Aquatic Humic Substances - ACS Publications - American Chemical

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Downloaded by UNIV OF TENNESSEE KNOXVILLE on September 4, 2014 | http://pubs.acs.org Publication Date: December 15, 1988 | doi: 10.1021/ba-1988-0219.ch005

Analysis of Humic Substances Using Flow Field-Flow Fractionation Ronald Beckett Water Studies Centre, Chisholm Institute of Technology, 900 Dandenong Road, Caulfield East, Victoria 3145, Australia James C. Bigelow, Zhang Jue, and J. Calvin Giddings Department of Chemistry, University of Utah, Salt Lake City, UT 84112

A new methodfor the determination ofmolecular-weight distributions of humic substances usingflowfield-flowfractionation (flow FFF) is outlined. Fairly good agreement between the results obtained by flow FFF and other methods for some humic reference substances was achieved using poly(styrene sulfonate) molecular-weight calibration standards. The molecular-weight distributions obtained for a variety of humic samples were all fairly broad and, in contrast to the data sometimes reported for gel permeation chromatography, did not show any indication of multiple peaks. The number- and weight-average molecular weights can be estimated, and these were shown to vary considerably (Mw from about 4400 to 19,000) for humic acids extracted from different environments. The method is also capable of fractionating a humic sample. However, because it is a very small -scale separation( x values decreasing by up to 15% in the range tested (crossflow = 1-4 mL/min). This result could have been caused by either intermolecular in teractions or membrane adsorption effects, both of which would be expected to be more pronounced as the field increased and the sample became more compressed against the accumulation wall. To minimize the influence of this effect on the molecular-weight de termination, the diffusion coefficients plotted on the calibration graph were those corresponding to about the same λ value (0.08) as attained in the humic-substance runs. This correspondence was achieved by plotting D versus λ for the data obtained at different crossflows and reading off the diffusion coefficient (D ) at λ = 0.08 for each PSS standard. The resulting calibration curves (log D versus log M) are shown in Figure 6. Both curves are linear, but with different slopes; the random-coil PSS molecules are displaced to lower diffusion coefficient, as would be expected. To test which of these curves may be best suited for the estimation of molecular weight of the humic substances, a few reference fulvic and humic substances were obtained whose molecular weights had been determined ma

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In Aquatic Humic Substances; Suffet, I., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1988.

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AQUATIC HUMIC SUBSTANCES

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LOG (MOLECULAR WEIGHT) Figure 6. Calibration lines obtained with either polystyrene sulfonate) or protein molecular-weight standards (solid symbols) with Millipore membrane. Aho plotted are points (open circles) corresponding to the reference Suwannee River and Mattole soil fulvic and humic acid samples. by a number of different methods (R. Malcolm, U . S . Geological Survey, Denver, C O , personal communication). The points corresponding to the diffusion coefficients at the F F F peak maximum and the molecular weight determined independently for these humic substances are also plotted on the calibration graph. Because these points fall satisfactorily on the PSS calibration line, it was chosen for use in all the work involving the Millipore 10,000-dalton polysulfone membrane, yielding A = 7.05 Χ 10" and b = 0.422 (with D expressed in square centimeters per second) for the constants in equation 4. Somewhat different calibration constants (A = 2.58 X 10 " , b = 0.320) were obtained subsequently when the Amieon 500-dalton cel lulose membrane was installed. 5

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Molecular-Weight Distributions. The fractograms were digitized (150 points) and the abscissa transformed from elution volume (V ) to mo lecular weight by using a calibration equation obtained as already described. Because the conversion between V and M is nonlinear, the ordinate must also be modified in order that the area under the curve is proportional to the mass of sample (m) between given molecular-weight limits (35). The ordinate of the molecular-weight distribution should represent dm/dM> whereas in the case of the original fractogram, the ordinate represents the detector signal, which is proportional to dm I dV. Because dm I dM = dm/dV - dVldM, the required transformation is achieved by multiplying the amplitude values of the digitized fractogram by AV/ΔΜ, where A V is the difference between the elution volume for consecutive points and ΔΜ is the difference in the molecular weight for the same points. r



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BECKETT ET AL.

Analysis Using Flow Field-Flow Fractionation

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The molecular-weight distributions were also normalized so that the total area under the curve was 100. Thus, the area between any two mo lecular-weight limits is the percentage by weight of the sample in that range. The minimum measurable molecular weight on these distributions is de termined by interference from the void volume peak and the deteriorating resolution as R increases toward unity. For the conditions used here, the lower limit is about 300 daltons. The effect of this molecular-weight transformation is illustrated in Figure 3B. The procedure heavily weights the points occurring at lower elution volumes; thus, the maximum in the fractogram is almost eliminated in the molecular-weight distribution. The result is a fairly broad molecular-weight distribution that decreases smoothly with increasing molecular weight, with out any indication of multiple peaks, and tails off toward 10,000 daltons or beyond. From these distributions it is possible to calculate both number (M ) and weight ( M J average molecular weights of the sample. However, a num ber of factors will restrict the accuracy of these parameters. The lower mo lecular-weight limit of 300 will particularly affect M , whereas uncertainties in the higher molecular-weight values will mainly affect M . Contributions to errors in the latter will be the difficulty in establishing the exact baseline in the long tail of the distribution and uncertainties in the calibration. n

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Comparison with Other Methods Various methods have been used to measure molecular size or weight of humic substances, primarily ultrafiltration, gel permeation chromatography (GPC), low-angle X-ray scattering (LAXRS), colligative properties, ultracentrifugation, and viscosity. A l l techniques have limitations, and the results must always be treated cautiously. Excellent critical appraisals of these meth ods can be found in the recent reviews by Wershaw and Aiken (18) and Thurman (19). One common problem with many of the available methods, including flow F F F , is the need to find an appropriate set of standards on which the molecular-weight determination can be based. Only colligative properties are free of this problem; unfortunately, they yield only the number average molecular weight, with no indication of the polydispersity of the sample. The work reported here showed that a random-coiled polyelectrolyte (PSS) could be used to give molecular-weight results for some reference fulvic and humic acids that were more consistent with data from other methods (ul tracentrifuge, L A X R S , vapor pressure osmometry) than when proteins are used as standards. This finding is not surprising, considering the likely struc ture of humic substances, which would be expected to behave more like the PSS molecules than the more rigid proteins. Nonetheless, proteins have been commonly used as molecular-weight standards in the past, particularly



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for ultrafilters and in G P C . Despite the apparent success obtained in using PSS standards, it is likely that even better reference materials could be found, as the humic molecules are probably somewhat more branched than the linear PSS molecules. The most comprehensive molecular-weight studies have been done us ing the International Humic Substances Society Suwannee River fulvic acid reference material. The results obtained with several methods are shown in Table I. The satisfactory agreement between the flow F F F results and the other methods (36) is evidence for the validity of the technique despite some of the potential difficulties, which still need further development to over come. Commercially available humic acids (e.g., Aldrich, Fluka) are commonly used in laboratory experiments on humic substances, although it is doubtful that they are a particularly good model, especially for aquatic humic sub stances (37). We can compare the results of flow F F F and G P C for Aldrich humic acid by using data of El-Rehaili and Weber (38). The raw fractionation output is given for each method in Figure 7A. The G P C elution volume was converted to molecular weight by using protein standards, whereas in the F F F determination, PSS standards were used. O f course, with G P C the molecular weight decreases with increasing elution volume. We have plotted it from right to left to be more comparable with the F F F fractogram. The most noticeable difference is the occurrence of two peaks in the G P C trace. The G P C curve was transposed to a molecular-weight distribution by using the same procedure as described for the F F F fractogram. This trans formation has rarely been attempted with the G P C results reported in the literature. The two molecular-weight distributions are compared in Figure 7B. The major difference is the occurrence of a quite sharp peak in the G P C curve. This peak was close to the column volume (77 mL) and is outside the range covered by the calibration standards. It probably does not accurately represent the true molecular-weight distribution in this region, and for this reason we show it as a dotted line in Figure 7B. F F F is generally charac terized by higher selectivity and resolution than G P C (39), so it is likely that this peak would also be resolved by flow F F F unless some nonideal phe nomenon (e.g., intermolecular interaction) was occurring with these humic samples. The G P C molecular-weight distribution still contains, in addition, a high-molecular-weight peak that is absent in the F F F distribution. This peak Table I. Molecular-Weight Studies for the Suwannee River Fulvic Acid Sample Method Reference M„ Flow FFF 33 1150 1910 36 Ultracentrifuge 655 1335 VP osmometry 823 36 —


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Analysis Using Flow Field-Flow Fractionation 77

BECKETT ET AL.

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Figure 7. Comparison offlowFFF (from ref 33) and GPC (from ref 38) data for Aldrich humic acid sample. (A) Flow FFF fractogram and GPC chromatogram. Volume scales are different. (B) Calculated molecular-weight distri butions. The dotted portion of the GPC distribution is subject to considerable uncertainty, as discussed in text. is much less pronounced than the first. Possibly this peak, which would correspond to a fairly high apparent molecular weight, is due to low-molec ular-weight components that have been excluded from the gel by charge repulsion. Alternatively, this peak may be missing in the F F F fractogram due to adsorption or aggregation phenomena. The presence of multiple peaks in G P C analysis of humic substances is a common occurrence that we believe may be an artifact of the method. However, further work is required before this matter can be resolved completely. We also stress that the plots of the raw output from fractionation techniques such as these can be rather mis-


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leading and may be a poor representation of the true molecular-weight distribution.


Potential Applications of Flow FFF Flow F F F offers the potential to provide molecular-weight distributions of humic substances. It could thus become a valuable additional tool in the quest to understand the origin, nature, and behavior of this important class of naturally occurring organic compounds. In combination with other ana lytical methods, it should be of help in unraveling the biogeochemical changes that occur in these materials, both in the short term in aquatic systems and in the longer term in ground water, soils, and sediments. We previously (33) reported the molecular-weight distributions of humic substances from a number of aquatic and terrestrial sources. Some of the data reproduced in Table II demonstrate a distinct trend toward increased molecular weights as we progress from stream, soil, and peat to coal humate types. O f course, this information alone will not answer the many questions that remain, and a multidisciplinary approach to these problems is most desirable. Preliminary experiments have indicated a shift to higher molecular weight as the solution p H is decreased or if calcium salts are added. The relatively high p H of 7.9 was used in this study in an attempt to repress any tendency of the humic substances to aggregate and thus to obtain estimates of the primary particle molecular weight. This possible aggregation of humic substances would have important consequences in their behavior in natural systems and their role in affecting the fate of pollutants. Certainly more work is warranted in this area of environmental research. The fact that flow F F F is also a fractionation technique should make it useful to study the interactions of humic substances with environmental pollutants. The small scale of the separation, which usually involves much less than 1 mg of sample, would be a limitation here. However, with the use of ultratrace analytical methods, a very powerful method for investigating the importance of different size fractions of humic substances responsible for binding pollutants could be developed. Radiotracer techniques and mass spectrometry are examples of methods that would provide detection systems sensitive enough for such experiments. Table II. Molecular Weights of Humate Samples as Determined by Flow F F F Data Sample Μ„/Μ„ M Suwannee stream humate 4,390 1,580 2.78 Mattole soil humate 6,140 3.16 1,940 Washington peat humate 17,800 5.89 3,020 Leonardite coal humate 18,700 5.01 3,730 n

NOTE: Data are from ref. 33.


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Analysis Using Flow Field-Flow Fractionation 79


Summary Flow F F F is capable of yielding the molecular-weight distributions of humic substances, thus providing more detailed molecular-weight information than any other method, with the possible exception of G P C . Flow F F F should be less prone to anomalous sample interaction effects than G P C , because with this method the separation is carried out in unpacked channels of low surface area. However, indications are that some sample interaction with the membrane on the accumulation wall of the F F F channel does still occur. We hope that further work will find membrane materials and solution con ditions that eliminate these undesirable effects. The availability of a quick and reliable method for molecular-weight determination should provide a useful addition to the tools available to assist with humic-substances research. Furthermore, the fractionating ability of flow F F F could be used to provide valuable information on the interaction of pollutants with humic substances.

Acknowledgments This work was supported by the Australian Research Grant Scheme and the Department of Science in Australia and Department of Energy Grant D E - F G 0 2 - 8 6 E R 6 0 4 3 1 in the United States.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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17. Aiken, G. R.; McKnight, D. M.; Wershaw, R. L.; MacCarthy, P., Eds. Humic Substances in Soil, Sediment and Water; Wiley-Interscience: New York, 1985. 18. Wershaw, R. L.; Aiken, G. R. In Humic Substances in Soil, Sediment, and x Wiley-Interscience: New York, 1985; p 477. 19. Thurman, Ε. M. Organic Geochemistry of Natural Waters; Martinus Nijhoff/ Junk: The Hague, Netherlands, 1985; p 304. 20. Malcolm, R. L.; MacCarthy, P. In Trace Organic Analysis: A New Frontier in Analytical Chemistry; Chester, S. N.; Hertz, H. S., Eds.; U.S. National Bureau of Standards: Gaithersburg, MD; U.S. National Bureau of Standards Special Publication No. 519, p 789. 21. MacCarthy, P. Geoderma 1976, 16, 179. 22. Wershaw, R. L. J. Contam. Hydrol. 1986, 1, 29. 23. Wershaw, R. L.; Thorn, Κ. Α.; Pinckney, D. J.; MacCarthy, P.; Rice, J. Α.; Hemond, H. F. In Peat and Water; Fuchsman, C. H., Ed.; Elsevier: New York, 1986; p 133. 24. Giddings, J. C.; Myers, M. N.; Caldwell, K. D.; Fisher, S. R. In Methods of Biochemical Analysis; Glick, D., Ed.; Wiley: New York, 1980; Volume 26, p 79. 25. Hovingh, M. E.; Thompson, G. H.; Giddings, J. C. Anal. Chem. 1970, 42, 195. 26. Giddings, J. C. Sep. Sci. Technol. 1984, 19, 831. 27. Giddings, J. C.; Yang, F. J.; Myers, M. N. Anal. Chem. 1976, 48, 1126. 28. Giddings, J. C.; Lin, G. C.; Myers, M. N. J. Colloid Interface Sci. 1978, 65, 67. 29. Giddings, J. C.; Yang, F. J.; Myers, M. N. J. Virol. 1977, 21, 131. 30. Giddings, J. C.; Yang, F. J.; Myers, M. N. Science (Washington, D.C.) 1976, 193, 1244. 31. Wahlund, K.-G.; Winegarner, H. S.; Caldwell, K. D.; Giddings, J. C. Anal. Chem. 1986, 58, 573. 32. Myers, M. N.; Giddings, J. C. Anal. Chem. 1982, 54, 2284. 33. Beckett, R.; Jue, Z.; Giddings, J. C. Environ. Sci. Technol. 1987, 21, 289. 34. Tanford, C. Physical Chemistry of Macromolecules; Wiley: New York, 1961; Chapter 6. 35. Giddings, J. C.; Myers, M. N.; Yang, F. J. F.; Smith, L.K. In Colloid and Interface Science; Kerker, M., Ed.; Academic: New York, 1976; Volume IV, p 381. 36. Averett, R. C., Ed. Humic Substances in the Suwannee River, Florida and Georgia: Interaction, Properties, and Proposed Structures; Water Supply Paper; U.S. Geological Survey, in press. 37. Malcolm, R. L.; MacCarthy, P. Environ. Sci. Technol. 1986, 20, 904. 38. El-Rehaili, A. M.; Weber, W. J. Water Res. 1987, 21, 573. 39. Gunderson, J. J.; Giddings, J. C. Anal. Chim. Acta 1986, 189, 1. RECEIVED for review November 3, 1987. ACCEPTED for publication February 29, 1988.


Aquatic Humic Substances - ACS Publications - American Chemical

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