A study of chemiluminescence from reaction of bis(2,4,6-trichlorophenyl)oxalate, hydrogen peroxide and diethyl-2-(cyclohexylamino)-5-[(E)-2-phenyl-1-ethenyl]-3,4-furandicarboxylate as a novel fluorescer

The chemiluminescence (CL) arising from reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with hydrogen peroxide in the presence of a diethyl-2-(cyclohexylamino)-5-[(E)-2-phenyl-1-ethenyl]-3,4-furandicarboxylate as a novel fluorescer (Flu) has been studied. The relationship between the chemiluminescence intensity and concentrations of TCPO, sodium salicylate, hydrogen peroxide and fluorescer is reported. The chemiluminescence parameters including intensity at maximum CL, time at maximum intensity, total light yield, theoretical maximum level of intensity and pseudo-first-order rate constants for the rise and fall of the CL burst (k_r and k_f) were evaluated from computer fitting of the resulting intensity-time plots. The activation parameters E_a, ΔH^Δ, ΔS^Δ and ΔG^Δ for the rise and fall steps were evaluated from the temperature dependence of k_r and k_f values.     

A study of peroxyoxalate-chemiluminescence of acriflavine

The chemiluminescence arising from the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with hydrogen peroxide in the presence of acriflavine has been studied. The relationship between the chemiluminescence intensity and concentrations of TCPO, H2O2, acriflavine and the base sodium salicylate are reported. The kinetic parameters for the peroxyoxalate-chemiluminescence (PO-CL) of acriflavine were evaluated from the computer fitting of the corresponding chemiluminescence intensity-time plots.     

Peroxyoxalate chemiluminescence of 1,4-dihydroxy-3-methyl-thioxanthone and quenching effect of β-cyclodextrin

The chemiluminescence generated from the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO), hydrogen peroxide and 1,4-dihydroxy-3-methyl-thioxanthone (DMT) was investigated. Effects of reacting components, solvent and concentrations of TCPO, sodium salicylate, hydrogen peroxide and DMT were studied and their optimal values were determined. In addition, the influences of β-Cyclodextrin (β-CD) on the peroxyoxalate chemiluminescence (PO-CL) system of DMT were examined at optimized condition. The results showed that the presence of β-CD causes both enhancing and quenching effects on PO-CL system of DMT based upon its concentration. The Stern–Volmer quenching constant (K _q) was evaluated as 2.32?×?10⁴?M^?1 (R ^2?= 0.991) by creating a linear regression plot on experimentally obtained data. This study resulted in satisfactorily determination of β-CD in the range 5.0?×?10^?6 to 1.0?×?10^?4?M.     

Quenching effect of some heavy metal ions on the fast peroxyoxalate-chemiluminescence of 1-(dansylamidopropyl)-1-aza-4,7,10-trithiacyclododecane as a novel fluorophore

The fast chemiluminescence (CL) arising from the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with hydrogen peroxide in the presence of 1-(dansylamidopropyl)-1-aza-4,7,10-trithiacyclododecane (L) as a novel fluorophore, and imidazole as catalyst, has been studied in ethyl acetate solution. The relationships between the chemiluminescence intensity and concentrations of TCPO, imidazole, hydrogen peroxide and L are reported. In the presence of imidazole as catalyst, the entire CL signal was completed in less than 3 s. The quenching effect of Cu²⁺, Pb²⁺, Cd²⁺, Hg²⁺ and Ag⁺ ions on the chemiluminescent system was investigated, the resulting Stern–Volmer plots were obtained and the K_Q values were calculated. It was found that the quenching effect of metal ions on the chemiluminescence of L decreases in the order Cu²⁺ > Pb²⁺ > Cd²⁺ > Hg²⁺ > Ag⁺.     

HPLC Determination of Hepatic Cholesterol 7α-Hydroxylase Activity Using Immobilized Cholesterol Oxidase and Chemiluminescence Detection

Abstract

This paper describes an HPLC method for the determination of cholesterol 7α-hydroxylase activity, at high or low activity levels, that is sensitive and specific for 7α-hydroxycholesterol. The method relies on the generation of hydrogen peroxide by oxidation of 7α-hydroxycholesterol using the enzyme cholesterol oxidase which has been immobilized on porous glass beads. The hydrogen peroxide is subsequently detected by chemiluminescence generated by reaction of peroxide with bis-(2, 4, 6-trichlorophenyl)-oxalate (TCPO), a commonly used chemiluminescence reagent specific for peroxides. In the procedure, sample preparation is limited to extraction of the incubation mixture and injection of the concentrated extract.     

Automated pre-column derivatization of amines in biological samples with dansyl chloride and with or without post-column chemiluminescence formation by using TCPO-H2O2.

On-line automation of two different liquid chromatographic procedures, a pre-column derivatization system and a pre- and post-column system, in order to generate chemiluminescence is reported. Dansyl chloride (Dns-Cl) was used as a pre-column reagent to form fluorophores and bis(2,4,6-trichlorophenyl) oxalate (TCPO) and hydrogen peroxide (H2O2) as a post-column reagent to generate chemiluminescence. This procedure is based on the employment of a primary column packed with C18 material inserted in a multi-dimensional assembly for sample clean-up and derivatization with Dns-Cl. The dansyl derivatives formed are transferred and separated in a LiChrospher 100 RP18 analytical column (125 x 4 mm id, 5 microns film thickness) using acetonitrile-imidazole buffer (pH 6.8) (70 + 30) as eluent. The separated derivatives were transferred to the detector for fluorescence detection or to the post-column system where the chemiluminescence response was generated by using TCPO-H2O2 and the products were detected by chemiluminescence. The procedure was optimised for amphetamine and related compounds. A comparison between the on-line pre-column and pre- and post-column systems was performed. The results show that the sensitivity of chemiluminescence detection can be higher than that of fluorescence detection. The recoveries obtained ranged from 98 +/- 8 up to 108 +/- 8% for amphetamine and methamphetamine, respectively. The accuracy and precision of these methods were evaluated.     

Peroxyoxalate chemiluminescence detection of condensates of malondialdehyde with thiobarbituric acids using a flow system.

The peroxyoxalate chemiluminescence(CL) detection method for the evaluation of the CL intensity of malondialdehyde(MDA) condensates with seven 2-thiobarbituric acid derivatives is described. The method consists of a flow injection technique together with a CL detection system using bis(2,4,6-trichlorophenyl) oxalate(TCPO) and hydrogen peroxide as chemiluminogenic reagents. Linear correlations between CL intensity and concentration are obtained for pmol levels of condensates. Among the condensates, 1,3-diethyl-2-thiobarbituric acid(DETBA)-MDA shows the largest CL intensity. High performance liquid chromatography (HPLC)/CL detection of DETBA-MDA and 1,3-diphenyl-2-thiobarbituric acid(DPTBA)-MDA using a mixture of TCPO and hydrogen peroxide in acetonitrile as a postcolumn reagent solution is also described. The detection limits for DETBA-MDA and DPTBA-MDA are 20 and 200 fmol, respectively, per 20 microL injection at a signal-to-noise ratio of 2. This HPLC/CL detection system was applied to the determination of MDA in rat brains by using DETBA as a fluorescent derivatizing reagent.     

A solid-state chemiluminescence detector for hydrogen peroxide based on an immobilized luminophore : Application to Rain Water

The construction and functioning of a chemiluminescence detector for hydrogen peroxide is described. It is based on peroxyoxalate chemiluminescence and consists of a two-bed reactor packed with solid trichlorophenyloxalate (TCPO) and 3-aminofluoranthene immobilized on controlled pore glass beads. Optimal conditions (pH, solvent, TCPO purity) for flow-independent operation are discussed. Samples can be injected into a moving stream or directly into the monitor with a syringe so as to provide a manually operated field monitor. The detection limit is 1.5 × 10^?8 M, and calibration graphs are linear over six orders of magnitude. The r.s.d. for the manual monitoring mode is ±3% for 17 μg l^?1 hydrogen peroxide. A sample throughput of 100 h^?1 is possible in the flow injection mode, and 40 samples h^?1 for manual injection.     

Peroxyoxalate chemiluminescence of N,N'-bistosyl-1H,4H-quinoxaline-2,3-dione and related compounds. Dependence on electronic nature of fluorophores

The title compound N,N'-bistosyl-1H,4H-quinoxaline-2,3-dione (TsQD) provides peroxyoxalate chemiluminescence (PO-CL) when reacted with hydrogen peroxide in the presence of fluorophores. The chemiluminescence (CL) efficiency of TsQD was superior to that of other related compounds such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), a typical oxalate for the peroxyoxalate PO-CL, under an aqueous condition. Factors affecting the PO-CL efficiency are discussed from the viewpoint of the structures of the substrates and the electronic nature of the fluorophores. A linear correlation of the logarithmic values evaluated from the CL quantum yields with the oxidation potentials of the aromatic fluorophores supports the involvement of the chemically initiated electron exchange luminescence (CIEEL) mechanism in both TsQD- and TCPO-CL systems. Also, an excellent Hammett relationship was derived from the correlation between the sigma(+) values and the relative singlet excitation yields in TsQD-CL enhanced by a series of fluorescent para,para'-disubstituted distyrylbenzenes.     

几种常用荧光探针的化学发光成像研究

利用双(2,4,6)三氯苯基过氧化草酸酯(TCPO)-过氧化氢(H2O2)-咪唑-荧光探针的化学发光体系,研究了荧光探针化学发光成像,对几种常用的荧光探针(丁基罗丹明、罗丹明B、罗丹明6G、荧光素及异硫氰酸荧光素等)进行了定量分析。本方法具有高灵敏度、成像分析高通量等优点,线性范围宽,检出限达10-11mol/L。对四甲基异硫氰酸罗丹明(TRITC)标记的单克隆羊抗人IgG的化学发光成像分析,比相同条件下荧光成像的检出限低一个数量级。     

Oxalate ester addition from a solid reagent bed for chemiluminescence detection in HPLC

Summary The combination of solid-state peroxyoxalate chemiluminescence detection and post-column chemical reaction systems in liquid chromatography is investigated. Bis-2,4,6-trichlorophenyloxalate (TCPO) is added from a solid reagent bed with the fluorophore, 3-aminofluoranthene, immobilized on glass beads. Hydrogen peroxide generated photochemically by quinone analytes is measured. Flow splitting is shown to be a simple means of reagent addition with a negligible band broadening effect. This system is compared to a more flexible dualpump design. To minimize band broadening in both systems, the reagent bed is located out of the path of the chromatographic effluent. Detection limits are in the sub-picomole range, an imporvement relative to those previously reported with TCPO added in the liquid phase. These systems can be easily be adapted for detection of peroxide generated by other post-column reactions.     

A post-column extraction system for the determination of tertiary amines by liquid chromatography with chemiluminescence detection

Summary The combination of an ion-pair extraction detection system with peroxyoxalate chemiluminescence detection has been investigated. Ion-pairs of protonated tertiary amines with a chemiluminescent counter ion are on-line post-column extracted to 1,2-dichloroethane containing bis(2,4,6-trichlorophenyl)oxalate (TCPO). Hydrogen peroxide is added to the organic phase by means of a solid-state perhydrit reactor. The influence of the base catalyst (imidazole) on the chemiluminescence reaction in apolar solvents was studied. Some sulfonated chemiluminophores were compared with respect to their chemiluminescence, ion-pairing and extraction properties, and 5-dimethylaminonaphthalene-1-sulfonate was found to be the most suitable reagent. The detection limit of the potential drug secoverine is in the sub-nanogram range.     

Chemiluminescence of bis(2,4,6-trichlorophenyl) oxalate in aqueous micellar systems

The chemiluminescent reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) in aqueous micellar systems is compared with the reaction in a mixture of acetonitrile and aqueous phosphate buffer. The chemiluminescence was studied in batch experiments with perylene as the fluorophore. The oxidation of TCPO produced the same intensity of chemiluminescence in the buffered acetonitrile as in Arkopal N-300 micelles, the best micellar system. The solubility of TCPO in an aqueous micellar system is greater than that in the acetonitrile/aqueous buffer (80:20, v/v), but TCPO is less stable in the former system.     

Effect of a dual-head short-stroke pump on post-column peroxyoxalate chemiluminescence detection

A dual-head short-stroke pump has two advantages in the post-column peroxyoxalate chemiluminescence (PO-CL) detection system. The first is to increase the mixing efficiency of solutions. The second is to increase the stability of the PO-CL reaction by keeping the aryl oxalate and hydrogen peroxide solutions separate. The detection sensitivities for six polycyclic aromatic hydrocarbons (PAHs) increased in the present system by using bis(2,4-dinitrophenyl) oxalate (DNPO) or bis(2,3,4,5,6-pentafluorophenyl) oxalate (PFPO) instead of such popular aryl oxalates as bis(2,4,6-trichlorophenyl) oxalate (TCPO) and bis[2-(3,6,9-trioxadecyloxycarbonyl)-4-nitrophenyl] oxalate (TDPO). Both DNPO and PFPO increased the sensitivities by factors of 4.1-10.2 and 3.5-8.1, respectively. In addition, DNPO was more stable than PFPO in acetonitrile. These results suggest that DNPO is the most useful aryl oxalate for the sensitive PO-CL detection of PAHs.     

Kinetics of two pathways in peroxyoxalate chemiluminescence

It has been shown that 1,1'-oxalyldiimidazole (ODI) is formed as an intermediate in the imidazole-catalyzed reaction of oxalate esters with hydrogen peroxide. Therefore, the kinetics of the chemiluminescence reaction of 1,1'-oxalyldiimidazole (ODI) with hydrogen peroxide in the presence of a fluorophore was investigated in order to further elucidate the mechanism of the peroxyoxalate chemiluminescence reaction. The effects of concentrations of ODI, hydrogen peroxide, imidazole (ImH), the general-base catalysts lutidine and collidine, and temperature on the chemiluminescence profile and relative quantum efficiency in the solvent acetonitrile were determined using the stopped-flow technique. Pseudo-first-order rate constant measurements were made for concentrations of either H2O2 or ODI in large excess. All of the reaction kinetics are consistent with a mechanism in which the reaction is initiated by a base-catalyzed substitution of hydrogen peroxide for imidazole in ODI to form an imidazoyl peracid (Im(CO)2OOH). In the presence of a large excess of H2O2, this intermediate rapidly decays with both a zero- and first-order dependence on the H2O2 concentration. It is proposed that the zero-order process reflects a cyclization of this intermediate to form a species capable of exciting a fluorophore via the "chemically initiated electron exchange mechanism" (CIEEL), while the first-order process results from the substitution of an additional molecule of hydrogen peroxide to the imidazoyl peracid to form dihydroperoxyoxalate, reducing the observed quantum yield. Under conditions of a large excess of ODI, the reaction is more than 1 order of magnitude more efficient at producing light, and the quantum yield increases linearly with increasing ODI concentration. Again, it is proposed that the slow initiating step of the reaction involves the substitution of H2O2 for imidazole to form the imidazoyl peracid. This intermediate may decay by either cyclization or by reaction with another ODI molecule to form a cyclic peroxide that is much more efficient at energy transfer with the fluorophore. The reaction kinetics clearly distinguishes two separate pathways for the chemiluminescent reaction.     

Determination of superoxide dismutase and SOD-mimetic activities by a chemical system: Co2/H2O2/lucigenin

The bright chemiluminescence has been observed in the system: Co²⁺/hydrogen peroxide/lucigenin. The chemiluminescence intensity was directly proportional to either cobalt, hydrogen peroxide, or lucigenin concentrations. A procedure of determination of superoxide dismutase (SOD) activity by the chemiluminescence method in the cobalt–hydrogen peroxide–lucigenin system at pH 8.5 is suggested. A linear dependence was established between a relative chemiluminescence intensity and SOD concentration in the range of SOD concentrations between 0 and 4.5 nM, c _1/2 = 0.8 nM. The determination of SOD activity was performed in several tissue samples (rat plasma, erythrocyte hemolysate, and liver mitochondria). A technique of tissue sample preparation with the use of thermal inactivation of interfering proteins at 60 °C was used. The method was successfully applied for comparison of the efficiency of SOD mimetics.     

Kinetics and Mechanism of the Nucleophilic Substitution Reaction of Imidazole with Bis(2,4,6-trichlorophenyl) Oxalate and Bis(2,4-dinitrophenyl) Oxalate

The kinetics of the imidazole-catalyzed decomposition of bis(2,4,6-trichlorophenyl) oxalate (TCPO) and bis(2,4-dinitrophenyl) oxalate (DNPO) was investigated by the stopped-flow technique. Pseudo-first-order rate constants were determined as a function imidazole concentration in the temperature range 6-45 degrees C by fitting the temporal changes in absorbance throughout the 245 to 345 nm wavelength range for TCPO and at 420 nm for DNPO. The reaction proceeds by release of two molecules of substituted phenol and formation of 1,1'-oxalyldiimidazole (ODI) for both esters. The identity of ODI was confirmed in the reaction of imidazole with TCPO by its UV absorbance spectrum and (13)C-NMR spectrum. The reaction of imidazole with TCPO has a second-order dependence on imidazole concentration and an observed negative activation energy of -6.2 +/- 0.3 kJ/mol, whereas the DNPO reaction has a first-order dependence on imidazole concentration and an observed positive activation energy of 12.0 +/- 0.6 kJ/mol. The differences in the temperature dependence and order of the reaction with respect to imidazole for the two oxalate esters are explained by a shift in the rate-determining step from addition to the acyl group for DNPO to imidazole-catalyzed release of the phenol leaving group for TCPO. These kinetics results are useful in interpreting the initial reaction steps in peroxyoxalate chemiluminescence.     

Evaluation of aryl oxalates for chemiluminescence detection in high-performance liquid chromatography

The chemiluminescence reaction between an aryl oxalate, hydrogen peroxide and a fluorescent compound is well known for use in h.p.l.c. post-column reactors. Here, several aryl oxalates are evaluated for this purpose in terms of intensity, rate of chemiluminescence decay, solubility in different solvents, and stability in the presence of hydrogen peroxide. Five oxalates are selected for different pH ranges of column eluates: bis(pentafluorophenyl) oxalate for pH < 2, bis(2,4-dinitrophenyl) oxalate for pH 2–4, bis(2-nitrophenyl) oxalate for pH 4–6, bis(2,4,6-trichlorophenyl) oxalate for pH 6–8, and bis(2,4,5-trichlorophenyl-6-pentyloxycarbonyl) oxalate for pH > 8.     

流动注射-鲁米诺-高碘酸钾-过氧化氢体系的化学发光行为及其应用

详细研究了流动注射－鲁米诺－高碘酸钾－过氧化氢体系化学发光行为，给出了反应的最佳条件。拟定了一种化学发光测定过氧化氢的新方法。方法的检测限为３．０Ｘ１０－８ｍｏｌ／ＬＨ２Ｏ２；线性范围为２．０×１０－７～６．０×１０－４ｍｏｌ／Ｌ。评价了该体系的应用前景。     

Mechanistic Studies on the Salicylate-Catalyzed Peroxyoxalate Chemiluminescence in Aqueous Medium

The peroxyoxalate reaction is one of the most efficient chemiluminescence transformations known and the only system occurring by an intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism with confirmed high quantum yields. The peroxyoxalate chemiluminescence (PO-CL) is mainly studied in anhydrous organic medium; however, for bioanalytical application, it should be performed in aqueous media. In the present work, we study the peroxyoxalate system in a binary 1,2-dimethoxyethane/water mixture with bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis[2-(methoxycarbonyl)phenyl] oxalate (DMO), catalyzed by sodium salicylate, in the presence of rhodamine 6G as activator. Reproducible kinetic results are obtained for all systems; emission decay rate constants depend on the salicylate as well as hydrogen peroxide concentration, and the occurrence of a specific base catalysis is verified. Although singlet quantum yields determined are lower than in anhydrous media in comparable conditions, they are still considerably high and adequate for analytical applications. The highest singlet quantum yields are obtained for the “ecologically friendly” derivative DMO indicating that this derivative might be the most adequate substrate for the use of the peroxyoxalate system in bioanalytical applications.