Chemical characterization of fractionated humic acids from lake and marine sediments

Chemical Geology, 12 (1973) 113-126 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

CHEMICAL CHARACTERIZATION OF FRACTIONATED HUMIC ACIDS FROM LAKE AND MARINE SEDIMENTS

R, ISHIWATARI

Department of Chemistry, Tokyo Metropolitan University, Setagaya-ku, Tokyo (Japan) (Accepted for publication July 17, 1973)

ABSTRACT lshiwatari, R., 1973. Chemical characterization of fractionated humic acids from lake and marine sediments. Chem. Geol., 12: 113-126. Humic acids from Recent lacustrine and marine sediments were divided into five components by extractions with organic solvents and characterized by elementary composition, ultraviolet, visible and infrared absorption spectra and n.m.r, spectra. The results suggest that sedimentary humic acids have a cyclic structure (40-50% of the total carbon), which is considered to be alicyclic rather than aromatic. No marked differences except for an absorption near 410 mu were observed between humic acids from lake and marine sediments. INTRODUCTION Organic constituents of dead organisms in aquatic environments undergo chemical and biological decomposition resulting in the formation of new compounds including darkcolored polymeric organic materials which are so-called humic substances. Studying humic substances in Recent sediments is very important since they are probably source materials for fossil organic materials such as coal, petroleum and dispersed organic materials in sedimentary rocks. Chemical studies on humic acids* from lake sediments have been performed by Karavaev and Budyak (1960), Karavaev et al. (1964) and the present author (Ishiwatari, 1970, 1971). Degens et al. (1964) have determined phenols and amino acids in the hydrolysate of humic acid from marine sediment. Rashid and King (1969) and the present author (1971 ) have measured molecular weight distribution of marine humic acids. Rashid and King (1970, 1971) and Rashid (1972a, 1972b) have studied oxygen-containing functional groups and amino acids in humic compounds from marine sediments. Hoering (1971) has reported chemical investigation of marine humic acids. Since humic acid is a polymer having a wide range of molecular weights, it is very

*The term humic acid as used here indicates a group of compounds which are extracted from sediments by alkaline solutions and then precipitated on acidification.

114

R. ISHIWATAR1

difficult to obtain precise structural information. One of the ways for overcoming this difficulty may be to separate humic acid into various components by an appropriate method and to study the chemical structure of each component. From the above point of view, the present author previously conducted chemical characterization of a lake-sediment humic acid, fractionating it into five components on the basis of solubility in an organic solvent (Ishiwatari, 1969). This paper is concerned with the chemical characterization of four kinds of lake and marine humic acids using the technique identical to that described in the previous paper (Ishiwatari, 1969). EXPERIMENTAL Materials

The sediment samples were collected from lacustrine (Lake Haruna and Lake Kizaki) and marine (offshore Kii Peninsula and Sagami Bay) environments. Lake Haruna is a mesotrophic fresh-water lake located in Gumma Prefecture, in the central part of Japan (altitude: 1084 m; area: 1.23 km2; maximum depth: 13.0 m;volume: 0.01 km3). The main source material of humic acid in bottom sediment in the lake is considered to be phytoplanktons, lake Kizaki is also a mesotrophic fresh-water lake, located in Nagano Prefecture (altitude: 764 m ; area: 1.4 km 2 ; maximum depth: 29.5 m; volume: 0.025 km3). A large percentage of organic materials in the bottom sediment is considered to be allochthonous. Saijo (1956)has estimated that the amount of autochtonous deposits in lakes Haruna and Kizaki is 93% and 24-40% of the total, respectively. The marine sediment sample M 1 was collected at 33°28'N, 135°57'E from a depth of 500 m (offshore Kii Peninsula). The sample M 2 was collected at 35°52'N, 139°07'E from a depth of 1100 m in Sagami Bay. Since these two sampling locations are near the shore, humic acids are considered to be significantly affected by allochthonous materials. Methods

Following collection, the samples were dried in air and stored until extraction. The humic acids were extracted and purified following the procedure reported in an earlier publication (Ishiwatari, 1969). A humic acid sample (0.4-3.2 g) was fractionated into five components by successive extractions with chloroform (Fr.1), methanol (Ft.2 and 3) and dimethylformamide (DMF: Fr.4) in a Soxhlet apparatus. Methanol extract was then dried in air and separated further into methylethylketone soluble and insoluble fractions by extraction at room temperature. Ultraviolet and visible absorption spectra were recorded on a Hitachi recording spectrometer (EPS-2). One mg of sample was dissolved in 10 ml of 0.1N sodium hydroxide solution. Infrared absorption spectra were recorded using a Shimazu IR-27A double-beam spectrometer by the KBr disk method (1 mg of sample per 100 mg of KBr). Nuclear

FRACTIONATEDHUMICACIDSFROM LAKE AND MARINESEDIMENTS

115

magnetic-resonance spectra were measured at a frequency of 60 Mc/sec with a JNMC-60H spectrometer at the laboratory of Japan Electron Optics Lab. Ltd., or at the Engineering Research Institute, University of Tokyo. Carbon, hydrogen and nitrogen were determined with a Yanagimoto CHN-corder at the Engineering Research Institute. Oxygen was calculated by difference. Average molecular weight of some fractionated humic acids was determined with a Mecrolab Model 301A vapor-pressure osmometer at the above Institute. RESULTS Table I shows the fractions expressed as percentage of the original humic acids. Approximately 30-40% of the original humic acid is in an organic solvent extractable form.

TABLE I Fractions of lake and marine humic acids Fr. No. 1 2 3 4 5

Solvent

chloroform methylethylketone methanol dimethylformamide remained

Percentage of the original humic acid Lake Haruna

LakeKizaki

M1

M2

3.2 2.2 5.4 14.5 74.7

1.1 7.3 5.2 17.2 69.2

2.2 4.2 9.4 12.7 71.5

3.0 3.6 22.9 14.3 56.2

Elementary composition Table II shows the elementary composition of fractionated humic acids. Fig.1 plots the results of Table II. The nitrogen content of the original humic acid calculated from those of the fractions are higher than those actually determined. The difference between the above two values (nitrogen content calculated minus nitrogen content determined) is 1.60, 2.68, 0.97 and 0.78% for the samples of Lake Haruna, Lake Kizaki, M 1 and M 2, respectively. This is due to contamination of DMF used as a solvent. N.m.r. has confirmed that the trace amount of DMF is not liberated completely from the samples of fractions 4 and 5 (see Fig.3). In particular, the N/C ratios determined for fraction 5 from the lake samples are considered to be much higher than the true values, judging from their nitrogen content. H/C ratio of the fraction in a humic acid generally decreases in the following order: Fr.1 ~ Ft.3 > Fr.2 ~ Fr.5 > Fr.4. This fact suggests that the humic acids consist of various groups of compounds similar in nature with respect to elementary composition. For fractions 2, 5 and 4, H/C ratios seem to depend on those of parent humic acid. For fraction 3 in sample M 2 the H/C is unusually high but the reason is unknown.

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TABLE II Elementary composition of fractionated humic acid s Sample

C%

H%

0%

N%

Atomic ratio H/C

N/C

E*400mt~

E*600 m~

Lake Haruna Original HA Fr.1 Fr.2 Fr.3 Fr.4 Ft.5

52.88 68.97 59.15 48.15 54.42 52.21

6.09 9.03 6.47 6.59 5.39 5.67

33.88 21.02 32.19 38.89 33.32 32.29

7.15 0.98 2.19 6.37 6.87 9.83

1,37 1,56 1.30 1,63 1,18 1.29

0.116 0.012 0.032 0.113 0.108 0.161

8.2 3.6 3.7 1.0 9.2 6.1

1.8 0.6 0.4 0.1 2.1 1.3

54.91 70.56 61.51 50.97 56.88 53.75

4.95 9.91 5.52 5.15 4.51 5.05

36.18 19.53 31.52 39.15 32.73 33.55

3.96 0.00 1.45 4.73 5.88 7.65

1.00 1.67 1.07 1.20 0.94 1.12

0.062 0.000 0.020 0.080 0.089 0.122

14.1 1.6 8.2 7.0 12.5 10.1

3.3 0.4 1.0 1.0 3.4 2.5

5.23 9.10 6.49 4.75 4.87 5.64

37.93 22.29 30.94 52.04 32.54 32.08

4.69 0.90 2.40 4.36 6.10 6.24

1.19 1.60 1.29 1.46 1.03 1.20

0.077 0.011 0.034 0,096 0.093 0.089

13.1 2.8 6.6 4.3 14.9 9.9

2.0 0.5 0.6 0.5 2.9 1.6

5.21 7.17 5.96 6.00 4.76 5.62

39.39 31.48 37.58 53.08 33.19 33.01

4.83 1.04 2.49 5.10 6.22 6.11

1.23 1.42 1,.32 2.00 1.02 1.21

0.082 0.015 0.040 0.122 0.096 0.095

13.0 3.5 4.5 1.7 23.8 10.8

2.1 0.5 0.4 0.4 4.3 1.7

Lake Kizaki Original HA Fr.1 Ft.2 Fr.3 Fr.4 Fr.5

M1 (offshore Kii Peninsula) Original HA Fr.1 Fr.2 Fr.3 Fr.4 Fr.5

52.15 67.71 60.17 38.85 56.49 56.04

M 2 (Sagami Bay) Original HA Fr.1 Fr.2 Fr.3 Fr.4 Fr.5

50.57 60.31 53.97 35.82 55.83 55.26

*Absorbance/mg-sample/ml-O.1N NaOH; light path = 10 mm.

Ultraviolet and visible absorption spectra Fig.2 shows t h e r e p r e s e n t a t i v e visible a b s o r p t i o n spectra o f t h e f r a c t i o n a t e d h u m i c acids. All f r a c t i o n s s h o w e d a nearly m o n o t o n i c curve w i t h a v e r y b r o a d s h o u l d e r in t h e region 260-290

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Infrared absorption spectra Table III gives chemical bonds suggested from i.r. spectra. Fig.3 gives quantitative data of 2920 cm -1 (methylene), 1380 cm -1 (methyl), 1640 cm -1 (Amide I; C = O of acid, ketone and/or quinone), 1720 cm -1 (C = O of aliphatic acid or its ester) and 1540 c m -1 (Amide II: Nit + C = N) (Ishiwatari, 1967). I.r. spectra (Table III, Fig.3) show the presence of a fair amount o f carboxylic groups in fractions 1 and 2. Absorptions at 1 6 4 0 - 1 6 1 0 cm -1 and 1540 cm -~ which may be due to a peptide-like bond (Ishiwatari, 1967) are remarkable for 3 and 5. E292o cm -~/E138o cm -l ratios for fraction 1 and 2 are larger than those for other fractions.

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TABLE III Chemical bonds suggested from i.r. spectra Fraction No.

Chemical bonds suggested

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Nuclear magnetic resonance spectra Fig.4 gives t h e r e p r e s e n t a t i v e n.m.r, spectra o f the f r a c t i o n a t e d h u m i c acids. Table IV s h o w s t h e t e n t a t i v e a s s i g n m e n t o f c h e m i c a l shifts. F o r the a s s i g n m e n t o f c h e m i c a l shifts, t h e f o l l o w i n g papers w e r e referred to: B r o w n et al. ( 1 9 6 0 ) , H o p k i n s a n d B e r n s t e i n (1959),

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TABLE IV Tentative assignment of chemical shifts of fractionated h u m i c acids Chemical shift (r, p.p.m.)

Assignment

9.50-9.00 9.00-8.65(8.75) 8.65-8.00 8.00-7.00 7.00-5.00

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FRACTIONATED HUMICACIDS FROM LAKE AND MARINE SEDIMENTS

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Oelert (1967), Purcell et al. (1966), Ludwig et al. (1964), Heredy et al. (1966). A shift from 8 to 7 p.p.m. (~- value) is generally considered to be due to the alpha methylene group adjacent to the carboxyl group or that on monocyclic aromatics. The signal may be due to the former methylene group, because the fractions showed no signal in the region 4-1 p.p.m. (aromatic hydrogen region). Table V shows the quantitative data of chemical shifts from 9.50 to 7.00 p.p.m. These data are expressed by taking the amount of methyl group as a unit. The amount of naphthene-ring carbon content (CN) was tentatively estimated by Williams' method (Williams, 1958). This method, which was first applied to the saturated fractions derived from petroleum, is based on the measurement of the relative height of n.m.r, signals due to the methyl and methylene groups. The relationship between CN and the methyl/ methylene ratio for saturates from petroleum was assumed to hold for the present samples. CN and CN/C values thus calculated are shown in column 7 and 8, respectively, in Table V. Clearly CN increases from Ft.1 to Fr.4 for all humic acid samples examined.

Molecular weights Table VI shows the average molecular weight of Fr.1 and Fr.2 as determined with a vapor-pressure osmometer. The result indicates that the molecular weights of Fr.2 are higher than those of Fr.1. Column chromatography through Sephadex G-75 indicated that the other fractions contain high molecular-weight components over 50,000 (11-35% of the total components in a fraction). Fig.5 shows representative molecular weight distribution patterns of fractionated humic acids. TABLE VI Average molecular weights of fractions 1 and 2

Fraction 1 Fraction 2

Lake Haruna

Lake Kizaki

M1

M2

570 770

480 1000

590 920

410 530

Each fraction was characterized as follows: Fraction 1: n.m.r, spectra indicate the presence of terminal methyl, acyclic and cyclic methylene, and C H 2 - a - C O O H . A calculated ratio of naphthene-ring carbon to total carbon is 0.2. Thus functional groups in this fraction are composed dominantly of aliphatic carboxylic acids. Samples from lake sediments and those from marine sediments showed similar chemical characteristics. Fraction 2: the ratio of naphthene-ring carbon to total carbon was estimated to be 0.2-0.4. The H/C ratio of this fraction (1.1-1.3) is lower than those of Fr.1 (1.4-1.7). This suggests a higher ring content. This fraction is considered to be composed of aliphatic (probably partly aromatic) carboxylic acid or esters. Aromatic protons were not detected.

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Fig.5. Elution patterns of fractionatexi humic acids from Lake Haruna through Sephadex G-75. Peak 1 = components with molecular weight over 50,000; peak 2 = components with molecular weight below 50,000; column = 450 X 10 mm (i.d.). Elution was conducted with 0.1N NH3/NH4C1 (4/1, pH = 9.0) at a flow rate of 17 ml/h. No marked difference was observed between samples from lakes and those from marine sediments, except for the 410-m/2 absorption peak. Fraction 3: i.r. spectra indicate that proteinous and carbohydrate-like materials are present in this fraction. The CN of this fraction ( 1 5 - 2 9 % ) is similar to that of Fr.2 (14-22%). CN of the fraction from marine sediments appears to be higher than those from lake sediments. High N/C ratio, relatively higher H/C ratio and low E4oo m~z are characteristic for this fraction. The latter two suggest the fraction to be of a lower degree of humification. Fraction 4: H/C ratios of this fraction (0.9-1.2) are the lowest of all fractions. Since CN is 3 9 4 2 % corresponding to 7 0 - 7 7 % of the total carbon, this fraction seems to have polycyclic structure. N/C ratios of the fractions are as high as those of Fr.3. Fraction 5: H/C ratio (1.1-1.3) of this fraction is slightly higher than those of Ft.4. I.r. spectra clearly indicate the presence of proteinous and carbohydrate-like materials in this fraction. The ratio of naphthene ring carbon to total carbon ranges from 0.3-0.5. Lakes have higher N/C ratios than oceans. DISCUSSION The components soluble in organic solvents are considered to have been derived from a humic acid (degradation products) and from other organic materials in mineral matrix

FRACTIONATEDHUMICACIDSFROM LAKE AND MARINESEDIMENTS

123

TABLE VII Benzene carboxylic acids found in the alkaline permanganate oxidation products of a sedimentary humic acid - Lake Haruna (After Ishiwatari, 1973a) Benzene carboxylic acid Mono-carboxylicacid Di-carboxylic acid Tri-carboxylic acid Tet ra-carboxylicacid Penta-carboxylic acid Hexa-carboxylic acid Total

Soluble fraction in MeOH after methylation (%)

Insoluble fraction in MeOH after methylation (%)

37.0 13.0 25.3 21.1 1.7 1.9

53.9 14.0 27.1 4.3 0.6 0

100.0

100.0

during extraction procedures by sodium hydroxide (Swift and Posner, 1972). Except for the H/C values for Ft.1 and the unusually high H/C ratio of Fr.3 for sample M 2, the ratio of each fraction is closely related with that of a parent humic acid. This suggests that the humic acids are not a simple mixture of specific compounds but that their larger portion is an assemblage of organic materials with different molecular weights which can be defined as humic acids. I.r. and n.m.r, spectroscopy of the fractionated humic acids showed clearly their aliphatic nature. N.m.r. spectroscopy showed no sign of the presence of aromatic protons in the fractionated humic acids. This fact could be due to: (a) low concentration of aromatic protons, either because of the sparing solubility of the humic acid, or because of heavily substituted benzene rings; or (b) the strong relaxation effect of the spin of unpaired electrons, as inferred by Atherton et al. (1967) for soil humic acid. A possibility for the presence of a fully substituted benzene ring is small, because the content of benzene hexacarboxylic acid was found to be extremely low in the alkaline permanganate oxidation products of a sedimentary humic acid (Ishiwatari, 1973a). Aromatic protons should be detected by n.m.r, spectra if the amount of aromatic rings, as suggested from our previous results (Table VII), is high. Therefore, the n.m.r, results may indicate a low concentration of aromatic rings in the fractionated humic acids. Semiquinone radicals in sedimentary humic acids were suggested to be present by e.s.r, spectroscopy (Ishiwatari, 1973b). Application of Williams' method suggests the highly alicyclic nature of sedimentary humic acids. However, this must be considered as only a tentative result, because we have not yet obtained direct evidence for the alicyclic structure of the humic acids. The CN/C value for the original humic acid was calculated to be 0.4-0.5. This value is higher than that expected from the result obtained by a densimetric method (Ishiwatari, 1970). The reason for this discrepancy is unknown. Since there is a possibility that CN includes C aromatic, it is necessary to determine the relative amounts of alicyclic and aromatic rings in the humic acids by a more precise method.

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Fig.6. Relation between H/C and E40omg or CN/C of fractionated humic acids. There is a nearly linear relationship between color intensity and H/C ratio for the fractionated humic acids (Fig.6; Ishiwatari, 1969). Moreover, there exists a strong correlation between H/C and CN/C when we exclude the values for Fr.3 from M 1 and M 2 since their carbon contents are extraordinally low as compared with those of other fractions. The relationship implies that the H/C ratio is closely associated with ring formation in fractionated humic acids. An absorption shoulder near 410 m/a was observed for all fractions from marine sediments, but the shoulder was obscure for the fractions from Lake Haruna (except for Fr.1) and was absent for the fractions from Lake Kizaki. In order to examine the 410 m/aabsorbing material in the lake sediment, the following experiment was conducted. Humic acid was extracted with 0.1N sodium hydroxide from a sediment of Lake Haruna without preliminary extraction of pignents with organic solvents. The extracted humic acid exhibited an absorption near 410 m/a. However, the 410 m/a-absorbing material could be extracted from the humic acid with methanol, and the residual humic acid hardly showed a shoulder near 410 m/a. The 410 m/a-absorbing material thus obtained showed characteristics of pheophytin a, indicating that pheophytin a is extractable with organic solvent even if it is mixed with humic acid. Therefore, the 410 m/a-absorbing material found in Fr.1 from Lake Haruna is interpreted to be pheopigrnent. In order to examine the nature of 410 m/a-absorbing material in a marine humic acid, Fr.2 from M 1 was sub-divided into

FRACTIONATEDHUMICACIDSFROM LAKE AND MARINESEDIMENTS

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EWTION CURVE Subf r, 2

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Fig.7. Visible absorption spectra of subfraction 1 and 2 of fraction 2 from M 1 humic acid. two components by column chromatography through Sephadex LH-20. The sample was dissolved in methanol (1 ml), applied to the column (23 cm X 2.5 cm i.d.) and eluted with methanol at a rate of 0.01-0.1 ml/min. Fig.7 shows the elution curve. Subfraction 1 exhibits a broad shoulder near 410 m/2; subfraction 2 a peak near 410,600 and 650 (very weak) m~t. It is well known that the ratio of the red band (600-700 m/~) to the Soret band (near 400 m/a) becomes lower as the degradation of chlorophyll pigment proceeds (Hodgson et al., 1967). Therefore, the 410 m/l-absorbing material is considered to be a degraded chlorophyll-type material. The content of 410 m/~-absorbing material in humic acid was estimated in terms of pheophytin a by using a 410-m/a peak. Its content of sedimentary humic acids was 0-1%. ACKNOWLEDGEMENTS The author thanks Prof. T. Hanya for his interest in the present work. The author is indebted to the crew of the "Tansei-maru" for help in collecting the marine sediment samples. REFERENCES Atherton, N.M., Cranwell, P.A., Floyd, A.J. and Haworth, R.D., 1967. Humic acid, 1. ESR spectra of humic acids. Tetrahedron, 23: 1653-1667.

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