Environ Sci Pollut Res DOI 10.1007/s11356-014-2646-9
RESEARCH ARTICLE
Determination of picogram quantities of chlortoluron in soil samples by luminol–chitosan chemiluminescence system Yajuan Li & Jingjing Zhang & Xunyu Xiong & Kai Luo & Jie Guo & Minxia Shen & Jiajia Wang & Zhenghua Song
Received: 20 November 2013 / Accepted: 10 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Based on the enhancing effect of chitosan (CS) on luminol-dissolved oxygen chemiluminescence (CL) reaction, a flow injection (FI) luminol–CS CL system was established. It was found that the increase of CL intensity was proportional to the concentrations of CS ranging from 0.7 to 10.0 μmol l−1. In the presence of chlortoluron (CTU), the CL intensity of luminol–CS system could be obviously inhibited and the decrements of CL intensity were linearly proportional to the logarithm of CTU concentrations ranging from 0.01 to 70.0 ng ml−1, giving the limit of detection 3.0 pg ml−1 (3σ). At a flow rate of 2.0 ml min−1, the whole process including sampling and washing could be accomplished within 36 s, offering a sample throughput of 100 h−1. The proposed FI–CL method was successfully applied to the determination of CTU in soil samples with recoveries ranging from 95.0 % to 105.3 % and the relative standard deviations (RSDs) of less than 4.0 %. Keywords Chlortoluron . Chitosan . Luminol . Flow injection . Chemiluminescence . Soil
Introduction Chlortoluron (CTU), a selective herbicide of the phenylurea family derivative, is one of the most widely used herbicides in Responsible editor: Gerald Thouand Y. Li : J. Zhang : X. Xiong : K. Luo : J. Guo : M. Shen : J. Wang : Z. Song (*) Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, 710069 Xi’an, China e-mail: [email protected] Z. Song e-mail: [email protected]
agricultural field with high efficiency to control annual grasses and broad-leaved weeds (Xu et al. 2012). As the adverse effect of CTU (Abdessalem et al. 2008; Moro et al. 2012; Caquet et al. 2013), it was designated “priority hazardous substances” by the European Union (European Commission 2001). A considerable number of research studies have focused on the determination of CTU in the field of environment and foodstuff analysis (Wang et al. 2010; Carrillo-Carrión et al. 2012; Li et al. 2012; Fenoll et al. 2012). The methods widely used for the determination of CTU are chromatography (LópezFeria et al. 2009; Wang et al. 2012; Amelin et al. 2013) and electrochemistry (Li and Jiang 2010; Jiang and Li 2010; Dejmkova et al. 2013; Li et al. 2013). Recently, chemiluminescence (CL) combined with flow injection (FI) technique, has increasingly attracted more attention from researchers in the field of environmental chemistry for its advantages of high sensitivity, low detection limit and simple instrument (Sánchez et al. 2009; López-Paz and Catalá-Icardo 2011; Xie et al. 2011; Chen et al. 2012). To the best of our knowledge, there has been no report on the determination of CTU using luminol–chitosan (CS) CL system up to now. CS ((1,4)-2-amino-2-deoxy-β-D-glucan; Fig. 1), obtained by the deacetylation of chitin, is a linear polysaccharide with widespread applications in the areas of biotechnology, biomedcine (Sogias et al. 2010; Dash et al. 2011; Kamari et al. 2011) and food ingredients (Dutta et al. 2009; Aider 2010; Tripathi et al. 2010) because of its many useful features such as hydrophilicity, biocompatibility and anti-bacterial property (Jia et al. 2009; Trapani et al. 2010). In this work, it was first found that CS could accelerate the electrons transferring rate of excited 3-aminophthalate with notable enhancing phenomenon of CL intensity of luminol-dissolved oxygen reaction. The increments of CL intensity were linear over the concentrations of CS ranging from 0.7 to 10.0 μmol l−1. It was also found that CTU could remarkably inhibit the CL signal of luminol–CS reaction, with the decrease of CL intensity
Environ Sci Pollut Res
Reagents
Fig. 1 The molecular structure of CS
linearly proportional to the logarithm of the CTU concentrations ranging from 0.01 to 70.0 ng ml−1. The limit of detection (LOD) for the determination of CTU was 3.0 pg ml−1 (3σ), and the relative standard deviations (RSDs) were less than 4.0 %. At a flow rate of 2.0 ml min−1, a complete analytical process including sampling and washing, could be performed within 36 s, offering a sample throughput of 100 h−1. This proposed method was successfully applied to the determination of CTU content in soil samples and the results were in good accordance with that obtained by high-performance liquid chromatography (HPLC).
Materials and methods Apparatus The FI–CL system used in this work was shown schematically in Fig. 2. A peristaltic pump of the IFFL-DD Luminescence Analyzer (Xi’an Remax Electronic Science-Tech. Co. Ltd., Xi’an, China) was applied to delivering all streams. The Poly Tetra Fluoro Ethylene (PTFE) tubing (1.0 mm i.d.) was used throughout the manifold for carrying the CL reagents. A sixway valve was used for quantitatively injecting 100.0 μL luminol into carrier stream. The CL signal produced in flow cell was detected by the photomultiplier tube (PMT), and the output was recorded by the computer. HPLC (HP 1100, Agilent, USA) with UV detector (λmax = 242 nm) measurements were performed under the following conditions: Column: C18 (150×4.6 mm), MP: methanol/ H2O=45:55 (v/v). Fig. 2 Schematic diagram of the present FI–CL system. Luminol: 2.5×10−5 mol l−1 (NaOH: 0.025 mol l−1); CS: 1.0× 10−4 mol l−1; flow rate: 2.0 ml min−1; mixing tube: 10.0 cm; high voltage: −750 V
All reagents used in this work were of analytical reagent grade. Doubly deionized water purified in a Milli-Q system (Millipore, Bedford, MA, USA; 18.2 MΩ cm) was used for the preparation of solutions in the whole procedure. Luminol (Fluka, Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station, China. CS (WM=9,000, DA=95 %; Wako Pure Chemicals Industry, Japan) was obtained from Xi’an Wolsen Biotechnology Co., Ltd. (Xi’an, China). Standard solution of CTU (5.00 mg ml−1) was supplied by the Evidence Identification Center of Ministry of Public Security, China. Working standard solutions of CTU were prepared daily from the above stock solution by appropriate dilution as required. Stock solution of luminol (2.5×10−2 mol l−1) was prepared by dissolving 0.44 g luminol with 100 ml 0.1 mol l−1 NaOH solution in a brown calibrated flask. CS solution was prepared by dissolving 0.45 g CS with water in a 500-ml calibrated flask. Experimental procedures As shown in Fig. 2, the luminol, carrier (purified water), CS and CTU solutions were inserted into flow lines by the peristaltic pump at a constant rate of 2.0 ml min−1. Purified water was first employed to wash the entire flow system, until a stable baseline had been recorded and the standard solution of luminol (100 μl) were injected into the premixed, homogeneous stream of CS and CTU. Then the whole mixed solutions were delivered into the CL cell in an alkaline medium, with the CL emission producing and detected by PMT at a high voltage of −750 V. The concentration of CTU could be quantified on the basis of the decrement of CL intensity, ΔICL =I0 −Is, where Is and I0 denote the CL signals in the presence and absence of CTU, respectively. Sample preparation Soil samples used in the experiment were collected in the upper layer (0–10 cm) of the fields, air-dried at room temperature, and passed through a 2-mm mesh sieve. The spiked
Environ Sci Pollut Res
Fig. 3 Effects of pH values on the CL response. NaOH: 1.0×10−5 to 3.0×10−1 mol l−1 (pH: 9.0–13.5)
samples were prepared by adding known quantities of CTU solutions in 1.0 g soil. Then the samples were treated according to reported method (Wang et al. 2009). The main procedure was as follows: 3.0 ml ethanol was added into the above spiked samples with an ultrasonic process for 3.0 min, followed by centrifugation at 3,000 rpm (~2,100×g) for 10 min, then transferring the supernatant solution into a 50.0-ml beaker. This process was repeated three times. All supernatants were collected together and then filtered through a 0.45-μm membrane filter using appropriate amount of ethanol to wash the filter, then the filtrate was evaporated to dryness. The solid CTU was dissolved in 0.1 ml ethanol solution and diluted to the mark in a 10.0 ml calibrated flask using deionized water.
Results and discussion
Fig. 5 Relative CL intensity–time profiles in different CL systems. Luminol: 2.5×10−5 mol l−1; CS: 1.0×10−4 mol l−1; CTU: 10.0 ng ml−1. Curve a: CL system of luminol-dissolved oxygen reaction; curve b: CL system of luminol–CS–CTU reaction; curve c: CL system of luminol–CS reaction
10−3 mol l−1) on the CL intensity were tested. It was found that the optimum concentration of luminol was 2.5 × 10−5 mol l−1. Because the luminol reaction was more favorable under alkaline conditions, NaOH was added into the luminol solution to increase the sensitivity of the CL system. The effect of NaOH solutions with different concentrations ranging from 1.0×10−5 to 3.0×10−1 mol l−1 (pH: 9.0–13.5) on CL intensity was tested. As shown in Fig. 3, it was clear that with the increase of NaOH concentration, the CL signal of luminol increased accordingly. When at the pH of about 12.0, the CL intensity could reach a maximum value and then
Optimization of CL experimental conditions The experimental conditions for the determination of CTU were optimized. The effects of luminol (5.0×10−7 to 5.0× 10−4 mol l−1) and CS concentrations (7.0×10−7 to 1.0×
Fig. 4 Effects of CS concentrations on CL intensity. Luminol: 2.5× 10−5 mol l−1; NaOH: 0.025 mol l−1
Fig. 6 The calibration curve of ΔІCL vs. ln CCTU. Linear range of CTU concentrations: 0.01–70.0 ng ml−1
Environ Sci Pollut Res Table 1 Stability and reproducibility test of the FI–CL system for CTU determinationa Time (day)
ICL blank
RSDb (%)
ICL (0.1 ng ml−1)
RSD (%)
ICL (10.0 ng ml−1)
RSD (%)
1st 2nd 3rd
343±4.1 346±4.8 344±7.9
1.2 1.4 2.3
318±4.1 315±8.5 319±7.3
1.3 2.7 2.3
259±5.2 256±5.6 257±7.2
2.0 2.2 2.8
a
The average of five determinations
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u n u t ∑ ðX i −X Þ2 i¼1
b
RSD: the relative standard deviation; RSD ¼
n−1
X
ðn ¼ 5Þ;
where Xi is the measured value and X is the average value of five determinations
decreased slowly. Thus, pH of 12.0 was selected in the subsequent experiments. The effect of CS concentrations on luminol CL intensity was also examined and optimized, as shown in Fig. 4. It could be seen that the increment of CL intensity was linearly proportional over the CS concentrations in the range of 7.0×10−7 to 1.0×10−4 mol l−1, giving a linear equation of ΔICL = 9.40CCS +2.07 (R2 =0.9990). Concerning the low consumption of CS solution, 1.0×10−4 mol l−1 was chosen as the optimum CS concentration. The flow rate and mixing tube length had substantial effects on the CL intensity. The influence of flow rate on CL intensity was examined in the range from 0.5 to 5.0 ml min−1. It was found that lower flow rates could broaden the peak of CL spectrum and slow down the sampling rate; while at higher flow rates, the signal-tonoise (S/N) ratio increased, and the baseline would become unstable. Thus, an optimum flow rate of 2.0 ml min−1 was chosen considering both good precision and low reagents consumption. The effect of the mixing tube length on the CL emission was examined from 5.0 to 20.0 cm. It was found that
10.0 cm mixing tube could afford the best result with good sensitivity and reproducibility. Relative CL intensity–time profiles The relative CL intensity–time profiles of different CL systems were shown in Fig. 5. It can be seen that the time Tmax for reaching maximum CL intensity (Imax) of luminol-dissolved oxygen system was 4.4 s with Imax of 134 (curve a); the Tmax for luminol–CS CL system was 4.0 s with Imax of 346 (curve b). In the presence of 10.0 ng ml−1 CTU, the Tmax for luminol– CS system was 4.0 s with Imax decreased from 346 to 257 by 25.7 % (curve c). The experiments were carried out under the optimum concentrations of luminol and CS, which were 2.5× 10−5 and 1.0×10−4 mol l−1, respectively. Analytical performance for the determination of CTU Under the optimum conditions described above, a series of CTU standard solutions was pumped into the flow line and determined accordingly. It was found that the decrease of CL intensity of luminol–CS system was proportional to the logarithm of CTU concentrations in the range of 0.01– 70.0 ng ml−1, giving the regression equation of ΔICL = 37.5lnCCTU +71.2 (R2 =0.9970) with an LOD of 3.0 pg ml−1 (3σ), and the calibration curve of CTU with the luminol–CS CL system is presented in Fig. 6. At a flow rate of 2.0 ml min−1, a complete analytical process including sampling and washing could be performed in 36 s, offering a sample throughput of 100 h−1. Operational stability of the FI–CL system
Fig. 7 Fluorescence spectra of luminol–CS system
The stability and reproducibility test of this FI–CL system for CTU determination was conducted in 3 consecutive days with the flow system being operated continuously over 8 h per day. The results of these replicate experiments were listed in Table 1, the CL intensity was the average of every five separate
Environ Sci Pollut Res Table 2 Results of determination of CTU in spiked soil samples
1
0/1.93±0.03 1.00/2.92±0.03
99.3
48.30±0.67
48.0±0.72
2
0/2.05±0.03 2.00/4.08±0.03 0/4.02±0.07 4.00/8.11±0.06 0/2.00±0.02 1.00/3.01±0.04 0/1.93±0.06 1.00/2.92±0.06 0/1.90±0.04 1.00/2.86±0.03 0/0.97±0.02 1.00/1.95±0.02 0/0.99±0.03 1.00/1.98±0.04 0/2.06±0.04 2.00/4.11±0.04 0/0.09±0.01 0.10/0.18±0.01 0/0.19±0.01 0.10/0.29±0.01 0/0.21±0.01 0.20/0.43±0.01
101.3
51.26±0.36
49.7±0.65
potential interfering species with increasing amounts were added to CTU standard solution (1.0 nmol l−1) and the relative error was controlled at 5 % level. The tolerable concentrations of foreign species were less than 10.0 nmol l−1 for Fe3+/Fe2+, Co3+/Co2+, Al3+, Cr3+, Cu2+; 500.0 nmol l−1 for Mg2+, Ca2+, NH4+; 1.2 μmol l−1 for I–, NO3–, Ac–, CO32–, HCO3–, PO43–, BrO3– and urea; 16.0 μmol l−1 for methanol, respectively.
102.1
50.28±0.40
48.5±0.87
The possible CL mechanism of luminol–CS–CTU reaction
101.2
5.02±0.06
4.9±0.06
99.7
4.95±0.14
4.7±0.14
98.3
4.91±0.09
4.8±0.09
98.0
0.98±0.02
NDc
99.0
0.99±0.03
ND
105.3
1.05±0.02
ND
95.0
0.095±0.002
ND
100.0
0.010±0.004
ND
103.5
0.011±0.003
ND
The possible luminescence mechanism of luminol–CS–CTU reaction is studied by FI–CL and fluorescence methods. According to the FI–CL results, it can be seen in Fig. 5 that the Tmax of the maximum CL intensity of luminol-dissolved oxygen system shifts from 4.4 to 4.0 s in the presence of CS with the corresponding intensity increasing from 134 to 346, demonstrating that CS can accelerate the electron transfer rate of excited 3-aminophthalate and enhance the CL intensity of luminol-dissolved oxygen system. According to the fluorescence results, it can be seen in Fig. 7 that the fluorescence intensity of 0.10 μmol l−1 luminol (λex/λem =350 nm/425 nm) is decreased in the presence of CS with its concentrations ranging from 0.01 to 5.0 μmol l−1. Using the Stern–Volmer quenching equation (Lakowicz 1999), τ0 of luminol is 10.1 ns (Vasilescu et al. 2003) and Kq of CS can be obtained as 2.5× 1013 l mol−1 s−1, which is far greater than the maximum scatter collision quenching constant of various quenchers with 2.0× 1010 l mol−1 s−1 (Lakowicz & Weber 1973). It is deduced that the inhibition effect of CS on luminol fluorescence intensity is not caused by a dynamic process, but results from the formation of a ground-state complex between CS and luminol. As also shown in Fig. 5, the Imax of luminol–CS CL system in the presence of CTU is obviously decreased with the same Tmax of luminol-dissolved oxygen system. By the homemade FI–CL model (He 2011), viz. the interaction equation of macromolecules with small molecules, the binding constant Ka of 2.79× 105 l mol−1 and number binding site n of 0.70 were obtained,
Samples
3 4 5 6 7 8 9 10 11 12
Added/Found (ng ml−1) (±SD)
Recovery (%)
a
Averaged from five determinations
b
Not detected by HPLC
−1
Amount of CTU (μg g ) by CLa
by HPLCb
determinations and the RSDs were less than 2.8 %, indicating this FI–CL system could exert a good stability. Interference studies In order to assess the influences of some common substances possibly existed in soil sample on CTU quantification, the Table 3 Comparison of different methods for the determination of CTU Method
Sample preparation
LOD (ng ml−1)
Samples
Refs.
GC-MS HPLC-DAD
SPEa GSEb DLLMEc
1.80±0.16 0.25±0.01 0.20±0.01
Olive oils Liquor Natural water
López-Feria et al. 2009 Wang et al. 2012 Amelin et al. 2013
Electrochemistry
– SPE Extraction – Extraction
4.70±0.13 0.51±0.32 72.31±3.9 21.27±0.49 (3.0±0.1)×10−3
Farmland water Irrigation water River water Farmland water Soil
Li and Jiang 2010b Li et al. 2013 Dejmkova et al. 2013 Jiang and Li 2010a This Work
The proposed CL a
Solid phase extraction
b
Gas–solid extraction
c
Dispersive liquid–liquid micro-extraction
Environ Sci Pollut Res
which indicate that a 1:1 CS–CTU complex can be formed online. The possible CL mechanism of luminol–CS–CTU reaction can be explained as follows: the formed ground-state complex of CS–luminol gives the effect of complexation enhancement of luminol CL; the formed 1:1 complex of CS–CTU might result in the inhibition of CL intensity from luminol–CS reaction. Determination of CTU in spiked soil sample CTU content in the soil samples prepared in the “Sample preparation” section were determined by the proposed FI– CL method. In order to validate the applicability of the method, recovery studies were carried out on samples to which known quantities of CTU standard solutions were added. The results were listed in Table 2 with recoveries ranging from 95.0 % to 105.3 %. By HPLC, the same soil samples were determined and the results obtained by the proposed CL method were in good agreement with those of HPLC.
Conclusion A comparison of previously reported methods, including GCMS, HPLC, electrochemistry and the presented FI–CL method for CTU determination is summarized in Table 3. Based on the inhibition effect of CTU on the CL intensity of luminol– CS system, a simple, rapid and sensitive FI–CL method for the determination of picogram level CTU in soil is developed. It can be seen that this FI–CL analysis exhibits a higher sensitivity and relatively low LOD, confirming this method is of practical value for the assay of CTU. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21275118) and the Open Fund from Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China. Conflict of interest There is no conflict of interest for our original unpublished work, which has not been submitted anywhere. This article does not contain any studies with human or animal subjects.
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RESEARCH ARTICLE
Determination of picogram quantities of chlortoluron in soil samples by luminol–chitosan chemiluminescence system Yajuan Li & Jingjing Zhang & Xunyu Xiong & Kai Luo & Jie Guo & Minxia Shen & Jiajia Wang & Zhenghua Song
Received: 20 November 2013 / Accepted: 10 February 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Based on the enhancing effect of chitosan (CS) on luminol-dissolved oxygen chemiluminescence (CL) reaction, a flow injection (FI) luminol–CS CL system was established. It was found that the increase of CL intensity was proportional to the concentrations of CS ranging from 0.7 to 10.0 μmol l−1. In the presence of chlortoluron (CTU), the CL intensity of luminol–CS system could be obviously inhibited and the decrements of CL intensity were linearly proportional to the logarithm of CTU concentrations ranging from 0.01 to 70.0 ng ml−1, giving the limit of detection 3.0 pg ml−1 (3σ). At a flow rate of 2.0 ml min−1, the whole process including sampling and washing could be accomplished within 36 s, offering a sample throughput of 100 h−1. The proposed FI–CL method was successfully applied to the determination of CTU in soil samples with recoveries ranging from 95.0 % to 105.3 % and the relative standard deviations (RSDs) of less than 4.0 %. Keywords Chlortoluron . Chitosan . Luminol . Flow injection . Chemiluminescence . Soil
Introduction Chlortoluron (CTU), a selective herbicide of the phenylurea family derivative, is one of the most widely used herbicides in Responsible editor: Gerald Thouand Y. Li : J. Zhang : X. Xiong : K. Luo : J. Guo : M. Shen : J. Wang : Z. Song (*) Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, 710069 Xi’an, China e-mail: [email protected] Z. Song e-mail: [email protected]
agricultural field with high efficiency to control annual grasses and broad-leaved weeds (Xu et al. 2012). As the adverse effect of CTU (Abdessalem et al. 2008; Moro et al. 2012; Caquet et al. 2013), it was designated “priority hazardous substances” by the European Union (European Commission 2001). A considerable number of research studies have focused on the determination of CTU in the field of environment and foodstuff analysis (Wang et al. 2010; Carrillo-Carrión et al. 2012; Li et al. 2012; Fenoll et al. 2012). The methods widely used for the determination of CTU are chromatography (LópezFeria et al. 2009; Wang et al. 2012; Amelin et al. 2013) and electrochemistry (Li and Jiang 2010; Jiang and Li 2010; Dejmkova et al. 2013; Li et al. 2013). Recently, chemiluminescence (CL) combined with flow injection (FI) technique, has increasingly attracted more attention from researchers in the field of environmental chemistry for its advantages of high sensitivity, low detection limit and simple instrument (Sánchez et al. 2009; López-Paz and Catalá-Icardo 2011; Xie et al. 2011; Chen et al. 2012). To the best of our knowledge, there has been no report on the determination of CTU using luminol–chitosan (CS) CL system up to now. CS ((1,4)-2-amino-2-deoxy-β-D-glucan; Fig. 1), obtained by the deacetylation of chitin, is a linear polysaccharide with widespread applications in the areas of biotechnology, biomedcine (Sogias et al. 2010; Dash et al. 2011; Kamari et al. 2011) and food ingredients (Dutta et al. 2009; Aider 2010; Tripathi et al. 2010) because of its many useful features such as hydrophilicity, biocompatibility and anti-bacterial property (Jia et al. 2009; Trapani et al. 2010). In this work, it was first found that CS could accelerate the electrons transferring rate of excited 3-aminophthalate with notable enhancing phenomenon of CL intensity of luminol-dissolved oxygen reaction. The increments of CL intensity were linear over the concentrations of CS ranging from 0.7 to 10.0 μmol l−1. It was also found that CTU could remarkably inhibit the CL signal of luminol–CS reaction, with the decrease of CL intensity
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Reagents
Fig. 1 The molecular structure of CS
linearly proportional to the logarithm of the CTU concentrations ranging from 0.01 to 70.0 ng ml−1. The limit of detection (LOD) for the determination of CTU was 3.0 pg ml−1 (3σ), and the relative standard deviations (RSDs) were less than 4.0 %. At a flow rate of 2.0 ml min−1, a complete analytical process including sampling and washing, could be performed within 36 s, offering a sample throughput of 100 h−1. This proposed method was successfully applied to the determination of CTU content in soil samples and the results were in good accordance with that obtained by high-performance liquid chromatography (HPLC).
Materials and methods Apparatus The FI–CL system used in this work was shown schematically in Fig. 2. A peristaltic pump of the IFFL-DD Luminescence Analyzer (Xi’an Remax Electronic Science-Tech. Co. Ltd., Xi’an, China) was applied to delivering all streams. The Poly Tetra Fluoro Ethylene (PTFE) tubing (1.0 mm i.d.) was used throughout the manifold for carrying the CL reagents. A sixway valve was used for quantitatively injecting 100.0 μL luminol into carrier stream. The CL signal produced in flow cell was detected by the photomultiplier tube (PMT), and the output was recorded by the computer. HPLC (HP 1100, Agilent, USA) with UV detector (λmax = 242 nm) measurements were performed under the following conditions: Column: C18 (150×4.6 mm), MP: methanol/ H2O=45:55 (v/v). Fig. 2 Schematic diagram of the present FI–CL system. Luminol: 2.5×10−5 mol l−1 (NaOH: 0.025 mol l−1); CS: 1.0× 10−4 mol l−1; flow rate: 2.0 ml min−1; mixing tube: 10.0 cm; high voltage: −750 V
All reagents used in this work were of analytical reagent grade. Doubly deionized water purified in a Milli-Q system (Millipore, Bedford, MA, USA; 18.2 MΩ cm) was used for the preparation of solutions in the whole procedure. Luminol (Fluka, Biochemika) was obtained from Xi’an Medicine Purchasing and Supply Station, China. CS (WM=9,000, DA=95 %; Wako Pure Chemicals Industry, Japan) was obtained from Xi’an Wolsen Biotechnology Co., Ltd. (Xi’an, China). Standard solution of CTU (5.00 mg ml−1) was supplied by the Evidence Identification Center of Ministry of Public Security, China. Working standard solutions of CTU were prepared daily from the above stock solution by appropriate dilution as required. Stock solution of luminol (2.5×10−2 mol l−1) was prepared by dissolving 0.44 g luminol with 100 ml 0.1 mol l−1 NaOH solution in a brown calibrated flask. CS solution was prepared by dissolving 0.45 g CS with water in a 500-ml calibrated flask. Experimental procedures As shown in Fig. 2, the luminol, carrier (purified water), CS and CTU solutions were inserted into flow lines by the peristaltic pump at a constant rate of 2.0 ml min−1. Purified water was first employed to wash the entire flow system, until a stable baseline had been recorded and the standard solution of luminol (100 μl) were injected into the premixed, homogeneous stream of CS and CTU. Then the whole mixed solutions were delivered into the CL cell in an alkaline medium, with the CL emission producing and detected by PMT at a high voltage of −750 V. The concentration of CTU could be quantified on the basis of the decrement of CL intensity, ΔICL =I0 −Is, where Is and I0 denote the CL signals in the presence and absence of CTU, respectively. Sample preparation Soil samples used in the experiment were collected in the upper layer (0–10 cm) of the fields, air-dried at room temperature, and passed through a 2-mm mesh sieve. The spiked
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Fig. 3 Effects of pH values on the CL response. NaOH: 1.0×10−5 to 3.0×10−1 mol l−1 (pH: 9.0–13.5)
samples were prepared by adding known quantities of CTU solutions in 1.0 g soil. Then the samples were treated according to reported method (Wang et al. 2009). The main procedure was as follows: 3.0 ml ethanol was added into the above spiked samples with an ultrasonic process for 3.0 min, followed by centrifugation at 3,000 rpm (~2,100×g) for 10 min, then transferring the supernatant solution into a 50.0-ml beaker. This process was repeated three times. All supernatants were collected together and then filtered through a 0.45-μm membrane filter using appropriate amount of ethanol to wash the filter, then the filtrate was evaporated to dryness. The solid CTU was dissolved in 0.1 ml ethanol solution and diluted to the mark in a 10.0 ml calibrated flask using deionized water.
Results and discussion
Fig. 5 Relative CL intensity–time profiles in different CL systems. Luminol: 2.5×10−5 mol l−1; CS: 1.0×10−4 mol l−1; CTU: 10.0 ng ml−1. Curve a: CL system of luminol-dissolved oxygen reaction; curve b: CL system of luminol–CS–CTU reaction; curve c: CL system of luminol–CS reaction
10−3 mol l−1) on the CL intensity were tested. It was found that the optimum concentration of luminol was 2.5 × 10−5 mol l−1. Because the luminol reaction was more favorable under alkaline conditions, NaOH was added into the luminol solution to increase the sensitivity of the CL system. The effect of NaOH solutions with different concentrations ranging from 1.0×10−5 to 3.0×10−1 mol l−1 (pH: 9.0–13.5) on CL intensity was tested. As shown in Fig. 3, it was clear that with the increase of NaOH concentration, the CL signal of luminol increased accordingly. When at the pH of about 12.0, the CL intensity could reach a maximum value and then
Optimization of CL experimental conditions The experimental conditions for the determination of CTU were optimized. The effects of luminol (5.0×10−7 to 5.0× 10−4 mol l−1) and CS concentrations (7.0×10−7 to 1.0×
Fig. 4 Effects of CS concentrations on CL intensity. Luminol: 2.5× 10−5 mol l−1; NaOH: 0.025 mol l−1
Fig. 6 The calibration curve of ΔІCL vs. ln CCTU. Linear range of CTU concentrations: 0.01–70.0 ng ml−1
Environ Sci Pollut Res Table 1 Stability and reproducibility test of the FI–CL system for CTU determinationa Time (day)
ICL blank
RSDb (%)
ICL (0.1 ng ml−1)
RSD (%)
ICL (10.0 ng ml−1)
RSD (%)
1st 2nd 3rd
343±4.1 346±4.8 344±7.9
1.2 1.4 2.3
318±4.1 315±8.5 319±7.3
1.3 2.7 2.3
259±5.2 256±5.6 257±7.2
2.0 2.2 2.8
a
The average of five determinations
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u n u t ∑ ðX i −X Þ2 i¼1
b
RSD: the relative standard deviation; RSD ¼
n−1
X
ðn ¼ 5Þ;
where Xi is the measured value and X is the average value of five determinations
decreased slowly. Thus, pH of 12.0 was selected in the subsequent experiments. The effect of CS concentrations on luminol CL intensity was also examined and optimized, as shown in Fig. 4. It could be seen that the increment of CL intensity was linearly proportional over the CS concentrations in the range of 7.0×10−7 to 1.0×10−4 mol l−1, giving a linear equation of ΔICL = 9.40CCS +2.07 (R2 =0.9990). Concerning the low consumption of CS solution, 1.0×10−4 mol l−1 was chosen as the optimum CS concentration. The flow rate and mixing tube length had substantial effects on the CL intensity. The influence of flow rate on CL intensity was examined in the range from 0.5 to 5.0 ml min−1. It was found that lower flow rates could broaden the peak of CL spectrum and slow down the sampling rate; while at higher flow rates, the signal-tonoise (S/N) ratio increased, and the baseline would become unstable. Thus, an optimum flow rate of 2.0 ml min−1 was chosen considering both good precision and low reagents consumption. The effect of the mixing tube length on the CL emission was examined from 5.0 to 20.0 cm. It was found that
10.0 cm mixing tube could afford the best result with good sensitivity and reproducibility. Relative CL intensity–time profiles The relative CL intensity–time profiles of different CL systems were shown in Fig. 5. It can be seen that the time Tmax for reaching maximum CL intensity (Imax) of luminol-dissolved oxygen system was 4.4 s with Imax of 134 (curve a); the Tmax for luminol–CS CL system was 4.0 s with Imax of 346 (curve b). In the presence of 10.0 ng ml−1 CTU, the Tmax for luminol– CS system was 4.0 s with Imax decreased from 346 to 257 by 25.7 % (curve c). The experiments were carried out under the optimum concentrations of luminol and CS, which were 2.5× 10−5 and 1.0×10−4 mol l−1, respectively. Analytical performance for the determination of CTU Under the optimum conditions described above, a series of CTU standard solutions was pumped into the flow line and determined accordingly. It was found that the decrease of CL intensity of luminol–CS system was proportional to the logarithm of CTU concentrations in the range of 0.01– 70.0 ng ml−1, giving the regression equation of ΔICL = 37.5lnCCTU +71.2 (R2 =0.9970) with an LOD of 3.0 pg ml−1 (3σ), and the calibration curve of CTU with the luminol–CS CL system is presented in Fig. 6. At a flow rate of 2.0 ml min−1, a complete analytical process including sampling and washing could be performed in 36 s, offering a sample throughput of 100 h−1. Operational stability of the FI–CL system
Fig. 7 Fluorescence spectra of luminol–CS system
The stability and reproducibility test of this FI–CL system for CTU determination was conducted in 3 consecutive days with the flow system being operated continuously over 8 h per day. The results of these replicate experiments were listed in Table 1, the CL intensity was the average of every five separate
Environ Sci Pollut Res Table 2 Results of determination of CTU in spiked soil samples
1
0/1.93±0.03 1.00/2.92±0.03
99.3
48.30±0.67
48.0±0.72
2
0/2.05±0.03 2.00/4.08±0.03 0/4.02±0.07 4.00/8.11±0.06 0/2.00±0.02 1.00/3.01±0.04 0/1.93±0.06 1.00/2.92±0.06 0/1.90±0.04 1.00/2.86±0.03 0/0.97±0.02 1.00/1.95±0.02 0/0.99±0.03 1.00/1.98±0.04 0/2.06±0.04 2.00/4.11±0.04 0/0.09±0.01 0.10/0.18±0.01 0/0.19±0.01 0.10/0.29±0.01 0/0.21±0.01 0.20/0.43±0.01
101.3
51.26±0.36
49.7±0.65
potential interfering species with increasing amounts were added to CTU standard solution (1.0 nmol l−1) and the relative error was controlled at 5 % level. The tolerable concentrations of foreign species were less than 10.0 nmol l−1 for Fe3+/Fe2+, Co3+/Co2+, Al3+, Cr3+, Cu2+; 500.0 nmol l−1 for Mg2+, Ca2+, NH4+; 1.2 μmol l−1 for I–, NO3–, Ac–, CO32–, HCO3–, PO43–, BrO3– and urea; 16.0 μmol l−1 for methanol, respectively.
102.1
50.28±0.40
48.5±0.87
The possible CL mechanism of luminol–CS–CTU reaction
101.2
5.02±0.06
4.9±0.06
99.7
4.95±0.14
4.7±0.14
98.3
4.91±0.09
4.8±0.09
98.0
0.98±0.02
NDc
99.0
0.99±0.03
ND
105.3
1.05±0.02
ND
95.0
0.095±0.002
ND
100.0
0.010±0.004
ND
103.5
0.011±0.003
ND
The possible luminescence mechanism of luminol–CS–CTU reaction is studied by FI–CL and fluorescence methods. According to the FI–CL results, it can be seen in Fig. 5 that the Tmax of the maximum CL intensity of luminol-dissolved oxygen system shifts from 4.4 to 4.0 s in the presence of CS with the corresponding intensity increasing from 134 to 346, demonstrating that CS can accelerate the electron transfer rate of excited 3-aminophthalate and enhance the CL intensity of luminol-dissolved oxygen system. According to the fluorescence results, it can be seen in Fig. 7 that the fluorescence intensity of 0.10 μmol l−1 luminol (λex/λem =350 nm/425 nm) is decreased in the presence of CS with its concentrations ranging from 0.01 to 5.0 μmol l−1. Using the Stern–Volmer quenching equation (Lakowicz 1999), τ0 of luminol is 10.1 ns (Vasilescu et al. 2003) and Kq of CS can be obtained as 2.5× 1013 l mol−1 s−1, which is far greater than the maximum scatter collision quenching constant of various quenchers with 2.0× 1010 l mol−1 s−1 (Lakowicz & Weber 1973). It is deduced that the inhibition effect of CS on luminol fluorescence intensity is not caused by a dynamic process, but results from the formation of a ground-state complex between CS and luminol. As also shown in Fig. 5, the Imax of luminol–CS CL system in the presence of CTU is obviously decreased with the same Tmax of luminol-dissolved oxygen system. By the homemade FI–CL model (He 2011), viz. the interaction equation of macromolecules with small molecules, the binding constant Ka of 2.79× 105 l mol−1 and number binding site n of 0.70 were obtained,
Samples
3 4 5 6 7 8 9 10 11 12
Added/Found (ng ml−1) (±SD)
Recovery (%)
a
Averaged from five determinations
b
Not detected by HPLC
−1
Amount of CTU (μg g ) by CLa
by HPLCb
determinations and the RSDs were less than 2.8 %, indicating this FI–CL system could exert a good stability. Interference studies In order to assess the influences of some common substances possibly existed in soil sample on CTU quantification, the Table 3 Comparison of different methods for the determination of CTU Method
Sample preparation
LOD (ng ml−1)
Samples
Refs.
GC-MS HPLC-DAD
SPEa GSEb DLLMEc
1.80±0.16 0.25±0.01 0.20±0.01
Olive oils Liquor Natural water
López-Feria et al. 2009 Wang et al. 2012 Amelin et al. 2013
Electrochemistry
– SPE Extraction – Extraction
4.70±0.13 0.51±0.32 72.31±3.9 21.27±0.49 (3.0±0.1)×10−3
Farmland water Irrigation water River water Farmland water Soil
Li and Jiang 2010b Li et al. 2013 Dejmkova et al. 2013 Jiang and Li 2010a This Work
The proposed CL a
Solid phase extraction
b
Gas–solid extraction
c
Dispersive liquid–liquid micro-extraction
Environ Sci Pollut Res
which indicate that a 1:1 CS–CTU complex can be formed online. The possible CL mechanism of luminol–CS–CTU reaction can be explained as follows: the formed ground-state complex of CS–luminol gives the effect of complexation enhancement of luminol CL; the formed 1:1 complex of CS–CTU might result in the inhibition of CL intensity from luminol–CS reaction. Determination of CTU in spiked soil sample CTU content in the soil samples prepared in the “Sample preparation” section were determined by the proposed FI– CL method. In order to validate the applicability of the method, recovery studies were carried out on samples to which known quantities of CTU standard solutions were added. The results were listed in Table 2 with recoveries ranging from 95.0 % to 105.3 %. By HPLC, the same soil samples were determined and the results obtained by the proposed CL method were in good agreement with those of HPLC.
Conclusion A comparison of previously reported methods, including GCMS, HPLC, electrochemistry and the presented FI–CL method for CTU determination is summarized in Table 3. Based on the inhibition effect of CTU on the CL intensity of luminol– CS system, a simple, rapid and sensitive FI–CL method for the determination of picogram level CTU in soil is developed. It can be seen that this FI–CL analysis exhibits a higher sensitivity and relatively low LOD, confirming this method is of practical value for the assay of CTU. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21275118) and the Open Fund from Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, China. Conflict of interest There is no conflict of interest for our original unpublished work, which has not been submitted anywhere. This article does not contain any studies with human or animal subjects.
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