Environ. Sci. Technol. 1996, 30, 3061-3065
Fluorescence Characterization of IHSS Humic Substances: Total Luminescence Spectra with Absorbance Correction JARAFSHAN J. MOBED, SHERRY L. HEMMINGSEN,† JENNIFER L. AUTRY, AND LINDA B. MCGOWN* Department of Chemistry, Gross Chemical Laboratory, Box 90346, Duke University, Durham, North Carolina 27708
Total luminescence spectroscopy was applied to the fluorescence characterization of humic substances obtained from the International Humic Substances Society (IHSS). Results show that total luminescence spectra, represented as excitation-emission matrices (EEMs), may be used to discriminate between soilderived and aquatic-derived IHSS humic substances and between humic and fulvic acids derived from the same source (soil or aquatic). Ionic strength in the range of 0-1 M KCl and humic substance concentration in the range of 5-100 mg/L had little effect on the fluorescence spectral characteristics of the humic substances, while pH had significant effects as expected. Absorbance correction was shown to be essential for accurate representation and comparison of the EEMs of the humic substances at high concentrations.
Introduction Humic substances are heterogeneous mixtures of high molecular weight organic compounds, both aromatic and aliphatic, that are rich in oxygen-containing functional groups (e.g., COOH, phenolic and/or enolic OH, alcoholic OH, and quinolic CdO) (1). They comprise approximately 60-70% of the total organic carbon in soils and 40-60% of dissolved organic carbon in natural waters (2). The vast diversity in structural components of humic substances is a result of the random polymerization of a variety of decomposed materials. Moreover, the composition of humic substances varies according to their source and method of extraction. It is this structural heterogeneity that has made the structural and conformational characterization of humic substances extremely challenging. Humic substances can be operationally defined according to their solubility in water: humic acids (HAs) are insoluble in aqueous solution with pH < 2 but soluble at * Corresponding author telephone: (919) 660-1545; fax: (919) 6601605; e-mail address:
[email protected]. † Present address: Chemistry Department, Ohio Wesleyan University, Delaware, OH 43015.
S0013-936X(96)00132-0 CCC: $12.00
1996 American Chemical Society
higher pH values; fulvic acids (FAs) are soluble in water under all pH conditions; and humins are insoluble in water at any pH (3). In spite of having many common functional groups and structural features, humic and fulvic acids differ significantly. Several degradative and spectroscopic techniques, including elemental analysis and 13C NMR, have been used to elucidate the structures of humic substances. Results show that FAs have lower molecular weights (5002000) and a smaller number of total and aromatic carbons than their HA counterparts (2000-5000), which in turn have longer chain fatty acid products and therefore a higher hydrophobicity than FAs (2). However, the utility of many analytical techniques for the study of humic substances is often subject to such limitations as poor sensitivity and the need for extractions or other physical treatment prior to measurement. Chemical and physical studies of humic substances and their interactions with environmental contaminants in the naturally occurring humic matrix are essential in order to elucidate their impact on the environmental fate, bioavailability, and toxicity of organic and metal contaminants (4). The sensitivity and nondestructive nature of fluorescence techniques are well suited to studies of the intact humic matrix. The intrinsic fluorescence of humic substances contains information relating to structure, conformation, and heterogeneity of humic substances as well as dynamical properties related to their intramolecular and intermolecular interactions (4, 5). Total luminescence spectroscopy provides a complete representation of the fluorescence spectral features of a sample in the form of an excitationemission matrix (EEM), in which fluorescence intensity is presented as a function of excitation wavelength on one axis and emission wavelength on the other. Previous work has shown that steady-state and phaseresolved total luminescence spectra provide good discrimination among commercial humic substances (6). In the present work, the application of total luminescence spectroscopy to the characterization and comparison of aquatic-derived and soil-derived humic substances from the International Humic Substance Society (IHSS) is demonstrated. Effects of humic substance concentration, ionic strength, and pH on the total luminescence spectra are described. Absorbance correction of the fluorescence spectra of solutions containing high concentrations of humic substances is shown to be essential for accurate representation. The fluorescence lifetime characterization of these samples will be described in a separate paper.
Experimental Section All samples were obtained from the International Humic Substances Society (IHSS). These included four aquaticderived samples: Suwannee River fulvic acid standard (SRFAS), Suwannee River humic acid standard (SRHAS), Nordic aquatic fulvic acid reference (NAFAR), and Nordic aquatic humic acid reference (NAHAR); and three soilderived samples: peat humic acid reference (PHAR), peat fulvic acid reference (PFAR), and soil humic acid reference (SHAR). The samples were used as received without further purification and dissolved in high-purity HPLC-grade water or aqueous KCl solutions. Concentration and pH studies were performed on all of the samples. In addition, ionic
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strength studies were performed on the soil-derived samples. All solutions were refrigerated, stored in the dark, and discarded after 1 week. For the ionic strength studies, a 100 mg/L stock solution was made for each humic sample by dissolving approximately 0.010 g of the humic substance in water in a 100-mL volumetric flask. The pH of the stock solution was adjusted to 6.5 using dilute HCl and NaOH solutions. Each stock solution was then filtered using a 0.22-µm Millipore filter to remove any undissolved particulate matter. A 2.0 M KCl stock solution was prepared and further diluted to give 0.20 and 0.020 M KCl solutions. Final solutions were obtained by diluting appropriate amounts of the humic stock solution with the KCl solutions to give 50 mg/L humic substance in 0, 0.010, 0.10, and 1.0 M KCl. Each solution was then adjusted to pH 6.5. For the concentration studies, a stock solution of 100 mg/L humic substance in 0.010 M KCl was prepared for each humic sample, and its pH was adjusted to 6.5. After the stock was filtered, appropriate dilutions were carried out to give additional concentrations of 5 and 50 mg/L. The pH of each solution was adjusted to 6.5. For the pH studies, solutions of 50 mg/L in 0.010 M KCl were prepared by dilution of the 100 mg/L stock solution. The pH was adjusted to 2.0, 6.0, or 10.0 using dilute HCl or NaOH. All spectral measurements were made using the SLM 48000S Phase-Modulation spectrofluorometer (Spectronics Inc.). A 450-W xenon arc lamp was used as the excitation source, and monochromators were used for excitation and emission wavelength selection. A series of emission spectra were collected over a range of excitation wavelengths to generate EEMs. The wavelength step size was 4 nm, and each intensity was measured as the internal average of five signal samplings. The excitation wavelength range was 300-500 nm. The emission wavelength range was 390590 nm for the ionic strength and concentration studies. For the pH studies, an emission range of 350-550 nm was used because of a blue-shift that was observed at pH 2 for some samples; exceptions are the two peat samples, for which an emission range of 390-550 nm was used. The entrance/exit slits on the monochromators were set to 16 nm/2 nm. The sample compartment temperature was maintained at 20.0 ( 0.1 °C by a Haake A81 temperature control unit. The EEMs were collected using both SLM and in-house macro programs and analyzed using SLM software and programs written in MATLAB. Results were presented graphically by using CorelDRAW (6). The EEMs were compared using the uncorrected matrix correlation (UMC) method, which compares two entire matrices (7). A UMC value of 1 indicates that the two matrices are identical and decreases to approach zero with decreasing correlation. Absorbance spectra were used for correction of both primary and secondary inner filtering effects (8, 9) in the EEMs. The absorbance spectra in the range of 200-600 nm were collected for all samples using a Perkin-Elmer UV/visible Lambda 6 spectrophotometer. Absorbance corrections were performed using an in-house program written in MATLAB (6).
Results and Discussion Absorbance Correction of Fluorescence Spectra. Since the solution concentrations in these studies were relatively high, it was necessary to correct all of the EEMs for primary and secondary inner filtering effects due to high absorbance.
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FIGURE 1. Uncorrected (left) and absorbance-corrected (right) EEMs of an aquatic fulvic acid (SRFAS) at various concentrations (pH 6, 0.010 M KCl).
FIGURE 2. Uncorrected (left) and absorbance-corrected (right) EEMs of a soil humic acid (PHAR) and an aquatic humic acid (SRHAS), (50 mg/L, 0.010 M KCl, pH 6.0).
High concentrations were used in order to obtain adequate signal-to-noise for fluorescence lifetime measurements of the humic samples, which will be described in a separate paper. Figures 1 and 2 illustrate the importance of absorbance correction for solutions containing high concentrations of absorbers. In the uncorrected spectra in
TABLE 1
UMC Values for Concentration Studies, Comparing EEMs of a Sample at Two Concentrations (pH 6.0, 0.010 M KCl) UMC values
concn compared (mg/L)
NAFAR
SRFAS 0.9979 0.9974 0.9996
NAHAR 0.9992 0.9982 0.9985
SRHAS 0.9991 0.9970 0.9975
PFAR 0.9994 0.9982 0.9994
PHAR 0.9990 0.9990 0.9987
SHAR
5/50 5/100 50/100
0.9993 0.9985 0.9990
0.9972 0.9958 0.9998
mean ( SD
0.9989 ( 0.0004 0.9983 ( 0.0012 0.9986 ( 0.0005 0.9979 ( 0.0011 0.9990 ( 0.0007 0.9989 ( 0.0002 0.9976 ( 0.0020
is increased. However, the UMCs did not indicate a statistical difference between the different ionic strengths for PFAR at the 96% confidence level, which suggests that the visual differences are within the measurement uncertainty. It appears from these results that comparisons among humic samples will generally be independent of ionic strength.
FIGURE 3. Absorbance-corrected EEMs of PFAR (50 mg/L, pH 6.0) at different ionic strengths.
Figure 1, as humic substance concentration increases, the fluorescence maxima shift to longer excitation and emission wavelengths. This apparent red shift is due to attenuation of the fluorescence emission in the shorter wavelength region due to inner filtering, which results from the high absorbance at the short wavelengths. The red shift is not observed in the corrected spectra. If absorbance correction were ignored, shifts in peak maxima with increasing concentration would be erroneously attributed to actual changes in the fluorescence spectral features of the humic substances instead of to the inner filtering effects. Figure 2 shows the uncorrected and corrected EEMs of PHAR and SRHAS at 50 mg/L. The uncorrected spectra, particularly of the SRHAS, are significantly different from the corrected spectra, and the differences between the two samples are greater in the corrected spectra. The UMC decreases from 0.9525 between the uncorrected EEMs of SRHAS and PHAR to 0.9297 between the corrected EEMs of the two samples. This illustrates the necessity of absorbance correction for accurate comparison between different humic samples. Ionic Strength Studies. Ionic strength in the range of 0-1 M KCl has no significant effect on the spectra of the aquatic humic samples nor on the spectra of the soil-derived humic acids PHAR and SHAR, as indicated both by visual examination of the EEMs and by comparison of the UMCs of the EEMs. A small effect is observed for the fulvic acid PFAR (Figure 3), in which the intensity of the small peak to the red of the dominant peak decreases as ionic strength
Concentration Studies. Spectra were collected for all of the samples at concentrations of 5, 50, and 100 mg/L. The spectral features are essentially independent of concentration in this range, as was illustrated in Figure 1 for the aquatic-derived SRFAS. The UMC values between the EEMs of a sample at different concentrations are shown in Table 1 for the seven humic samples. None of the UMCs indicate a statistical difference between any of the concentrations. Changes in spectral features due to aggregation of humic substances at high concentrations have been reported for a commercial fulvic acid (5). Such changes were not observed in the present work, possibly due to the relatively low ionic strength (0.010 M KCl) of the sample solutions used in the concentration studies. It is also possible that different humic materials have different spectral responses to intermolecular aggregation. pH Studies. Figure 4 shows the EEMs for NAHAR and PFAR at a pH of 2, 6, and 10. For both samples, increasing pH causes a red shift in the longer wavelength peak region. Similar trends were observed for the other samples. The red shift, which was previously observed for IHSS humic substances (10-14), may be attributed to the changes in fluorescence characteristics of the acidic functional groups (phenols and phenolates) in the humic molecules. Phenols are known to exhibit two fluorescence maxima, of which the one at longer wavelengths becomes dominant in high pH solutions (15). Spectral shifts may also reflect changes in the conformations of the humic molecules at different pH, which could cause changes in the exposure of functional groups to the solvent. For NAHAR, increasing pH also causes a blue shift in the short wavelength peak near 320 nm excitation. The blue shift was observed for all of the aquatic samples and may be attributed to increased isolation of certain fluorophores from the bulk aqueous solvent (14). In contrast to the aquatic samples, the soilderived samples tend to show a red shift in the short wavelength region. For all samples, the fluorescence intensity increases with increasing pH, which has been previously reported (13, 14). Table 2 shows the comparison at a given pH between HAs and FAs from the same source and between FAs or HAs derived from different sources. By using the UMCs from Table 1 as replicates, since there was no significant change with concentration, it is possible to perform a Q-test
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FIGURE 4. Absorbance-corrected EEMs of an aquatic humic acid (NAHAR) and a soil fulvic acid (PFAR) at different pHs (50 mg/L, 0.010 M KCl).
FIGURE 5. Absorbance-corrected EEMs of aquatic (top) and soil (bottom) humic substances (50 mg/L, 0.010 M KCl, pH 6.0).
for significant differences between samples. Significant differences between EEMs at the 96% confidence level are indicated in Table 2 by the values given in boldface type. All of the comparisons show significant differences except for the soil and peat humic acids at pH 6. The pH yielding best discrimination is strongly dependent on which samples are being compared. For example, pH 10 provides best discrimination between FAs from different sources, while pH 6 provides best discrimination between aquatic and soil HAs. In some cases, all three pHs provide similar discrimination. A pH of 6 was selected as the standard condition for subsequent experiments. Comparison of Humic Substances. The EEMs of all of the IHSS samples (50 mg/L, 0.010 M KCl, pH 6.0) are shown in Figure 5. The HAs have fluorescence maxima at longer excitation and emission wavelengths than the corresponding FAs. These results are in agreement with those of
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previous studies (11-14). A red shift in the fluorescence maxima in the HAs relative to the FAs from similar sources is attributed to the presence of high molecular weight fractions, electron-withdrawing substituents, and a higher degree of conjugation in the HAs. The presence of electrondonating substituents in the FA structure contributes to the relatively short wavelengths of the fluorescence maxima. Intensities cannot be compared among the spectra in Figure 5 because each sample was measured under different detector settings in order to optimize the signal. However, qualitative comparison did show that the FAs are generally more intensely fluorescent than the HAs. This has been previously attributed to the highly substituted aromatic structure of HAs, inter- and/or intramolecular bonding of HAs, or self-quenching in the HA solutions (13, 16). Figure 5 also shows that the fluorescence spectral maxima of the soil samples are at longer excitation and emission wave-
TABLE 2
UMC Values for Comparison between Different Samples at a Given pH (50 mg/L, 0.010 M KCl)a Comparing a Humic Acid and a Fulvic Acid from the Same Source aquatic samples soil samples pH SRFAS/SRHAS PFAR/PHAR 2 6 10
0.9851 0.9865 0.9860
0.9324 0.8774 0.8622
Comparing Two Humic Acids or Two Fulvic Acids from Different Aquatic or Soil Sources aquatic samples soil samples pH SRFAS/NAFAR SHAR/PHAR 2 6 10
0.9900 0.9915 0.9790
0.9815 0.9967 0.9832
Comparing Two Humic Acids or Two Fulvic Acids from an Aquatic Source and a Soil Source humic acids fulvic acids pH SRHAS/SHAR NAFAR/PFAR 2 6 10
0.9505 0.8775 0.9404
0.9925 0.9943 0.9734
a
A significant difference at the 96% confidence level is indicated by UMC in boldface type.
TABLE 3
UMC Values for IHSS Samples (50 mg/L, 0.010 M KCl, pH 6.0)a solutions compared
UMC values
comments
NAFAR/SRFAS NAFAR/PFAR SRFAS/PFAR NAHAR/SRHAS NAHAR/PHAR NAHAR/SHAR SRHAS/PHAR SRHAS/SHAR PHAR/SHAR
0.9877 0.9943 0.9945 0.9949 0.9583 0.9553 0.9297 0.9252 0.9967
aquatic FAs from different sources aquatic vs soil FAs aquatic vs soil FAs aquatic HAs from different sources aquatic vs soil HAs aquatic vs soil HAs aquatic vs soil HAs aquatic vs soil HAs soil HAs from different sources
a A significant difference at the 96% confidence level is indicated by UMC in boldface type.
lengths than those of the aquatic samples. Other investigators made a similar observation when comparing the uncorrected synchronous excitation spectra of humic substances from various sources (10, 12). Table 3 shows the UMCs between the various EEMs that are shown in Figure 5. Significant differences were obtained for most comparisons; the exceptions were SRFAS and PFAR (aquatic vs soil FAs), NAHAR and SRHAS (aquatic HAs from different sources), and PHAR and SHAR (soil HAs from different sources). The results of these studies show that fluorescence EEMs may be used to discriminate between humic substances
from different sources and between humic acids and fulvic acids. A pH of 6 provides significant discrimination among most humic substances. A pH of 2 or 10 can provide good discrimination as well, but these represent extreme solution conditions that generally are not representative of thenatural environment of the humic matrix. Ionic strength in the range of 0-1 M KCl and humic substance concentration in the range of 5-100 mg/L do not significantly affect the fluorescence spectral features of the humic materials. This could simplify the collection and analysis of environmental samples by eliminating the need to exactly control these two factors. Absorbance correction for elimination of inner-filtering effects in the EEMs of high concentration solutions of humic substances was shown to be essential for accurate comparison between EEMs.
Acknowledgments This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, United States Department of Energy (Grant DEFG0588ER13931) and by the Office of Exploratory Research of the United States Environmental Protection Agency (Grant R822251-01).
Literature Cited (1) Stevenson, F. J. Humus Chemistry: Genesis, Compositions, Reactions, 2nd ed.; John Wiley and Sons, Inc.: New York, 1994; pp 31-33. (2) Senesi, N. In Organic Substances in Soil and Water: Natural Constituents and Their Influences on Contaminant Behavior; Beck, A. J., Jones, K. C., Hayes, M. H. B., Mingelgrin, U., Eds.; The Royal Society of Chemistry: Cambridge, 1993; pp 74-77. (3) MacCarthy, P. In Aquatic Humic Substances: Influence in Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry Series 219; American Chemical Society: Washington, DC, 1989; p xx. (4) Leenheer, J. A.; Brown, P. A.; Noyes, T. I. In Aquatic Humic Substances: Influence in Fate and Treatment of Pollutants; Suffet, I. H., MacCarthy, P., Eds.; Advances in Chemistry Series 219; American Chemical Society: Washington, DC, 1989; pp 25-39. (5) Lochmuller, C. H.; Saavedra, S. S. Anal. Chem. 1986, 58, 1978. (6) Hemmingsen, S. L. Dissertation, Duke University, 1995. (7) Burdick, D. S.; Tu, X. M. J. Chemom. 1989, 3, 431. (8) Tucker, S. A.; Amszi, V. L.; Acree, W. E., Jr. J. Chem. Educ. 1992, 69, A8. (9) Acree, W. E., Jr.; Tucker, S. A.; Fetzer, J. C. Polycyclic Aromat. Compd. 1991, 2, 75. (10) Senesi, N. Anal. Chim. Acta 1990, 232, 77. (11) Senesi, N.; Miano, T. M.; Provenzano, M. R.; Brunetti, G. Soil Sci. 1991, 152, 259. (12) Senesi, N.; Miano, T. M.; Provenzano, M. R.; Brunetti, G. Sci. Total Environ. 1989, 81-82, 143. (13) Miano, T. M.; Sposito, G. Soil Sci. Soc. Am. J. 1988, 52, 1016. (14) Pullin, M. J.; Cabaniss, S. E. Environ. Sci. Technol. 1995, 29, 1460. (15) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum: New York, 1983. (16) Seitz, W. R. In Treatise on Analytical Chemistry, Part 1, 2nd ed.; Elving, P. J., Meehan, E. J., Kolthoff, I. M., Eds.; Wiley: New York, 1981; Vol. 7, p 159.
Received for review February 13, 1996. Revised manuscript received May 20, 1996. Accepted May 28, 1996.X ES960132L X
Abstract published in Advance ACS Abstracts, August 1, 1996.
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