ANALYTICAL SCIENCES FEBRUARY 2000, VOL. 16 125 2000 © The Japan Society for Analytical Chemistry

Simultaneous Determinations of the Concentration and Molecular Weight of Humic Substances in Environmental by Gel Chromatography with a Fluorescence Detector

Etsu YAMADA,*,**† Kenichi DOI,* Katsuya OKANO,* and Yasuro FUSE**

*Department of Chemistry and Material Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606Ð8585, Japan **Center for Environmental Science, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606Ð8585, Japan

Simultaneous determinations of the concentration and molecular weight of humic substances in concentrated and unconcentrated environmental water samples by gel chromatography with a fluorescence detector were carried out. The calibration curves of humic and fulvic acid were linear in the concentration range of 0.1 to 10 mg/l. The relative standard deviations of humic substances were less than 10%. The concentrations of aquatic humic substances in rivers were high in the summer and low in the winter. The molecular weight of most humic substances in Katsura, Uji, Kidzu and Yodo Rivers was estimated to be about 3000 Ð 10000. The ratio of the higher molecular weight of humic substances was larger in the warmer season than that in the cooler season. The main origin of aquatic humic substances in river water may be humic substances from around the rivers.

(Received August 17, 1999; Accepted November 12, 1999)

Yodo rivers. Introduction

Dissolved humic substances, the main constituents of the Experimental pool in surface water (freshwater and marine water), groundwater, and porewater, commonly Reagents and apparatus impart a yellowish-brown color to the water system.1 Humic Diethylaminoethyl(DEAE)-cellulose (Wako Pure Chemicals, substances strongly interact with toxic heavy metals2,3 and may Osaka, Japan) was washed with 0.5 M for 1 h be the precursors of trihalomethane (THM) formed during water and distilled water, sequentially washed with 0.5 M sodium treatment with chlorine.4Ð7 However, few studies about the hydroxide for 1 h and distilled water, and stored in distilled behavior of humic substances in environmental water have been water at pH 7. Humic acid (Aldrich Chemicals, extracted from reported. In our previous study, humic and fulvic in soil) was purified by repeated dissolution in 0.1 M sodium environmental water were determined by UV spectrophotometry hydroxide and precipitation in 0.1 M hydrochloric acid until the after preconcentration with diethylaminoethyl(DEAE)-cellulose color of the supernatant solution was eliminated. Fulvic acids and separation at pH 1 by centrifugation; the relationship obtained from A-horizons of a brown forest soil (Dystric between aquatic humic substances and THM formation was Cambisol, Dando, Aichi, Japan) and an ando soil (Humic further examined.6 In order to clarify the behavior of humic Andozol, Inogashira, Shizuoka, Japan) were used. Fulvic acids substances in environmental water, it is important to determine prepared according to the IHSS (International Humic not only the concentration, but also the molecular weight of Substances Society) method were supplied by the Japan Humic humic substances, because they are mixed molecular Substances Society and used as a standard without compounds with hundreds to several hundred thousand of purification.12 A Pharmacia Biotech low-molecular-weight gel- molecular weight. Many studies about the sizes and shapes of filtration calibration kit (blue dextran, 200000; bovine serum humic substances by gel chromatography (such as Sephadex albumin, 67000; ovalbumin, 43000; chymotrypsinogen a, gel) have been reported.8 However, few studies of humic 25000; ribonuclease a, 13700), cytochrome c (12400) and substances by HPLC have been carried out, except for those by vitamin B12 (1355) were used as standards for determining the Susic.9,10 Fluorescence in humic substances, which contain molecular weight of humic and fulvic acids. All of other aromatic carboxyl and phenolic groups, is very sensitive.11 chemicals were of the best commercial grade. In this study, simultaneous determinations of the concentration The apparatus used for HPLC was a Shimadzu LC-10AD and molecular weight of humic substances in environmental water chromatography pump equipped with a Shimadzu SPD-6A were investigated by gel chromatography with a fluorescence ultraviolet detector and a Shimadzu RF-535 fluorescence detector. The present methods were applied to the direct detector. The wavelength of the ultraviolet detector was set at determination of humic substances in Katsura, Uji, Kidzu and 280 nm. The excitation and emission wavelengths of the fluorescence detector were 340 nm and 435 nm, respectively. A †To whom correspondence should be addressed. Shimadzu C-R7A chromatopac was used for data analysis. The 126 ANALYTICAL SCIENCES FEBRUARY 2000, VOL. 16 test samples were applied to a gel-filtration column (Pharmacia Biotech Superose12 HR10/30 (300×10 mm i.d.)) by a sample injector (Rheodyne 7125) with a 100 µl loop. A 0.0l M solution was used as an eluent at a flow rate of 0.4 ml minÐ1. Two centrifuges, a Kubota KN-70 and a Millipore Tibitan, were used to separate humic substances. Ultrafilter membranes (Millipore ultrafree-LC centrifugal filter, available in 3000, 5000 and 10000 nominal molecular weight limit) were used for the fractionation of humic and fulvic acids.

Procedure for the determination of humic and fulvic acids in river-water samples after preconcentration Water samples were collected from the Katsura, Uji, and Kidzu rivers and the Yodo river. The sampling stations were previously described.6 In the laboratory, all samples were filtered through a membrane filter (0.45 µm, Millipore) as soon as possible to avoid biodegradation. Membrane filters were used after washing with 1 M hydrochloric acid and distilled water. The preconcentration of humic and fulvic acids in river-water samples was carried out according to a previous study.6 A filtered 500-ml water sample was passed through a DEAE- cellulose column (25 mm×6 mm i.d.) at a flow rate of 3 ml minÐ1 in order to sorb humic substances. The humic substances were then desorbed by backward-flow elution with 10 ml of 1 M sodium hydroxide at a flow rate of 0.3 ml minÐ1. The effluent collected in a centrifuge tube was acidified to pH 1 with 2 ml of hydrochloric acid to precipitate humic acid. The precipitated humic acid and supernatant solution were separated by centrifugation at 4600 rpm for 1 h. Ten milliliters of a supernatant solution was carefully pipetted out and diluted to 25 ml with distilled water after neutralizing with 10 M sodium hydroxide. To the remaining solution, 8 ml of 1 M sodium Fig. 1 Chromatograms of Aldrich humic acid (A) and Inogashira hydroxide was added to dissolve the precipitated humic acid, fulvic acid (B) with a fluorescence detector and a UV detector at 280 neutralized with hydrochloric acid, and then diluted to 25 ml. nm. Eluent: 0.01 M NaOH. After an aliquot of both solutions was adjusted to a 0.01 M sodium hydroxide solution, 100 µl of each solution was measured by gel chromatography with a fluorescence detector. This peak position corresponds to those of Aldrich humic acid The concentration of humic acid was calculated by using the total and fulvic acids (Inogashira and Dando). The effects of the pH peak areas of all peaks, except for the peak at Rt=56 min. and ionic strength on the fluorescence of humic substances were However, the concentration of fulvic acid was calculated by using investigated. The fluorescence intensities of humic substances the total peak areas of all peaks. Moreover, the concentrations reached their maximum at pH 6 Ð 10, but those at pH 12 were 80 of humic and fulvic acids were calculated by considering the Ð 90% of the maximum values. Furthermore, the fluorescence remaining humic acid in the supernatant solution. intensities of humic substances were almost constant at ionic strengths of 0.01 to 0.2 M, which were adjusted with sodium Procedure for the direct determination of humic substances in chloride. These results were similar to those by Nagano et al.13 river-water samples When the fluorescence intensity of Aldrich humic acid was 1.0, A 100 µl aliquot of filtrated river-water samples without those of the Inogashira and Dando fulvic acids were 1.88 and preconcentration after adjusting to 0.01 M sodium hydroxide 1.04, respectively. The difference in the fluorescence between solution was measured by gel chromatography with a humic substances may be dependent on the difference of the fluorescence detector (chromatogram A). Further, a filtered functional groups and the organic compounds which make up river-water sample which had passed through a DEAE-cellulose humic substances. Although the calibration curves of humic column was similarly measured (chromatogram B). The acid and fulvic acids used as standard samples were linear in the concentration and molecular weight of humic substances in concentration range of 0.1 to 10 mg/l, the fluorescence river-water samples were calculated by a chromatogram (C), intensities became lower at above 20 mg/l. The relative obtained from the difference between (A) and (B). The standard deviations of humic substances were less than 10%. concentration of humic substances was calculated with reference to the standard of fulvic acid, because fulvic acid is Optimum conditions for the gel-filtration method of humic predominant in river water. substances Many different kinds of gel chromatography media are commercially available, including dextran gels, polyacrylamide Results and Discussion gels, agarose gels and porous glass beads.8 In this study, an agarose gel, Pharmacia Biotech Superose12 HR10/30, was used. Fluorescence characteristics of humic substances Sorption and other problems due to interactions between humic Humic substances in river-water samples exhibit one peak at substances and gel have been reported. Therefore, the effects of an excitation of 340 Ð 350 nm and emission at 430 Ð 450 nm. ion exclusion and sorption on the separation of humic ANALYTICAL SCIENCES FEBRUARY 2000, VOL. 16 127

Table 1 Analytical results of fulvic and humic acids in river water by gel chromatography with a fluorescence detector (FD) after preconcentration (December, 1995)

Sampling station Fulvic acid/mg lÐ1 Humic acid/mg lÐ1

Katsura River 11 0.42 0.029 12 0.40 0.042

Uji River 21 0.40 0.029

Kidzu River 31 0.72 0.052 3F 0.69 N.D. 3G 1.01 N.D. 3H 0.94 0.023

Yodo River 41 0.81 N.D.

substances by this gel were studied. Five kinds of eluents, namely distilled water, a 0.1 M ammonia solution, 0.001 M and 0.01 M sodium hydroxide solutions, and a 0.5 M Tris buffer were examined. The sorption of humic substances occurred in Fig. 2 Chromatograms of humic substances in the Kidzu river elution with distilled water. Ion exclusion of humic substances without preconcentration in August, 1995. (A) direct analysis of was observed in elution with a 0.1 M ammonia solution and a river water after filtration; (B) analysis of river water through a 0.001 M sodium hydroxide solution. Although neither the DEAE-cellulose column; (C) the difference between chromatograms sorption nor ion exclusion of humic substances took place in (A) and (B). elution with 0.01 M sodium hydroxide solution and the 0.5 M Tris buffer, the separation and sensitivity with 0.01 M sodium hydroxide were higher than those with the 0.5 M Tris buffer. Furthermore, it was found that the concentrations of humic Rt=32 min were estimated to be 5000 Ð 10000 and 3000 Ð 5000, substances in the range of 0.5 to 200 mg/l have no effect on the respectively. These results were almost the same as those by retention time and profile of the humic substance fractions. The gel chromatography. The molecular weights of small peaks at molecular weight of fulvic acid by gel chromatography eluted 18 min, 35 min, and 56 min with a UV detector at 280 nm were from Sephadex G-75 with 0.01 M sodium hydroxide solution about 200000, 5000, and 500, respectively. The molecular was confirmed to be the same as that determined by vapor- weight of a broad peak was in the range of 5000 Ð 11000. pressure osmometry.14 Since this result indicated that the molecular weight of humic substances may not change with this Application for the determination of humic substances in Yodo eluent, 0.01 M sodium hydroxide was chosen as the eluent. Rivers after preconcentration The concentrations of humic and fulvic acids in the Katsura, Determination of the molecular weights of humic substances Uji, Kidzu, and Yodo rivers were determined by the present The chromatograms of humic and fulvic acids with a method after preconcentration (Table 1). The analytical results fluorescence detector and a UV detector at 280 nm are shown in of fulvic acid by gel chromatography with a fluorescence Fig. 1. In the case of humic acid, two comparatively large peaks detector were in good agreement with those by UV at 29 min and 30 min, and an additional three small peaks at 32 spectrophotometry.6 On the other hand, the values of humic min, 35 min, and 56 min were detected with a fluorescence acid by UV spectrophotometry were always higher than those detector. On the other hand, with a UV detector at 280 nm, a by gel chromatography with a fluorescence detector. These small peak of high molecular weight at 18 min was detected and results indicate that besides humic acid several substances with three peaks at 29 min, 30 min, and 32 min became a broad peak an absorbance at 350 nm may be present in river water. at around 30 min. This result suggests that the fluorescence intensity of the humic-acid fraction with a high molecular Evaluation of the direct determination of the concentration and weight may be lower than those with a low molecular weight. molecular weight of humic substances in river-water samples In the case of fulvic acid, a large peak at around 29 min and two The direct determination of the concentration and molecular small peaks were detected with a fluorescence detector. The weight of humic substances in river-water samples was retention time and profile of the fulvic-acid fractions with a UV examined, because the properties of humic substances, such as detector at 280 nm are similar to those with a fluorescence the molecular weight and fluorescence, may change during the detector. process of preconcentration. Chromatograms of humic The molecular weights of humic substances were determined substances in the Kidzu river (without preconcentration) are by means of a calibration graph obtained by plotting the elution shown in Fig. 2 as an example. Several small peaks were volumes of proteins of known molecular weight against the detected after Rt=32 min in chromatogram (B) of river water logarithm of their molecular weight. The molecular weights of through a DEAE-cellulose column. Chromatogram (C), separated peaks with a fluorescence detector were estimated to obtained from the difference between chromatograms (A) and be 7500 for peak 1 at Rt=29 Ð 30 min and 6000 for peak 2 at (B), was similar to that of fulvic acid after preconcentration and Rt=32 min. The molecular size of humic substances was also separation. Because the standard humic substances used in this determined by fractionation with ultrafilter membranes. The study were quantitatively sorbed on a DEAE-cellulose column, molecular weights of peak 1 at Rt=29 Ð 30 min and peak 2 at several fluorescent organic compounds passing through the 128 ANALYTICAL SCIENCES FEBRUARY 2000, VOL. 16

Fig. 3 Chromatograms of humic substances in river water samples without preconcentration. (A) August 1995, (B) December 1995.

DEAE-cellulose column may be present in river water. The Table 2 Seasonal changes in the ratio of peak1/peak2 calculated by amounts of these compounds were larger at the stations where chromatograms of humic substances in river anthropogenic contamination enters the rivers. In the , Ratio of peak 1/peak 2 several types of fluorescent signals were observed, including Sampling humic-like, tyrosine-like, and triptophan-like signals.15 Several station Aug. 1995 Sep. 1995 Dec. 1995 Oct. 1996 Dec. 1996 fluorescent compounds in rivers should be further investigated. The concentrations (y) of humic substances directly determined Katsura River by gel chromatography with a fluorescence detector were in 11 0.97 — 0.74 1.32 1.08 13 — — 0.44 0.34 0.60 good agreement with those (x) of fulvic acid after preconcentration; y=0.991x+0.022 (r=0.933, n=16). The Uji River present method makes it possible to determine both the 21 0.84 0.79 0.77 1.18 1.14 concentration and molecular weight of aquatic humic substances in environmental water rapidly. Kidzu River Figure 3 shows the chromatograms of humic substances in 31 1.34 0.92 0.71 0.80 0.63 water samples of the Yodo rivers collected in August and Yodo River December, 1995. The concentrations of humic substances in 41 1.37 — 0.63 0.76 0.58 these rivers were larger in the following order: Uji river Katsura river < Yodo river < Kidzu river. Three peaks (peak 1 at Rt=29 Ð 30 min, peak 2 at Rt=32 min, and peak 3 at Rt=35 min) were detected in all river-water samples. From these rivers were higher in warmer seasons and lower in cooler results, the molecular weight of most humic substances in seasons. Next, seasonal changes in the molecular weight of Katsura, Uji, Kidzu and Yodo rivers was estimated to be about humic substances were investigated. The seasonal changes in 3000 Ð 10000, which is the same as that of standard humic the ratio of peak 1/peak 2, calculated by chromatograms of substances extracted from soil. These results suggest that most humic substances in Katsura, Uji, Kidzu and Yodo rivers, are humic substances in river water may originate from soil around listed in Table 2. The ratio of peak 1/peak 2 in 1995 was larger the rivers.6 In rivers such as the Katsura river, several peaks in the following order: December < September < August. The besides the three peaks mentioned above were detected, ratio of higher molecular weight of humic substances in the indicating that the aquatic humic substances with lower warmer season was larger than that in the cooler season of the molecular weight present in it may have a different origin, such same year. The seasonal changes in the ratio of peak 1/peak 2 as anthropogenic contamination. The chromatograms of humic in the Uji river were 0.77 Ð 1.18, while those in the Katsura river substances collected at the sampling station (Kudzuha) in the (station 11) and the Kidzu river were 0.74 Ð 1.32 and 0.63 Ð Yodo River were similar to those from the Kidzu river. Since 1.34, respectively, which were relatively large compared with the sampling station is located immediately past the place where those in the Uji river. Furthermore, the seasonal changes in the the Katsura, Uji and Kidzu rivers meet, it was considered that Yodo river, 0.58 Ð 1.37, were similar to those in the Kidzu river. the contribution of the Kidzu river to the water quality of the However, those at station 13 in the Katsura river were 0.34 Ð Yodo river is relatively large. 0.60; a similar trend was not observed. It seems that the difference in the seasonal changes of the concentration and Seasonal changes in the concentrations and molecular weight of molecular weight of humic substances at station 13 may have humic substances been due to anthropogenic contamination entering the river, The concentrations of fulvic and humic acids in all measured because this sampling point was located downstream of a ANALYTICAL SCIENCES FEBRUARY 2000, VOL. 16 129 sewage-disposal plant. These results suggest that the main 1988, J. Wiley & Sons, New York, 165. origin of aquatic humic substances in rivers may be humic 4. J. J. Rook, J. Am. Water Works Assoc., 1976, 68, 615. substances from soil around the rivers, rather than those from 5. J. J. Rook, Environ. Sci. Technol., 1977, 11, 478. anthropogenic contamination. 6. E. Yamada, T. Ozaki, and M. Kimura, Anal. Sci., 1998, 14, 327. 7. E. Yamada, Anal. Sci., 1997, 13, 385. Acknowledgement 8. M. D. Nobili, E. Gjessing, and P. Sequi, in “Humic Substances II in Search of Structure”, ed. M. H. B. Hayes, The authors would like to thank Mr. Daisaku Yoshida and Mr. P. MacCarthy, R. L. Malcolm, and R. S. Switt, 1989, J. Motoki Ohno for their help in collecting environmental water Wiley & Sons, New York, 562. samples. We also thank Mr. Hisao Yoneyama, Mr. Takashi 9. M. Susic and K. G. Goto, J. Chromatogr., 1989, 482, 175. Inaba and Mr. Motoaki Tokukura for their help with 10. M. Susic and L. G. Armstrong, J. Chromatogr., 1990, 502, measurements of humic substances in water sample. The 443. present study was supported by a Grant-in-Aid for Scientific 11. P. R. Bloom and J. A. Leenheer, in “Humic Substances II in Research from the Ministry of Education, Science and Culture Search of Structure”, ed. M. H. B. Hayes, P. MacCarthy, R. (No. 07680554, No. 10680501). L. Malcolm, and R. S. Switt, 1989, J. Wiley & Sons, New York, 409. 12. A. Watanabe, K. Itoh, S. Arai, and S. Kuwatsuka, Soil Sci. 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