Molar mass and size of Suwannee River natural organic matter using

Humic materials from soil, freshwater, and marine systems can be distinguished from one another in terms of elemental (2) and functional group content...
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Environ. Sci. Technol. 1997, 31, 937-941

Molar Mass and Size of Suwannee River Natural Organic Matter Using Multi-Angle Laser Light Scattering DAVID B. WAGONER AND RUSSELL F. CHRISTMAN* Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7400 GREG CAUCHON InterMol Group, 369 Paseo de Playa, No. 310, Ventura, California 93001 ROBERT PAULSON Wyatt Technology Corporation, 802 East Cota Street, Santa Barbara, California 93103

Introduction The determination of molar mass (M) is a valuable tool for understanding the fundamental properties of many classes of organic substances. In particular, it is necessary for establishing stoichiometric relationships for reactions involving these molecules and also as a basis of comparison for complex mixtures from different natural sources (1). Humic materials from soil, freshwater, and marine systems can be distinguished from one another in terms of elemental (2) and functional group content (3), and perhaps size (4). However, it is not clear that humic materials from one environmental compartment can be distinguished from another sample from a similar compartment. Previous efforts at estimating the size of humic materials by size exclusion (SEC) chromatography (5-9), vapor pressure osmometry (1, 10), small angle X-ray scattering (4), and ultracentrifugation (11) have all required either a priori assumptions about fundamental molecular properties or the use of artificial standards (Table 1). Some of the techniques require that the humic substances be dissolved in organic solvents, a condition markedly different from the natural state. Thus, no absolute or unambiguous data regarding the molar mass or size of humic materials are available. Nevertheless, the available data suggest that aquatic humic acids are 2-3 times larger than aquatic fulvic acids, and both are smaller than their soil organic matter counterparts. For soil and aquatic humic materials, estimates of the number-average molar masses obtained by colligative property measurements appear to be smaller than weight-average estimates (transport property measurements), suggesting polydispersity, although aquatic humic samples appear to exhibit less polydispersity than soil humic materials. The inherent polydispersity of natural aquatic humic mixtures tends to render “average” molar mass values meaningless, unless the polydispersity is reduced by prior size separation, i.e., fractionation by SEC. Scattering of EM radiation has also been used to investigate the size and shape of humic materials, although more data exists for X-ray scattering than for light scattering (4). For macromolecular solutions of known structure and uniform size, it is possible to use X-ray scattering to estimate the root mean square (rms) radius, molecular volume, molar mass, and particle size. For polydisperse solutions of humic materials, however, the only unambiguous data available are * Corresponding author telephone: (919) 966-1683; fax (919) 9667911; e-mail address: [email protected].

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the high degree of polydispersity and the rms radius of scattering particles from Guinier plots of X-ray data. Previous work with small-angle X-ray scattering has established that solutions of soil humic materials are polydisperse and have a degree of aggregation that is pH dependent (12-14). Aquatic fulvic acids appear to be less polydisperse than aquatic humic acids (15). Wershaw (4) has reviewed the use and limitations of X-ray scattering of humic materials. Very limited reports are available in the literature regarding light scattering measurements of humic solutions. Black and Christman (16) reported the detection of scattering signals from aquatic fulvic acid solutions using 436 and 546 nm light and secondary filters to stop fluorescent emissions from reaching the detector. Wershaw (4) has also described the central problem with light scattering measurements of humic solutions, i.e., they strongly absorb UV light, fluorescing at wavelengths in the visible region. This has necessitated the use of higher wavelengths that are scattered much less intensely owing to the dependence of scattering on 1/λ4. Thus, this approach has not been feasible until the advent of commercial laser-based instrumentation, operating at higher wavelengths. An important technique for determining molar masses and rms radius distributions of polymers and other macromolecules is multi-angle laser light scattering (MALLS), which measures the classical, time-averaged angular dependence of the scattering of light by molecules in solution. MALLS differs from the conventional techniques listed in Table 1 as it does not require a priori assumptions about molecular conformation; the constants required for determining MALLS molar masses and rms radii are measured experimentally. Recent advances in instrumentation, including the incorporation of laser light sources and computerized data reduction, have greatly improved the practical applications of light scattering, particularly when combined with highperformance size exclusion chromatography (HPSEC) (17). The ability to separate complex molecules by size, combined with on-line laser light scattering detection, now permits the extraction of considerable information regarding the distribution of both size and molar mass. The calculation of concentration and M in light scattering experiments requires measurement of the specific refractive index increment (dn/dc), which is previously unreported for humic substances. It represents the change in solution refractive index (dn) as a function of the solute concentration (dc). It is used in the calculation of the light scattering instrumental optical constant, K*, and in the calculation of the mass-based concentration of the analyte at each slice in the chromatogram. Molecular radius is measured independently of the dn/dc value. We report here measurements of dn/dc for unfractionated (not separated into humic and fulvic acid fractions) Suwannee River natural organic matter (SRNOM) in two different buffers from two different laboratories and preliminary results of our evaluation of the applicability of the HPSEC/MALLS combination to the determination of the absolute molar mass and rms radius distributions.

Experimental Section The unfractionated Suwannee River (Georgia) natural organic matter was isolated using reverse osmosis by Professor Michael Perdue of The Georgia Institute of Technology (18). It was stored in amber glass, wrapped in metal foil, and refrigerated until use. Experimental procedures were jointly evaluated by the authors on samples of SRNOM provided by UNCsChapel Hill. The data in Table 1 were generated in laboratories at

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TABLE 1. Limitations of Selected Molecular Size/Mass Methods method size exclusion (gel permeation) chromatography ultrafiltration ultracentrifugation, using sedimentation velocity viscometry osmometry

parameter

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ref

molecular size

sorption, electrostatic interactions, need for calibration molecular size membrane interactions, need for calibration molar mass (Mw, Mn, Mz) diffusion coeff; swamping of charge effects; absorptivity varies with M molar mass (Mn) and shape must assume K′ and R values or calculate from known M molar mass (Mn) corrections for ionizable compds

Wyatt Technology, Santa Barbara, CA (samples 1 and 2) and UNCsChapel Hill (samples 3 and 4). A stock HPSEC sample (8.000 mg/mL) of solid SRNOM in 2.5 mM aqueous phosphate buffer was sent to Wyatt Technology along with additional solid SRNOM. The dn/dc value was measured using dilutions of a separate solution of SRNOM in 2.5 mM aqueous phosphate buffer and 200 ppm sodium azide with a Wyatt Technology Optilab DSP interferometric refractometer operating at 23 °C and 690 nm. The dn/dc samples were not filtered before being introduced into the RI detector. A value of 0.176 mL/g was obtained using DNDC version 5.10 (Wyatt Technology) software. Chemicals were obtained from Aldrich Chemical (Milwaukee, WI), and HPLC-grade water was from Burdick & Jackson (Muskegon, MI). The HPSEC instrumentation consisted of a Waters Corp. (Milford, MA) LC-625 metal-free gradient HPLC system with a Hewlett-Packard (San Fernando, CA) 1050-series on-line vacuum degasser and a Rheodyne (Cotati, CA) 7125i manual six-port injector. The HPSEC column set consisted of a Shodex (Showa-Denko, Tokyo, Japan) SB-G guard column, a Shodex 8 × 300 mm SB-804 HQ OHPak, and a Shodex 8 × 300 mm SB-802.5 HQ OHPak column, all packed with poly(hydroxy methacrylate). Previous use of this column set revealed an exclusion volume (Ve) around 10.5 mL and an inclusion volume (Vi) around 19.2 mL. On-line detection was accomplished with a Wyatt Technology miniDAWN laser light scattering photometer, using a 690-nm laser, and a Waters 410 differential refractive index (RI) detector. Normalization of the miniDAWN detectors at 41.5° and 138.5°, relative to the 90° detector, was accomplished with isotonic bovine serum albumin (BSA) monomer (Sigma, St. Louis, MO). The RI detector was operated at 35.0 °C with a 940-nm light source during HPSEC analysis. The HPSEC mobile phase consisted of isocratic aqueous 2.5 mM sodium phosphate buffer, prepared with Na2HPO4 and NaH2PO4, and final pH adjusted to 7.0 with H3PO4 and NaOH. The flow rate was 1.0 mL/min. It was vacuum-filtered before use through a 0.1-µm Whatman (Maidstone, England) Anodisc 47-mm filter in an all-glass filtration apparatus and also filtered on-line through a 25-mm Millipore (Bedford, MA) 0.1-µm type VV poly(vinylidene difluoride) filter. Injections consisted of 25.0 µL (200 µg of SRNOM) of the undiluted stock SRNOM filtered through a 0.45-µm Gelman Acrodisc LC poly(vinylidene difluoride) (PVDF) syringe-tip filter. Duplicate analyses were obtained for this sample and averaged. The data were collected at 1 Hz, despiked, smoothed, and processed with Astra version 4.2 software (Wyatt Technology) using a dn/dc of 0.176 mL/g. Measurements of the SRNOM (samples 3 and 4, Table 2) were also made at the Department of Environmental Sciences and Engineering at the University of North Carolina at Chapel Hill. A sample was prepared by dissolving 94.81 mg of dry SRNOM in 10 mL of freshly prepared 0.1 M aqueous phosphate buffer to yield a concentration of 9.481 mg/mL. The sample was briefly sonicated to aid dissolution and then vacuum filtered through a 47-mm 0.45 µm Supor (Gelman Sciences, Ann Arbor, MI) membrane filter. The HPSEC mobile phase

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Swift and Posner, 1971 Buffle et al., 1978 Cameron et al., 1972 11 Reuter and Perdue, 1981

TABLE 2. Number-, Weight-, and z-Average Molar Masses and rms Radii for Samples of Suwannee River Natural Organic Matter buffer ionic strength (M) buffer pH sample concn (mg/mL) Mn (g/mol) Rn (nm) Mw (g/mol) Rw (nm) Mz (g/mol) Rz (nm) polydispersity (Mw/Mn) dn/dc (mL/g) no. of samples a

samples 1 and 2a

samples 3 and 4b

0.00688 7.0 8.000 16 595 ( 3500 55.8 ( 17.7 25 715 ( 7500 59.6 ( 20.1 78 830 ( 45 000 68.1 ( 20.4 1.550 ( 0.574 0.176 2

0.275 6.8 9.481 15 050 ( 1500 29.9 ( 12.7 20 185 ( 1500 29.0 ( 11.2 28 390 ( 3500 27.8 ( 9.3 1.342 ( 0.160 0.135 2

In 2.5 mM phosphate buffer.

b

In 0.1 M phosphate buffer.

consisted of isocratic aqueous 0.1 M phosphate buffer (pH 6.8), prepared with Na2HPO4 and KH2PO4. It was vacuumfiltered before use through a 0.02-µm Whatman (Maidstone, England) Anodisc 47-mm filter in an all-glass filtration apparatus and also filtered on-line through a 25-mm 0.1-µm Whatman (Maidstone, England) Anodisc filter in a Millipore (Bedford, MA) stainless steel in-line filter holder positioned between the pump and the injector. The flow rate was 1.0 mL/min. The HPLC instrumentation consisted of a Waters Corp. (Milford, MA) 616 HPLC pump and 600S system controller, 717 autosampler, and temperature control module with column heater. The silica-packed HPSEC column set consisted of Progel-TSK 4000 SWXL, Progel-TSK 3000 SWXL, and Progel-TSK 2000 SWXL, columns connected in series and mounted in the column heater box at 30 °C. Each of the three columns were 300 mm × 7.8 mm and were obtained from Supelco (Bellefonte, PA). The measured Ve for this column set was around 14 mL, using aqueous polystyrene sulfonate (Mw ) 990 000 g/mol), and the Vi was around 37 mL, using ethylene glycol (62 g/mol) in water. A Wyatt Technology miniDAWN laser light scattering photometer, using a 690-nm laser, and OptiLab DSP interferometric refractometer with 690-nm light source operated at 30 °C were used for on-line detection during HPSEC analysis. The data were collected at 1 Hz and processed with Astra version 4.2 (Wyatt Technology). Normalization of the miniDAWN detectors at 41.5° and 138.5°, relative to the 90° detector, was accomplished with isotonic bovine serum albumin (BSA) monomer (Sigma, St. Louis, MO) in 0.1 M aqueous phosphate buffer on two separate occasions, and the values averaged. Duplicate HPSEC injections consisted of 200 µL (1.9 mg of SRNOM) of the stock sample. The dn/dc value for unfractionated SRNOM in 0.1 M aqueous phosphate buffer was measured off-line using the OptiLab refractometer at 30 °C on two separate occasions and gave a mean value 0.135 mL/ g.

FIGURE 1. Chromatograms of the SRNOM samples 1 and 2 in 2.5 mM phosphate buffer illustrating signals from the 90° light scattering and RI detectors; Ve ) exclusion volume, Vi ) inclusion volume.

FIGURE 2. Chromatograms of the SRNOM samples 3 and 4 in 0.1 M phosphate buffer illustrating signals from the 90° light scattering and RI detectors; Ve ) exclusion volume, Vi ) inclusion volume.

Results and Discussion Number-average (Mn), weight-average (Mw), and z-average (Mz) molar masses, moments of the rms radius distribution, and polydispersity values are reported in Table 2 (see ref 17 for discussion of the calculations). Traditionally the Mw and Rz are considered to be the most reliable measurements on samples that are not exactly defined with respect to uniform shape and polydispersity (19). The precision values reported with molar masses and radii are statistical calculations based on the baseline data in the chromatograms from each of the three light scattering detectors and the refractive index detector. These translate into standard deviations for Rθ and c for each slice in each peak. The difference between dn/dc values measured in the two buffers was probably due largely to the fact that the sample in 2.5 mM phosphate was unfiltered and contained sodium azide.

The HPSEC chromatograms of samples 1 and 2 in 2.5 mM phosphate are shown in Figure 1. The small amount of mass injected resulted in a lower LS signal (90° detector) as compared to the RI signal, but it was of sufficient strength for the calculations. The general peak selection for both chromatograms is marked with vertical dashed lines, and the exclusion and inclusion volumes for the column set are shown. Figure 2 shows the corresponding information for samples 3 and 4 in 0.1 M phosphate. The differences in the elution patterns shown in Figures 1 and 2 are primarily due to the use of two different column sets; the polymer-packed twocolumn set for the samples in 2.5 mM phosphate and the silica-packed three-column set for the samples in 0.1 M phosphate. The three-column set produced, of course, a larger total elution volume.

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FIGURE 3. Differential molar mass distributions for the SRNOM samples in 2.5 mM (SRNOM 1 and SRNOM 2) and 0.1 M (SRNOM 3 and SRNOM 4) phosphate buffers. The preliminary data in Table 2 suggest that laser light scattering measurements of SRNOM HPSEC fractions at two different laboratories produced similar results, which indicate significantly higher molar masses and rms radii for this material than heretofore reported (22 000-26 000 g mol-1 and 30-70 nm vs 800-1100 g mol-1 and 0.88-1.14 nm). The differences in total ionic strength for samples 1 and 2 (compared to samples 3 and 4) are more pronounced than are differences with respect to pH, concentration, and ionic composition. It is attractive to assume that the reason the rms radii for samples 3 and 4 is approximately one-half of the value for samples 1 and 2 is increased macromolecular coiling in the higher ionic strength solutions. Theoretically, the molar mass of humic substances could be affected by analyte concentration, solution pH, ionic composition, and total ionic strength. The concentration effect could be exerted through aggregate formation, suggesting that the molar mass values might be interpreted as “apparent molar masses”, as humic solutions are believed to be composed of heterogeneous molecules. The heterogeneity of aqueous humic solutions with respect to molar mass is clearly evident in Figure 3, which shows differential molar mass distributions for SRNOM samples in 0.025 and 0.1 M buffers. Thus, these preliminary data support the random coil hypothesis (20) for macromolecular natural organic matter species in aqueous solution, for which there has previously been only indirect evidence (21). Furthermore, the technique may be useful in systematic studies of size and shape variations as a function of solution composition. Because no interference filters were used in these experiments, the possibility of a fluorescence contribution to the detected light cannot be ruled out. However, since humic materials typically absorb around 320-460 nm and emit around 420-540 nm (22), it would appear unlikely that fluorescence caused a significant problem with the 690-nm miniDAWN light source. For similar reasons, photocatalytic polymerization also appears unlikely to have contributed to the large M values observed. There are two disadvantages to this technique, one real and the other hypothetical. As the overall LS response is a

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product of organic substrate concentration and molar mass, it would appear that LS detection would be useful for in situ measurements only for natural waters containing at least 25 mg/L TOC (approximately 50 mg/L NOM), assuming that molar masses do not vary significantly among sources and that the level of precision reported here is satisfactory. Insitu measurements might become possible with the development of rapid and effective in-line concentration devices, after the effect of concentration on size and shape is systematically evaluated. In the work reported here, the stock solutions were very concentrated to permit injection of very small volumes, i.e., the largest concentrations reaching the RI detector were approximately 70 and 500 mg L-1, respectively, for the peak maxima in Figures 1 and 2. The demonstrable fact that aquatic humic solutions are polydisperse (Figure 3) is a hypothetical disadvantage and has meaning only when these measurements are compared to relatively pure solutions of synthetic polymers. The laser light scattering technique documents the polydispersity of natural humic samples, and changes in polydispersity values with solution composition may eventually turn out to be predictive of humic behavior. Humic sample polydispersity may ultimately prevent, however, the determination of molecular shape and branching that has been measured for some polymers using light scattering (19). The significant advantage of this technique is that it does not require the use of calibration standards to measure molar masses and radii of humic substances. It is also amenable to aqueous solvents and buffers that more closely approximate the solution environment in which these substances are formed and found in nature. On the whole, these data suggest that multi-angle laser light scattering has potential for improving our understanding of fundamental properties of humic materials in solution and the solution parameters affecting them, such as solute concentration, ionic strength, interacting ions (e.g., metals), and pH.

Acknowledgments The project described was supported by Grants 5-P42ESO5948-05 and 5-41431 from the National Institute of Environmental Health Sciences, NIH, and the Water Resources Research Institute of the University of North Carolina, respectively. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH, or the Water Resources Research Institute of the University of North Carolina. We thank Celine Gallon for assistance with sample preparation and Phil Wyatt for constructive comments on the text.

Literature Cited (1) Cronin, C.; Aiken, G. R. Geochim. Cosmochim. Acta 1985, 49, 1697. (2) Wershaw, R. L.; Aiken, G. R. In Humic Substances in Soil, Sediment, and Water; Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; Wiley: New York, 1985; pp 477-492. (3) Malcolm, R. L. Anal. Chim. Acta 1990, 23, 19. (4) Hayes, M. H. B.; MacCarthy, P.; Malcolm, R. L.; Swift, R. S. In Humic Substances II: In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; Wiley: New York, 1989; pp 690-731. (5) Gjessing, E. Nature 1963, 208, 1091. (6) Christman, R. F.; Ghassemi, M. Limnol. Oceanogr. 1968, 13, 583. (7) Stuermer, D. H.; Harvey, G. R. Nature 1974, 250, 480. (8) Tuschall, J. R.; Brezonik, P. L. Limnol. Oceanogr. 1980, 25, 495. (9) Davis, J. A.; Gloor, R. Environ. Sci. Technol. 1989, 15, 1223. (10) Aiken, G. R.; Gillam, A. H. In Humic Substances II: In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; Wiley: New York, 1989; pp 515-544.

(11) Visser, S. Plant Soil 1985, 87, 209. (12) Wershaw, R. L.; Pinckney, D. J. J. Res. U.S. Geol. Surv. 1973, 1, 70. (13) Wershaw, R. L.; Pinckney, D. J. In Contaminants and Sediments; Baker, R. A., Ed.; Ann Arbor Science: Ann Arbor, MI, 1980; p 207. (14) Wershaw, R. L.; Pinckney, D. J. J. Res. U.S. Geol. Surv. 1977, 5, 571. (15) Thurman, E. M.; Wershaw, R. L.; Malcolm, R. L.; Pinckney, D. J. Org. Geochem. 1982, 4, 27. (16) Black, A. P.; Christman, R. F. J. Amer. Water Works Assoc. 1963, 55, 897. (17) Wyatt, P. J. Anal. Chim. Acta 1993, 272, 1. (18) Serkiz, S. M.; Perdue, E. M. Water Res. 1990, 24, 911-916. (19) Kratochvil, P. In Light Scattering from Polymer Solutions; Huglin, M. B., Ed.; Academic Press: London, 1972; pp 333-384. (20) Swift, R. S. In Humic Substances II: In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; Wiley: New York, 1989; pp 549-565. (21) Murphy, E. M.; Zachara, J. M.; Smith, S. C.; Phillips, J. L.; Wietsma, T. W. Environ. Sci. Technol. 1994, 28 (7), 1291-1299. (22) Visser, S. A. In Aquatic and Terrestrial Humic Materials; Christman, R. F., Gjessing, E. T., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; pp 183-202. (23) Aiken, G. R.; Brown, P. A.; Noyes, T. I.; Pinckney, D. J. In Humic Substances in the Suwannee River, Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. M., Thorn, K. A., Eds.; USGS Report 87-557; USGS: Denver, CO, 1989; pp 163-178.

Received for review July 9, 1996. Revised manuscript received December 2, 1996. Accepted December 11, 1996. ES960594Z

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