Environ. Sci. Technol. 2001, 35, 4301-4306
Determination of Electrophoretic Mobilities and Hydrodynamic Radii of Three Humic Substances as a Function of pH and Ionic Strength M. HOSSE AND K. J. WILKINSON* CABE (Analytical and Biophysical Environmental Chemistry), University of Geneva, Sciences II, 30 Quai Ernest Ansermet, Geneva 4, CH-1211, Switzerland
Capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) were employed to determine electrophoretic mobilities and hydrodynamic sizes of three humic substances (IHSS aquatic fulvic acid (FA), IHSS aquatic humic acid (HA), and IHSS peat humic acid (PHA)) as a function of pH and ionic strength. A slight aggregation corresponding to the formation of dimers and trimers was observed at low pH using fluorescence correlation spectroscopy (FCS). For example, for the peat humic acid, diffusion coefficients decreased from 2.1 × 10-10 m2 s-1 at pH 4 to 2.4 × 10-10 m2 s-1 at pH 11. For all three humic substances, electrophoretic mobilities were also shown to decrease significantly below pH 6. Calculated zeta potentials observed at high pH of -69 mV (FA), -62 mV (HA), and -63 mV (PHA) decreased to -39, -50, and -47 mV, respectively, under slightly acidic pH (4.5-4.8) conditions. No evidence of ionic strength induced aggregation was found using fluorescence correlation spectroscopy (FCS): diffusion coefficients increased slightly ( pKa, since at lower pH values, it would be difficult to observe mobility differences for the complexation of a relatively small, noncharged buffer molecule with the HS. Nonetheless, replacement of the MES buffer by a malonate buffer eliminated the peak splitting and gave mobility values which were identical to those obtained for the MES buffer below its pKa. In another experiment, the complexation of the HS by NaBES buffers was verified by using two experimental protocols: in the first, NaBES concentrations were increased from 5 to 40 mM with a concurrent increase in ionic strength; in the second, NaBES concentrations were maintained constant while ionic strength was increased with NaCl. Although an increase in buffer concentration was expected to result in an increase in negative EPM due to an increased complexation of the HS with the buffer, this was not observed in Figure 7A. EPM values calculated from the second peak were generally more negative with increasing pH, but the effect was much more significant at 5 mM than at 40 mM. At 40 mM, there was no significant increase in electrophoretic mobility as a function of increasing pH over the examined pH range. Furthermore, only a single peak was observed under these conditions. This result suggested that the ionic strength of the medium might also play an important role in the complexation or ion association reaction. Indeed, similar curves were observed when the NaBES concentration was maintained at 5 mM and ionic strength was adjusted using NaCl (Figure 7B), indicating that the complexation by the buffers needed only to be taken into account at low ionic strengths. In this paper, all data for which there was evidence of chemical complexation by the buffers were excluded from further consideration.
Discussion Role of Ionic Strength. As mentioned above, the observed reduction of up to 25% in the EPM could either be due to a decreasing charge at the shear plane or an increasing hydrodynamic radius, or a combination of the two factors.
FIGURE 7. A) Electrophoretic mobilities of the FA in varying concentrations of NaBES (9) 5 mM, (O)10 mM, (2) 20 mM, and (]) 40 mM. (B) Electrophoretic mobilities of the FA in 5 mM NaBES with the ionic strengths adjusted with NaCl to give (9) 5 mM, (O)10 mM, (2) 20 mM, and (]) 40 mM. Because diffusion coefficients were observed to remain constant or increase slightly as a function of ionic strength (Figure 2), the observed decrease in EPM can only be attributed to a charge screening effect since no evidence of aggregation was observed as a function of increasing ionic strength for any of the three HS. The observation of a limited ionic strength effect on the hydrodynamic radii suggests that the assumption of the HS as rigid spheres is reasonable. The observed slight compression of the three HS at high ionic strength is also in agreement with the viscometric measurements made by Avena et al. (10) on several humic acids. Although viscometry is not sensitive to aggregation, their determinations of molecular volume decreases of 35-65% for an increasing ionic strength (1-100 mM) are in line with our measurements of slightly decreasing hydrodynamic radii (recall that the volume depends on ca. rH3). Furthermore, because the HS examined here are relatively small, branched molecules with an internal structure that limits any potential swelling or shrinking (49), only relatively small changes in the diffusion coefficient should be expected under these conditions of pH and ionic strength. Although the lack of ionic strength induced aggregation may be contrasted with our recent observation of the U.K. Geological Survey peat humic acid (PPHA) at 500 mM (11), we postulate that differing hydrophobicities of the HS are largely responsible for the observed differences. Role of pH. Small but significant decreases in the diffusion coefficients of the HS were observed as a function of decreasing pH. For the IHSS fulvic and humic acids, the corresponding hydrodynamic radii were between 0.8 and 1.0 nm in buffers, in good agreement with the results of Lead et al. (24) who worked in unbuffered solutions. The rH of the PHA is in the same range; the diffusion coefficient was slightly smaller than the other HS and increased slightly with
increasing pH. The decrease in the diffusion coefficient at low pH is attributed to the formation of small aggregates due to a reduction in intermolecular repulsion. Although this result would appear to contradict the viscosity measurements of Avena et al. (10) which demonstrated a 15-82% decrease in molecular volumes for a series of HS over the pH range 3-11, it does not imply that molecular compression is not important. In fact, in light of the viscosity result, it would appear that the estimate of an aggregate containing 2-3 molecules is likely a lower limit. These results suggest that the increase in radius due to coagulation overwhelms the decrease in radius due to molecular shrinking. The EPM results as a function of pH were consistent with the FCS results and the known properties of the HS. The FA is known to be more highly charged with a slightly smaller diameter than the HA and PHA which is reflected in higher negative mobilities. For the PHA, mobilities were of the same order of magnitude as for the HA, again consistent with the available values of titratible charge (50). On the basis of these results, it is possible that the mobility dependence once again was largely derived from a charge effect since the observed 1020% increase in rH with decreasing pH could not account for the observed reductions in negative mobility (up to 40% for the FA). Above pH 6, no significant increase in electrophoretic mobilities was observed. This is somewhat counterintuitive considering that the phenolic protons correspond to 25% of the protonable functional groups in the FA with an approximate pKa value of 9.9 (51) which is well within the limits of our technique. Furthermore, our results do not correspond to the results obtained by Schmitt-Kopplin et al. (16) who carried out mobility measurements on the IHSS FA and IHSS HA as a function of pH. In their work, they found curves with a rather sharp inflection point at pH 11 and the beginning of another inflection point at pH 5. In fact, in their work, most of the observed charge neutralization appeared to be due to the protonation of phenolic groups (decrease in negative mobility from (-3 to -2.5) × 10-8 m2 V-1 s-1 between pH 12 and 9 corresponding to ca. 70% of the total decrease in EPM). We have no unambiguous explanation for the discrepancy in results. Electrophoretic mobility measurements are more difficult to perform at very high (or very low) pH values due to the deprotonation (or protonation) of the fused silica capillaries using in CE, but these effects are generally taken into account by the determination of the electroosmotic mobility. The lack of a phenolic inflection point in this study is attributed to the fact that for large values of molecular charge, the bulk pH differs significantly from the pH at the surface of the HS macromolecule. This observation is also supported by theoretical calculations based upon the equations given above. To verify if our electrophoretic mobilities for the FA were reasonable, we compared calculated electrokinetic charge (eqs 2-4) with values of titratable charge obtained from data supplied by Ritchie and Perdue (50). In these calculations, it is assumed that the HS are polyelectrolytic, hard spheres with all of their charge on the surface. Molar masses of 990, 1136, and 1264 g mol-1 and ionic radii of 0.73, 0.79, and 0.84 nm calculated (52) from the mean diffusion coefficients determined between pH 8-11 were used for the FA, HA, and PHA, respectively. For the FA, at 5 mM ionic strength, an electrokinetic charge of -294 C g-1 was determined at pH 7 and a value of -173 C g-1 at pH 4.5. These values corresponded to zeta potentials of -69 and -39 mV, respectively. In a similar manner, a minimum value of -221 C g-1 (ζ ) -50 mV, pH 4.6) and a maximum value of -257 C g-1 (ζ ) -62 mV, pH 7) were calculated for the HA in 5 mM buffer. As expected, the calculated charges for the PHA was slightly lower ranging from -191 to -248 C g-1 (ζ ) -47 to -63 mV, pH 4.8-6.9). VOL. 35, NO. 21, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Values of charge derived from EPM values using eqs 2-4 reflect the electrokinetic charge (at the shear plane) and are necessarily smaller than the total titratable charge of the molecule. Therefore, the values are reasonably coherent with literature values of charge. For example, by using the same molar masses as were employed earlier, Ritchie and Perdue’s values of titration determined surface charge would be -608 C g-1 for the FA, -538 C g-1 for the HA, and -487 C g-1 for the PHA. Furthermore, de Wit et al. (4) determined a charge which varied from -145 to -530 C g-1 for the IHSS fulvic acid for ionic strengths between 0.001 and 1 M. Future Potential for CE Measurements of HS. A priori, the determination of EPM values using CE would appear to be a good manner to better understand the nature of the HS, especially under the conditions resembling those found in natural waters. Nonetheless, as was demonstrated here, some care must be taken in this approach. For example, the complexation of the humics by the pH buffers (results given here and in refs 16, 17) is possible and must be carefully verified. Furthermore, even with valid results, the determination of zeta or surface potentials from EPM values is difficult. Nonetheless, measurements of EPM coupled to determinations of charge by titration and hydrodynamic radii by FCS and other techniques will continue to advance our knowledge with respect to these heterogeneous and analytically difficult substances.
Acknowledgments Unpublished titration data for the Suwannee River standard fulvic acid were graciously provided by Jason D. Ritchie and E. Michael Perdue of the Georgia Institute of Technology. We thank M. Avena, M. Brynda, J. Buffle, and the four journal referees for helpful discussion and comments on previous versions of the manuscript. This work was supported by the Swiss National Funds (Projects 2000-050629.971 and 2100055 668.981).
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Received for review February 7, 2001. Revised manuscript received July 30, 192001. Accepted August 23, 2001. ES010038R
