Flow Injection Chemiluminescence Analysis Research Articles

Field Quantification of Hydroxyl Radicals by Flow-Injection Chemiluminescence Analysis with a Portable Device.

Hydroxyl radical (•OH) is a powerful oxidant abundantly found in nature and plays a central role in numerous environmental processes. On-site detection of •OH is highly desirable for real-time assessments of •OH-centered processes and yet is restrained by a lack of an analysis system suitable for field applications. Here, we report the development of a flow-injection chemiluminescence analysis (FIA-CL) system for the continuous field detection of •OH. The system is based on the reaction of •OH with phthalhydrazide to generate 5-hydroxy-2,3-dihydro-1,4-phthalazinedione, which emits chemiluminescence (CL) when oxidatively activated by H2O2 and Cu3+. The FIA-CL system was successfully validated using the Fenton reaction as a standard •OH source. Unlike traditional absorbance- or fluorescence-based methods, CL detection could minimize interference from an environmental medium (e.g., organic matter), therefore attaining highly sensitive •OH detection (limits of detection and quantification = 0.035 and 0.12 nM, respectively). The broad applications of FIA-CL were illustrated for on-site 24 h detection of •OH produced from photochemical processes in lake water and air, where the temporal variations on •OH productions (1.0-12.2 nM in water and 1.5-37.1 × 107 cm-3 in air) agreed well with sunlight photon flux. Further, the FIA-CL system enabled field 24 h field analysis of •OH productions from the oxidation of reduced substances triggered by tidal fluctuations in coastal soils. The superior analytical capability of the FIA-CL system opens new opportunities for monitoring •OH dynamics under field conditions.

Rapid and selective screening of organic peroxide explosives using acid-hydrolysis induced chemiluminescence

Organic peroxide explosives (OPEs) are unstable, non-military, contemporary security threats often found in improvised explosive devices. Chemiluminescence (CL) can be used to detect OPEs, via radical formation consisting of peroxide moieties (-O-O-) under acidic conditions. However, selectivity for specific OPEs is hampered by the ubiquitous background of H2O2. Herein, we report the differentiation of hexamethylene triperoxide diamine (HMTD), triacetone triperoxide (TATP), and methyl ethyl ketone peroxide (MEKP) by specific flow injection analysis–CL (FIA-CL) signal profiles, after H2SO4 treatment. The radical degradation pathway of each structure, and its corresponding FIA-CL profile, was explored using mass spectrometry to reveal the rapid loss of -O-O- from TATP and HMTD structures, while MEKP formed CL signal-sustaining oligomers, as opposed to the immediate attenuation of H2O2. The CL response for OPEs in an aqueous media, measured via the described FIA-CL method, enabled ultra-trace limits of detection down to 0.40 μM for MEKP, 0.43 μM for HMTD, and 0.40 μM for TATP (combined linear range 1–83 μM with 95% confidence limit, n = 12). Expanded uncertainties of measurement (UM) of MEKP = ±0.98, HMTD = ±1.03, and TATP = ±1.1 (UM included probabilities of false positive and false negative as well as standard deviations of % recoveries and limit of detections of OPEs). Direct aqueous sample introduction via FIA-CL thus offers the prospect of rapid and selective screening of OPEs in security-heightened settings (e.g., airports), averting false positives from more ubiquitous H2O2.

From the Ocean to the Lab-Assessing Iron Limitation in Cyanobacteria: An Interface Paper.

Iron is an essential, yet scarce, nutrient in marine environments. Phytoplankton, and especially cyanobacteria, have developed a wide range of mechanisms to acquire iron and maintain their iron-rich photosynthetic machinery. Iron limitation studies often utilize either oceanographic methods to understand large scale processes, or laboratory-based, molecular experiments to identify underlying molecular mechanisms on a cellular level. Here, we aim to highlight the benefits of both approaches to encourage interdisciplinary understanding of the effects of iron limitation on cyanobacteria with a focus on avoiding pitfalls in the initial phases of collaboration. In particular, we discuss the use of trace metal clean methods in combination with sterile techniques, and the challenges faced when a new collaboration is set up to combine interdisciplinary techniques. Methods necessary for producing reliable data, such as High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS), Flow Injection Analysis Chemiluminescence (FIA-CL), and 77K fluorescence emission spectroscopy are discussed and evaluated and a technical manual, including the preparation of the artificial seawater medium Aquil, cleaning procedures, and a sampling scheme for an iron limitation experiment is included. This paper provides a reference point for researchers to implement different techniques into interdisciplinary iron studies that span cyanobacteria physiology, molecular biology, and biogeochemistry.

Multicommuted Flow Injection Analysis Using Chemiluminescence Detection (MCFIA-CL) for Olive Oil Analysis

This article describes the applicability of multicommuted chemiluminescence flow injection analysis (MCFIA-CL) to the determination of olive oil quality parameters. Two automated MCFIA methods were developed and validated for the determination of peroxide value (PV) and phenolic compounds in commercial extra virgin olive oil samples. The PV method proposed is based on the chemiluminescent (CL) signal produced from luminol oxidation by the olive oil hydroperoxides in basic conditions using Co(II) as catalyst, while the determination of phenolic compounds is based on CL emission generated from permanganate reduction in acidic media by hydroalcoholic extracts of olive oil. The flow assemblies for automated liquid handling consisted, respectively, of 2 and 3 three-way solenoid valves, which were controlled by a homemade electronic interface and Java-written software together with a CL detector. The signals obtained correlated well against the PV and total phenolic contents measured by the official and the Folin-Ciocalteu methods, respectively. The linear response range observed for PV allows checking whether the samples are in compliance with European Union regulations. On the other hand, the phenolic compounds content also correlated well with data reported by the well-known FC method across the entire typical concentration range of these compounds in virgin olive oils. The proposed methods are faster, safer, and more environmentally friendly than the respective official or classical ones involving lower consumption of solvents, reagents, and sample, as well as less waste generation together with a high sample throughput. Moreover, they are straightforward and cost-effective to implement in routine analysis, and can be regarded as a feasible alternative to the official or classic methods.

Investigation on Ruthenium(II) Bipyridine/AgIII Complexes Chemiluminescence System and Its Application for Sensitive Norfloxacin and Ofloxacin Detection

The reaction mechanism of AgIII complex ([Ag(HIO6)2]5–) with ruthenium(II) bipyridine (Ru(bpy)32+) in acid medium was investigated. A novel flow injection chemiluminescence (CL) analysis method was developed for the detection of norfloxacin (NFLX) and ofloxacin (OFLX) in commercial drug, milk and human urine samples based on AgIII-ruthenium(II) bipyridine in acidic solution. Under optimal conditions, CL intensities were proportional to drug concentrations in the range of 1.6 × 10-9 to 8 × 10-6 g mL-1 for NFLX and 1.0 × 10-9 to 4 × 10-6 g mL-1 for OFLX. The detection limit was 1.0 × 10-9 g mL-1 for NFLX and 0.8 × 10-9 g mL-1 for OFLX. On the basis of the CL, UV and fluorescence spectra, possible reaction mechanisms were proposed.

Quantitative Monitoring of Rutin in Human Urine by Flow Injection-Chemiluminescence Analysis

In this paper, a rapid and sensitive flow injection-chemiluminescence (FI-CL) method is proposed for the quantitative determination of rutin based on the inhibitory effect of rutin on the chemiluminescence intensity from the luminol–chymotrypsin (CT) system. The decrease of CL intensity was found to be proportional to the logarithm of rutin concentration in the range 0.1–30.0 ng/mL. A method for the quantification of rutin is proposed, with the limit of detection (LOD) of 0.03 ng/mL (3σ). A complete analytical process including sampling and washing for rutin determination, which was conducted at a flow rate of 2.0 mL/min, could be performed completely within 30 s, yielding a sample efficiency of 120 h−1. The proposed procedure was successfully applied for the determination of rutin in human urine after oral intake, with recoveries varying from 93.9 to 108.1% and relative standard derivation <4.0% (n = 5). Results showed that urine reached the maximum concentration at ~2.5 h, and the total excretion ratios were (83.5 ± 0.6) and (86.8 ± 0.7)%, respectively, for two volunteers in 8 h. The pharmacokinetic parameters, including the half-life (1.05 ± 0.02 h), absorption rate constant (1.18 ± 0.01 h−1), and elimination rate constant (0.70 ± 0.01 h−1), were obtained. The possible CL mechanism of the luminol–CT–rutin reaction is discussed by FI-CL, fluorescence, and molecular docking methods.

Study on the luminescence behavior of lanthanide ions with luminol by flow injection chemiluminescence analysis

This work first described the linear quenching of LnIII (including LaIII, CeIII, PrIII, NdIII, SmIII, EuIII, GdIII, TbIII, DyIII, HoIII, ErIII, TmIII, YbIII and LuIII) on luminol–dissolved oxygen chemiluminescence system and possible quenching mechanism by flow injection analysis. The general equation of the decrement of CL intensity with LnIII concentration, ΔI=AlnCLn+B, was given and the interaction model of LnIII with luminol, lg[(I0−I)/I]=rlg[Ln]+lgK, was established. The sensitive factor A was found to be linear with the physical parameters (Z, γ±, Eo and ΔHhyd). By plotting A against Z, it could be seen light lanthanides (LL) and heavy lanthanides (HL) presented a good symmetry. The results showed that the reaction of LnIII with luminol was a spontaneous process by the electrostatic force with the association constants K at 106–107 level. The binding constants KD (about 104–105) and the number of binding sites n (about 1) of LnIII (LaIII, EuIII, GdIII, TbIII and LuIII) to bovine serum albumin were first given by FI–CL, and the binding ability of LnIII followed the pattern: LaIII<EuIII<GdIII<TbIII<LuIII.

Ultrasensitive Determination of Diphacinone by Flow Injection Chemiluminescence: Application to Quantification in Biofluids and Photodegradation Monitoring in Water Samples

An ultrasensitive, quick, and simple approach for the determination of pg levels of diphacinone (DPN) by flow injection chemiluminescence (CL) analysis is proposed for the first time. It is based on the quenching effect of DPN on the CL intensity from a luminol-bovine serum albumin (BSA) CL system, for which the CL intensity decrease was linearly proportional to the logarithm of DPN concentration in the range of 5.0 to 5000 pg/mL. The LOD for DPN determination was as low as 2.0 pg/mL (3α a), and the RSD values were less than 5.0%. One determination cycle that included sampling and washing could be performed in 0.5 min with a sample throughput of 120/h under the optimum experimental conditions. This proposed method was successfully applied to determining DPN in human gastric juice and serum samples with recoveries from 91.8 to 114.3%, and to continuous monitoring of the degradation of DPN in water samples exposed to sunlight during 43 h with a variation ratio of 99.99%. The possible interaction behavior of BSA-DPN is briefly discussed.

Hollow-fiber liquid-phase microextraction for the direct determination of flumequine in urban wastewaters by flow-injection analysis with terbium-sensitized chemiluminescence.

A flow-injection analysis chemiluminescence method based on the enhancement effect of the flumequine-Tb(III) complex on the weak native emission of the Ce(IV)-Na2SO3 system has been developed for the determination of flumequine. The method includes a cleanup and preconcentration stage (750-fold) of the sample by hollow-fiber liquid-phase microextraction using an Accurel(®) Q 3/2 polypropylene hollow fiber impregnated with 1-octanol as the supported liquid membrane. The obtained 50 μL acceptor phase was injected in a 1 mM Tb(III) + 4 mM Ce(IV) in 5% v/v H2 SO4 stream and mixed with a 2 mM Na2 SO3 stream before its introduction into the flow cell. The chemiluminescence signal was linear in the 0.3-15 ng/mL range, with detection and quantitation limits of 0.1 and 0.3 ng/mL, respectively. The method allows the selective extraction and determination of flumequine in wastewater samples, using simpler and lower-cost instrumentation and with shorter extraction and analysis times than traditional high-performance liquid chromatography analysis.

Study on the interaction of catalase with pesticides by flow injection chemiluminescence and molecular docking

The interaction mechanisms of catalase (CAT) with pesticides (including organophosphates: disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate, dipterex, methamidophos and acephate; carbamates: carbaryl and methomyl; pyrethroids: fenvalerate and deltamethrin) were first investigated by flow injection (FI) chemiluminescence (CL) analysis and molecular docking. By homemade FI–CL model of lg[(I0−I)/I]=lgK+nlg[D], it was found that the binding processes of pesticides to CAT were spontaneous with the apparent binding constants K of 103–105Lmol−1 and the numbers of binding sites about 1.0. The binding abilities of pesticides to CAT followed the order: fenvalerate>deltamethrin>disulfoton>isofenphos-methyl>carbaryl>malathion>isocarbophos>dimethoate>dipterex>acephate>methomyl>methamidophos, which was generally similar to the order of determination sensitivity of pesticides. The thermodynamic parameters revealed that CAT bound with hydrophobic pesticides by hydrophobic interaction force, and with hydrophilic pesticides by hydrogen bond and van der Waals force. The pesticides to CAT molecular docking study showed that pesticides could enter into the cavity locating among the four subdomains of CAT, giving the specific amino acid residues and hydrogen bonds involved in CAT–pesticides interaction. It was also found that the lgK values of pesticides to CAT increased regularly with increasing lgP, Mr, MR and MV, suggesting that the hydrophobicity and steric property of pesticide played essential roles in its binding to CAT.

Ultrasensitive Determination of Cadmium in Rice by Flow Injection Chemiluminescence Analysis

Based on the enhancing effect of Cd2+ on luminol–Co2+ chemiluminescence (CL) system, a sensitive method for determining picogram Cd2+ in rice by flow injection (FI)–CL was proposed. It was found that the CL intensity increments were proportional to the concentrations of Cd2+, giving a calibration graph linear over the Cd2+ concentrations ranging from 7.0 to 5,000.0 pmol L−1, with a detection limit of 2.0 pmol L−1 (3σ) and relative standard deviations (RSDs) of 3.2, 2.8, and 2.5 % for 30.0, 300.0, and 1,000.0 pmol L−1 Cd2+ (n = 5), respectively. At a flow rate of 2.0 mL min−1, a complete determination of Cd2+ including sampling and washing could be accomplished within 36 s, giving a sample throughput of 100 h−1. The contents of Cd2+ in rice were found to be 0.07–0.10 mg kg−1. The proposed method was also applied to the determination of Cd2+ in spiked human serum samples with recoveries from 95.9 to 106.3 % and RSDs less than 4.0 %.

Study on the interaction behavior of lysozyme with lanthanide ions by flow injection chemiluminescence analysis

The interaction behavior of lysozyme with trivalent lanthanide ions (LnIII) was investigated by flow injection chemiluminescence (FI-CL) analysis. It was found that lysozyme with LnIII could form a 1 : 1 association complex that remarkably quenched CL intensity from the luminol–lysozyme reaction, and the quantitative correlation equation of CL intensity decrements versus the logarithm of the concentration of LnIII ΔI = A ln CLn + B was established. The results showed the sensitive factor A varied as atomic number Z increased monotonically, and ALL of light lanthanides (LL) from LaIII to EuIII was less than AHL of heavy lanthanides (HL) from GdIII to LuIII. The relationships of A vs. Z plot for LL and HL with a GdIII break at f7 were clearly presented, and the correlations of A with physical parameters (γ±, ΔHhyd and Eo) of LnIII were discussed. Using a homemade FI-CL model, the binding constants (K = 105 to 106 level) and binding sites (n = 1.0) were obtained, and the binding affinity abilities increased with increasing Z along the LnIII series. The thermodynamic parameters of lysozyme with LnIII were obtained, and the results showed that the reaction was a spontaneous process by electrostatic interaction. The possible mechanism and application of the luminol–lysozyme–LnIII CL system were given.

Relationship investigation of molecular structure–binding affinity of antibiotics to bovine serum albumin using flow injection chemiluminescence analysis and molecular docking

Luminescently probed by luminol, the interaction behaviors of bovine serum albumin (BSA) with different antibiotics (macrolides, tetracyclines and sulfonamides) at picomolar levels were investigated using flow injection chemiluminescence (FI-CL) analysis. It was found that BSA and antibiotics formed complexes with the binding ratio of 1 : 1, with the binding constants K within the range of 103 to 105 L mol−1 generally following the order of macrolides < tetracyclines < sulfonamides. Results showed that the ester groups of –OOCC3H7 or –OOC(CH2)2COOC2H5 in macrolides led to increased K by factors of 3.1–42.8 times, –OH or –Cl in tetracyclines increased K by 1.1–1.3 times, and the additional –C(NH)NH2 in sulfonamides increased K about 1.3 times. The thermodynamic parameters demonstrated that the BSA–antibiotic binding process should be spontaneous mainly through hydrophobic interaction for macrolides, hydrogen bonding and van der Waals force for tetracyclines, and electrostatic interaction for sulfonamides. The further antibiotics to BSA molecular docking study revealed that the pocket at subdommain IIA of BSA should be the principle binding site for antibiotics, showing that the interaction parameters including K and ΔG agreed well with the results from the proposed FI-CL analysis. The interesting phenomenon that the log K had good linear correlation to molecular volume MV, molar refractivity MR, polarizability POL and partition coefficient log P of antibiotics was also observed.

Investigation of a Novel Ag(III) Chemiluminescence System and its Mechanism for Determination of Uric Acid in Human Urine

A simple and sensitive method based on flow-injection chemiluminescence analysis coupled with luminol-Ag(III) complex was developed for the determination of uric acid (UA) in human urine. The method was based on the chemiluminescence (CL) reaction of luminol with Ag(III) in alkaline solution. UA in the urine can dramatically enhance CL intensities. In optimum conditions, the relative CL intensity had a linear relationship with UA concentration in the range of 1.0 × 10-8 to 5.0 × 10-6 mol L-1 with a detection limit of 2.0 × 10-9 mol L-1 (3σ). The relative standard deviation (n = 11) was 0.71% for 1.0 × 10-7 mol L-1 UA. Possible reaction mechanisms for the CL system were suggested based on the UV absorption spectra, CL spectra, and the free radical trapping experiment performed in this work.

A rapid and sensitive method for the determination of dibutyl phthalate in wine by flow-injection chemiluminescence analysis

A sensitive method for the determination of picogram level dibutyl phthalate (DBP) in wine by flow-injection chemiluminescence (FI–CL) analysis is presented for the first time, which was based on the quenching effect of DBP on the luminol–myoglobin (Mb) CL system. The decrement of CL intensity was linearly proportional to the logarithm of DBP concentration in the range of 0.1–100pgmL−1 with the detection limit of 0.03pgmL−1 (3σ). At a flow rate of 2.0mLmin−1, a complete determination of DBP including sampling and washing could be accomplished in 0.5min, giving the maximum sample throughput of 120h−1. The proposed method was successfully applied to the determination of DBP in wine, human serum and urine samples with the relative standard deviations (RSDs) of less than 3.0% (n=5). The molecule docking results showed that DBP interacted with the amino acid residues near the heme moiety of Mb. The possible CL mechanism of luminol–Mb–DBP reaction should be that the binding of Mb with DBP forming a 1:1 complex (binding constant K=1.55×104Lmol−1) led to the conformational change of Mb and resulted in the quenching of CL intensity.

Determination of ferulic acid by flow injection chemiluminescence analysis based on enhancement of theN-bromobutanimide-eosin-CrCl3system in alkaline solution

A simple and sensitive flow injection chemiluminescence method has been developed for the determination of ferulic acid (FA) based on the significant enhancement effect of FA on the CL signal of the N-bromobutanimide (NBS)-eosin-CrCl3 system in alkaline solution. Under optimum conditions, the enhanced CL intensity is linearly related to the concentration of FA in its pharmaceutical preparations and human plasma samples. The corresponding linear regression equations were established over the 4.0 × 10(-10)-1.0 × 10(-7) g/mL for FA tablets and 2.0 × 10(-10)-1.0 × 10(-7) g/mL for plasma samples. The limit of detection for FA tablets and limit of quantification for plasma samples were 2.8 × 10(-10) g/mL (3 σ) and 3.04 × 10(-10) g/mL (10 σ), respectively. A complete analysis could be performed within 40 s, including washing and sampling, giving a throughput of ≈90/h. The proposed method was successfully applied to the determination of FA in pharmaceutical preparations and human plasma samples with satisfactory results. The recoveries of pharmaceutical preparations and human plasma samples at three different concentrations were 97.8-102.6% and 96.7-104.0%, respectively. Furthermore, the possible mechanism of CL reactions was also discussed briefly.

Quantification of Iron in Seawater at the Low Picomolar Range Based on Optimization of Bromate/Ammonia/Dihydroxynaphtalene System by Catalytic Adsorptive Cathodic Stripping Voltammetry

A new analytical protocol for the challenging analysis of total dissolved iron at the low picomolar level in oceanic waters suitable for onboard analysis is presented. The method is based on the revision of the adsorptive properties of the iron/2,3-dihydroxynaphthalene (Fe/DHN) complexes on the hanging mercury drop electrode with catalytic enhancement by bromate ions. Although it was based on a previously proposed reagent combination, we show here that the addition of an acidification/alkalinization step is essential in order to cancel any organic complexation, and that an extra increment of the pH to 8.6-8.8 leads to the definition of a preconcentration-free procedure with the lowest detection limit described up to now. For total dissolved iron analysis, samples were acidified to pH 2.0 in the presence of 30 μM DHN and left to equilibrate overnight. A 10 mL sample was subsequently buffered to a pH of ∼8.7 in the presence of 20 mM bromate: a 60 s deposition at 0 V led to a sensitivity of 34 nA nM(-1) min(-1), a 4-fold improvement over previous methods, that translated in a limit of detection of 5 pM (2-20 fold improvement). Several tests proved that a nonreversible reaction in the time scale of the analysis, triggered by the acidification/alkalinization step, was behind the signal magnification. The new method was validated onboard via the analysis of reference material and via intercalibration against flow injection analysis-chemiluminescence on Southern Ocean surface samples.

Continuous, Quantitative Monitoring of Roxithromycin in Human Saliva by Flow Injection Chemiluminescence Analysis

Human saliva quantitative monitoring of roxithromycin (ROX) at picomolar-level by flow injection (FI) chemiluminescence (CL) analysis is described for the first time, to our knowledge. Monitoring was based on the CL intensity from luminol-BSA reaction, which can be quenched in the presence of ROX, with the decreasing CL intensity linearly proportional to the logarithm of the ROX concentration, ranging from 0.6 to 1000 pmol·L(-1). The detection limit of the proposed method for the determination of ROX was as low as 0.2 pmol·L(-1) (3σ), and the relative standard deviations were less than 4.0% (n = 7). A complete analytical process, including sampling and washing for ROX determination, conducted at a flow rate of 2.0 mL·min(-1), was performed completely within 30 s, yielding a sample efficiency of 120 h(-1). The proposed method was successfully applied to the determination of ROX in human saliva and serum samples with recoveries from 90.9% to 110.1%. The continuous monitoring of ROX in human saliva after oral intake showed that the total elimination ratio was 87.1% during 24 h, and the pharmacokinetic parameters were 0.97 ± 0.18 h(-1) for the absorption rate constant K(a), 0.082 ± 0.010 h(-1) for the elimination rate constant K(e), and 8.56 ± 1.11 h for the elimination half-life time t(1/2). It was also found that ROX in human saliva and urine simultaneously reached the maximum at 2 h with the concentration correlate ratio of 0.97.

Comparison of uric Acid quantity with different food in human urine by flow injection chemiluminescence analysis.

Based on the inhibitory effect of uric acid (UA) on luminol-Co2+ chemiluminescence (CL) system, a sensitive method for the determination of UA at nanomolar level by flow injection (FI) CL was proposed. The proposed method was successfully applied to real-time monitoring of UA excretion in human 24 h urine with different food intake, showing that meats, vegetables, and porridge intake caused differential UA excretions of 879, 798, and 742 mg, respectively. It was also found that UA concentrations in urine under the three kinds of food intake simultaneously reached maximum at 2 h after meals with the values of 417, 318, and 288 μg mL−1, respectively. The UA concentration in human serum was also determined by this approach, and the possible mechanism of luminol-Co2+-UA CL reaction was discussed in detail.

Determination of Picogram Amounts of Dihydroxybenzenes in Water Samples with Luminol–lysozyme Chemiluminescence System

AbstractAn ultrasensitive method for the determination of dihydroxybenzens by flow injection (FI) chemiluminescence (CL) analysis was proposed for the first time. It was found that the CL intensity of luminol–lysozyme system could be significantly inhibited by dihydroxybenzens. The CL intensity decrements were linear with the logarithm of dihydroxybenzens concentrations over the ranges of 1.0 ∼ 700 pg mL‐1 for hydroquinone (HQ), 5.0 ∼ 700 pg mL‐1 for catechol (CT) and 10 ∼ 7000 pg mL‐1 for resorcinol (RS), with the corresponding limits of detection of 0.7, 3.0 and 7.0 pg mL‐1, respectively. The proposed method was successfully applied to the determination of CT in tap water, rain water, river water and HQ in waste photographic developer samples, with recoveries from 93.5 to 105.8% and relative standard deviations (RSDs) less than 4.0% (n = 5). The possible interaction mechanism of lysozyme with dihydroxybenzens was discussed, and CT to lysozyme's binding constant and the thermodynamic parameters were given by the homemade FI–CL model. The results shown that the binding of dihydroxybenzens to lysozyme was spontaneous with the hydrophobic force.