


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 3, No. 4, 2019 

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142 
 

Original Paper 

A Synergistic Sensitized Fluorescent Determination of 

2,4-Dichlorophenoxyacetic Acid in Vegetable Samples Based on 

the Derivatives of Calix[4]arene 

Xiashi Zhu1,2* & Qiuyi Ren2 

1 College of Guangling, Yangzhou University, Yangzhou 225002, PR China 

2 College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, PR China 

* Xiashi Zhu, College of Guangling, Yangzhou University; College of Chemistry & Chemical 

Engineering, Yangzhou University, Yangzhou 225002, PR China 

 

Received: October 24, 2019   Accepted: November 5, 2019   Online Published: November 18, 2019 

doi:10.22158/fsns.v3n4p142        URL: http://dx.doi.org/10.22158/fsns.v3n4p142 

 

Abstract 

A novel fluorescent determination of 2,4-dichlorophenoxyacetic acid (2,4-D) based on the derivatives 

of calix[4]arene (SAX) in β-cyclodextrin(β-CD)/ sodium dodecyl sulfate (SDS) synergistic sensitized 

system was developed. The results were shown that the fluorescence intensity of SAX could be quenched 

by 2,4-D, and the fluorescence quenching (∆F=FSAX-F2,4-D -SAX) was synergistic sensitized in β-CD/ SDS 

medium. Under the conditions of λex/em=332/468 nm and pH 7.0, the linear range for 2,4-D were found 

to be 0.020-4.00 μg/mL. The mechanism of determination was discussed with quenching type analysis, 

inclusion interaction and sensitizing effect. This method has been applied for the determination of 

2,4-D in vegetable samples with satisfactory results.  

Keywords 

2,4-dichlorophenoxyacetic acid, β-cyclodextrin, SDS, synergistic sensitization, fluorescence quenching 

 

1. Introduction  

2,4-dichlorophenoxyacetic acid (2,4-D, Figure 1(a)) belongs to the category of benzoic acid pesticides, 

which has the biological activity of auxin and can be used as plant growth regulator and preservative for 

vegetable and fruit (Jiang, Zha, & Tie, 2015). Nevertheless, 2,4-D residues in agricultural products and 

environment have great harm to human health due to its carcinogenic, mutagenic and estrogenic activity 

(Garabrant & Philbert, 2002). Up to now the reported techniques for 2,4-D determination have been 

performed on LC-MS (Jiang, Zha, & Tie, 2015), fluorescence spectroscopy (Wang, Yua, & Wu, 2016; 



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Boroduleva & Eremin, 2016; Atta, Bera, & Chattopadhyay, 2015), high performance liquid 

chromatography(HPLC) (Wu, Ee, & Lee, 2005), capillary electrophoresis (CE) (Zhu & Lee, 2001) and 

gas chromatography(GC) (Rezazadeh, Yamini, Seidi, Tahmasebi, & Rezaei, 2014). Although these 

techniques have good performance, they are complicated, expensive and time-consuming. So it is 

necessary to establish a rapid, simple and high selectivity method for 2,4-D detection.  

Calixarenes are macrocyclic compounds through a series of phenol connected with the ortho methylene 

units. In calixarene molecule, the upper edge is composed of para-position substituent of benzene; the 

lower edge is formed by neatly arranged phenolic hydroxyl group; the middle hydrophobic cavity is 

composed of benzene rings. Schiff base calix[4]arene (SAX, Figure 1(b)) has been followed with great 

interests due to the simple structure and high symmetry. The cavity of SAX is composed of four 

benzene rings, meeting the size and stability required for the inclusion interaction. The analytical 

method based on host-guest chemistry of calix[4]arene derivatives have been reported (Ma & Zhu, 

2012; Yang, Yan, & Zhu, 2014; Wang, Zhu, & Yan, 2013; Yang, Qin, Yan, & Zhu, 2015; Li, X. Y., Li, M., 

& Chen, 2011; Khan, Shah, & Ahmed, 2016). But the fluorescence quenching methods using schiff 

base calix[4]arene derivatives as a fluorescent chemosensor for the determination of 2,4-D seems to be 

lacking. 

 

      

(a)                                 (b) 

Figure 1. Chemical Structure of (a)2,4-D and (b)SAX 

 

The sensitivity of spectral analysis could be improved in suitable medium, such as surfactant (Ma & 

Zhu, 2012; Yang, Yan, & Zhu, 2014; Wang, Zhu, & Yan, 2013; Yang, Qin, Yan, & Zhu, 2015; Li, X. Y., 

Li, M., & Chen, 2011), β-cyclodextrin (β-CD) (Sanchez, Rubio, & Blanco, 1988; Márquez, Hernández, 

& García, 1990; Zhang, Liu, & Fan, 2009; Sánchez, Lopez, & Gómez, 1987; Zhu, Sun, Bao, & Guo, 

2006; Sun, Zhu, & Wu, 2007) room temperature ionic liquid (Liu & Zhao, 2008; Berton & Martinis, 

2009; Martinis & Olsina, 2008; Zhu & Jiang, 2011). In our previous publications, the sensitizing effects 

of surfactant (Wu, Ee, & Lee, 2005; Zhu & Lee, 2001; Rezazadeh, Yamini, Seidi, Tahmasebi, & Rezaei, 

2014; Ma & Zhu, 2012), β-CD and its derivatives (Zhu, Sun, Bao, & Guo, 2006; Sun, Zhu, & Wu, 

2007), ionic liquids (ILs) (Zhu & Jiang, 2011) on the ultraviolet spectrometry and spectrofluorimetry 



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were developed. Further research showed that mixed medium (such as surfactant/ILs, β-CD/surfactant) 

could synergistically sensitize fluorescence method, which has a better sensitization effect than that 

single medium (Zhu, Sun, Bao, & Guo, 2006; Zhu & Jiang, 2011; Ren & Zhu, 2016). 

In this study, the fluorescence intensity of SAX could be quenched by 2,4-D, the fluorescence 

quenching value (∆F=FSAX-F2,4-D-SAX) were enhanced in β-CD/SDS due to the synergistic sensitization, 

which has a much better quenching effect than that in single β-CD or SDS medium. There was a linear 

relationship between fluorescence quenching value (∆F) and concentration of 2,4-D, a novel β-CD/SDS 

synergistic sensitized fluorescence quenching method for the determination of 2,4-D was successfully 

developed. The mechanism of determination was also investigated. The proposed method was applied 

to analyte 2,4-D in real samples with satisfactory results. 

 

2. Experimental Reagents and Instruments 

Schiff base calix[4]arene was synthesized according to the published methods (Bi, Sun, & Yan, 2012). 

0.01% SAX (M=1328.7 g/mol, c = 7.5×10-6 mol/L) was prepared in ethanol. 

100.0 µg/mL stock solution of 2,4-dichlorophenoxyacetic acid (2,4-D) was prepared by dissolving 

0.100g 2,4-D in 100 mL volumetric flask and diluting with anhydrous ethanol to scale. The stock 

solutions were further diluted with anhydrous ethanol to obtain a standard working solution of 10.0 

µg/mL for experiment. 

1.0% β-CD solution was prepared by dissolving 1.00g of β-CD in 100.0 mL with distilled water. 1.0% 

SDS solution was prepared by dissolving 1.00g of SDS in 100.0 mL volumetric flask with distilled 

water. And pH=7.0 CH3COONH4 buffer solution was employed. 

All the fluorescence measurements were performed on a Hitachi F-4500 spectrofluorimeter (Japan) 

with excitation and emission slits at 10.0 nm and 5.0 nm, λex=332 nm. The pH was measured on a pH 

FE20 pH meter (Mettler Toledo). A UV 2501 spectrophotometer (Shimadzu, Japan) was used for all 

absorption spectral recordings and absorbance measurements . 

 

3. Experiment Method 

Fluorescence measurements. In centrifuge tube (5.0 mL), 2.0 mL 0.01% SAX, 1.0 mL CH3COONH4 

buffer solution (pH = 7.0), 0.5 mL 1.0% β-CD solution, 0.5 mL 1.0% SDS and 0.5 mL reference 

substance solution of 2,4-D (10.0 μg/mL) were added and then diluted to the scale with distilled water. 

Then fluorescence spectra was recorded in the range of 300- 650 nm with excitation at 332 nm. 

Quenching type analysis (Gong, Zhu, & Hu, 2007). 2.0 mL 0.01% SAX solution, 1.0 mL buffer 

solution, 0.5 mL 1.0% β-CD, 0.5 mL 1.0% SDS and different amount of 10.0 μg/mL 2,4-D solutions 

were added into 5.0 mL centrifuge tube, then diluted to the scale with distilled water and mixed 

completely. The fluorescence intensity of SAX was measured at different temperature (288 K, 298 K 

and313 K), respectively. Quenching type could be analyzed by Stern-Volmer Eq.(1):  



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QQ CKKC
F

F
0q

0 11   

F0 and F were the fluorescence intensities of SAX in the absence and presence of 2,4-D respectively, K 

was the Stern–Volmer quenching constant, CQ was the concentration of quencher 2,4-D, Kq was the 

quenching rate constant, τ0 was the average lifetime of the SAX without 2,4-D. 

If the quenching type is single static or dynamic quenching, the curve of F0/F versus CQ (Stern–Volmer 

curve) would be linear within certain concentration. 

Absorption spectrum titrations (Fu, Zeng, & Mu, 2012). The recognition ability of SAX and β-CD to 

2,4-D can be evaluated through the change of absorption spectrum. The absorption spectrum titrations 

of 2,4-D with SAX and β-CD was made in the range of 200.0-600.0 nm. The absorbance of 2,4-D was 

measured with nSAX: n2,4-D and nβ-CD: n2,4-D. 

Inclusion interaction. The solution of a certain amount of SAX, 1.0 mL buffer solution and different 

amount of 10.0 μg/mL 2,4-D solutions were added into 5.0 mL centrifuge tube, then diluted to the mark 

with distilled water and mixed thoroughly. The fluorescence intensity was measured at 25℃, then the 

Benes-Hildebrand method (Vimal, Ajay, & Narinder, 2008) (double reciprocal plot) was used to 

calculate the inclusion constant (K) of SAX-2,4-D and β-CD-2,4-D assuming a 1:1 inclusion model. 

The Benesi-Hildebrand method is a spectroscopic method to determine the inclusion constants of the 

host guest complexes (including fluorescence spectroscopy and absorption spectroscopy). The equation 

is as follows (SAX as an example): 

       00 14,2111 SAXDSAXKF    

where [SAX]0 was the concentration of SAX, ∆F was the quenching value of fluorescence intensity, α 

was a constant. Thus, the inclusion constant (K) of the 1:1 inclusion complex could been calculated by 

dividing the intercept by the slope of the double reciprocal plot. 

Determination of critical micelle concentration (cmc). cmc values of the medium were measured by 

conductivity measurements. The cmc was obtained from the inflection point of the straight lines of 

before and after micellar concentration range (Kumaraguru & Santhakumar, 2006; Mehta, Bhawna, & 

Ram, 2010).  

Determination of fluorescence quantum yield. Fluorescence quantum yields of SAX were measured 

using 1.0×10−6 g/mL quinine sulfate as reference substance (Zhao & Wei, 2006; Zhu, Gong, & Yu, 

2008). Under the same apparatus conditions, the quantum yield of the SAX was calculated. 

Kinetics of the reaction. The kinetics of the reaction could be described by the first order kinetic 

model, the second kinetic model and the Weber Maurice diffusion model (Azizian & Fallah, 2010; 

Wang, Wei, & Li, 2015). In this study, the first order kinetic fitting of the quenching process was 

carried out.  

Sample preparation. Vegetable samples (green vegetables, Chinese cabbage and chrysanthemum 

coronarium) were purchased from local market. A certain amount of each vegetable sample was cut up 



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and homogenized. Then, 20 g of each sample was weighed and placed in a 50 mL centrifuge tube and 

50 mL ethanol was added as well. Then tighten up the lid, dipped for 1 hour, shaken thoroughly for 20 

min. After centrifugation 10 min (5000 r/min), the test sample solution was prepared after the upper 

solution was filtered. 

In order to reduce the fluorescence background, the sample solution was diluted 15 times and then used 

as an analytical sample (Farokhcheh & Alizadeh, 2013). 

 

4. Results and Discussion 

Choice of medium. The effect of different medium on ∆F (∆F = FSAX− F2,4-D–SAX) was studied. As can 

be seen in Figure 2 that the sequence of ∆F was ∆Fβ-CD-SDS>∆FSDS>∆Fβ-CD > ∆FH2O. The fluorescence 

quenching value in β-CD/ SDS synergistic sensitized medium was greater than that in single β-CD or 

SDS medium. So β-CD-SDS medium was selected for further experiment. 

 

1 2 3 4

0

100

200

300

400

500

 

 

F

H
2
O

SDS

-CD

-CD-SDS

 

Figure 2. Effect of Different Medium on Fluorescence Intensity 

 

Fluorescence spectra. The fluorescence emission spectrum of SAX (present or absent of 2,4-D) in 

β-CD-SDS and H2O medium were shown in Figure 3. It can be seen that (1) the fluorescence intensity 

of SAX (FSAX) was enhanced in β-CD/SDS medium (curves a and c); (2) the fluorescence intensity of 

SAX (FSAX or FSAX-β-CD-SDS) was quenched when 2,4-D was added (curves b and d) and gradually 

diminished with the increase concentration of 2,4-D (inset Figure 3); (3) ∆F= FSAX-β-CD-SDS – 

F2,4-D-SAX-β-CD-SDS was larger than that ∆F’=FSAX-F2,4-D-SAX with the same concentration of 2,4-D, which 

was the synergistic sensitizing effect in β-CD/SDS. 

 



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350 400 450 500 550 600 650

0

500

1000

1500

2000

2500

3000

350 400 450 500 550 600 650

0

500

1000

1500

2000

2500

3000

F

nm

1

8

F '

 

 

 

nm

F

a

d

F

 

Figure 3. Fluorescence Spectra 

(a). SAX-β-CD-SDS   (b). 2,4-D-SAX-β-CD-SDS    (c). SAX-H2O   (d). 2,4-D-SAX-H2O 

Inset 1-8: 2,4-D-SAX-β-CD-SDS, [2,4-D]: (1) 0 µg/mL, (2) 0.02µg/mL, (3) 0.08µg/mL, (4) 0.1µg/mL, 

(5)0.2µg/mL, (6) 0.4µg/mL, (7) 0.8µg/mL, (8) 1.0 µg/mL 

 

Effect of pH. The influence of pH on ∆F was investigated. As could be seen in Figure 4, ∆F gradually 

increased with the increase of pH and reached maximum at pH = 7.0, but it diminished at pH > 7.0. The 

reason may be related to the formation of SAX-2,4-D inclusion, which will be discussed in section 

3.13.2. So 1.0 mL of pH = 7.0 CH3COONH4 buffer solution was chosen for the further study. 

 

4 5 6 7 8 9 10

0

20

40

60

80

100

 

 

 

pH

F(a.u)

 

Figure 4. Effect of pH on Fluorescence Quenching Value 

 

Effect of SAX amount. The effect of the amount of SAX was studied in Fig. 5. It was shown that the 

∆F was increased and reached a maximum value at a SAX (7.5×10-6 mol/L) amount of 2.0 mL, and 

then decreased (curve 1). This was because that the FSAX-β-CD-SDS gradually decreased with the increase 

of SAX amount due to the self-quenching of SAX at higher concentration (curve 2). Thus, 2.0 mL SAX 

(7.5×10-6 mol/L) was selected for the optimized method. 

 



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0.0 0.5 1.0 1.5 2.0 2.5 3.0

0

20

40

60

80

100

120

140

160

180

200

 

SAX/mL

F(a.u)

0

500

1000

1500

2000

2500

3000

F

2

1

 

Figure 5. Effect of the Amount of SAX on Fluorescence Quenching Value (Left Axis) and 

Fluorescence Intensity of SAX (Right Axis) 

(1: fluorescence quenching value, 2: fluorescence intensity of SAX) 

 

Effect of β-CD amount. Effect of β-CD was investigated. As is shown in Fig. 6, with the increase of 

β-CD (1.0%) amount, ∆F gradually increased and reached the maximum when the β-CD amount was 

0.50 mL, but decreased when β-CD amount was more than 0.50 mL.  

The change trend of ∆F could be explained from the change of FSAX-β-CD-SDS and F2,4-D-SAX-β-CD-SDS with 

β-CD amount (inset Figure 6). (1) FSAX-β-CD-SDS gradually increased with β-CD amount while 

F2,4-D-SAX-β-CD-SDS slowly decreased (Vβ-CD = 0-0.50 mL) ( ∆F↑= FSAX-β-CD-SDS↑ -F2,4-D-SAX-β-CD-SDS↓); (2) 

FSAX-β-CD-SDS gradually decreased, while FCR-SAX-M-β-CD-Tx-100 remain unchanged ( Vβ-CD = 0.50-0.80 mL) 

(∆F↓= FSAX-β-CD-SDS↓ -F2,4-D-SAX-β-CD-SDS). Therefore, 0.50 mL 1.0% β-CD was chosen for the following 

experiments. 

 

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0

20

40

60

80

100

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

500

1000

1500

2000

2500

3000

3500

2,4-D-SAX--CD-SDS

SAX--CD-SDS

-CD/mL

F

 

 

F(a.u)

-CD/mL  

Figure 6. Effect of the Amount of β-CD on Fluorescence Quenching Value 

 

Effect of SDS amount. Effect of SDS was studied. As is shown in Figure 7, ∆F gradually rose up with 

the increase of SDS (1.0 %) amount and up to the maximum when the SDS amount was 0.50 mL, but 

∆F decreased when SDS amount was greater than 0.50 mL.  



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The change of FSAX-β-CD-SDS and F2,4-D-SAX-β-CD-SDS with SDS amount (inset Figure 7) could declare the 

change of ∆F with SDS. (1) FSAX-β-CD-SDS obviously increased with SDS amount was larger than 

F2,4-D-SAX-β-CD-SDS ( VSDS = 0-0.30 mL)( ∆F↑= FSAX-β-CD-SDS↑↑ - F2,4-D-SAX-β-CD-SDS↑); (2) FSAX-β-CD-SDS 

continuously increased, while F2,4-D-SAX-β-CD-SDS gradually decreased ( VSDS= 0.30–0.50 mL) (∆F↑= 

FSAX-β-CD-SDS↑-F2,4-D-SAX-β-CD-SDS↓); (3) FSAX-β-CD-SDS gradually decreased, while F2,4-D-SAX-β-CD-SDS changed 

rarely (VSDS= 0.50–0.80 mL) (∆F↓= FSAX-β-CD-SDS↓ -F2,4-D-SAX-β-CD-SDS). Therefore, 0.50 mL 1.0 % SDS 

was chosen for the further study.  

In conclusion, the ∆F was biggest when mass ratio or molar ratio of β-CD and SDS are 1:1 and 1:4, 

respectively, which illustrated that β-CD and SDS showed the synergistic sensitized effect on ∆F. 

Either excess β-CD or SDS will weaken the synergistic sensitized effect, resulting the decrease of ∆F.  

 

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0

20

40

60

80

100

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

500

1000

1500

2000

2500

3000

3500

2,4-D-SAX--CD-SDS

SAX--CD-SDS

SDS/mL

F

SDS/mL

F(a.u)  

 

 

Figure 7. Effect of the Amount of SDS on Fluorescence Quenching Value 

 

Effect of reaction time. The effect of reaction time on ∆F was tested. It was found from Figure 8 that 

∆F obviously increased from 10 min to 30 min, then ∆F remained unchanged after 30 min. Therefore, 

30 min of reaction time was chosen for the following experiments. 

According to the data ∆F-t, the fluorescence quenching reaction kinetics could be discussed. In this 

study, the first order kinetic fitting of the quenching process was carried out. As shown in inset Fig.8, 

the curve of lnC2,4-D versus t was linear and R2 was 0.9960. The reaction rate constant was 1.14×10-1 

min-1. The results show that the fluorescence quenching process followed the first order kinetic model. 

The reason may be that the amount of SAX is excess in this system, so the rate of fluorescence 

quenching reaction is only in relation to the concentration of 2,4-D. 

 



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0 20 40 60 80 100

0

20

40

60

80

100

5 10 15 20 25

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

 

 

t/min

ln(CA)

F(a.u)  

 

t/min

First order kinetics model 

 

Figure 8. Effect of Reaction Time on Fluorescence Quenching Value 

 

Effect of temperature. The effect of temperature (5-50℃) on ∆F was investigated. As can been seen 

from Figure 9 that ∆F was increased from 5 to 25℃ then it decreased with the increase of temperature. 

Thus, the suitable temperature of 25℃ was chosen for the study. 

 

0 10 20 30 40 50

0

20

40

60

80

100

 

 

T/℃

F(a.u)

 

Figure 9. Effect of Temperature on Fluorescence Quenching Value 

 

Effect of foreign substances. The effect of foreign substrates was discussed for the determination of 

1.0 μg/mL 2,4-D. With a relative error of less than ±5%, the majority of these substances showed no 

remarkable interference in the determination of 2,4-D (Table 1). 

 

Table 1. Effect of Interfering Substances 

Foreign substances 
Foreign substances / 

2,4-D(w/w) 
Foreign substances 

Foreign substances / 

2,4-D(w/w) 

K+ 500 Al3+ 20 

Na+ 1200 Mn2+ 2 

Ca2+ 1000 Cl- 1800 



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Mg2+ 300 NO3
- 700 

Ba2+ 100 SO4
2- 300 

Zn2+ 200 Glucose 500 

Fe3+ 2 Sucrose 500 

Cu2+ 2 Glycine 200 

 

Analytical performance. Under the optimum conditions, the linear regression equations were 

determined to be: ΔF = 10.14 + 2672.1c (μg/mL) in the range of 0.020~0.10 μg/mL, R = 0.9972, the 

detection limit estimated (S/N = 3) was 0.23 ng/mL; ΔF = 331.08 + 290.87c (μg/mL) in the range of 

0.10μg/mL ~ 4.0 μg/mL, R = 0.9918, the detection limit estimated was 2.8 ng/mL. The relative standard 

deviation (RSD) was 0.96% (n = 3, c = 1.0μg/mL). 

Sample analysis. The proposed method was successfully applied for the determination the amount of 

2,4-D in vegetable samples. The data were listed in Table 2. The recovery ratio ranged from 95.0% ~ 

105.5% which was satisfactory.  

 

Table 2. Determination Results of 2,4-D 

Samples Added (μg/g) Found（μg/g） Recovery (%) 

Chinese cabbage 

0.0 ND - 

0.50 0.53 105.5 

1.0 0.95 95.5 

2.0 1.9 98.2 

green vegetables 

0.0 ND - 

0.50 0.48 95.0 

1.0 2.0 95.7 

2.0 2.1 102.0 

chrysanthemum 

coronarium 

0.0 ND - 

0.50 0.52 104.9 

1.0 0.96 96.5 

2.0 2.1 103.4 

 

Comparison of different methods. The results obtained from this experiment were compared with 

those previously reported methods for 2,4-D determination (Table 3). The advantages of the proposed 

method are: easy operation, high sensitivity, low detection limit and high recovery rate.  

 

 

 



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Table 3. Comparison with Previously Reported Methods 

Methods Linearity range LOD Recovery Reference 

HPLC-MS 0.025-1.0 mg/L 0.005 mg/kg 88.3-95.4% (Jiang, Zha, & Tie, 2015) 

SHPLC 1.0-500 ng/mL 0.3 ng/mL 105-116 % (Wu, Ee, & Lee, 2005) 

CE 3.0-500 ng/mL 0.02 ng/mL - (Zhu & Lee, 2001) 

GC 10-500 ng/mL 5.0 ng/mL - (Rezazadeh, Yamini, Seidi, 

Tahmasebi, & Rezaei, 2014) 

Fluorescence quenching 

calix[4]arene methods 
0.020-4.0 µg/mL 2.8 ng/mL 95.0-105.5% This method 

 

5. Discussion of Mechanism  

In this paper, the discussion of mechanism was included quenching type analysis, inclusion interaction 

and sensitizing effect.  

Quenching type. Quenching types can be divided into static quenching and dynamic quenching. The 

static quenching is caused by the formation of non or weak fluorescent compound (Gong, Zhu, & Hu, 

2007). The dynamic quenching is initiated from the collision of fluorescence substance and quencher, 

resulting in the decrease of fluorescence intensity and quantum yield.  

Quenching type could be discussed with Ksv and Kq. With the increasing temperature, Ksv would be 

decreased for static quenching, while Ksv would be increased for dynamic quenching (Gong, Zhu, & Hu, 

2007). The Stern-Volmer plots of SAX with 2,4-D at different temperature (288 K, 298 K and 313 K) 

was shown in Figure 10. The order of Ksv at different temperature were Ksv 
288K =3.90×104 > Ksv 

298K 

=3.45×104 > Ksv 
313K=3.04×104 L/mol. It demonstrated that the fluorescence quenching mechanism of 

SAX by 2,4-D was a static quenching procedure and a complex was formed between SAX and 2,4-D.  

 

0.0 0.5 1.0 1.5 2.0

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

1.40

 

 

C
2,4-D

(g/mL)

F0/F

a

b

c

 

Figure 10. The Stern-Volmer Curves for the Binding of 2,4-D with SAX at 288 K (a), 298 K (b) 

and 313 K (c) 

 



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Inclusion interaction. From the previous discussion, the type of fluorescence quenching was static 

quenching, which may be caused by the formation of non or weak fluorescent compound. SAX and 

β-CD have a cavity structure, both of them are likely to form inclusion interaction with 2,4-D. 

The recognition ability of SAX and β-CD to 2,4-D can be evaluated through the change of absorption 

spectrum. The results of an absorption titration at λ = 281.0 nm (characteristic absorption peak of 2,4-D) 

(Figure 11) was shown that the absorbance of 2,4-D was gradually increased with the increase of SAX 

or β-CD and was unchanged when nSAX:n2,4-D = 1:1 or nβ-CD:n2,4-D = 1:1, which implied a 1:1 

stoichiometry for binding between SAX and 2,4-D or β-CD and 2,4-D. 

 

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

0.04

0.05

0.06

0.07

0.08

0.09

0.10

0.11

0.12

 

 

 

n/n2,4-D

A

1

2

 

Figure 11. Absorption Spectrum Titrations of 2,4-D with SAX and β-CD 

(1: 2,4-D-SAX, 2: 2,4-D-β-CD) 

 

The inclusion constant (K) of SAX-2,4-D and β-CD-2,4-D could be obtained by Benesi-Hildebrand 

method (Vimal, Ajay, & Narinder, 2008). The larger the value of K, the more steady the inclusion 

complex. As is shown in Figure 12, double reciprocal plots of SAX-2,4-D and β-CD-2,4-D have good 

linear relationship, which supports the formation of a 1:1 complex. The calculated inclusion constant 

(K) was listed in Table 4. 

As can be seen in Table 4: (1)K SAX-2,4-D >>Kβ-CD-2,4-D, which suggested that inclusion complex of 

SAX-2,4-D is more stable than β-CD-2,4-D. SAX plays a major role in inclusion interaction with 2,4-D, 

which bringing about static fluorescence quenching. (2) K SAX-2,4-D > K SAX-2,4-D-β-CD , the inclusion 

constant of SAX-2,4-D was decreased in presence of β-CD, which is caused by the formation of 

β-CD-2,4-D complex; (3) K SAX-2,4-D, pH 7.0 > K SAX-2,4-D, pH 4.5 > K SAX-2,4-D, pH 10.0, which indicated that pH 

had a significant effect on the formation SAX-2,4-D and the inclusion complex of SAX-2,4-D is more 

stable at pH 7.0 than in acidic or basic solution; (4) the larger the K, the greater the ∆F, the largest 

fluorescence quenching value ∆F was at pH=7.0, which is in accordance with the discussion about 

effect of pH on ∆F in section 3.3. 

 



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0 200000 400000 600000 800000 1000000 1200000

0.000

0.002

0.004

0.006

0.008

0.010

0.012

 

 

1/c

1/F

a

0 200000 400000 600000 800000 1000000 1200000

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

 

 

 1/F

1/c

b

 

0 1000 2000 3000 4000 5000

0

200

400

600

800

1000

 

 

 

1/C

A

c

 

Figure 12. Benesi-Hildebrand Plot 

(a: SAX-2,4-D, b: SAX-2,4-D-β-CD, c: β-CD-2,4-D) 

 

Table 4. Results of Inclusion Constant  

System 
Inclusion constant K（L/mol） 

pH 4.5 pH 7.0 pH 10.0 

SAX-2,4-D 5.71×104 5.55×1010 2.00×104 

SAX-2,4-D-β-CD  2.21×109  

β-CD-2,4-D  262.47  

 

Sensitizing effect. The discussion of sensitizing effect was included the formation of the ordered 

molecular assembly and fluorescence quantum yield. 

The formation of the ordered molecular assembly 

The synergistic sensitized effect of β-CD/SDS may be from the formation of the ordered molecular 

assembly. The interaction of SDS and β-CD can change the critical micelle concentration (cmc). By 

measuring the cmc of a single and mixed medium, it can be determined whether the β-CD/SDS has 

formed a new ordered molecular assembly (Zhu & Jiang, 2011). The value of cmc can be obtained from 

the inflection point of the straight lines of before and after micellar concentration range (Kumaraguru & 



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Santhakumar, 2006). The results were summarized in Figure 13 and Table 5. 

As is shown in Figure 13, the cmc of β-CD and SDS/β-CD were 3.0-5.0 mmoL/L and 5.0-6.0 mmoL/L, 

respectively. The cmc of the mixed medium model could be calculate by the clint model based on the 

assumption of ideal mixture behavior. As is shown in Table 5, the cmc of SDS is 8.4 mmoL/L (Mehta, 

Bhawna, & Ram, 2010), the molar ratio of SDS and β-CD was 4:1. Thus, the cmc of the mixed solution 

should between 7.8-8.1 mmoL/L. There was a difference between the cmc of the mixed medium 

(β-CD/SDS) in determined value and the clint model calculated value. This result illustrated that the 

β-CD/SDS mixed medium formed a new ordered molecular assembly. What’s more, the sensitivity of 

the determination could be enhanced by the new ordered molecular assembly. 

 

0 2 4 6 8 10 12 14 16 18

0

200

400

600

800

1000

 

 

concentration mmoL.L-1

th
e 

el
ec

tr
ic

 c
o

n
d

u
ci

ti
vi

ty
 (


s.
cm

-1
)

a

b

 

Figure 13. Electrical Conductivity vs. the Concentration of β-CD 

(a: Electrical conductivity vs. the concentration of β-CD, b: Electrical conductivity vs. the 

concentration of SDS/β-CD) 

 

Table 5. cmc of the Different Medium 

Medium 
cmc (mmoL/L) 

Determination value Clint model value 

SDS 8.4a  

β-CD 3.0-5.0  

SDS/β-CD (4:1) 5.0-6.0 7.8-8.1b 

a: the literature (Mehta, Bhawna, & Ram, 2010); b: calculate by Clint model 

 

Fluorescence quantum yield. The fluorescence quantum yield represents the ability of translation of 

absorption energy to fluorescence. It is one of the basic and significant parameters for fluorescence 

substances. What’s more, ∆F = FSAX – F2,4-D-SAX, where ∆F must rise up with the increase of 

fluorescence quantum yield of SAX. The fluorescence quantum yield of SAX (Y) in the medium of 



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H2O，β-CD，SDS and β-CD-SDS were listed in Table 6. As can be seen in Table 6, the order of 

fluorescence quantum yield of SAX in different medium is Yβ-CD-SDS > YSDS > Yβ-CD > YH2O. It is in 

accord with the sensitivities in different medium. The results could be considered with internal and 

external environment. The fluorescence quantum yield of SAX in β-CD-SDS medium was increased 

obviously. It was because that the external environment provided by β-CD/SDS mixed medium is more 

favorable on solubilization than that in single medium (β-CD or SDS). Moreover, the 

microenvironment which was formed by the new ordered molecular assembly β-CD/SDS could provide 

the protective environment for the singlet excited state and could reduce the non-radiation of the 

fluorescent substance SAX. In other words, both self fluorescence quenching of SAX and fluorescence 

quenching of external quenchers can be decreased in the medium of β-CD/SDS. As a result, the 

fluorescence quantum yield of SAX in the mixed medium is largest due to the synergistic sensitizing 

effect of β-CD-SDS. 

 

Table 6. The Fluorescence Quantum Yield of 2,4-D 

Medium  Y 

H2O 0.011 

β-CD 0.038 

SDS 0.094 

β-CD-SDS 0.108 

 

In summary, the mechanism of this method was (1) the fluorescence quenching of SAX is due to the 

inclusion interaction of SAX-2,4-D; (2)the sensitizing effect is that SDS/β-CD medium could form a 

new ordered molecular assembly, in which fluorescence quantum yield of SAX increased obviously, 

resulting synergistic sensitizing effect on fluorescence quenching value. 

 

6. Conclusion 

In this study, a novel fluorescence quenching method for the determination of 2,4-D has been 

developed. The fluorescence intensity of SAX was quenched due to inclusion interaction between SAX 

and 2,4-D, and the fluorescence quenching value (∆F) was increased in β-CD-SDS medium. The 

proposed method has been applied for the determination of 2,4-D in vegetable samples with 

satisfactory results. 

 

Funding 

The National Natural Science Foundation of China (21375117) and the Priority Academic Program 

Development of Jiangsu Higher Education Institutions are acknowledged for funding this research. 

 



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References 

Atta, S., Bera, M., & Chattopadhyay, T. (2015). Nano-pesticide formulation based on fluorescent organic 

photoresponsive nanoparticles: For controlled release of 2,4-D and real time monitoring of 

morphological changes induced by 2,4-D in plant systems. RSC Adv., 5, 86990-86995. 

https://doi.org/10.1039/C5RA17121K 

Azizian, S., & Fallah, R. N. (2010). A new empirical rate equation for adsorption kinetics at 

solid/solution interface. Appl. Surf. Sci., 256, 5153-5156. 

https://doi.org/10.1016/j.apsusc.2009.12.080 

Berton, P., & Martinis, E. M. (2009). Room temperature ionic liquid based micro extraction for 

vanadium species separation and determination in water samples by electrothermal atomic 

absorption spectrometry. Anal. Chim. Acta., 640, 40-46. https://doi.org/10.1016/j.aca.2009.03.028 

Bi, X., Sun, J., & Yan, C. G. (2012). Efficient construction and structure determination of novel 

macrocycles with calixarene bishydrazones. Chin. J. Chem., 30(7), 1539-1543. 

https://doi.org/10.1002/cjoc.201200072 

Boroduleva, A.Y., & Eremin, S. A. (2016). Determination of 2,4-dichlorophenoxyacetic acid in cereals 

by fluorescence polarization immunoassay. J. Anal. Chem., 71, 949-955. 

https://doi.org/10.1134/S1061934816090045 

Farokhcheh, A., & Alizadeh, N. (2013). Determination of diphenylamine residue in fruit samples using 

spectrofluorimetry and multivariate analysis. LWT - Food Sci. Technol., 54(1), 6-12. 

https://doi.org/10.1016/j.lwt.2013.05.032 

Fu, Y., Zeng, X., & Mu, L. (2012). Use of a new thiacalix[4]arene derivative bearing two 

4-chloro-7-nitrobenzofurazan groups as a colorimetric and fluorescent chemosensor for Ag+ and 

AcO-. Sens. Actuators B., 164, 69-75. https://doi.org/10.1016/j.snb.2012.01.071 

Garabrant, D. H., & Philbert, M. A. (2002). Review of 2,4-docholorophenoxyacetic acid (2,4-D) 

epidemiology and toxicology. Crit. Rev. Toxicol., 32, 233-257. 

https://doi.org/10.1080/20024091064237 

Gong, A. Q., Zhu, X. S., & Hu, Y. Y. (2007). A fluorescence spectroscopic study of the interaction 

between epristeride and bovin serum albumine and its analytical application. Talanta, 73(4), 

668-673. https://doi.org/10.1016/j.talanta.2007.04.041 

Jiang, Z. H., Zha, W. L., & Tie, Y. J. (2015). Determination of 4-Chlorophenoxyacetic Acid and 

2,4-Dichlorophenoxyacetic Acid in Strawberry by Liquid Chromatography- Tandem Mass 

Spectrometry. J. Chin. Inst. Food Sci. Technol., 15(11), 192-198. 

Khan, B., Shah, M. R., & Ahmed, D. (2016). Synthesis, characterization and Cu2+ triggered selective 

fluorescence quenching of bis-calix[4]arene tetra-triazole macrocycle. J. Hazard. Mater., 309, 

97-102. https://doi.org/10.1016/j.jhazmat.2016.01.074 

Kumaraguru, N., & Santhakumar, K. (2006). Synthesis, characterization and micellization behaviour of 

some surface active mixed-ligand complexes of cobalt(III). Polyhedron, 25, 3253-3260. 



www.scholink.org/ojs/index.php/fsns                Food Science and Nutrition Studies                     Vol. 3, No. 4, 2019 

158 
Published by SCHOLINK INC. 

https://doi.org/10.1016/j.poly.2006.05.038 

Li, X. Y., Li, M., & Chen, Q. F. (2011). Determination of rhodamine B in red wine by solid phase 

extraction-high performance liquid chromatography. Food Sci., 32(8), 238-243. 

Liu, F., & Zhao, G. P. (2008). Ionic liquids as a new spectrophotometric determination of aluminum 

sensitizing. Chem. Res. Appl., 20, 611-616.  

Ma, L. N., & Zhu, X. S. (2012). Determination of emodin by hexadecyl trimethyl ammonium bromide 

sensitized fluorescence quenching method of the derivatives of calix[4]arene. Spectrochim. Acta A., 

95(5), 246-251. https://doi.org/10.1016/j.saa.2012.04.084 

Márquez, J. C., Hernández, M., & García, S. F. (1990). Enhanced spectrofluorimetric determination of 

the pesticide warfarin by means of the inclusion complex with β-cyclodextrin. Analyst, 115(7), 

1003-1005. https://doi.org/10.1039/AN9901501003 

Martinis, E. M., & Olsina, R. A. (2008). Sensitive determination of cadmium in water samples by room 

temperature ionic liquid based preconcentration and electrothermal atomic absorption 

spectrometry. Anal. Chim. Acta., 628, 41-50. https://doi.org/10.1016/j.aca.2008.09.001 

Mehta, S., Bhawna, K., & Ram, G. (2010). Behavior of papain in mixed micelles of anionic-cationic 

surfactants having similar tails and dissimilar head groups. J.Colloid Interf. Sci., 344, 105-111. 

https://doi.org/10.1016/j.jcis.2009.12.036 

Ren, Q. Y., & Zhu, X. S. (2016). Methyl-β-cyclodextrin /cetyltrimethyl ammonium bromide synergistic 

sensitized fluorescence method for the determination of levofloxacin. J. Fluoresc., 26, 671-677. 

https://doi.org/10.1007/s10895-015-1753-4 

Rezazadeh, M., Yamini, Y., Seidi, S., Tahmasebi, E., & Rezaei, F. (2014). Electromembrane surrounded 

solid phase microextraction followed by injection port derivatization and gas 

chromatography-flame ionization detector analysis for determination of acidic herbicides in plant 

tissue. J. Agric. Food Chem., 62, 3134-3142. https://doi.org/10.1021/jf500017r 

Sánchez, F. G., Lopez, M. H., & Gómez, J. C. M. (1987). Fluorimetric determination of scandium using 

the cyclodextrin-1,4-dihydroxyanthraquinone inclusion complex. Analyst, 112(7), 1037-1040. 

https://doi.org/10.1039/AN9871201037 

Sanchez, F. G., Rubio, A. L. R., & Blanco, C. C. (1988). Enhanced fluorimetric determination of 

procaine in pharmaceutical preparations by aqueous β-cyclodextrin inclusion and non-aqueous 

competitive action. Anal. Chim. Acta., 205, 139-144. 

https://doi.org/10.1016/S0003-2670(00)82323-5 

Sun, J., Zhu, X. S., & Wu, M. (2007). Hydroxypropyl-β-cyclodextrin enhanced determination for the 

vitamin B12 by fluorescence quenching method. J. Fluoresc., 17(3), 265-270. 

https://doi.org/10.1007/s10895-007-0168-2 

Vimal, K. B., Ajay, P. S. P., & Narinder, S. (2008). Synthesis of new tripodal receptors-a ‘PET’ based 

“off-on” recognition of Ag+. Tetrahedron, 64(22), 5384-5391. 

https://doi.org/10.1016/j.tet.2008.03.013 



www.scholink.org/ojs/index.php/fsns                Food Science and Nutrition Studies                     Vol. 3, No. 4, 2019 

159 
Published by SCHOLINK INC. 

Wang, J. J., Wei, J., & Li, J. (2015). Rice straw modified by click reaction for selective extraction of 

noble metal ions. Bioresource Technol., 177, 182-186. 

https://doi.org/10.1016/j.biortech.2014.11.092 

Wang, W. J., Zhu, X. S., & Yan, C. G. (2013). Determination of safranine T in food samples by CTAB 

sensitised fluorescence quenching method of the derivatives of calix[4]arene. Food Chem., 141(3), 

2207-2212. https://doi.org/10.1016/j.foodchem.2013.05.036 

Wang, X. Y., Yua, J. L., & Wu, X. Q. (2016). A molecular imprinting-based turn-on ratiometric 

fluorescence sensor for highly selective and sensitive detection of 2,4-dichlorophenoxyacetic acid 

(2,4-D). Biosens. Bioelectron., 81, 438-442. https://doi.org/10.1016/j.bios.2016.03.031 

Wu, J. M., Ee, K. H., & Lee, H. K. (2005). Automated dynamic liquid-liquid microextraction followed 

by high-performance liquid chromatography-ultraviolet detection for the determination of phenoxy 

acid herbicides in environmental waters. J. Chromatogr. A., 1082, 121-127. 

https://doi.org/10.1016/j.chroma.2005.05.077 

Yang, Q., Qin, X. X., Yan, C. G., & Zhu, X. S. (2015). A novel fluorescent chemosensor for safranine T 

based on calixarene-1,3-diacyl hydrazine. Sens. Actuators B., 212, 183-189. 

https://doi.org/10.1016/j.snb.2015.02.020 

Yang, Q., Yan, C. G., & Zhu, X. S. (2014). A fluorescent chemosensor for paeonol based on tetramethoxy 

resorcinarene tetraoxyacetic acid. Sens. Actuators B., 191(2), 53-59. 

https://doi.org/10.1016/j.snb.2013.09.044 

Zhang, X. G., Liu, J. X., & Fan, Z. J. (2009). Advances of research on cyclodextrin and its derivatives 

applied in pesticides. Chin. J. Pesticide Sci., 11(3), 291-297. 

https://doi.org/10.1016/j.carres.2008.11.015 

Zhao, J., & Wei, Y. J. (2006). Fluorescence spectra and fluorescence quantum yield of Triton X- 00. 

Spectrosc. Spect. Anal., 26(8), 1523-1530.  

Zhu, L. Y., & Lee, H. K. (2001). Field-amplified sample injection combined with water removal by 

electroosmotic flow pump in acidic buffer for analysis of phenoxy acid herbicides by capillary 

electrophoresis. Anal. Chem., 73, 3065-3072. https://doi.org/10.1021/ac001313f 

Zhu, X. S., & Jiang, R. R. (2011). Determination of iron (III) by room temperature ionic 

liquids/surfactant sensitized fluorescence quenching method. J. Fluoresc., 21(1), 385-391. 

https://doi.org/10.1007/s10895-010-0727-9 

Zhu, X. S., Gong, A. Q., & Yu, S. H. (2008). Fluorescence probe enhanced spectrorimetric method for 

the determination of gatifloxacin in pharmaceutical formulations and biological fluids. Spectrochim. 

Acta A., 69, 478-482. https://doi.org/10.1016/j.saa.2007.04.026 

Zhu, X. S., Sun, J., Bao, L., & Guo, R. (2006). Effect of β-cyclodextrin-microemulsion on 

determination of trace Bi(Ⅲ) by spectrofluormietry. Chin. J. Appl. Chem., 23, 323-328.  

 


