Thermodynamic Study on the Protonation Reactions of Glyphosate in

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Thermodynamic Study on the Protonation Reactions of Glyphosate in Aqueous Solution: Potentiometry, Calorimetry and NMR spectroscopy Bijun Liu, Lan Dong, Qianhong Yu, Xingliang Li, Fengchang Wu, Zhaoyi Tan, and Shunzhong Luo J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b11550 • Publication Date (Web): 10 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016

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The Journal of Physical Chemistry

Thermodynamic Study on the Protonation Reactions of Glyphosate in Aqueous Solution: Potentiometry, Calorimetry and NMR spectroscopy Bijun Liua, Lan Donga, Qianhong Yua, Xingliang Lia,*, Fengchang Wub,*, Zhaoyi Tana, Shunzhong Luoa,*

a, Institute of Nuclear Physics and Chemistry, China Academy of Engineering Physics, Mianyang, Sichuan 621999, China b, State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China

Address correspondence to Xingliang Li, E-mail: [email protected] Fengchang Wu, E-mail: [email protected] Shunzhong Luo, E-mail: [email protected]

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Abstract: Glyphosate [N-(phosphonomethyl)glycine] has been described as the ideal herbicide because of its unique properties. There is some conflicting information concerning the structures and conformations involved in the protonation process of glyphosate. Protonation may influence the chemical and physical properties of glyphosate, modifying its structure and the chemical processes in which it is involved. To better understand the species in solution associated with changes in pH, thermodynamic study (potentiometry, calorimetry and NMR spectroscopy) about the protonation pathway of glyphosate is performed. Experimental results confirmed that the order of successive protonation sites of totally deprotonated glyphosate is phosphonate oxygen, amino nitrogen, and finally carboxylate oxygen. This trend is in agreement with the most recent theoretical work in the literature on the subject (J. Phys. Chem. A 2015, 119, 5241-5249). The result is important because it confirms that the protonated site of glyphosate in pH range 7-8, is not on the amino, but on the phosphonate group, instead. This corrected information can improve the understanding of the glyphosate chemical and biochemical action.

Key Words: Glyphosate; Protonation; Potentiometry; Calorimetry; NMR Spectroscopy

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1 Introduction

Glyphosate [N-(phosphonomethyl)glycine] is a broad spectrum herbicide and has been world widely applied. Glyphosate is a weak acid (H3L, where L3- stands for the fully deprotonated glyphosate anion) with three functional groups (amine, carboxylate, and phosphonate) which may be coordinated as a tridentate ligand through the amine group nitrogen, the carboxylate group oxygen, and the phosphonate group oxygen or as tetradentate ligand when the phosphonate group coordinates through two oxygen atoms. Past decades studies indicate that glyphosate is a strong chelator for a variety of metal ions1-6. The ability to form metal complexes is the key to understanding the environmental and biochemical behaviors of glyphosate. For instance, foliar absorption is widely reduced when glyphosate is applied in solution with calcium, iron, magnesium, manganese, and zinc7. In the soil, glyphosate is microbiologically degraded and consequently deactivated8. This process is inhibited by complex with metal cations in the soil, which dramatically affects its half-life by forming insoluble complexes that are virtually inaccessible to microbial and chemical degradation routes. For example, addition of Fe3+ and Al3+ cations to the soil evidently decreases the degradation of glyphosate9. These cations form new active sites in the soil which bind glyphosate and make it more resistant to biodegradation. Thus, glyphosate can withstand degradation for more times and remain in the soil for much longer. Complex formation of glyphosate with metal cations in aqueous solution will increase the solubility and the mobility of glyphosate in the soil, which allows it to leach into deep layers and eventually into groundwater. Divalent nickel is also known to form stable complexes with glyphosate in both acidic and basic solutions10. An undesired effect of using glyphosate in glyphosate-resistant genetically modified soybean crops is that the bioavailability of nickel is lowered by complex formation with glyphosate11. This decrease in available nickel inhibits symbiotic microbial nitrogen fixation and affects the growth of soybean plants. In this line, the complexation step with metal ions modulates the glyphosate action. Our group is especially interested in the thermodynamic studies because of their importance in providing fundamental information on the nature (e.g., ionic bonding vs covalence bonding, outer sphere vs inner sphere), energetics (e. g., free energy, enthalpy, entropy and heat capacity), structures and stabilities of complexes. Despite these health and environmental factors associated with glyphosate and its metal complexes, there are very few thermodynamic investigations on the glyphosate and of its metal complexes in aqueous solution1, 12. Thermodynamic parameters other than stability constants, e.g., enthalpy of complex formation13, are rarely available for complexation of glyphosate. As a result, it is difficult to predict the chemical behaviour of complexes of glyphosate in environmental solution. Formation of complexes of glyphosate with metal depends on the pH of the reaction medium, i.e. the degree of deprotonation of glyphosate. Despite a large body of data from research, there is 3



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conflicting information concerning the study on the protonation processes of glyphosate in the literature. The debate is as to whether the first protonation reaction site of glyphosate involves the phosphonate oxygen atom or the central amine group nitrogen atom14, 15. Almost all reports suggest that the protonation reaction of glyphosate (L3-) starts with amine group nitrogen atom8, 15-26

. Only one report from Silva et al. considers that the first protonation step occurs on the one of

oxygen atoms in the phosphonate group using computer simulation14. To help address these contradictory results, this paper presents a thermodynamic study on the protonation reaction of glyphosate.

2 Experiment 2.1 Chemicals

All chemicals were reagent-grade or higher. Milli-Q water was used in preparations of all solutions. Glyphosate (≥96%, Aldrich) was purchased as the fully protonated acid (H3L). A stock solution of H3L was prepared by dissolving the desired amount of H3L in the solution of NaClO4 (≥98%, Aladdin). Deuterium oxide (99.8 atom % D, J＆K) was used to prepare NMR sample solution. Working solutions of NaOH and HClO4 in NaClO4 were standardized by titration with potassium hydrogen phthalate ( primary standard, 99.95-100.05%, Alfa Aesar) and Trizma® base (crystalline, Sigma), respectly. Carbonate contamination in NaOH titrant solution is less than 0.5% after blank acid-base titrations by Gran’s method27. The ionic strength of all working solutions was maintained at 1.0 mol∙L-1 (NaClO4). 2.2 Potentiometry

Potentiometric experiments were carried out using a specially designed vessel as previously described28. The potentiometric vessel consists of a 100 mL glass cell with a lid. Both the cell and the lid are water-jacketed so that the cell temperature can be maintained at 25oC by water circulating from a constant temperature bath. Experiments are protected by argon throughout the titration to avoid the contamination of carbon dioxide. Argon was passed through a series of solutions including 1.0 mol∙L-1 NaOH and 1.0 mol∙L-1 NaClO4 solution before entering the titration cup. Electromotive force (EMF, in millivolts) was measured by a potentiometric titrator (888 Titrando, Metrohm) equipped with a combination pH electrode (6.0259.100 Unitrode, Metrohm). The original 3.0 mol∙L-1 KCl salt bridge solution of the electrode was replaced with a 1.0 mol∙L-1 NaCl solution to avoid formation of insoluble KClO4 in the electrode joint sleeve that is in contact with the working solution. All the EMF data were corrected for a small contribution from the contact junction potential of hydrogen or hydroxide ion. Corrections for the contact

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junction potential of the glass electrode in the acidic and basic regions can be expressed by Eq. 1 and 2. E = E°+ RT/F ln[H+] + ɣH[H+]

(1)

E = E°+ RT/F ln(Qw/[OH-]) + ɣOH[OH-]

(2)

where R is the gas constant, F is the Faraday constant, and T is the temperature in K. Qw = [H+][OH-].

The last terms are the electrode junction potentials (∆Ej,H+ or ∆Ej,OH-) for the

hydrogen ion (Eq.1) or the hydroxide ion (Eq.2), assumed to be proportional to the concentration of the hydrogen or hydroxide ions. The electrode was calibrated before each titration by an acid/base titration with standard HClO4 and NaOH solution to obtain the electrode parameters of E°, ɣH, and ɣOH. These parameters allow the calculation of hydrogen ion concentrations from the electrode potential in the subsequent titration. Multiple potentiometric titrations were conducted using different H3L concentrations. Usually, about 50 points were collected for each titration. The protonation constants (logβ) were caculated by non-linear regression program.

2.3 Microcalorimetry

Calorimetric titrations were conducted with an isothermal microcalorimeter (TAM III, TA Instruments) at 25 °C as previously described28. The TAM III system consists of a nanocalorimeter, a removable titration ampoule (1.0 ml) with stirring facilities, and a precision syringe pump for titrant delivery. The performance of the calorimeter has been tested by measuring the enthalpy of protonation of tris(hydroxymethyl)-aminomethane. A dynamic electrical calibration of the calorimeter was performed prior to each experiment to determine the gain, offset, and time constants τ1 and τ2 for the calorimeter to calibrate the measured heat flow during titrations. Multiple titrations were conducted with solutions of different concentrations of H3L. For each titration, n additions were made (usually n = 50), resulting in n experimental values of the heat generated in the reaction cell (Qex,j, where j = 1 to n). These values were corrected for the heat of dilution of the titrant (Qdil,j), which was determined in separate runs. The net reaction heat at the j-th point (Qr,j) was obtained from the difference: Qr,j = Qex,j − Qdil,j. Details of the titration conditions are provided in Table S1 and S2 in the Supplementary Information. These data, in conjunction with the protonation constant obtained by potentiometry, were used to calculate the enthalpy of protonation with the computer program.

2.4 NMR spectroscopy

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A 400 MHz Bruker Avance spectrometer was applied to conduct

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P-NMR measurements.

Samples were prepared by dissolving 3.0 mg glyphosate in 2.5 mL D2O. After the glyphosate was completely dissolved, the whole solution was adjusted to a design pH. At each pH value, an aliquot was pipeted into a NMR sample tube. The same protonation constants from H2O solution were used. The acidities (expressed as pD = -log[D+]) in D2O were obtained by using pD=pH+0.4029. 3 Results and discussion 3.1 Protonation constants of glyphosate

Representative potentiometric titrations at 25°C and the fitting curves are shown in Fig. 1. There are three successive protonation reactions that are observed in the pH range of this study. The protonation reaction on the second oxygen atom of the phosphonate group occurs outside the normal pH range and is therefore not considered here. The speciation of glyphosate during the course of these titration experiments is superimposed on these plots. From multiple titrations, the protonation constants (logβ) of glyphosate were calculated and shown in Table 1. Other comparable values at different ionic strengths from the literature are also listed in table 1.

Figure 1 Multiple potentiometric titrations of the protonation of glyphosate at 25°C. I=1.0 mol∙L-1 NaClO4. (a) Initial solution: V = 22.06 mL, nL3- = 131.31 mmol, nH+ = 593.57 mmol, Titrant: 115.43 mmol·L-1 NaOH; (b) Initial solution: V = 22.60 mL, nL3- = 127.17 mmol, nOH- = 22.66 mmol, Titrant: 99.73 mmol·L-1 HClO4; Symbols: blue solid square – experimental data (−log[H+]), blue dashed line – fit (−log[H+]), solid lines – percentages of species relative to the total glyphosate concentration (black: H3L, red: H2L-, green: HL2−, pink: L3-, where H3L stands for the neutral glyphosate). 6


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Table 1 Thermodynamic parameters for the protonation of glyphosate. Reaction H + L3- = HL2+

2H+ + L3- = H2L-

3H+ + L3- = H3L

logβ 9.66±0.06 9.76 9.96 9.98 10.28 9.91 10.13 10.14 10.86 10.25 10.25 14.86±0.12 14.84 15.18 15.26 15.50 15.36 15.57 15.60 16.72 15.93 15.83 16.44±0.15 16.61 17.35 17.22 17.89 17.61 17.79 17.83 19.07 18.46 18.10

∆H, kJ∙mol-1 -32.0±0.3

-34.1±0.3

-59.0±2.0

Solution 1.0 mol∙L-1 NaClO4, this work 1.0 mol∙L-1 NaCl16 0.6 mol∙L-1 NaCl2 0.5 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl2 0.1 mol∙L-1 NaCl30 0.1 mol∙L-1 KNO318 0.05 mol∙L-1 KCl31 No control of ion strength 32 No control of ion strength 20 1.0 mol∙L-1 NaClO4, this work 1.0 mol∙L-1 NaCl16 0.6 mol∙L-1 NaCl2 0.5 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl2 0.1 mol∙L-1 NaCl30 0.1 mol∙L-1 KNO318 0.05 mol∙L-1 KCl31 No control of ion strength32 No control of ion strength 20 1.0 mol∙L-1 NaClO4, this work 1.0 mol∙L-1 NaCl16 0.6 mol∙L-1 NaCl2 0.5 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl16 0.1 mol∙L-1 NaCl2 0.1 mol∙L-1 NaCl30 0.1 mol∙L-1 KNO318 0.05 mol∙L-1 KCl31 No control of ion strength 32 No control of ion strength 20

3.2 Enthalpy of protonation of glyphosate

Data of the calorimetric titrations for the protonation of glyphosate are shown in Fig. 2. The observed reaction heat (“partial” or stepwise Q) is a function of a number of parameters, including the concentrations of reactants (CH, CL), the protonation constants (logβ) and the enthalpy of protonation of glyphosate (ΔH). Using the stoichiometric concentrations of the reactants and the protonation constants measured by potentiometry in this work, the enthalpies of protonation of glyphosate at 25oC are calculated from the calorimetric titration data, and are presented in Table 1.

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Figure 2 Calorimetric titrations of the protonation of glyphosate at 25°C. I = 1.0 mol∙L-1 NaClO4. (a) thermogram (solid line, using bottom x-axis and left y-axis) and stepwise heat (blue solid square, using top x-axis and right y-axis). (b) stepwise heat (right y axis, blue solid square experimental Q, blue dashed line – fitted Q) and speciation of Glyphosate (solid line, left y axis, black: H3L, red: H2L-, green: HL2−, pink: L3-, where H3L stands for the neutral glyphosate) vs. the titrant volume. Initial cup solutions: V = 750 µL, nL3- = 4.214 µmol, nH+ = 17.164 µmol, Titrant: 57.7 mmol·L-1 NaOH, injection volume: 5.0 μL. The cumulative enthalpies of protonation measured by microcalorimetry at 25oC are -32.0±0.3 kJmol-1 (for H+ + L3- = HL2-), -34.1±0.3 kJ∙mol-1 (for 2H+ + L2- = H2L-), and -59.0±2.0 kJ∙mol-1 (for 3H+ + L- = H3L), indicating that the all three protonation steps are exothermic. It was reported that the successive protonation sites of totally deprotonated glyphosate (L3-) are the amino nitrogen atom (HL2-), the phosphonate oxygen atom (H2L-), and finally the carboxylate oxygen atom (H3L) in order as scheme 18, 15-26.

Scheme 1 Successive protonation sites of totally deprotonated glyphosate reported previously 8


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However, the stepwise enthalpies of protonation are -32.0±0.3 kJmol-1 (for H+ + L3- = HL2-), -2.1±0.4 kJ∙mol-1 (for H+ + HL2- = H2L-), and -24.9±2.0 kJ∙mol-1 (for H+ + H2L- = H3L). It should be pointed out the exact value of enthalpy of protonation reaction for the second step is much smaller than for the first and third steps. The N-H bond lengths (0.962 and 0.914Å ) in the amino group of neutral crystal of glyphosate are much longer than O-H bond lengths in the carboxyl groups (0.711 Å ) and phosphono groups (0.743 Å ) (Fig. S1)33. It well known that amine nitrogen atoms are less hydrated than the oxygen atoms in carboxylate or phosphate groups in aqueous solution. Less energy is released upon amino nitrogen protonation reaction comparing to protonation reaction on the carboxyl groups or phosphono groups. For example, the molecule of glutaroimide-dioxime(H2A, Fig. S2), a cyclic imidedioxime moiety that can form during the synthesis of the poly(amidoxime)sorbent and is reputedly responsible for the extraction of uranium from seawater34. There are three steps of protonation, from A2-, through HA- and H2A, to H3A+. The first two stepwise protonated sites are oxygen atoms of oxime group and the third protonation reaction occurs on the nitrogen atom of middle imide group. In crystal of glutarimidedioxime, the bond lengths for the O-H bonds are 0.885Å while those for the N-H bonds are 0.907Å. The first two stepwise enthalpies for the protonation of glutarimidedioxime are -36.1 kJmol-1 (for H+ + A2- = HA-) and -33.6 kJ∙mol-1 (for H+ + HA- = H2A), while enthalpy for last step protonation reaction is 7.3 kJ∙mol-1 (for H+ + H2A = H3A+)34. The variation of enthalpies for the protonation of glyphosate suggest that protonation upon amino nitrogen atom is the second step. The 31P NMR studies described in the next section help to further confirm that the order in which the functional groups of the fully deprotonated form of glyphosate (L3-) becomes protonated starts with one of the oxygen atoms in the phosphonate group (HL2-), followed by amine group nitrogen atom (H2L-), and finally the carboxylate group oxygen atom (H3L). Scheme 2 descripts the successive protonation sites in order.

Scheme 2 Successive protonation sites of totally deprotonated glyphosate reported by this work 3.3 31P NMR

Earlier works have attempted to investigate protonation reaction and complex formation of glyphosate by means of infrared spectrometry15,

16, 20, 25, 30

. However, the conclusions on the

importance of hydrogen bonds and dispersive forces were only indirect. NMR spectroscopy is a powerful technique for studying both the qualitative and quantitative relations among different organic molecules2, 17, 35, 36. Direct NMR information has already been obtained to understand the glpyphosate role in physiological and ecotoxicological processes in human and plant biology36-38. 9



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P NMR spectra of four glyphosate solutions (Solutions a, b, c, and d) were collected to help

identify the protonation sites of glyphosate (Fig. 3). Values of pD (pD = -log[D+]) of the solutions are 13.0, 7.5, 3.4, and 1.0, for solutions (a), (b), (c), and (d), respectively. The dominant species of glyphosate in these solutions were calculated to be: solution (a): L3- (100%), solution (b): HL2(99%), solution (c): H2L- (97%), and solution (d): H3L (82%). Therefore, the variations in the NMR spectra from solution (a) to (d) could be discussed as reflecting the stepwise protonation of glyphosate. The trends in the chemical shift of protonation for glyphosate are consistent with those observed in the literature17, 19, 21, 39, 40. The chemical shift dependence on pH is shown in Fig. 4 along with duplicated data recorded in previously.

Figure 3 31P spectra of four Glyphosate solutions

Figure 4 pH dependence of 31P-NMR phosphorus shifts 10


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Usually, the largest chemical shift occurs closest to the site of protonation. As shown in Fig.3, from solution (a) to (b), the dominant species changes from L3- to HL2-, representing the first protonation step (H+ + L3- = HL2-). The largest chemical shift (~ 8.83 ppm) suggests the first protonation occurs on the phosphonate oxygen atom. From solution (b) to (c), the dominant species changes from HL2- to H2L-, representing the second protonation step (H+ + HL2- = H2L-). The chemical shifts (~ 1.48 ppm) is smaller than the first protonation step, but larger than the chemical shifts (~ 0.18 ppm) of the third protonation step, suggesting that the second protonation occurs on amino nitrogen atom and the third protonation occurs on the carboxylate oxygen atom. Therefore, successive protonation sites of totally deprotonated glyphosate in order should be expressed as scheme 2. Someone argues that three successive protonation reactions of fully deprotonated form of glyphosate should be written as scheme 1. It is known that glyphosate exist in aqueous solutions as zwitter ions, where the amine nitrogen is protonated and possess a positive charge, leaving the phosphonate oxygen unprotonated with a negative charge. The largest shift of

31

P signal on the

first protonation reaction can be explained by the intramolecular interaction between the hydrogen atom bonded to the amine nitrogen atom and one of the oxygen atoms in the phosphonate group. If this hypothesis is true, the second chemical shift of

31

P signal caused by protonation reaction on

the one of the phosphonate oxygen atoms should be larger than that of first step, or at least equal to the shift of protonation reaction occurring on amine group nitrogen atom because the first shift is caused by intramolecular interaction rather than direct bonding. The fact is that the shift upon the second protonation reaction is much smaller than the first step. So, we suggest that process of stepwise protonation reaction of totally deprotonated glyphosate should be written as scheme 2. To further illuminate the stepwise protonation process of glyphosate, X-ray photoelectron spectroscopy (XPS) of N1s for glyphosate species is presented in Fig. S3. The high binding energy (BE) component at 401.9-402.0 eV corresponds to a protonated amine group (NH2+), and the low BE component at 399.9 eV corresponds to a deprotonated amine group (NH)41. In Fig S3 (d), the dominant species is HL2- (HL2- 90%, L3- 9%), NH group governs N1s line in XPS spectrum. From Fig S3 (d) to Fig S3 (a), the N1s XPS spectra show protonation of the amine group (NH2+) with increasing acidity. The dominant species in Fig S3 (a) change from HL2- to H2L- (H2L- 94%, HL2- 5%), representing the second protonation step (H+ + HL2- = H2L-), and NH2+ group can be observed obviously. The changes of N1s XPS spectra in the different pH values show the second protonation site of glyphosate occurs on the on amino nitrogen atom. In brief, N1s XPS and

31

P NMR spectra further confirm that the first protonation site of

glyphosate occurs on the one of phosphono oxygen atoms. These results help to interpret the exothermic enthalpies of the second step is much smaller than the first and the third steps of

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protonation. Our results is in good agreement with a recent report using computer simulation to study the possible glyphosate conformations in aqueous solution14. The pH value is 7~8 for most biochemical solutions, surface waters, and ground waters. For these aqueous solutions, the dominant species of glyphosate is HL2-. Our study suggests that this speciation should be written as scheme 3a rather than 3b. The results will help to explain mechanism of glyphosate.

Scheme 3 Structural formula of glyphosate (HL2-)

4 Summary

The protonation of glyphosate was evaluated by potentiometry, calorimetry and

31

P NMR

spectroscopy. Stepwise, the protonation reaction of fully deprotonated glyphosate starts with one of the oxygen atoms in the phosphono group, followed by amine group nitrogen atom, and finally the carboxyl oxygen atom. The energetics of the protonation can be well explained by the difference in the nature of N-H and O-H bonds. The data from this work will help to understand the role of glyphosate in physiological and ecotoxicological processes in environmental and biochemical solution.

Acknowledgements

The authors thank the National Natural Science Foundation of China (Grant No. 41573122) and China Academy of Engineering Physics for financial support. Supporting Information

Details of the calorimetric titration conditions,

molecular structures of glyphosate and

glutaroimide-dioxime, N1s XPS spectra are available free of charge via the Internet at http://pubs.acs.org

Reference

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Thermodynamic Study on the Protonation Reactions of Glyphosate in

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