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

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Simultaneous Determinations of the Concentration and Molecular Weight of Humic Substances in Environmental Water by Gel Chromatography with a Fluorescence Detector 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 Water 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 606–8585, Japan **Center for Environmental Science, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606–8585, 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 acid 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 – 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 soils around the rivers. (Received August 17, 1999; Accepted November 12, 1999) Yodo rivers. Introduction Dissolved humic substances, the main constituents of the Experimental dissolved organic carbon pool in surface water (freshwater and marine water), groundwater, and soil 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 hydrochloric acid 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.4–7 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 acids in peat 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 sodium hydroxide solution was used as an eluent at a flow rate of 0.4 ml min–1. 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 min–1 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 min–1. 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 – 10, but those at pH 12 were 80 of humic and fulvic acids were calculated by considering the – 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.
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