probes were immobilized onto a membrane and labelled with biotinylated glucose oxidase by a streptavidin bridge and placed in a microtitre well. The glucose oxidase-labelled sample was incubated in a solution of glucose and the enzymatically produced hydrogen peroxide was determined using flow injection with a peroxyoxalate chemiluminescence reaction. The peroxide generated is related to the amount of the labelled DNA probe. The important factors for increasing the sensitivity of the CL measurement are to maximize the production of hydrogen peroxide and to reduce the non-specific binding of the enzyme label on the membrane. This CL-flow-injection measurement using biotinylated glucose oxidase-labelled DNA probes was applied to a DNA dot-blot hybridization assay and compared with a visual colorimetric method using 5-bromo-4-chloroindolyl phosphate and nitro blue tetrazolium as a chromogenic substrate for detection of an alkaline phosphatase label. The CL method was quantitative over the range 100 pg-100 ng of target DNA with a precision (r.s.d.) of 4–11%. In comparison, the colorimetric method was useful for the range 10 pg-1 ng.

Photographic Detection of Hydrogen Peroxide and Glucose by Peroxyoxalate Chemiluminescence

A simple, rapid chcmiluminescent photographic method was developed for the assay of hydrogen peroxide or glucose using a camera luminometer. Some chemiluminogenic reagents were tested regarding their ability by using a visual method. 4,4′-Oxalyl-bis[(trifluoromethylsulfonyl)imino]trimethulene-bis(4-methylmorpholinium)trifluoromethane-sulfonate (MPTQ) was found to be a suitable oxamide for the photographic method. Pyrimido[5,4-d]pyrim-idines, dipyridamole (DP), 2,4,6,8-tetramorpholinopyrimido[5,4-d]pyrimidine (MP) and 2,4,6,8-tetrathiomorpholino-pyrimido[5,4-d]pyrimidine (TMP) were effective enhancers, as were perylene and Rhodamine B, on the peroxyoxalate chemiluminescent reaction examined. Fifty picomoles of hydrogen peroxide per 200 μl on a micro-titer plate well could be detected as a white spot on instant film. Glucose was detected after being enzymatically oxidized to hydrogen peroxide with glucose oxidase. The detection limit of glucose was 100 pmol per well using MPTQ and MP as chemiluminogenic reagents. By the proposed method, glucose in 50 μl of 100-fold diluted human serum could be detected.

Background emission from the peroxyoxalate chemiluminescence reaction in the absence of fluorophores

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBackground emission from the peroxyoxalate chemiluminescence reaction in the absence of fluorophoresB. Mann and Mary Lynn. GrayeskiCite this: Anal. Chem. 1990, 62, 14, 1532–1536Publication Date (Print):July 15, 1990Publication History Published online1 May 2002Published inissue 15 July 1990https://doi.org/10.1021/ac00213a038RIGHTS & PERMISSIONSArticle Views194Altmetric-Citations30LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (691 KB) Get e-Alerts Get e-Alerts

Studies on the effects of imidazole on the peroxyoxalate chemiluminescence detection system for high performance liquid chromatography

The catalytic effect of bases (imidazole, pyridine, Tris and triethylamine) on the peroxyoxalate chemiluminescence (PO-CL) reaction for high performance liquid chromatography (HPLC) was investigated. Imidazole increased PO-CL intensity extraordinarily, whereas the other bases (pyridine, Tris and triethylamine) did not. The peak heights of dipyridamole (coronary vasodilator) obtained using the eluents containing buffers were largest at pH 7.0, a few times less at pH 6.0 and pH 5.0, 100 times less at pH 4.0 and a few hundred times less at pH 3.0. The eluents containing buffers at pH 3, 4, 5, 6 or 7 each with imidazole increased the peak heights by a few to ten times as compared with those without imidazole, and those peak heights were within one order of magnitude. On the other hand, the eluent containing buffer at pH 2 did not affect the peak heights with or without imidazole. Bis(4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) alone and bis(2,4-dinitrophenyl)oxalate (DNPO) plus TDPO were recommended to be used against eluents containing buffers of pH 5-7 and pH 3-4, respectively. Dipyridamole and benzydamine hydrochloride (anti-inflammatory drug) were separated on the ODS column and detected by the present system. The detection limits of dipyridamole and benzydamine hydrochloride were 40 amol and 270 fmol, respectively.

Sensitive detection of oxo-steroids and oxo-bile acids by liquid chromatography with peroxyoxalate chemiluminescence detection

Oxo-steroids and oxo-bile acid ethyl esters were derivatized with 5- N,N-dimethylamino-naphthalene-1-sulphonohydrazide (DNS-hydrazine) to DNS-hydrazone in the presence of hydrochloric acid in ethanol or trifluoroacetic acid in benzene, purified by high-performance gel-permeation chromatography, separated on an ODS column with an eluent containing tetrahydrofuran and detected via a peroxyoxalate chemiluminescence reaction using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO). Sensitive detection (femtomole level) of each oxo-steroid which appeared as a single peak was achieved. The procedure for the isolation of oxo-bile acids developed for GC-MS allowed the detection by this system of an unusual oxo-bile acid, 7α-hydroxy-3-oxo-5β-cholanic acid, at the nanomole l −1 level in urine from a patient with cholestatic liver disease.

Synthesis and evaluation of aryl oxalates as peroxyoxalate chemiluminescence reagents

Eight aryl oxalates having an alkyoxy moiety were synthesised to improve the solubility in acetonitrile for the peroxyoxalate chemiluminescence reaction system. Of these, bis[2-(3,6-dioxaheptyloxycarbonyl)-4-fluorophenyl] oxalate showed the highest solubility (1685 mM). To evaluate these esters together with some known aryl oxalates as chemiluminescence reagents, a simple and rapid method using a flow injection system was developed. The chemiluminescence reaction system consisted of an oxalate, hydrogen peroxide, triethylamine and 9,10-diphenylanthracene in acetonitrile. Of the oxalates synthesised, bis[2-(3,6-dioxaheptyloxycarbonyl)-4-bromophenyl] oxalate gave the highest intensity, which was about 100 times lower than that of bis(2,4-dinitrophenyl) oxalate. However, the chemiluminescence intensities of the oxalates synthesised continued for a far longer period of time than those of commercially available oxalates.