Anal. Chem. 2005, 77, 4194-4200
Characterizing Dissolved Organic Carbon Using Asymmetrical Flow Field-Flow Fractionation with On-Line UV and DOC Detection Thorsten N. Reszat* and M. Jim Hendry
Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK, Canada, S7N 5E2
A method of characterizing dissolved organic carbon (DOC) by asymmetrical flow field-flow fractionation with on-line UV and DOC detection is described and applied to standards and natural water samples. Poly(styrenesulfonate) polymer standards, Suwannee River humic standards, and naturally occurring surface water and groundwater DOC were analyzed using this coupled detection technique. Molecular weight determinations in the samples and standards were 6-30% lower with DOC analysis than UV analysis. This difference was attributed to the insensitivity of the latter technique to nonaromatic carbon and suggests the molecular weight determined with the DOC detector is a more accurate representation of the actual molecular weight of the DOC. A normalized intensity comparison (NIC) method was proposed to distinguish differences in the relative amounts of aromatic and aliphatic carbon in DOC by comparing the two detector responses. The NIC method was applied to yield an average aromatic content of the bulk DOC and to detail the aromatic content over a range of molecular weights in a single DOC fraction. Naturally occurring dissolved organic matter (DOM) is ubiquitous in terrestrial and aquatic environments.1-4 DOM is frequently reported as dissolved organic carbon (DOC, e0.45 µm filtrate) and is dominantly composed of aquatic humic colloids in the form of higher molecular weight humic acids (HA), fulvic acids (FA), and lower molecular weight hydrophilic organic acids.5 Types of DOC (e.g., aquatic FA) play an important role in the geochemistry of pollutants because they can form metal-ligand complexes,6 enhance the solubility of nonpolar organic contami* Corresponding author. Phone: (306) 966-5683. Fax: (306) 966-8593. E-mail:
[email protected]. (1) Thurman, E. M. Organic Geochemistry of Natural Waters; Martinus Nijhoff/ Dr W. Junk Publishers: Dordrecht, 1985. (2) McCarthy, J. F.; Williams, T. M.; Liang, L.; Jardine, P.; Jolley, L. W.; Taylor, D. L.; Palumbo, A. V.; Cooper, L. W. Environ. Sci. Techno. 1993, 27, 667676. (3) Aravena, R.; Wassenaar, L.; Plummer, L. N. Water Resour. Res. 1995, 31, 2307-2317. (4) Crum, R. H.; Murphy, E. M.; Keller, C. K. Water Res. 1996, 30, 13041311. (5) Thurman, E. M. In Humic substances in soil sediment and water: Geochemistry, isolation, and characterization; Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; John Wiley and Sons: New York, 1985; pp 87103.
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nants,7 influence colloid stability,8 and participate in redox reactions.1,3,4,9 If various components of DOC (with different chemistry) are significantly different in molar mass, then the molecular weight of DOC components can be indirectly attributed to distinct structure and functional group concentrations. Determination of the molecular weights of components comprising the DOC yields information on the type of DOC present. This, in turn, can indicate how the DOC will function in the environment For example, determining the type of DOC present in natural waters can help identify the complexation ability of DOC fractions.10 Determining the molecular weight of DOC in groundwater systems may yield valuable insight into the effective porosity of the associated porous media.11 The molecular weight of DOC has been measured using a variety of methods. The most common of these methods are size exclusion chromatography,12,13 gel permeation chromatography,14 sequential ultrafiltration,15 and field-flow fractionation.15-19 The field-flow fractionation (FFF) technique is the gentlest method (6) Means, J. L.; Maest, A. S.; Crear, D. S. In The Technology of High-Level Nuclear Waste Disposal; National Technical Information Service, U.S. Department of Energy: Springfield, VA, 1987; pp 215-247. (7) Chiou, C. T.; Malcolm, R. L.; Brinton, T. I.; Kile, D. E. Environ. Sci. Technol. 1986, 20, 502-508. (8) Ranville, J. F.; Macalady, D. L. In Geochemical Processes, Weathering and Groundwater Recharge in Catchments; Sather, O., de Caritat, P., Eds.; Balkema: Rotterdam, 1997. (9) Scott, D. T.; McKnight, D. M.; Blunt-Harris, E. L.; Kolesar, S. F.; Lovely, D. R. Environ. Sci. Technol. 1998, 32, 2984-2989. (10) Stevenson, F. J. In Humic Substances in Soil, Sediment, and Water: Geochemistry, Isolation, and Characterization; Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; John Wiley & Sons: New York, 1985; pp 13-52. (11) Hendry, M. J.; Ranville, J. F.; Boldt-Leppin, B. E. J.; Wassenaar, L. Water Resour. Res. 2003, 39, 1194-1203. (12) Her, N.; Amy, G.; Foss, D.; Cho, J. Environ. Sci. Technol. 2002, 36, 33933399. (13) Perminova, I. V.; Frimmel, F. H.; Kudryavtsev, A. V.; Kulikova, N. A.; AbbtBraun, G.; Hesse, S.; Petrosyan, V. S. Environ. Sci. Technol. 2003, 37, 24772485. (14) Kim, J. I.; Buckau, G.; Li, G. H.; Duschner, H.; Psarros, N. Fresenius J. Anal. Chem. 1990, 338. (15) Assemi, S.; Newcomb, G.; Hepplewhite, C.; Beckett, R. Water Res. 2004, 38, 1467-1476. (16) Dycus, P. J. M.; Healy, K. D.; Stearman, G. K.; Wells, M. J. M. Sep. Sci. Technol. 1995, 30, 1435-1453. (17) Benedetti, M.; Ranville, J. F.; Ponthieu, M.; Pinheiro, J. P. Org. Geochem. 2002, 33, 269-279. (18) Beckett, R.; Jue, Z.; Giddings, C. J. Environ. Sci. Technol. 1987, 21, 289295. (19) Schimpf, M.; Wahlund, K.-G. J. Microcolumn Sep. 1997, 9, 535-543. 10.1021/ac048295c CCC: $30.25
© 2005 American Chemical Society Published on Web 05/18/2005
available for separations, being relatively nondestructive to samples. Other advantages include flexibility and ease of use with different carriers and membranes, rapid measurements, minimal cleanup between sample runs, and the widest dynamic operating range in terms of Mw of any separation method. Identification and molecular weight characterization of DOC is commonly determined using UV detection in the range of 254and 270-nm adsorption is associated with chromophores such as CdC and CdO double bonds, aromatic rings, and phenolic functional groups.5,20 However, UV spectroscopy may not be an accurate method to characterize molecular weights of DOC as the majority of DOC contains unsaturated bonds that cannot be resolved with UV detection.21-23 An alternate approach to UVbased detection systems was to couple a DOC analyzer with HPLC-size exclusion chromatography and gel chromatography.12,24,25 To date, studies using FFF have used only UV methods for DOC characterization and identification.16-18,26-29 Our primary objective was to develop a method to accurately characterize DOC in surface water and groundwater samples by modifying and optimizing a conventional total organic carbon (TOC) analyzer for use with a FFF-UV system. Secondary objectives were to compare results of the FFF-DOC to those of FFF-UV and develop and apply an interpretive method to utilize both continuous DOC and UV data. The objectives were attained using surface water and groundwater samples with concentrations of DOC ranging from 18 to 136 mg L-1. METHOD DEVELOPMENT Instrumentation Setup. Separation experiments were conducted using an asymmetrical flow field-flow fractionation (AsFlFFF) system (model HRFFF 10 000) from Postnova Analytics (FFFractionation, Salt Lake City, UT). The PC-controlled instrumentation consisted of an arrangement of pumps, an AsFlFFF channel, and a vacuum degasser (to remove air bubbles from the carrier solution that may interfere with separation efficiency in the AsFlFFF channel). The AsFlFFF system primarily separates particles by differences in their aqueous diffusion coefficient. Aqueous diffusion coefficient can be related to molecular weight by determining the retention times of known molecular weight standards having properties similar to DOC and through FFF theory.18 Particles, in this case, DOC are detected as they elute from the system after fractionation. The resulting plots of detector intensity versus time, called fractograms, consist of two regions: (20) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds, 5th ed.; John Wiley and Sons: New York, 1991. (21) Harvey, G. R.; Boran, D. A.; Chesal, L. A.; Tokar, J. M. Mar. Chem. 1983, 12, 119-132. (22) Tan, K. H. Humic Matter in Soil and the Environment: Principles and Controversies; Marcel Dekker Inc.: New York, 2003. (23) Malcolm, R. L. In Humic Substances in Soil, Sediment and Water; Aiken, G. R., McKnight, D. M., Wershaw, R. L., MacCarthy, P., Eds.; John Wiley & Sons: New York, 1985; pp 181-210. (24) Her, N.; Amy, G.; Foss, D.; Cho, J.; Yoon, Y.; Kosenko, P. Environ. Sci. Technol. 2002, 36, 1069-1076. (25) Huber, S. A.; Frimmel, F. H. Environ. Sci. Technol. 1994, 28, 1194-1197. (26) Hassellov, M.; Lyven, B.; Haraldson, C.; Sirinawin, W. Anal. Chem. 1999, 71, 3497-3502. (27) Lyven, B.; Hassellov, M.; Turner, D. R.; Haraldson, C.; Andersson, K. Geochim. Cosmochim. Acta 2003, 67, 3791-3802. (28) Geckeis, H.; Mahn, T. N.; Bouby, M.; Kim, J. I. Colloids Surf. A 2003, 217, 101-108. (29) Thang, N. M.; Geckeis, H.; Kim, J. I.; Beck, H. P. Colloids Surf. A 2001, 181, 289-301.
the void peak, which may contain some undifferentiated low molecular weight ligands, and the colloidal peak(s). To ensure organic material of interest was not lost through the membrane into the cross-flow during analysis, the AsFlFFF channel was fitted with regenerated cellulose acetate membranes (Millipore Corp.) with a nominal molecular weight cutoff of 1000 Da. A Rheodyne 100-µL manual sample injection loop was used to inject filtered ( groundwater). NIC values exhibit a strong linear correlation with molecular weight (r2 ) 0.96 for Mw and r2 ) 0.92 for Mn). If NIC reflects aromaticity, as suggested above, this relationship indicates aromaticity is a function of molecular weight for DOC. E4/E6 ratios (ratio of absorbance at 465 and 665 nm), used to determine characteristics of HA and FA, also exhibit a relationship between degree of aromaticity and molecular weight.5,38,39 Thurman showed humic acids in groundwaters have a consistently lower E4/E6 ratio than fulvic acids.5
Molecular Weight Specific NIC Analysis. The strong linear correlation between molecular weight and NIC values suggests a change in the chemical composition of DOC (increasing aromaticity) over the range of molecular weights in samples. To substantiate this molecular weight trend, we performed continuous (38) Kononova, M. M. Soil Organic Matter: Its Nature, Its Role in Soil Formation and in Soil Fertility, 2nd ed.; Pergamon Press: London, 1966. (39) Chen, Y.; Senesi, N.; Schnitzer, M. Soil Sci. Soc. Am. J. 1977, 41, 352358. (40) Aiken, G. R.; Malcolm, R. L. Geochim. Cosmochim. Acta 1987, 51, 21772184. (41) Janos, P. J. Chromatogr., A 2003, 983, 1-18.
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Figure 4. Continuous molecular weight specific NICc analysis on representative samples (a) SRNOM and (b) King Site 2.2 m. UV analyses are represented by ], DOC detector results by 4, and NIC values by O.
molecular weight specific NIC analysis (NICc) on all samples. We divided UV absorbance by DOC intensity at a given molecular weight, resulting in a continuum of NICc values over the entire range of measurable molecular weights in a sample. The NICc increased with increased molecular weight in all groundwater and surface water samples. The NICc values for SRNOM and a representative groundwater sample (Figure 4) increased from 0.53 to 0.95 from 600 to 5200 Da and from 0.52 to 0.96 from 600 to 2900 Da, respectively. All NICc values became unstable as the peak intensities approached baseline values, at about 5000 Da for SRNOM and at 2500 Da for the 2.2-m groundwater sample. The NICc profiles exhibited the same trend to increasing aromatic content with increasing in molecular weight of DOC as the NIC analysis. CONCLUSIONS A method of characterizing DOC by AsFlFFF with on-line UV and DOC detection was developed and applied to standards and natural groundwater and surface water samples. DOC and UV fractograms obtained with this technique were highly reproducible and can be interpreted through a simple molecular weight
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calibration approach. Our testing suggests DOC detection may be a more accurate method to characterize molecular weight of the DOC than UV detection; UV detection may bias molecular weights high because of the method’s inability to resolve aliphatics (usually of lower molecular weight). Coupling of the two detectors provides information on the dominant structures (aliphatic vs aromatic content) in DOC fractions. Distinct and repeatable differences observed with a NIC analysis of different samples support the ability of this method to characterize the aromaticity of DOC. DOC aromaticity was positively correlated with molecular weight, in both a bulk and molecular weight specific analysis. ACKNOWLEDGMENT This research was funded by research grants from the Natural Sciences and Engineering Council of Canada (NSERC) and the Potash Corporation of Saskatchewan.
Received for review November 17, 2004. Accepted April 12, 2005. AC048295C
