Anal. Chem. 2006, 78, 873-882
A Stepwise Stoichiometric Representation To Confirm the Dependence of Pesticide/Humic Acid Interactions on Salt Concentration and To Test the Performance of a Silica Bonded Humic Acid Column C. Andre´,† M. Thomassin,† A. Berthelot,‡ and Y. C. Guillaume*,†
Equipe Sciences Se´ paratives et Biopharmaceutiques (2SB/EA-3924), Laboratoire de Chimie Analytique, and Equipe Optimisation Me´ tabolique et Cellulaire (OMC/ EA-3921), Laboratoire de Physiopathologie Cardiovasculaire et Pre´ vention, Faculte´ Me´ decine Pharmacie, Place Saint-Jacques, 25030 Besanc¸ on Cedex, France
In a previous paper (Andre´ et al., in press), a novel chromatographic column was developed in our laboratory for studying the binding of pesticides with humic acid (HA), the main organic component in soil. It was demonstrated that this column supported a low fraction of organic modifier in the aqueous mobile phase ( 0.6 M), a decrease of the pesticide-HA binding was observed due to a restriction of the HA accessible surface to the pesticide due to a change of the HA structure, which was in a random coil rather than in a flexible conformation.28,29 This confirmed that this chemically bonded HA on silica did not alter the HA conformation change capacity. As well, for the charged pesticides at pH 7 (i.e., chlordimeform and paraquat), these plots could be divided into three domains (Figure 5) as was previously observed on the AHAC due to the low salt concentrations (x < 0.3 M) and to the formation of a ion pair between the Cl- anion of the salt and the amino group of the charged pesticides, which decreased the ionic attraction between the cationic pesticides and the HA- stationary phase. Equation 52 was fitted to the chromatographic data (0.1 M < x < 0.6 M) for the charged species. The nonlinear regression r2 and F for each pesticide molecule were at least equal to 0.987 and 123. The values of the parameters A and ∆n are presented in Table 1. All these values are in accordance with the one obtained with other ligand-receptor binding.30 Affinity Energy Distribution at 20 °C. The AEDs were calculated for a neutral pesticide, i.e., lindane (Figure 6) and a cationic pesticide, i.e., paraquat (Figure 7) for four NaCl concentrations in the medium, i.e., 0, 0.3, 0.6, and 0.8 M, and at 20 °C. For the neutral pesticide, three distinct peaks are clearly seen. The association constant of high-energy sites (peak.3) increases with increasing concentration of NaCl until 0.6 M and decreases above this value while the number of these sites did not change significantly. For a more clear visualization of this variation, the values of K and ln K for peak 3 are indicated above this peak in (28) Cameron, R. S.; Thornton, B. K.; Switft, R. S.; Posner, A. M. J. Soil Sci. 1972, 23, 394. (29) Ghosh, K.; Schnitzer, M. Soil Sci. 1980, 129(5), 266.
Figure 8. Chromatogram obtained for the paraquat pesticide at 20 °C for a methanol/phosphate buffer 0.15/0.85 (v/v) mobile phase containing (a) 0.05 M NaCl and (b) 1 M NaCl.
Figure 6 for each value of NaCl concentration in the mobile phase. This result followed the hypothesis for the existence of hydrophobic pockets that can change their conformation with the NaCl concentration in the medium. For the intermediate and the lowest energy sites (peaks 1 and 2) their association constants increased slowly for the entire salt concentration in the medium (when the NaCl concentration in the mobile phase increased from 0 to 0.8 M, the association constant increased by 8%) while the number of these sites remained relatively stable. As well, as the silica surface is not completely covered, these last peaks may be due to non-humic acid interactions. For the charged pesticides, an additional peak (peak 4) of high energy was observed (peaks 1-3 have variations similar to those observed for the neutral pesticides). For these sites (peak 4), the association constant and the number of the sites (i.e., density) decreased when the NaCl concentration in the bulk solvent increased. As above, for a more clear visualization of this variation, the values of K and ln K for peak 4 are indicated above this peak in Figure 7 for each value of NaCl concentration in the mobile phase. To confirm this result, the chromatogram of the paraquat molecule was analyzed. For a NaCl concentration in the mobile phase equal to 0.05 M, the paraquat peak distortion was reflected by an asymmetry factor around 1.6 (ideal is 1) (Figure 8). Sodium chloride addition to 1 M resulted in an improvement of the asymmetry factor to ∼1.1 (Figure 8). The decrease of the asymmetry factor was a consequence of a decrease of the number of the binding sites, i.e., the sites of high energy. Moreover, this confirmed at pH 7 the existence of anionic sites on the HA macromolecule and electro(30) Andre´, C., Guillaume, Y. C. Electrophoresis. 2003, 24, 1620.
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static attractions between the positive charge of the cationic pesticide and the negative charge of the HA, which decreased when the NaCl concentration in the medium increased (reduction of the debye length). CONCLUSION The results reported in this paper demonstrated that this HA immobilized stationary phase allowed determination of binding affinity for neutral and charged pesticides. As well, it was clearly demonstrated that this procedure for the immobilization of the HA molecule on the silica support allowed its utilization in a large methanol fraction range (
