Humic and Fulvic Acids - American Chemical Society

Chapter 10

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on August 24, 2015 | http://pubs.acs.org Publication Date: November 14, 1996 | doi: 10.1021/bk-1996-0651.ch010

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Investigation of Fulvic Acid—Cu Complexation by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography with Post-Column Fluorescence Quenching Titration G. Christopher Butler and David K. Ryan Department of Chemistry, University of Massachusetts, Lowell, MA 01854 A n ion-pair reversed-phase high performance liquid chromatography (IP-RP-HPLC) separation with post-column addition of Cu and simultaneous U V and fluorescence detection is used to investigate the interactions between Cu and separated fractions of soil and water fulvic acids. In previous steady-state fluorescence experiments, binding characteristics for humic materials have been determined based on fluorescence quenching data from titrating the sample with paramagnetic metal ions. In the present study, the humic material fractions from the IP-RP-HPLC separation are "titrated on-line" with incrementally increasing concentrations of Cu and the extent of quenching of the fluorescence peaks is measured. A titration curve of percent fluores cence vs. metal concentration is plotted for each fraction and the ligand concentrations (C ) and conditional stability constants (K) are calculated from nonlinear regression of a one-site model applied to the data. 2+

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Humic substances such as humic and fulvic acid have been shown to affect transport, bioavailability, and toxicity of metals in natural waters (/). Fluorescence quenching measurements have been used to determine binding characteristics of these naturally occurring organic substances under conditions typically found in aquatic environments (2-4). These studies have employed titrations of humic materials with metal ion, yielding results which describe the overall binding properties of the humic substance. However, since humic substances are polydisperse, comprising a wide range of compounds with differing structures, molecular weights, spectroscopic characteristics, and chemical and complexing properties, the resulting binding parameters, Κ and C , are average values for the many fractions in the material being tested. L

0097-6156/96/0651-0140S15.00/0 © 1996 American Chemical Society

In Humic and Fulvic Acids; Gaffney, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Investigation of Fulvic Acid-Cu Complexation

In order to investigate the properties of individual fractions of humic substances, various modes of high performance liquid chromatography (HPLC) have been employed. Hydrophobic interaction chromatography (5) has proved to be an effective separation technique, resulting in five distinct humic fractions from one sample. Structural analysis of these fractions was subsequently performed by infrared and C nuclear magnetic resonance spectroscopy, and molecular weight distribution was also measured. Size exclusion chromatography (SEC) has been used to measure molecular weight (MW) distribution of humic substances (3, 6-9). Coupled with detection methods such as molecular fluorescence spectroscopy and dissolved organic carbon analysis (7), electrochemical detection (9), and atomic emission spectroscopy (3), SEC has been used extensively to study humic-metal complexes. A major disadvantage of SEC is that it does not provide adequate resolution for separating humic materials as they do not appear to be made up of distinct fractions with large differences in MW. Naturally-occurring humic-metal complexes have been isolated from estuarine systems and seawater using solid phase extraction (SPE) onto a C H P L C column to preconcentrate the sample (10-12). Samples were subsequently eluted from the SPE column at a much higher concentration and injected onto another H P L C column and detected by U V absorbance and a metal-sensitive detector, such as atomic fluorescence spectroscopy. The concentration of metal-humic complexes in natural aquatic environments was then calculated. However, there was some evidence of competitive binding of the metal ion between the organic matter and free silanol groups in the stationary phase resulting in a loss of metal in the column and erroneously low metal values (10). More recent developments in the study of humic substances by RP-HPLC include the use of stepwise gradients (13-15) and fluorescence emission and synchronous spectra (75, 16). Separations using stepwise gradients resulted in improved resolution and a greater number of fractions when compared with previous RP-HPLC experiments. The use of fluorescence emission (75, 16) and synchronous spectra (16) have lead to better understanding of the differences in spectroscopic characteristics of humic fractions. In one such study (75) it was observed that for humic-like marine dissolved organic matter fluorescing material was mainly present in the fractions of intermediate polarity, while non-fluorescing material was predominantly present in the more non-polar fractions. Studies have been performed utilizing HPLC coupled with fluorescence quenching to examine the metal-binding characteristics of humic fractions separated by size-exclusion chromatography (3) and IP-RP-HPLC (77) in an effort to determine if fractions had different binding and/or fluorescing characteristics. In these experiments, the modified Hummel-Dreyer mode of HPLC (18) was utilized, in which a constant concentration of the metal of interest is introduced in the mobile phase to prevent dissociation of the humic-metal complexes (9, 18). In HPLC-fluorescence quenching experiments, U V and fluorescence detectors were used to simultaneously collect chromatographic data. Fluorescence detection was utilized to measure free ligand since complexation of humic materials with


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HUMIC AND FULVIC ACIDS

paramagnetic transition metal ions has been shown to quench fluorescence. "Total" ligand was measured by U V detection, since complexation of the metal by the humic substance is not known to effect the absorbance of the molecule in the U V . In a study utilizing SEC and fluorescence, retention time increased with increased concentration of C u which in this mode of HPLC would indicate that there had been a decrease in molecular size (3). It was also hypothesized that ionic interactions between the humic-metal complex and charged surfaces such as free silanol groups in the column packing may have also contributed to the increased retention time. In addition to changes in retention time, there was a decrease in peak area with increased copper concentration for both the U V and fluorescence chromatograms. This may have been due to an irreversible binding (in the timescale of the separation) of copper-humic complexes to the stationary phase in the presence of increased Cu . In the case of IP-RP-HPLC (77) marked improvements in the separation of distinct fractions of fulvic acid were observed when no metals were present. However, when metal-ion containing mobile phase was introduced into the system, drastic changes occurred to both the U V and fluorescence chromatograms showing peaks merging or disappearing altogether and changing retention times. Since similar changes were shown for both the U V and fluorescence chromatograms, it was difficult to determine i f decreases in the fluorescence peaks were due to quenching or to loss of the humic metal complexes to the column. It is clear from the above studies that coupling HPLC and fluorescence using the Hummel-Dreyer mode leads to confusing results due to changes in the retention characteristics of the humic molecules in the column in the presence of metal ion. Ideally, the study of fluorescence quenching of fractions of humic materials on-line using HPLC and fluorescence detection should include a mode of H P L C which provides adequate ability to resolve fractions of humic materials as well as a means of introducing metals into the system in such a way that the chromatography of the humic sample is not disrupted. In the present study, humic samples are first separated by IP-RP-HPLC run in the isocratic mode. The column effluent is then mixed on-line with a copper reagent solution via a post-column mixing Τ connected to a reaction coil. In this way, eluting fractions of the humic sample are exposed to metal ion only after being separated, thereby eliminating the possibility that addition of metal will disrupt chromatographic performance.


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Experimental In the current study, IP-RP-HPLC was performed using a metal-free Dionex Gradient Pump Module (GPM) connected to a glass-lined C H P L C column (Scientific Glass Engineering model 250 GL4-ODS2-30/5) and a model PF-1 photodiode array (PDA) detector (Groton Technology, Inc.) and a dual monochromator fluorescence detector (model FD-300, Groton Technology, Inc.). A l l tubing, fittings, and detector flow cells were metal-free to minimize metal contamination. The post-column reagent system consisted of a second G P M pump capable of delivering known concentrations of C u by mixing predetermined 1 8

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proportions of the two reagent solutions. The outlet of the column and the reagent pump were connected by tubing to a mixing Τ followed by a reaction coil 12 inches in length. The reaction coil allowed mixing of the eluent and metal reagent streams with minimal band-broadening as well as providing time for complexation between the sample and metal ions. The mobile phase was 37% acetonitrile in H 0 with 6.0 m M tetrabutylammonium perchlorate and 0.05 Μ Ν^οφηο1ΐηο-βΐ1^68υ1Αοηΐο acid (MES) with a pH of 6.00 + 0.05. The reagent solutions were, A) 0.05 M MES, pH = 6.00 + 0.05 and B) 2.0 m M copper perchlorate and 0.05 M MES, pH = 6.00 + 0.05. The HPLC was run isocratically with an eluent flow rate of 0.7 mL/min and a post-column reagent flow rate of 0.1 mL/min. The monitor wavelength of the P D A was set at 254 nm and the fluorescence excitation and emission wavelengths were set at 332 nm and 442 nm, respectively. The concentration of C u in the postcolumn reagent was set by entering the percentage of solution A and percentage of solution Β on the reagent pump prior to beginning the chromatographic run and was held constant throughout. Subsequent sample runs were performed with the concentration of C u in the post-column reagent increased in increments of 0.1 mM, up to a maximum of 2.0 mM C u (100% reagent B). Humic samples were dissolved in a solution of 0.05 M MES and adjusted to pH = 6.00 + 0.05 for a final concentration of 500 mg/L. The injection volume was 80 μ ί .


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Results and Discussion Samples used to demonstrate this method were soil fulvic acid (SFA) and water fulvic acid (WFA), both well-characterized materials obtained from Dr. James H . Weber at the University of New Hampshire (19). Figure 1 shows that the chromatographic method resulted in four fractions separated for the SFA. The use of IP-RP-HPLC with the biological buffer MES resulted in sufficient separation to eliminate the need for gradients as have been used in previous studies (13-17). Simultaneous collection of U V (254 nm) and fluorescence (λ = 332 nm and λπτώβίοη 442 nm) data showed similar chromatograms with peaks at the same retention times except in the case of the more non-polar (later-eluting) fractions which did not exhibit measurable fluorescence. This result is similar to that reported by Lombardi et al. (15) for marine D O M . Figure 2 shows a very similar separation for WFA. An example of the quenching of fluorescence chromatograms of SFA appears in figure 3. A l l chromatograms are shown on the same scale with the concentration of C u in the post-column reagent given above the respective chromatogram. Peak heights and areas decrease with increased C u concentration as is expected, while the retention times and relative positions of the peaks remain unchanged (figure 3). However, a maximum decrease of approximately 10 % in U V peak area for each peak was observed at the higher C u concentrations. This may be due to precipitation of SFA in the presence of high concentrations of C u or other loss of SFA in the post-column system. Fluorescence chromatograms for each titration experiment were integrated 6Χ0ί1β1ίοη

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I Fluorescence

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Figure 1. Fluorescence (top, λπ = 332 nm, λ = 442 nm) and U V (bottom) (254 mn) chromatograms of SFA (500 mg/L) with an injection volume of 80 Eluent and post-column reagent (0.0 m M Cu ) flow rates were 0.7 mL/min. and 0.1 mL/min., respectively. 0ήΛύοη

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Figure 2. Fluorescence (top, λ ^ ^ ^ = 332 nm, λ ^ , ^ = 442 nm) and U V (bottom) (254 mn) chromatograms of W F A (499 mg/L) with an injection volume of 80 |iL. Eluent and post-column reagent (0.0 m M Cu ) flow rates were 0.7 mL/min. and 0.1 mL/min., respectively. 2+




0.0 mM Cu(ll)

0.2 mM Cu(ll)

0.5 mM Cu(ll)

2.0 mM Cu(ll)

Figure 3. Quenching of SFA fluorescence chromatograms with addition of Cu . Post-column reagent concentration of C u is listed above corresponding chromatogram. Conditions are the same as listed in figure 1. 2+

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and the areas of each individual peak, expressed as percent relative fluorescence for each experiment, were plotted against the corresponding concentration of C u in the reagent. The resulting quenching data was subjected to non-linear regression using the data treatment of Ryan and Weber (2) resulting in best-fit curves for each set of quenching data based on calculated values of /„., the residual fluorescence, C , the concentration of ligand, and K, the 1:1 conditional stability constant. The best-fit quenching curves for SFA in figure 4 shows similar quenching for the three peaks, although peak 1 quenches more steeply relative to C u concentration than peaks 2 and 3. The quenching of the three fluorescence peaks separated from W F A shown in figure 5 differs more widely in terms of steepness and residual fluorescence. Peak 1 of the W F A shows a similar shaped quenching curve compared with peak 1 of SFA while curves for peaks 2 and 3 of the W F A are more broad and shallow showing less quenching for a given C u concentration. This may be due to the less polar fractions being weaker binders of Cu . Similar quenching trends have been observed for Suwannee Stream fulvic acid and Aldrich humic acid. Table I lists binding parameters for SFA based on total C u concentration, correcting for the dilution of the C u ion when post-column reagent stream was mixed with the column eluent. While these values show that this data treatment works well for SFA, binding parameters for W F A showed negative values for C and / ^ , indicating that the quenching behavior of W F A is not fit by the model on which the data treatment is based. The fact that quenching of the W F A is not modelled well may be a result of the fundamental assumption of solution equilibrium not being met (2). 2+


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Table I. Fitted Binding Parameters for HPLC Post-Column Fluorescence Titration of SFA with Cu 2+

C μΜ (std dev)

K(* 10^) (std dev)

/RES

Peak 1

22.5 (+ 2.3)

101.2 (+55.8)

5.6 (+ 1.1)

Peak 2

33.3 (±6.2)

39.9 (+32.8)

10.2 (+2.2)

Peak 3

46.2 (+ 5.3)

26.07 (+ 13.0)

6.6 (+ 1.9)

Sample

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(std dev)

SFA

Since the quenching of the fluorescence of fractions eluting from the chromatographic column occurs as C u is added on-line, there may not be sufficient time for equilibrium to occur. This may also explain why data points in this study show greater deviation from the best-fit curves than has been observed in experiments performed under equilibrium conditions (2). However, based on a stability constant of 1.08 χ 10 for complexation at pH 6 (2), a rate constant in the 2+

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REAGENT Cu2+ CONC. (M)

Figure 4. Fluorescence peak area quenching (X^m™ = 332 nm, = 442 nm) of SFA chromatographic peak 1 (Δ), peak 2 (O), and peak 3 (•), and bestfit quenching curves for peak 1 (-), peak 2 (—) and peak 3 (...).

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Figure 5. Fluorescence peak area quenching (λ^^οη = 332 nm, λ = 442 nm) of W F A chromatographic peak 1 (Δ), peak 2 (O), and peak 3 (•), and best-fit quenching curves for peak 1 (-), peak 2 (—) and peak 3 (...). βπώβίοη


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Investigation ofFulvic Acid-Cu Complication


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range of 311.0 to 2948.4 s" (20) and a mixing time in the reaction coil and tubing of approximately 2.3 seconds, greater than 99 % complexation should occur before the humic material reaches the fluorescence detector, assuming a first-order rate constant and 1:1 humic:metal binding. Although it is evident from the quenching of fluorescence that binding does occur, it is possible in the time-scale of this experiment that some secondary effects of the binding on the humic molecules, such as intramolecular hydrogen bonding and conformational changes may not have time to occur. The complicated nature of the instrumentation required for these experiments may also result in artifacts which may cause the fluorescence quenching to not conform to the model. Fluctuations in the proportioning of solutions A and Β in the post-column reagent pump may result in error in the total C u concentration reported as well as variation in concentration of C u in the post-column system throughout an experiment. Likewise, fluctuations in pump flow rate of both the eluent and post-column reagent pumps may also effect the total C u concentration in the post-column system. Since each experiment was conducted over nine to twelve hours, temperature of the system and the fluorescence detector lamp output may also have contributed to variations in fluorescence quenching. A decreased run time for the chromatographic method and a decrease in the number of sample runs for each experiment may help to reduce this artifact. 2+

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Summary A new method for combining HPLC with fluorescence quenching titrations has been demonstrated and provides information on the binding characteristics of individual fractions of humic materials. By mixing the metal titrant with the column effluent post-column, the chromatographic separation of the sample is not disrupted by the addition of metal, as shown by the fact that the retention time and relative positions of the peaks do not change with increasing C u concentration in the solution. However, some small decreases in U V peak height and area were observed with increased Cu concentration. This method yields values for K, C , and /RES similar to those reported in previous studies, but also provides information about the metal binding behavior of the individual fractions which make up the humic material. This method may also be adapted to other modes of HPLC, resulting in greater understanding of the effects of metal-complexation on other properties of humic substances. 2+

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Literature Cited 1. Paulauskis, J. D.; Winer, R. W. Aquat. Toxicol. 1988, 12, 273. 2. Ryan, D. K.; Weber, J. H. Anal. Chem. 1982, 54, 986. 3. Ventry, L. S. K. Ph.D. Thesis, Northeastern University, 1989. 4. Ventry, L. S. K.; Ryan, D. K.; Gilbert, T. R. Microchem. J. 1991, 44, 201.


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7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20.

Kalinowski, E.; Blondeau, R. Mar. Chem. 1988, 24, 29. Knuutinen, J.; Virkki, L.; Mannila, P.; Mikkelson, P.; Paasivirta, J.; Herve, S. Water Res. 1988, 22, 985. Frimmel, F. H.; Gremm, T.; Huber, S. Sci. Total Environ. 1992, 117-118, 197. Chin, Y.; Aiken, G.; O'Loughlin, E. Environ. Sci. Technol. 1994, 28, 1853. Adamic, M. L.; Bartak, D. E. Anal. Chem. 1985, 57, 279. Mackey, D. J. Mar. Chem. 1985, 16, 105. Mills, G. L.; McFadden, E.; Quinn, J. G. Mar. Chem. 1987, 20, 313. Mackey, D. J.; Higgins, H. W. J. Chromatogr. 1988, 436, 243. Saleh, F. Y.; Chang, D. Y. Sci. Total. Environ. 1987, 62, 67. Lombardi, A. T.; Morelli, E.; Balestreri, E.; Seritti, A. Environ. Technol. 1992, 13, 1013. Lombardi, A. T.; Seritti, Α.; Morelli, E. in Humic Substances in the Global Environment and Implications on Human Health; Senesi, N.; Miano, T. M., Eds., Elsevier: Amsterdam, 1994, 851. Morelli, E.; Puntoni, F.; Seritti, A. Environ. Technol. 1993, 14, 941. Liu, X. M.S. Thesis, University of Massachusetts Lowell, 1993. Williams, R. F.; Aivaliotis, M. J.; Barnes, L. D.; Robinson, A. K. J. Chromatogr. 1983, 266, 141. Weber, J. H.; Wilson, S. A. Water Res. 1975, 9, 1079. Rate, A. W.; McLaren, R. G.; Swift, R. S. Environ. Sci. Technol. 1993, 27, 1408.


Humic and Fulvic Acids - American Chemical Society

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