Allelochemicals: Role in Agriculture and Forestry - ACS Publications

C h a p t e r 35

Studies on the Fulvic and Humic Acids of Minnesota Peat Durga Kumari , S. A. Spigarelli , and George R. Waller 1,3

1

2

Center for Environmental Studies, Bemidji State University, Bemidji, M N 56601 Department of Biochemistry, Oklahoma State University, Stillwater, OK 74078

1

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 23, 2018 | https://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch035

2

Fulvic and humic acids have been investigated with carbon-13 and proton nuclear magnetic resonance spectrometry, GC/MS, and IR spectroscopy. The fulvic and humic acids were found to be predominantly carboxylic and aromatic with a high proportion ofO-and N-substituted carbon atoms, although aliphatic ones were also observed. Humic substances constitute a very important class of natural products, especially fulvic and humic acids. These are present in soil, water, and coal throughout the world (1_) and participate in many significant agricultural, geochemical, and environmental processes (1-4). Examination of recent publications shows that there is an increasing interest in these materials by chemists, soil scientists, hydrologists, organic geochemists, and others in environmental sciences. Chemical investigations on humic substances have occupied the attention of scientists for more than 200 years (1,2); but relatively l i t t l e progress has been made in the elucidation of their chemical nature as compared to that of other natural products such as proteins, polynucleotides, and polysaccharides. However, this is not to imply that no progress has been made at all in the study of these substances. A vast amount of information has been accumulated on the chemical, physical, biological, geochemical and agricultural aspects of humic substances, but i t has not been possible to integrate this knowledge within a satisfactory conceptual framework of the nature of these substances. Although soil organic matter has been extensively studied by spectroscopic methods, very few investigators have studied peat as a source of humic substances (5-8). This paper presents carbon-13, proton nuclear magnetic resonance (^C-NMR and ^-H-NMR) spectra, 3

Current address: Department of Forest Products, University of Minnesota, St. Paul, MN 55108

0097-6156/87/0330-0384$06.00/0 © 1987 American Chemical Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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p r o t o n n u c l e a r magnetic resonance (^c-NMR and ^H-NMR) s p e c t r a , GC/MS, and IR a b s o r p t i o n s p e c t r a of f u l v i c and humic a c i d s of M i n n e s o t a p e a t , and c o n c l u s i o n s about t h e n a t u r e of both t y p e s of acids.


Materials

and Methods

Materials. The p e a t , taken from S t . L o u i s C o u n t y , M i n n e s o t a , i s d e r i v e d from mosses of t h e genus Sphagnum. I t a l s o c o n t a i n s remains of some e r i c a c e o u s shrubs and few f o r b s and sedges. I t was a i r d r i e d t o a p p r o x i m a t e l y 15% m o i s t u r e , c o a r s e - s c r e e n e d t o remove woody p a r t i c l e s , and p u l v e r i z e d i n a l a b o r a t o r y W i l e y m i l l f i t t e d w i t h a 20-mesh s c r e e n . A l l s o l v e n t s were d i s t i l l e d b e f o r e u s e . P r e p a r a t i o n of F u l v i c and Humic A c i d s . Waxes, r e s i n s , and o t h e r s u b s t a n c e s s o l u b l e i n o r g a n i c s o l v e n t s were removed by s u c c e s s i v e e x t r a c t i o n s w i t h p e t r o l e u m e t h e r ( 3 5 - 6 0 ° ) , c h l o r o f o r m , and e t h y l acetate. These e x t r a c t i o n s removed 4% of t h e o r i g i n a l m a t e r i a l . The r e s i d u a l peat was a i r - d r i e d t o remove s o l v e n t s . Wax and r e s i n f r e e peat (50 g) was s t i r r e d w i t h 1 l i t e r of 0.5N NaOH f o r 24 h. The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g i n g at 5000 rpm f o r 15 min i n a Beckman Model No J - 6 B c e n t r i f u g e . T h i s e x t r a c t i o n with a l k a l i was r e p e a t e d t w i c e more and t h e s o l u t i o n s of sodium s a l t s of a c i d s were c o l l e c t e d . Humic a c i d s were p r e c i p i t a t e d from the com b i n e d NaOH s o l u t i o n s by a d j u s t i n g the pH t o 1 w i t h 2N HC1 s l o w l y w i t h s t i r r i n g and t h e m i x t u r e was l e f t o v e r n i g h t . The p r e c i p i t a t e d humic a c i d s were c o l l e c t e d by f i l t r a t i o n through Whatman 1MM paper and washed w i t h 0.1N HC1. The f i l t r a t e s were e x t r a c t e d t h r e e t i m e s w i t h e t h y l a c e t a t e and the e x t r a c t s d r i e d over sodium s u l f a t e and e v a p o r a t e d , the r e s i d u e c o n s t i t u t i n g t h e f u l v i c a c i d s . Buth f u l v i c and humic a c i d s ( p r e c i p i t a t e s ) were a i r - d r i e d , and then d r i e d i n a vacuum d e s i c c a t o r over phosphorus p e n t o x i d e at room t e m p e r a t u r e . The y i e l d s were 3.5 g and 18.9 g r e s p e c t i v e l y . Samples of both f u l v i c and humic a c i d s were suspended i n methanol and m e t h y l a t e d w i t h diazomethane. Both *H and 13c s p e c t r a of t h e f r e e a c i d s were o b t a i n e d , at 299.94 MHz and 75.42 MHz r e s p e c t i v e l y , on a V a r i a n XL-300 s p e c t r o m e t e r h a v i n g a N i c o l e t TT-100 PET a c c e s s o r y . S p e c t r a were o b t a i n e d i n D2O, i n a 12-mm t u b e , w i t h d e u t e r a t e d TSP (sodium 3-(trimethylsilyl)propionate2 ^ , 3 , 3 - d 4 ) added as i n t e r n a l r e f e r e n c e . GC/MS of m e t h y l a t e d a c i d s was conducted on a H e w l e t t - P a c k a r d Model No 5995 GC/MS/DA system equipped w i t h a f u s e d s i l i c a c a p i l l a r y column (12 m χ .020 mm ID, Hewlett Packard) i n t e r n a l l y c o a t e d w i t h c r o s s l i n k e d methylene silicone. I n f r a r e d s p e c t r a were o b t a i n e d w i t h s o l i d samples d i s p e r s e d i n KBr p e l l e t s , by u s i n g a Beckman IR-33 s p e c t r o p h o t o meter. The v a r i o u s a b s o r p t i o n peaks i n IR and NMR were i n t e r p r e t e d conventionally (9-10).


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Results The ^ C - N M R s p e c t r a of t h e f u l v i c and humic a c i d s are p r e s e n t e d i n F i g u r e s 1 and 2 r e s p e c t i v e l y . An i m p o r t a n t r e g i o n f o r a b s o r p t i o n was t h e 171-184 ppm r a n g e ; a p a r t i c u l a r l y sharp s i g n a l at 176 ppm i n f u l v i c a c i d i s c h a r a c t e r i s t i c of c a r b o x y l i c g r o u p s . The r e g i o n between 171 and 184 ppm i s r e p r e s e n t a t i v e of c a r b o n y l c a r b o n s . These can be c o n t a i n e d i n f r e e a c i d s , e s t e r s , s a l t s , amides, a l d e h y d e s , and k e t o n e s . The r e g i o n between 113 and 145 ppm r e p r e s e n t s a r o m a t i c , h e t e r o a r o m a t i c , and o l e f i n i c c o n s t i t u e n t s . A l t h o u g h o l e f i n i c carbons are g e n e r a l l y not major c o n s t i t u e n t s t o t h e s e a c i d s , a r o m a t i c ones are expected t o be major c o n t r i b u t o r s t o peaks i n t h i s r e g i o n . The l a r g e s t peaks are p r e s e n t i n t h e a r o m a t i c c a r b o n r e g i o n , which spans t h e r e g i o n from 119 t o 132 ppm i n both a c i d s ; t h u s they appear t o r e p r e s e n t p r e d o m i n a n t l y a r o m a t i c structures. The peaks at =58 ppm are most l i k e l y due t o methoxy g r o u p s , w h i l e t h o s e around 59-64 ppm would a l s o i n c l u d e c o n t r i b u t i o n s from N - s u b s t i t u t e d c a r b o n s of amino a c i d s (_11). Both s p e c t r a show r e s o n a n c e s i n t h e 19-40 ppm r e g i o n c h a r a c t e r i s t i c of a l i p h a t i c moieties. S i g n i f i c a n t d i f f e r e n c e s e x i s t between t h e two t y p e s of a c i d s , f u l v i c a c i d s p e c t r a showing more prominent r e s o n a n c e s . Both a c i d s , however, are r i c h i n carbon r e s o n a t i n g at - 3 0 ppm. These may i n c l u d e -CH2groups i n l o n g - c h a i n f a t t y a c i d s . The peak at 19 ppm i n f u l v i c a c i d ( F i g u r e 1) i s a s s i g n e d t o the t e r m i n a l methyl i n aliphatic chains. T h e r e i s l e s s a l i p h a t i c carbon than a r o m a t i c i n both a c i d s . The s p e c t r a , p a r t i c u l a r l y t h a t of f u l v i c a c i d ( F i g u r e 1 ) , a l s o show t h e p r e s e n c e of a c e t a l groups at 101 ppm. The 1-H-NMR spectrum of f u l v i c a c i d i s shown i n F i g u r e 3. The s i g n a l at 0.94 ppm i s t h e methyl *H r e s o n a n c e , w h i l e t h a t at 1.24 ppm i s due t o methylene p r o t o n s . The s t r o n g s i g n a l at - 1 . 9 7 ppm can a r i s e from t h e resonance of methylene groups to Ph, OH, - 0 - , OCOR, COPh, CHO, COOH, COOR, or groups. Although a l i p h a t i c c a r b o n s α t o c a r b o x y l , c a r b o n y l , or carboxamide groups y i e l d s i g n a l s t h a t are not s h i f t e d d o w n f i e l d of t h e a l i p h a t i c carbon r e g i o n , they produce a ^H-NMR s i g n a l at - 2 . 2 4 ppm t h a t i s c l e a r l y r e s o l v e d from t h a t of o t h e r a l i p h a t i c p r o t o n s , such as from amino acids. A wide r e g i o n between 2.99 ppm and 4 . 0 ppm shows t h e p r e s e n c e of methylene and methyl groups l i n k e d t o e l e c t r o n e g a t i v e atoms, v e r y l i k e l y oxygen, and/or the p r o t o n of a Ph-CH2 fragment t o c a r b o n y l and/or c a r b o x y l i c groups ( 1 2 - 1 3 ) . The broadness i s a t t r i b u t a b l e t o the l a r g e v a r i e t y of oxygen and n i t r o g e n compounds i n f u l v i c a c i d s , t h e most l i k e l y ones c o n t a i n i n g e t h e r , amino a c i d , and p e p t i d e g r o u p s . A r o m a t i c or c o n j u g a t e d o l e f i n i c p r o t o n s i g n a l s were p r e s e n t between 6.64 t o 8.54 ppm. The sharp s i g n a l at 8.54 ppm can be a t t r i b u t e d t o a r o m a t i c p r o t o n s . The prominent peak at a p p r o x i m a t e l y 5 ppm o b v i o u s l y shows t h e p r e s e n c e of OH p r o t o n s i n D 0.

COCH3,

β CONH2

Crl=CH2*

α

2

The 1 H - N M R spectrum of humic a c i d s i s shown i n F i g u r e 4. It i s s i m i l a r t o t h a t of f u l v i c a c i d s except t h e r e i s no sharp peak at 8.54 ppm c o r r e s p o n d i n g t o a r o m a t i c p r o t o n s . The peaks f o r a l i p h a t i c p r o t o n s were at 0 . 9 0 , 1.28, 1.34, 1.93, 1.94, 2 . 1 9 , 2 . 2 2 , 3.37 t o



i

2 500

500 m

I

2 000

13

F i g u r e 1. C-NMR of F u l v i c A c i d of instrumental artifact)

Minnesota

Peat

(*

indicates


^1

00

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY


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Fulvic and Humic Acids of Minnesota

Peat


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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY


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Peat

3.83 ( b r o a d ) , and 3.97 ppm. O l e f i n i c and a r o m a t i c p r o t o n s were i n d i c a t e d by peaks at 6 . 0 5 , 6 . 2 4 , 6 . 3 0 , 6 . 6 5 , 7 . 2 0 - 7 . 5 2 and 7 . 7 2 - 7 . 7 7 ppm. The GC/MS s t u d y of m e t h y l a t e d f u l v i c a c i d showed t h e presence of ^ - h y d r o x y b e n z o i c a c i d (M * 166), v a n i l l i c a c i d ( M 196), a m e t h y l d i h y d r o x y b e n z o i c a c i d (NT "' 2 1 0 ) , coumaric a c i d (M * 192), s y r i n g i c a c i d (M * 2 2 6 ) , c a f f e i c a c i d ( M 2 2 2 ) , f e r u l i c a c i d (M * 2 2 2 ) , and s t e a r i c a c i d (M * 2 9 8 ) . The same compounds were p r e s e n t i n humic a c i d , a l o n g w i t h p a l m i t i c a c i d (M * 270). The p r e s e n c e of t h e s e compounds was c o n f i r m e d by T L C , u s i n g s t a n d a r d compounds. +

+e

4

+

+

+ e

+

+


+

A t y p i c a l IR spectrum of f u l v i c a c i d ( F i g u r e 5a) shows t h e following features: a wide and i n t e n s e band from s t r e t c h i n g of the Η-bonded OH groups at 3280 c m " , and a weak band near 2960 c m " due t o a l i p h a t i c C-H s t r e t c h i n g . A 1740 c m a b s o r p t i o n i s due t o C-0 s t r e t c h i n g i n c a r b o x y l i c and/or c a r b o n y l g r o u p s . The 1615 c m " band i s a t t r i b u t e d t o v i b r a t i o n s of a r o m a t i c C=C and/or c a r b o n y l C=0 groups t h a t form hydrogen bonds, and/or of double bonds c o n j u g a t e d with carbonyl groups. The a b s o r p t i o n at 1520 c m " i s p r o b a b l y due t o a r o m a t i c C=C s t r e t c h i n g , w h i l e the bands at 1445 c m " and 1380 c m " are l i k e l y due t o CH2 s c i s s o r d e f o r m a t i o n and CH3 symmetric deformation. Bands were a l s o e x h i b i t e d near 1215 c m " and 1025 c m " , l i k e l y due t o C-0 s t r e t c h i n g of e s t e r s and e t h e r s and OH deformation i n c a r b o x y l i c groups. 1

1

- 1

1

1

1

1

1

1

The IR spectrum of humic a c i d i s shown i n F i g u r e 5b. As f o r f u l v i c a c i d , i t shows broad bands i n t h e 3380 c m " r e g i o n (hydrogen-bonded OH g r o u p s ) , a weak band near 2920 c m " (aliphatic C-H s t r e t c h ) , a w e l l d e f i n e d peak near 1710 c m " (C=0 s t r e t c h i n g f r e q u e n c y of C00H; C=0 s t r e t c h of k e t o n i c c a r b o n y l ) , m e d i u m - s i z e d bands near 1650 c m " ( p r o b a b l y due t o a r o m a t i c C=C bonds c o n j u g a t e d w i t h C=0 and/or C00"), 1500 c m " ( a r o m a t i c C=C s t r e t c h i n g ) , and 1445 c m " (CH2 deformation). The bands at 1220 c m " and 1020 c m " are due t o C-0 s t r e t c h i n g of e s t e r s and e t h e r s and t o OH d e f o r m a t i o n of c a r b o x y l i c group; t h o s e at 825 c m " and 685 c m " might be a l i p h a t i c CH2 chain>or a r o m a t i c bands. 1

1

1

1

1

1

1

1

1

1

Di s c u s s i o n A number of i n v e s t i g a t o r s (14-20) have r e c o r d e d 1 3 -NMR s p e c t r a of humic a c i d and observed s i g n a l s from a r o m a t i c c a r b o n s , but o t h e r s (21-22) found no such s i g n a l s i n the *H and C - N M R s p e c t r u m . C

13

S i g n a l s i n t h e a r o m a t i c or c o n j u g a t e d o l e f i n i c r e g i o n between 6 and 8 . 5 ppm i n J-H-NMR and 101-145 ppm i n C - N M R are p r e s e n t . The presence of a r o m a t i c s was c o n f i r m e d by GC/MS of m e t h y l a t e d samples showing t h e p r e s e n c e of a number of p h e n o l i c a c i d s , and t h e p r e s e n c e of 1650, 1500, 1520 c m " bands i n the IR spectrum. Aromatic r e s o n a n c e s have been observed f o r both a c i d s and are predominant i n both C spectra. The c o n f i r m e d presence of p h e n o l i c a c i d s i s i n agreement w i t h t h e d i s c o v e r y of l i g n i n i n humic s u b s t a n c e s ( 2 3 ) . H resonances i n t h e 0 - 2 . 4 ppm r e g i o n c h a r a c t e r i z e a l i p h a t i c p r o t o n s , 13

1

1 3

1




:

36£ AHisaaod QNV aannnDRiDV NI HIOH sivDM3HDOianv

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while resonances at 0-50 ppm show the corresponding aliphatic carbons. The presence of fatty acids, palmitic and stearic, was shown by GC/MS and IR spectra. It is clear from the work of Schnitzer and Vendette (24) that fatty acids were present in arctic soil samples. Other spectra of soil humic acids obtained in NaOD solution (2J5) were quite informative, as both aromatic and aliphatic protons were shown present. No thorough study of fulvic and humic acids was conducted by Hatcher and associates (26-28) using *H and !3c-NMR. The experiments reported here show that there are more aromatic and less aliphatic components in fulvic and humic acids of Minnesota peat. Conclusion 13

Both *H and C NMR showed the presence of aromatic and aliphatic components. In 1C-NMR, resonances at -58 ppm indicate the presence of many OCH3 groups, such as those occurring in syringic, vanillic, and ferulic acids. Spectra clearly show the presence of palmitic and stearic acids by GC/MS, IR, and NMR data. The fulvic and humic acids are predominantly made up of phenolic and fatty acid units. These are highly aromatic because lignin residues have been incorporated in the humification process. 3

Acknowledgments This work was supported by a grant from Minnesota Department of Natural Resources, St. Paul, Minnesota. Literature Cited 1. Schnitzer, M.; Khan, S.U. "Humic Substances in the Environment"; Marcel Dekker: New York, 1972. 2. Kononova, M. "Soil Organic Matter", 2nd ed.; Pergamon Press: Oxford, 1966. 3. Gjessing, E.T. "Physical and Chemical Characteristics of Aquatic Humus"; Ann Arbor Science: Ann Arbor, 1976. 4. Felbeck, G.T., Jr. In "Soil Biochemistry"; McLaren, A.D.; Skujins, J.J., Eds.; Marcel Dekker: New York, 1971; Vol. 2, pp. 36-59. 5. Arnold, C.L.; Lowy, Α.; Thiessen, R. Fuel 1935, 14, 107-112. 6. Lindberg, B.; Theander, O. Acta Chem. Scand. 1952, 6, 311-312. 7. Farmer, V.C.; Morrison, R.I. Sci. Proc. Roy. Soc. Dublin, Ser. A 1960, 1, 85-105. 8. Kosonogova, L.V.; Evdokimova, G.A.; Rakovski, V.E. Khim. Tverd. Topl. 1976, 10, (3), 97-101; Chem. Abstr. 86, 75698t. 9. Chamberlain, N.F. "The Practice of NMR Spectroscopy"; Plenum: New York, 1974. 10. Levy, G.C.; Lichter, R.L.; Nelson, G.L. "Carbon-13 Nuclear Magnetic Resonance Spectroscopy"; Wiley-Interscience, New York, 1980.



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11. Preston, C.M.; Mathur, S.P.; Rauthan, B.S. Soil Sci. 1981, 131, 344-352. 12. Neyroud, J.A.; Schnitzer, M. Can. J. Chem. 1974, 52, 4123-4133. 13. Sciacovelli, O.; Senesi, N., Solinas, V.; Testini, C. Soil Biol. Biochem. 1977, 9, 287-293. 14. Vila, F.J.G.; Lentz, H.; Lüdemann, H.D. Biochem. Biophys. Res. Commun. 1976, 72, 1063-1070. 15. Wilson, M.A.; Jones, A.J.; Williamson, B. Nature 1978, 276, 487-489. 16. Ogner, G. Soil Biol. Biochem. 1979, 11, 105-108. 17. Grant, D. Nature 1977, 270, 709-710. 18. Ruggiero, P.; Interesse, F.S.; Sciacovelli, O. Geochim. Cosmochim. Acta 1979, 43, 1771-1775. 19. Newman, R.H.; Tate, K.R.; Barron, P.F.; Wilson, M.A. J. Soil Sci. 1980, 31, 623-631. 20. Wilson, M.A.; Collins, P.J.; Tate, K.R. J. Soil Sci. 1983, 34, 297-304. 21. Schnitzer, M.; Barton, D.H.R. Nature 1963, 198, 217-219. 22. Wilson, M.A.; Goh, K.M. J. Soil Sci. 1977, 28, 645-652. 23. Hurst, H.M.; Burges, N. In "Soil Biochemistry"; McLaren, A.D.; Peterson, G.H., Eds.; Marcel Dekker: New York, 1967. 24. Schnitzer, M.; Vendette, E. Can. J. Soil Sci. 1975, 55, 93-103. 25. Lentz, H.; Lüdemann, H.D.; Ziechmann, W. Geoderma 1977, 18, 325-328. 26. Hatcher, P.G.; Rowan, R.; Mattingly, M.A. Org. Geochem. 1980a, 2, 77-85. 27. Hatcher, P.G.; VanderHart, D.L.; Earl, W.L. Org. Geochem. 1980b, 2, 87-92. 28. Hatcher, P.G.; Macial, G.E.; Dennis, L.W. Org. Geochem. 1981, 3, 43-48. RECEIVED June 16,1986


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