A study of peroxyoxalate-chemiluminescence of 4,4'-bis{[4,6-bis (2-hydroxyethyl)amino-1,3,5-triazin-2-yl]amino}stilbene-2,2'-disulfonic acid-disodium salt as a novel blue fluorescer

The chemiluminescence arising from the reaction of bis(2,4,6-trichlorophenyl)oxalate (TCPO) with hydrogen peroxide in the presence of brightener 4,4'-bis{[4,6-bis(2-hydroxyethyl)amino-1,3,5-triazin-2-yl]amino}stilbene-2,2'-disulfonic acid-disodium salt (Triazinyl) has been studied. The influence of concentration of TCPO, hydrogen peroxide, Triazinyl, base catalysts and temperature on the resulting chemiluminescence was investigated. The kinetic parameters for the peroxyoxalate-chemiluminescence (PO-CL) of Triazinyl were evaluated from computer fitting of the resulting intensity-time plots. The activation energies, E(a), were evaluated from temperature dependence of the corresponding rise and fall rate constants.     

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.     

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.     

Chemiluminescence detection in capillary electrophoresis using an ultra-fast co-catalyzed peroxyoxalate chemiluminescent reaction and electrokinetic reagent delivery

Summary Despite the impressive limits of detection and inherent selectivity afforded by peroxyoxalate chemiluminescence (POCL) detection, efficient coupling of POCL to capillary electrophoresis (CE) remains limited by the relatively slow kinetics of the reactions that drive imidazole-catalyzed chemiluminescence. Moreover, oxalate esters, used in POCL, are sparingly soluble in polar solvents and hydrolyze rapidly, presenting an additional challenge with respect to detection following aqueous phase separations. In this paper, a novel method for coupling an ultrafast POCL reaction to CE is presented. Post separation electrokinetic delivery of the POCL reagent bis(2,4,6-trichlorophenyl)oxalate (TCPO) was accomplished using a commercially available micro tee. Electrokinetic addition of TCPO allowed for precise control of the ratio of TCPO to the chemiluminescence (CL) reagents 1,2,2,6,6-pentamethylpiperidine (PMP) and 1,2,4-triazole (triazole), spiked into the running buffer. This novel method for CL reagent delivery avoided the problems and costs associated with using pressure or mechanical pumps to deliver reagents post separation. Use of this dual-component system (PMP and triazole) resulted in intense CL with half-lives of less than 2 seconds. Optimum conditions for CE-POCL detection were investigated using stopped-flow kinetics. The detection limit for 3-aminofluoranthene, following separation by CE, was<0.95 nM.     

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。     

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.     

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⁺.     

Inhibition of peroxyoxalate chemiluminescence by intercalation of fluorescent acceptors between DNA bases

We have examined the ability of different fluorescent DNA dyes to become chemically excited by the peroxyoxalate chemiluminescent reaction. The intercalating dyes ethidium bromide and propidium iodide, and the bis-intercalating dyes ethidium homodimer-1, benzoxazolium-4-pyridinium dimer-1 and benzoxazolium-4-quinolinium dimer-1, exhibit an intense chemiluminescence when they are excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction in the absence of DNA. However, the chemiluminescence of these dyes is very low when they are bound to double-stranded DNA (dsDNA). In contrast, the minor groove-binding dye Hoechst 33258 excited by the TCPO-H2O2 reaction shows approximately the same chemiluminescence intensity when it is free in solution or complexed with dsDNA. Structural alterations or partial dissociation of dsDNA-bis-intercalating dye complexes produced by the addition of acetone, NaCl, MgCl2 or the cationic surfactant cetyltrimethylammonium bromide increases the chemiluminescence intensity. A moderate chemiluminescence intensity is observed when bis-intercalating dyes are complexed with single-stranded DNA. Our results indicate that the energy from the intermediates produced in the peroxyoxalate chemiluminescent reaction cannot be efficiently transferred to fluorescent dyes complexed with DNA; chemiexcitation is almost completely inhibited when dyes are buried in the dsDNA structure by intercalation between the base pairs.     

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.     

Enhancement of peroxyoxalate chemiluminescence intensity by surfactants and its application to detect detergent

Enhancement of peroxyoxalate chemiluminescence (PO-CL) intensity by a surfactant in the H₂O₂/bis(2,4,6-trichlorophenyl)oxalate (TCPO)/rhodamine B system was described. The effects of 15 surfactants were evaluated by comparing the ratio of a relative CL intensity (RCI) with surfactant to that of the blank in each system. In preliminary study, H₂O₂/imidazole-HNO₃ buffer/TCPO/rhodamine B system was used to study the effects of surfactants on PO-CL intensity. Fourteen surfactants reduced the CL intensity at the 2% concentration, where their relative CL intensities ranged from 0.6 to 93.5%. Some of these phenomena may be caused by a notable change of pH that was occurred by adding the surfactant.Additionally, enhancement of PO-CL intensity was studied by using system (1) H₂O₂/TCPO/rhodamine B and (2) H₂O₂/imidazole-HNO₃ buffer/TCPO/systems. In the system 1, the favorable enhancement of CL intensity (ranged from 124 to 472%) was observed with 9 surfactants at the 0.5% concentration. This result suggested that several surfactants might play a role as a catalyst in the PO-CL reaction. There was no tendency to enhance CL intensity among the surfactant types. In the system 2, the enhancement of CL intensity was also observed by adding with 11 surfactants, which might be mainly caused by the fluorescent impurities of surfactants used.Furthermore, detection of detergent commercially available was applied by using the system 1.     

A new sensitive determination method of estradiol in plasma using peroxyoxalate ester chemiluminescence combined with an HPLC system

A new sensitive determination method of estradiol in a plasma sample using peroxyoxalate ester chemiluminescence was developed. Estradiol, which was extracted by liquid-liquid extraction using ethyl acetate from plasma, was derivatized with dansyl-chloride (DNS-Cl) and separated by reverse-phase HPLC. The performance of four oxalates, bis(trichlorophenyl)oxalate (TCPO), bis(2,4-dinitrophenyl)oxalate (DNPO), bis(pentafluorophenyl)oxalate (PFPO), and bis[4-nitro-2-(3,6, 9-trioxadecyloxycarbonyl)phenyl] oxalate (TDPO), were evaluated using the static system, and DNPO was found to have the most sensitive and stable chemiluminescence at a H(2)O(2) concentration of 30 mM. HPLC-chemiluminescence system using DNPO for the determination of estradiol was established. The detection limit of dansylated-estradiol (DNS-E2) was 15 fmol (4 pg) in the standard solution and 44 fmol (12 pg) in the rat plasma sample at S/N = 3.     

Sensitive determination of aliphatic amines in water by high-performance liquid chromatography with chemiluminescence detection

A sensitive method has been developed for liquid chromatographic determination of short aliphatic amines in water samples. Analytes are preconcentrated and dansylated on solid sorbents (C18 solid-phase extraction cartridges). The dansyl derivatives are chromatographed and post-column mixed with peroxyoxalate (TCPO) and H2O2 in order to perform chemiluminescence detection. Optimal results have been obtained using a sample volume of 5 ml. The method has been applied to the quantification or screening of several aliphatic amines: methylamine, ethylamine, butylamine, diethylamine, pentylamine and hexylamine. The screening procedure has been developed including also polyamines (putrescine, cadaverine, spermidine and spermine). The results obtained by using chemiluminescence (CL) detection have been compared with other detection systems (fluorescence and UV). The sensitivity can increase from 3 to 75 times respect UV detection and from 2 to 10 times respect fluorescence detection depending on the amine. The detection limits achieved were between 0.15 and 0.9 microg/l.     

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 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.     

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.     

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.     

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.     

Fast peroxyoxalate chemiluminescence for minimized analytical separation systems

The maximum intensity, Imax, and time required to reach the maximum emission, taumax, for 1-aminopyrene monitored in 1,1'-oxalyldi-4-methylimidazole (OD4MI) chemiluminescence (CL) reactions are approximately 61 times higher and 16 times faster than their respective values for bis(2,4,6-trichlorophenyl)oxalate (TCPO) CL reactions in the presence of imidazole (ImH).     

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.