Glyphosate (GFT) is a widely used herbicide, considered toxic and a probable carcinogen. The main challenge is its detection, usually requiring expensive and laborious methodologies. Herein, we report a colorimetric detection of GFT, using a derivatization reaction with 2,4-dinitrofluorobenzene (DNFB) that leads to a yellow-colored product. This is undertaken under mild conditions (weakly basic aqueous medium and ambient conditions). A thorough kinetic study was carried out, showing that the derivatization reaction with GFT predominates over the hydrolysis of DNFB. Hence, the colorimetric product is the major product formed, which was fully characterized by nuclear magnetic resonance. Finally, a portable, handmade and cheap colorimeter was used to detect and quantify GFT, relying on the colorimetric reaction proposed. Simulating real contaminated samples, it was possible to analyze in just 10 min, with less than 7 % of error of the nominal concentration. Overall, a highly sustainable approach is shown for an herbicide monitoring, with a simple and mild derivatization reaction that does not require purification and leads to a colorimetric product. Moreover, a simple apparatus with low time analysis is proposed that uses a problematic electronic trash: cellphone chargers. This cheapens the process and allows field analysis that can be extended to other agrochemicals.
Organophosphates englobe the class of highly toxic compounds present in many agrochemicals and threatening chemical weapons . Concerns with chemical security have raised alerts towards safe handling of these compounds . Specifically in the case of agrochemicals, which continues to be the most effective cultivation method for guaranteeing food worldwide, a lot of attention is directed towards their effective neutralization (i.e. destruction of stockpiles)  and fast field and food monitoring. Some examples of phosphorus based agrochemicals are shown in Fig. 1, primarily known for their stable nature, hence do not reactive easily . Chemical approaches can be ideal for strategically designing reactions that can fulfill both optimal neutralization, by safe processes that lead to less toxic products, as well as in the detection of these substances by various responses (e.g. colorimetric, electrochemical) originated from targeted reactions.
Glyphosate (GFT), N-(phosphonomethyl) glycine, is a phosphonate belonging to the class of organophosphates  first synthetized in 1950 . GFT is one of the main herbicides currently used worldwide for agricultural purposes . From 1995 to 2014 its overall use increased over 1000%  and the global demand for 2020 is estimated as 1000 kt . GFT do not react easily, belonging to toxicological class IV according to the US Environmental Protection Agency (EPA) . However, since 2015, the herbicide has been considered a probable carcinogen by the International Agency for Research on Cancer, with an increase in liver and kidney tumors. In addition, epidemiological studies suggest a direct relationship with non-Hodgkin’s lymphoma and the capacity to cause stress .
Concomitant to these health issues (human and animal), the abusive use of GFT is concerning that requires effective monitoring  methods. There are studies showing that agrochemicals are among the leading causes of human poisoning, with organophosphates being one of the most evident classes , . In addition, use of GFT is a concern environmental contamination. GFT monitoring is challenging and not straightforward, despite its relatively simple chemical structure. The usual methods of detection are laborious and longstanding. These facts lead to flaws in government control measures. Due to this, an important concept known as “glyphosate paradox” arises: in spite of that herbicide being one of the most used in the world, there are no techniques that allow its systematic analysis. Thus, until this day, the real effects of GFT in the economy or health system are uncertain .
Currently, most of the techniques used to detect and quantify GFT are based on chromatographic methods. However, these techniques are relatively expensive, it needs laboratorial infrastructure and laborious methods, although can detect very low concentrations, which is highly desirable. The techniques found in the literature can be classified in techniques that use derivatization reactions and methods without derivatization . Derivatization reactions are interesting and among the most reported methods, because they can alter the structure of the GFT forming a more volatile, stable or with different molar absorptivity, allowing its spectroscopic or chromatographic analysis . Some of the most used derivatizing reagents are Isobutyl chloroformate (IBC), isopropyl chloroformate (IPC), 4-methoxybenzenesulfonyl fluoride (MBS) and 9-fluorenylmethylchloro-formate (FMC) (Fig. 2).
For the reaction with IBC and IPC, the products can be analyzed by the gas chromatography (GC) technique, being detected by a flame photometer . For the determination with MBS, it is possible to apply liquid chromatography (LC), which is one of the most common techniques for the detection and quantification of GFT . For FMC, the product can be detected using fluorescence by high performance liquid chromatography (HPLC) . Another way of determining GFT is by methods that do not use derivatization reactions. Since GFT is a compound capable of ionizing, ionic chromatography (IC) can be used, which is based on the attraction of the solute ions to those of the stationary phase . In addition, another very applied method is chromatography-mass spectrometry, which, because of its good sensitivity, does not require derivatization reactions . Although this technique detects GFT satisfactorily, it requires a longer time in the analysis. Further, another technique that is greatly encouraged in many analyses is the colorimetric detection, which is usually fast, low-cost and simple. Although in the case of GFT, this is still poorly exploited, with almost no reports.
In a study carried out in 1986 , 2,4-dinitrofluorobenzene (DNFB) was used for the analysis of GFT, also known as Sanger’s reagent (usually used with N-terminal amino acid of polypeptides to sequence proteins). Samples of GFT-containing soil were quantified from this derivatization reaction and the HPLC technique was used. However, a detailed kinetic and mechanistic study of the reaction was not carried out and the chemical structure of the product was not confirmed. Although very promising, it was a preliminary study that couldn’t solidly confirm the derivatization product formed nether the ideal reaction conditions.
Therefore, herein we focused in developing a feasible and fast method of detection and quantification of GFT using a portable and handmade colorimeter. Particularly, we explored and deepen the derivatization reaction of GFT using DNFB, forming the colored product N-(2,4-dinitrophenyl)-N-(phosphomethyl) glycine (GFTD). This reaction is performed under mild conditions requiring no further separation or purification methods. A thorough kinetic, mechanistic and product characterization study was carried out, including use of nuclear magnetic resonance (NMR) spectral analysis. This reaction shows to be highly promising and depending on the reaction conditions such as the concentrations of the reactants, single or parallel reactions occur. Moreover, to the best of our knowledge this is the first report characterizing the product GFTD, derived from this reaction. Further we propose a cheap compact apparatus for detecting and quantifying GFT, relying on this simple colorimetric reaction, which could be used for field analysis. In just 10 min, it is possible to infer on the abusive presence of GFT, with a handmade colorimeter that costs under US$5 (without the multimeter) that uses a common electronic trash: cellphone chargers. By the green and sustainable view, the present study complies in many aspects, from the straightforward colorimetric reaction to the portable analyzer and its construction with simple pieces.
GFT was obtained from a commercial sample (Roundup WG®), containing GFT (720 g kg−1), isopropylamine (72.5 g kg−1) and other inert components (207.5 g kg−1). In a fume hood, 2 g of the commercial sample were mixed with 15 mL of water in a beaker under magnetic stirring. After, 15 mL of sodium hydroxide solution (4 mol L−1) was added dropwise and the solution was heated (80°C) until isopropyl amine vapor ceases. Then, at room temperature, 5.6 mL of hydrochloric acid (37%) was added dropwise under stirring and after 30 min the solution was dried under reduced pressure and a solid was obtained. The solid was boiled in ethanol, filtered, solubilized with minimum boiling water amount, decolorized with active charcoal, filtered again and evaporated until half water volume. The precipitate was filtered, washed with acetone and dried in a high vacuum pump. It was obtained 0.55 g of N-phosphomethylglicine hydrochloride, GFT salt (37% yield). 1H NMR (200 MHz, D2O, TMSP): δ=3.24 (duplet JHP=12.6, 2H) and 3.93 (singlet, 1H). 31P NMR (80 MHz, D2O, H3PO4) δ=8.7 (triplet JPH=12.6) .
The reactions were monitored by UV-vis spectroscopy (Agilent Cary 60) by following the absorbance between 200 and 700 nm, under controlled temperature with an ultra-thermostatic bath (SP Labor SP-152). Two experimental conditions were carried out: (i) Condition 1 (excess of GFT): the reaction started with the addition of an aliquot of 20 μL of a DNFB solution (1.5×10−3 mol L−1 in ethyl acetate) in a 3 mL cuvette (1 cm optical path), giving a final concentration of 1×10−5 mol L−1. The cuvette contained aqueous solution of GFT (5×10−2 mol L−1), buffered with bicarbonate (0.1 mol L−1), at 25 C; (ii) Condition 2 (excess of DNFB): the reaction started with the addition of an aliquot of 30 μL of a GFT solution (1×10−3 mol L−1 in water) and an aliquot 30 μL of a DNFB solution (0.1 mol L−1 in ethyl acetate), in a 300 μL quartz cuvette (0.1 cm optical path), giving a final concentration of 1×10−4 mol L−1 and 1×10−2 mol L−1 of GFT and DNFB, respectively. The media was buffered with bicarbonate (8×10−2 mol L−1) at 25°C. The hydrolysis reaction of DNFB was also followed for comparison purposes. The reaction started with the addition of an aliquot of 20 μL of a DNFB solution (1.5×10−3 mol L−1 in ethyl acetate) in 3 mL quartz cuvette, giving a final concentration of 1×10−5 mol L−1 in a buffered solution (pH 8, bicarbonate 0.1 mol L−1), at 35–55°C range. The alkaline hydrolysis was evaluated similarly by varying the concentration of KOH at 25°C. The kinetic profiles were fitted using iterative least-squares software. In the case of reactions with excess of GFT, reactions followed single reaction pseudo-first order equations given in the literature. In the case of reactions with excess of DNFB, two parallel reactions of pseudo-first order were observed (hydrolysis reaction of DNFB and reaction of GFT with DNFB) which were fitted with eq. 1.
where Ainf(1) is the infinite absorbance for the hydrolysis reaction of DNFB; kH2O is the rate constant for the hydrolysis reaction of DNFB; Ainf(2) is the infinite absorbance for the reaction of DNFB with GFT; kGFT is the rate constant for the reaction of GFT with DNFB; A0 is the absorbance at the beginning of the reaction and t is the time of the reaction.
The equipment used was an NMR Bruker DPX 200 operating with 4.7 tesla, 200 MHz for 1H and 80 MHz for 31P, with 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TMSP) and H3PO4 capillary as reference, respectively. The reactions were performed directly in the NMR tube, where GFT (1 mg) and DNFB (1 mg) were added to 500 μL of a D2O buffer solution (bicarbonate 0.1 mol L−1), pD 9 and consecutive spectra were obtained.
The colorimeter construction was based on a previous report using Styrofoam, which was proposed as an experiment for graduate course in physical chemistry . Herein, we improved this apparatus, as follows and applied for the analysis of the herbicide GFT. In a wood box (8 cm×6 cm×6 cm), two holes (1 mm) with 4 cm of height in both sides (A and B), to connect the LEDs, were made (Fig. 3; Side A and Side B). In the side A, two additional holes (1 mm) were made with height of 3 and 5 cm to connect a (100 Ω) resistor. A styrofoam material was placed inside the box with a hole to place the cuvette. The blue LEDs (430 nm) were situated in front of each other and a resistor (100 Ω) was placed in the 3 and 5 cm height holes. In this case, the objective is that LED 2 illuminates the cuvette and the amount of transmitted light at LED 1 is measured by the potential using a multimeter. Indeed, the wires from LED 1 were welded in a plug to couple to a multimeter. Then a power supply (5V; 1A), for example an old cell phone charger was connected to the colorimeter. It is important to notice the sustainable appeal in the construction of the colorimeter in this sense, since we were able to reuse cell chargers that are widely discarded and could be considered an electronic trash problem. The positive wire from the power supply was welded to the resistor (a), the second wire from resistor was welded in the positive pole of LED 2 (b) and the negative wire from energy supply was welded in the negative pole of LED 2 (5V; 1A), (Fig. 3).
Determination of the molar absorptivity of the GFTD
The molar absorptivity of the product GFTD at pH 9 was determined using the colorimeter under conditions with excess of GFT, since under this condition the only product formed is GFTD. The absorbance was correlated to the potential (Volts) of LED 1, measured using a multimeter. The potential value was converted to absorbance using eq. 2 and the molar absorptivity (εGFTD) was calculated using the Lambert-Beer equation , considering that the concentration of GFTD is equal to the initial concentration of DNFB (totally consumed). This was repeated for different reactions and lead ε values with less than 10% error.
where A is the absorbance, V0 is the potential obtained with 100% transmittance (solvent only) and V is the potential obtained for any given solution.
Detection with handmade colorimeter
The detection and quantification of GFT was carried out using the colorimeter as follows and under conditions with excess of DNFB. The reaction started with the addition of an aliquot of 30 μL of a GFT solution (1×10−3 mol L−1) and 30 μL of a DNFB solution (0.1 mol L−1) to a 300 μL cuvette (0.1 cm of optical path) containing 240 μL of a aqueous buffered solution (bicarbonate 0.1 mol L−1), giving a final concentration of 1×10−4 mol L−1 and 1×10−2 mol L−1 of GFT and DNFB, respectively. The reaction was carried out at 25°C and pH 9. The reaction progress was monitored by measuring the potential (Volts) of LED 1 and converting to absorbance using eq. 2, after 10 min. The concentration of the product GFTD was obtained considering its the molar absorptivity (εGFTD). Finally, it was possible to determine the concentration of GFT in the sample using eq. 3, obtained from combining a first order equation with the Lambert-Beer equation. The concentration of the product (GFTD) at infinity is equal to the concentration of GFT at the beginning of the reaction which is calculated considering the rate constant for this reaction (kGFT) at the given time (10 min). Thus, the kinetic studies are fundamental for obtaining the rate constant. It should be noted that 10 min was the ideal condition for quantifying GFT, but varying values of times could also be used.
where [GFT]0 is the initial concentration of GFT of the analyzed sample, A is the absorbance with 10 min of reaction, kGFT is the rate constant for the reaction of GFT with DNFB (excess of DNFB) and t is the time of reaction, i.e. 10 min, but could be adapted to other times.
Results and discussion
Herein, we explore the derivatization reaction between GFT and DNFB, shown in Fig. 4. Firstly, the equilibria of GFT should be considered, since the totally deprotonated species should be the most reactive in the nucleophilic substitution reaction (pKaH~10) . This species reacts with DNFB via the nitrogen atom towards the aromatic carbon atom, leading to GFTD. This product is highly stable and does not hydrolyze or lead to any other products under the reaction conditions. Fortunately, while the reactants GFT and DNFB are colorless, GFTD presents a yellow color, yielding a simple and fast colorimetric signal for this reaction. In addition, DNFB could also hydrolyze under aqueous medium, leading to 2,4-dinitrophenol (DNP, also yellow but with different maximum wavelength) as a byproduct in these reactions , . Although, we will show that the reaction conditions (concentration ratio) can favor one pathway.
Kinetic studies were first carried for the hydrolysis reaction of DNFB, since it can occur concomitantly with the reaction of GFT. Therefore, it is necessary to evaluate its contribution to the overall reaction rate. The hydrolysis reaction is slow at 25°C, hence solely the rate constant at pH 8 was calculated from a temperature profile (35, 45, 55°C) using the Arrhenius equation (see the Supporting Information, SI). For the alkaline hydrolysis, rate constants were obtained by varying the concentration of hydroxide. Typical UV-Vis spectra are presented in Fig. 5a, showing the characteristic consumption of DNFB around 230 nm and the formation of the phenolic product DNP with maximum wavelengths at 360 and 400 nm . Pseudo-first order kinetic profiles were obtained at 230 and 400 nm, as shown in the inset of Fig. 5a. Figure 5b shows the hydroxide concentration profile for the alkaline hydrolysis of DNFB, which was fitted with eq. 4, that considers that reaction with water (kH2O) and the alkaline hydrolysis (kOH). The slope of the linear plot in Fig. 5b gave the value of kOH=7.05 min−1 L mol−1. Since obtaining kH2O from the linear coefficient is not so reliable, this value was obtained from the value of kobs calculated at pH 8 (25°C) and using eq. 4. A value of kH2O=1.82×10−5 min−1 was obtained for the hydrolysis of DNFB. Hence, the hydrolysis reaction is very slow and would take nearly 4 months to complete (5 half lifetimes) at 25 °°C.
In the following, the reaction of DNFB with GFT (excess of GFT) was studied and its typical kinetic profiles are shown in Fig. 6a. In contrast to the hydrolysis of DNFB, the reaction with GFT occurs readily (reaction time under 1 h) at 25°C, already suggesting that is the predominant reaction occurring. Figure 6a shows the known consumption of DNFB and the formation of a new product at 400 nm (distinct from the DNP profile), that was attributed to the product GFTD. The same profile was obtained in all the pH range ~6–11. The kinetic profile for GFTD formation at 400 nm also obeys a pseudo-order behavior, shown in the inset of Fig. 6a. This reaction forms the product GFTD which presents a yellow colour. Indeed, during the reactions which initially was colorless (only GFT and DNFB), the yellowish color appeared (picture shown in Fig. 6a). This color change enables a simple straightforward colorimetric detection of GFT, which can be done visually, in a short period of time and under mild conditions (weakly basic aqueous medium and room temperature). Even though the hydrolysis reaction of DNFB also leads to a colored product (DNP, yellow), the kinetic studies rule out the significant contribution of this reaction under the reaction conditions evaluated (reaction takes up to 4 months). In addition the spectrum profile for the products GFTD and DNP are different. Figure 6b presents the typical spectrum at the end of the reactions for the hydrolysis of DNFB (forms DNP) and the reaction of GFT with DNFB (forms GFTD). Clearly, it is possible to differentiate the profiles of the two products, reiterating that the hydrolysis reaction of DNFB does not contribute in the colorimetric reaction proposed with GFT.
Figure 7 presents the pH rate profile for the reaction of GFT with DNFB. The results show an increase in the rate constant with higher pH, evidencing that the most reactive species is formed at higher pH (totally deprotonated; pKa3~10; GFT species in Fig. 4). Interestingly, even at pH 6.5–8.5 where there are very low amounts of the GFT species, the reaction is appreciably faster than the hydrolysis of DFNB, indicating that the monoprotonated species (GFT+ pKa2~6 Fig. 4) also reacts with DNFB forming GFTD. Indeed, all spectra profiles are characteristic for GFTD. Hence, eq. 5 was used to fit the pH profile, which was deduced based on the schematic presented in Fig. 8 . It considers the contribution of the hydrolysis (kH2O, kOH, agrees with Fig. 5) reaction and the reaction with the two species: GFT and GFT+, related to the second-order rate constants and respectively. The terms χGFT and χGFT+ refer to their molar fraction (calculated using pKas), while [GFT]t is the total concentration of GFT. Fitting agreed with previous values of kH2O and kOH and (i) pKas of 6.0 and 10.18, consistent with the literature ; (ii) rate constants of and This evidences that the reaction with GFT is even more efficient that the alkaline hydrolysis of DNFB Comparing the reaction of GFT with DNFB with its spontaneous reaction, rate enhancements (comparing second-order constants, ) of 5×107 and 4×104-fold are observed for GFT and GFT+ species, respectively. This means that the proposed reaction is highly effective and fast. Thus, the reaction completion (estimated as 5 half lifetimes at 0.05 M) reaches 4 min for GFT species and 77 h for GFT+. At pH 9, where there is a mixture of the species, the reaction takes 2 h, in contrast to 27 days for the spontaneous hydrolysis of DNFB under the same conditions. Hence, the hydrolysis should not contribute significantly in the reaction of DNFB with GFT, under the reaction conditions, guaranteeing the formation of one product: GFTD.
Henceforth, the kinetic for the reaction of DNFB with GFT is presented herein for the first time, showing that the predominant product formed is the colored GFTD, which is compared to the hydrolysis of DNFB. The results show that the reaction of DNFB with GFT is highly effective, fast and pH-dependent, with two possible reactive species of GFT: totally deprotonated and monoprotonated. The first species is expected for the proposed nucleophilic substitution, while the second is more intriguing. Possibly, the hydrogen of the amine moiety of GFT+ can be abstracted by a general basic catalyst (water or the anionic sites of GFT+), concomitant to the nucleophilic substitution with DNFB. A similar explanation for the biprotonated species of GFT was discarded, since it would not fit the pH rate profile.
NMR spectroscopy analysis
The chemical structure of the product formed (GFTD) from the reaction of DNFB with GFT has already been mentioned in the literature as a possible product formed , but its chemical structure has not been confirmed by any techniques. Previous kinetic studies herein show the UV-Vis characterization but seeking deeper understanding, consecutive 1H NMR spectra were acquired for the reaction of DNFB with GFT. Figure 9 presents the 1H NMR spectra obtained in the aromatic and aliphatic region, where the typical signals for DNFB and GFT were attributed. Additional peaks were attributed to GFTD that will be discussed in the following. Table 1 details all attributions.
|GFT||2.97 (d, 2H), 3.72 (s, 1H)||7.9|
|DNFB||7.70 (dd, 1H, Ar), 8.66 (ddd, 1H, Ar), 9.09 (dd, 1H, Ar)|
|GFTD||3.97 (d, 2H), 4.07 (s, 2H), 7.42 (d, 1H, Ar), 8.21 (dd, 1H, Ar), 8.68 (d, 1H, Ar)||12.4|
Analysis of Fig. 9 shows the consumption of GFT and DNFB with the decay of the characteristic signals of these compounds in the aliphatic and aromatic region, respectively ,  (2.97 and 3.72 ppm for GFT; 7.70, 8.66 and 9.09 ppm for DNFB). In addition, it is possible to observe the appearance of new signals corresponding to the formation of GFTD (3.97, 4.07, 7.42, 8.21 and 8.68 ppm). The multiplicity of the –CH2– groups belonging to GFTD is the same of the –CH2– moiety of GFT. Hence, corresponding to the doublet at 3.72 ppm and a singlet at 4.07 ppm for GFTD. These signals are upshifted relative to the –CH2– of GFT due to the insertion of the aromatic nitro group which is highly electron withdrawing, which is expected. In the aromatic region, the characteristic signals of 2,4-substituted compounds were observed for the GFTD (a signals set with 2 duplets and 1 doublet of doublets). For DNFB, these signals are more complex due to the hydrogen-fluoride coupling . It is important to note the small intensity of the signals referent to the hydrolysis reaction DNFB that lead to the product DNP (6.44, 8.12 and 8.92 ppm) , evidencing the small contribution of this parallel reaction. (See SI).
For 31P NMR analysis, a spectrum was acquired only at the end of the reaction that corroborates with 1H NMR analysis. Figure 10 evidences only two signals due to GFT in 7.9 ppm and a signal upshifted in 12.4 ppm attributed to GFTD. This signal is a triplet with a coupling constant of JPH 10 Hz, which agrees with the coupling constant of the doublet of doublet in the 1H spectra (4.07 ppm) attributed to –CH2– of GFTD (hydrogen-phosphorus coupling). NMR analyzes solidly confirm the structure of the GFTD product, supporting the proposed reaction proposed between DNFB and GFT. Table 1 shows the chemical shifts and multiplicity pattern for each compound signal.
Colorimetric detection of GFT with a handmade colorimeter
Seeking the detection and quantification of GFT using the handmade colorimeter, a kinetic study was carried out with excess of DNFB, that would be the optimal condition for field analysis (GFT<DNFB). UV-Vis spectra obtained for the reaction of DNFB with GFT (excess of DNFB) at pH 9 and 25°C are shown in Fig. 11. The results show the band characteristic of GFTD, overlapped with the band of DNP, indicating that under these conditions the formation of DNP is quite significant in the overall reaction with GFT. The kinetic profile obtained at 400 nm, given in the inset of Fig. 11, shows a distinct behavior from the case with excess of GFT (Fig. 6), indicating two concomitant pseudo-first order reactions which are attributed to the GFTD formation (faster reaction) and also to the hydrolysis product DNP (slower reaction). The data was fitted with eq. 1 for parallel reaction, which enabled obtaining the rate constants for the reaction of GFT with DNFB (kGFT) and its hydrolysis (kH2O). Thus, it was possible to kinetically separate the contribution of the parallel reactions, which differ significantly from each other. Values of kGFT=1.6×10−2 min−1 and kH2O=1.82×10−5 min−1 were obtained, showing than even with two reactions occurring, the reaction with GFT is faster than the hydrolysis by nearly 104-fold. Further the hydrolysis constant obtained agrees with the value obtained in the kinetic studies for the hydrolysis (Fig. 5). Although, the rate constant obtained with GFT (with excess of DNFB) cannot be correlated to the one obtained in the kinetic study for the analogous reaction with excess of GFT (Fig. 7). It is important to notice that the thorough understanding in distinguishing the kinetics of the proposed reaction under different conditions is fundamental in the elucidation of the reaction and also for the next steps regarding the detection of GFT with the portable analyzer. Indeed, we showed previously for the reaction of GFT with DNFB that solely the product GFTD is formed under excess of GFT and the pH-dependence was interpreted. In the case of excess of DNFB, which would be the conditions in real field/food analysis that have low amounts of GFT, two parallel reactions occur, predominating the reaction that leads to GFTD, in contrast to the DNP product. Under excess of DNFB, the pH was also varied (pH 8-9.5, see SI), but the competing reactions are difficult to discriminate since in pH<9, the overall reaction is too slow and for pH>9, the alkaline hydrolysis contributes more significantly. Thereby, the optimal conditions considered for the detection was at with excess of DNFB and pH 9, that will be shown with the handmade colorimeter.
Aiming towards obtaining the handmade colorimeter for the detection and quantification of GFT, firstly the reaction of DNFB with GFT (excess of DNFB) was carried out using the proposed handmade colorimeter, in order to compare with the commercial equipment. Figure 12 shows the kinetic profile obtained using the handmade colorimeter with the blue led (maximum wavelength 430 nm)  and the commercial UV-Vis spectrophotometer. They present very similar behaviors and the fits using eq. 1 give similar values, validating the proposed portable colorimeter, even for complex kinetics, such as parallel reactions. These results prove that the colorimeter can successfully substitute the commercial equipment, and further be applied for the GFT quantification as a simple, compact, portable and cheap apparatus.
Figure 13 shows a photograph of the handmade colorimeter used in the detection of the GFT. It’s simple, portable, cheap and easy to use. Moreover, the power supply is accomplished by recycling a problematic electronic trash: a cellphone charger. Indeed, cellphone chargers are widely discarded and can be sustainably reused in the proposed apparatus, not only cheapening the analyzer but also comprising an environmentally friendly appeal. The detection is done by inserting the cuvette containing the reaction medium and measuring the potential variation with the multimeter (converting to absorbance with eq. 2). For that initially, the molar absorptivity of GFTD (εGFTD) was obtained, as described in the methodology giving a value of 5.4×104 L mol−1cm−1. In the following the reaction between DNFB (1×10−2 mol L−1) and GFT (1×10−4 mol L−1) at pH 9 was carried out for 10 min. For such short time, the contribution of the hydrolysis reaction is very low (less than 1%), hence in the beginning of the reaction the absorbance is only related to GFTD product. Then the absorbance was measured with 10 min of reaction and the concentration of initial GFT in the reaction medium was calculated using eq. 3. It is not necessary that the reaction completes, since the previous kinetic studies allows to confidently obtain the distinct rate constants. Therefore, using kinetic integrated equations (eq. 1), it is possible to determine the concentration of GFT as any given time (eq. 3).
Accordingly, the handmade colorimeter showed to be effective for detecting GFT, since the product GFTD could easily be measured by the reaction proposed. Further, for validating its quantification with eq. 3, we repeated the reaction in triplicate and the results are presented in Table 2. The results show a successful quantification of GFTY with the analyzer proposed with a mean error of 3% of the initial concentration of GFT.
|Initial concentration of GFT=1.00×10−4 mol L−1
|Exp 1||Exp 2||Exp 3|
|GFT determined with the colorimeter, mol L−1 a||1.07×10−4||1.00×10−4||9.75×10−5|
|Relative error, %||7.0||–||2.5|
aUsing eq. 2 and 3.
It should be noted that even the report on a previous version of the colorimeter  had not been used for the analysis of agrochemicals, as proposed herein. The obtained values of concentration of the GFT in the solutions are very close to the nominal value prepared, having a low relative error. These results show that the colorimeter is capable of performing quantitative GFT analyzes under these conditions at short times. We believe that the proposed apparatus can be applied in the field more easily and can systematically monitor the use of GFT in food samples, avoiding its indiscriminate use. Further, the portable analyzer can surely be extended to other agrochemicals and even chemical warfare, which have analogous chemical structures. This does not restrict the detection just using DNFB and can be adapted relying on other reactions that produce colored products that can be detected by different LED detectors.
Overall, a novel approach is shown for a simple and straightforward colorimetric detection of the herbicide GFT, a worldwide concern. The reaction of GFT with DNFB is thoroughly exploited, where the product formed GFTD is colored. GFTD was fully characterized by 1H and 31P NMR, confirming its structure. Kinetic studies were carried out for the reactions of GFT with DNFB under various conditions and the pH-dependence was elucidated indicating that two important species can contribute in the reaction: totally deprotonated (most reactive) and monoprotonated specie. The competing hydrolysis reaction of DNFB was studied and results show that in reaction with excess of GFT, the hydrolysis does not occur significantly but in conditions with excess of DNFB, the hydrolysis does indeed occur to a higher extend. The contributions of both parallel reactions were calculated using equation for concomitant parallel reactions. Finally, a handmade colorimeter was proposed as a cheap, portable and fast analyzer of GFT, triggered by the formation of the colored product GFTD. The reaction of GFT with DNFB was carried using the colorimeter, showing that with only 10 min, it is possible to determine confidently the initial concentration of GFT. Figure 14 presents a summary of the results presented herein. The apparatus is reliable, relying on monitoring a simple reaction that is performed in aqueous medium under mild conditions (room temperature and weakly basic medium). This analyzer can be easily handheld, enabling facile field analysis and also the proposed procedure can be extended to other contaminants (agrochemicals and chemical warfare). In summary, many green and sustainable aspects are found in the proposed approach, such as the simple and mild derivatization reaction that leads to a colorimetric product and does not require purification. The analyzer is cheap and compact, with low time analysis and reuses cellphone chargers, a problematic electronic trash. Finally, chemical security can be accomplished by guaranteeing a safe detection and quantification procedure.
Authors acknowledge the financial support from UFPR, CNPq, CAPES, L’Oréal-UNESCO-ABC, PhosAgro/UNESCO/IUPAC, Fundação Araucária and National Institute of Science and Technology of Carbon Nanomaterials (INCT-Nanocarbon). This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brasil, finance code 001.
A collection of invited papers based on presentations at the 8th IUPAC International Conference on Green Chemistry (ICGC-8), Bangkok, Thailand, 9–14 September 2018.
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