Presence and potential significance of aromatic-ketone groups in aquatic humic substances

Org. Geochem.Vol. II, No. 4, pp. 273-280, 1987 Printed in Great Britain

0146-6380/87 $3.00+0.00 Pergamon Journals Lid

Presence and potential significance of aromatic-ketone groups in aquatic humic substances J. A. LEENHEER', M. A. WILSON: and R. L. MALCOLMI 'U.S. Geological Survey, Denver Federal Center, Denver, CO 80225, U.S.A. 2CSIRO Division of Fossil Fuels, P.O. Box 136, North Ryde, N.S.W. 2113, Australia (Received 18 September 1986; accepted 18 February 1987) Abstract--Aquatic humic- and fulvic-acid standards of the International Humic Substances Society were characterized, with emphasis on carbonyl-group nature and content, by carbon-13 nuclear-magneticresonance spectroscopy, proton nuclear-magnetic-resonance spectroscopy, and infrared spectroscopy. After comparing spectral results of underivatized humic and fulvic acids with spectral results of chemically modified derivatives, that allow improved observation of the carbonyl group, the data clearly indicated that aromatic ketone groups comprised the majority of the carbonyl-group content. About one ketone group per monocyclic aromatic ring was determined for both humic and fulvic acids. Aromatic-ketone groups were hypothesized to form by photolytic rearrangements and oxidation of phenolic ester and hydrocarbon precursors; these groups have potential significance regarding haioform formation in water, reactivity resulting from active hydrogen of the methyl and methylene adjacent to the ketone groups, and formation of hemiketal and lactoi structures. Aromatic-ketone groups also may be the point of attachment between aliphatic and aromatic moieties of aquatic humic-substance structure. Key words: humic acid, fulvic acid, nuclear magnetic resonance spectroscopy, infrared spectroscopy, water

INTRODUCTION Aromatic-ketone groups have not been reported as a significant structural component of humic substances extracted from soil or water. However, 3,4-dihydroxyacetophenone and 3,4,5-trihydroxyacetopbenone were reported as products of sodium-amalgam.reduction of a soil humic acid (Schnitzer and Ortiz de Serra, 1973); they also were detected after base hydrolysis of soil humic and fulvic acids (Schnitzer and Neyround, 1975). The yields of these substituted acetophenones were small, and not much significance was attached to their presence. An aromatic-ketone group is a reactive functional group. Under basic conditions, methyl or methylene groups adjacent to the ketone form carhanions that are readily chlorinated, methylated, acetylated, or condensed via the aldol condensation. These groups are readily oxidized to aromatic-carboxyl groups or reduced to aliphatic-phenyl carbinol or aryl-aliphatic hydrocarbons. If the aromatic-ketone group is ortho to a carboxyl, or to a benzyl-hydroxyl group, it may cyclize to form a lactol or a hemiketal. As most organic-matter extraction and degradation procedures utilize a combination of alkaline conditions, oxidation, or reduction (Hayes and Swift, 1978), the identity of the aromatic-ketone group likely has been lost through these degradation procedures. The presence of active hydrogens attached to carbon in a variety of aquatic and soil humic substances was noted by Thorn (1984), who found extensive carbon-methylation of these substances by sodium hydride-methyl iodide. Leenheer and Noyes (1986)

suspected the presence of significant quantities of methyl ketones in a stream fulvic acid by interpreting the changes in the proton nuclear-magneticresonance (IH-NMR) spectrum after oximation. This preliminary evidence of active hydrogen and methylketone contents led to a systematic study of the nature of the ketone functional group in the stream humic- and fuivic-acid standards of the International Humic Substances Society. The objective of this report is to provide qualitative evidence and semiquantitative estimates of the aromatic ketone groups in the humic- and fulvic-acid standards, and to comment on the significance of the aromatic-ketone group regarding diagnetic processes and reactive properties. METHODS AND MATERIALS The aquatic humic substances selected for study were the stream humic- and fulvic-acid standards of the International Humic Substances Society. These standards were isolated from the Suwannee River near Fargo, Georgia, from November 1982 to February 1983, by procedures described by Thurman and Malcolm (1981). These procedures utilized brief contact with 0.I N sodium hydro~de, which may have caused some slight alteration in ketone content. A Varian FT-80A N M R Spectrometer was used to determine IH-NMR spectra for characterization of standards and samples (the use of trade names in this report is for identification purposes only, and does not constitute endorsement by the U.S. Geological Survey). Samples were dissolved in deuterium oxide 273

274

J.A. Lr~I-l~a et al.

or deuterated dioxane, in concentrations ranging from 20 to 100 mg/ml. Proton N M R spectra, operating at 79.5 MHz, were generated with a pulse width of 3 sec and a pulse delay of 5 sec. Solid-state ~3C-NMR spectra were obtained at 22.6 MHz, using a cross-polarization technique. A Bruker instrument, equipped with a CXP100 console and a 90-MHz ('H) iron magnet was used. The sample was spun at the magic angle (54.7 ° to the applied field) at about 3.5 kHz, in rotors constructed from boron nitride with a KeI-F base. The dipolardephasing technique was used to distinguish protonated from non-protonated carbon. Details are given in Wilson et al. (1983). Liquid-state z3C-NMR on the Suwannee River fulvic acid was obtained at 300 MHz on a Varian XL300 N M R spectrometer. The sample was dissolved at 300 mg/ml in :3C-depleted dimethyl sulfoxide, and a trace of acetone was added as an internal standard to reference the chemical shift range of sample ketone peaks. The inverse gated decoupled pulse sequence was used to obtain a quantitative spectrum; a pulse delay of 8 sec and a flip angle of 90 ° (25-Fs pulse width) was used. Total transients were 9479 which were Fourier-transformed to give the carbon-13 nuclear-magnetic-resonance (~3C-NMR) spectrum. Infrared spectra were determined on a PerkinElrder 580 infrared spectro-photometer. Samples for infrared analysis were prepared by evaporating a sample solution on a silver-chloride pellet to give a cast film of sample. The humic and fulvic acids had their infrared spectra determined as tetrabutylammonium salts, that were prepared by titration of the samples in water to pH 10 with tetrabutylammonium hydroxide, vacuum evaporation of the water, and solution of the salt in acetonitrile, from which the cast film was prepared. The carbonyl group of the samples was selectively reduced by sodium-borohydride reduction. Equal weights (20--100 rag) of sample and sodium borohydride was dissolved in 10 ml of water, then heated to 60°C for 4 hr. The reduced sample was isolated by adjusting the pH to 2 with hydrochloric acid to decompose excess borohydride, passing the reaction solution through a 20-ml column of XAD-8 resin, followed by 40 ml of 0.01 M hydrochloric-acid rinse, and eluting the sample with 50 ml of acetonitrile, which was removed by vacuum evaporation. Hydroxyl and carboxyi groups in the samples were derivatized by acetylation followed by methylation. From 20 to 100 mg of sample were dissolved in 2 ml of 1:1 pyridine-acetic anhydride, to which 10 mg of 4-dimethylaminopyridine acetylation catalyst was added. After setting at room temperature for 24 hr, the reaction mixture was added to an aqueous solution of sodium bicarbonate, then was stirred until excess acetic anhydride and mixed acetyi anhydrides in the sample had hydrolyzcd. The sample was solvent extracted with methylene chloride to remove pyridine and acetylated pyridine by-products. The sample was

adjusted to pH 1 with hydrochloric acid, and solvent extracted with methyl-ethyl ketone, into which the sample partitioned. The methyl-ethyl ketone was removed by vacuum evaporation, and the residue was taken up in 5 ml of 4:1 acetonitrile-methanol. Diazomcthane gas was passed into the acetonitrilemethanol mixture containing the acylated sample until carboxyl-group methylation was complete; the solvents were removed by vacuum evaporation. Solid-state ~3C cross polarization/magic-angle spinning (CP/MAS) spectra of the humic and fulvic acid are shown in Fig. 1. The spectra show resonances from carboxyl carbon centered at around 170 ppm; aromatic carbon centered at about 130 ppm; oxygenated aliphatic centered at about 80 ppm; and alkyl carbon centered around 44, 29 and 20 ppm. An additional peak occurs at about 200 ppm in both spectra. A major problem with obtaining ~3C CP-MAS spectra is the need to spin at sufficient speeds to re~aove both the chemical-shift anisotropy of highly anisotropic carbons (that is, aromatic and carboxyl carbons) and the spinning sidebands from the chemical-shift range. Hence, in principle, the peak at about 200 ppm could be a spinning sideband of the resonance from aromatic or carboxyl carbon. However, because sidebands occur at integral multiples of the spinner speed, for the peak at about 200 ppm to be a sideband, the rotor must be spinning at 1580 Hz or less; clearly this is not the case. Hence, the peak

/

29.5 44

(a)Humicacid

(b)~ ~ J 107

I 3o0

J

1 200

IO0

0

(PPM) Fig. 1. Solid-state z3C nuciear-magnetic-resonance spectrum of: (a) Suwannee River standard humic acid; and Co) Suwann~ River standard fulvic acid. Contact time, 1.0 reset; recycle time, I sec. Spectra were collected in 4 K points and Fourier-transformed, using a linebroadening factor of 20Hz; about 3 x I0' scans were collected.

Aromatic-ketone groups in aquatic humic substances 171

1GO

130

100

(o) Humic acid

(b) Fuivic acid

[ 300

~f,~j

,/-

i

I 200

J

I 100

I

I 0

b (PPM)

Fig. 2. Dipolar-dq)hased, solid-state t3C nuclvax-magneticresonance spectrum of: (a) Suwannce River standard humic acid; and Co) Suwannee River standard fulvicacid. Contact time, 1.0 msec; recycle time, I sec. A iinebroadening factor of 25 Hz was used; about 3 x 104 scans were collected. A dipolar.
at about 200 ppm probably is caused by quinone, ketone, or aldehyde carbon. Because quinones normally resonate at less than 200 ppm, the peak at about 200ppm probably represents ketone or aldehyde carbon. By exploiting the relaxation-rate differences among carbons having different ~3C-IH dipolar interactions, spectra can be obtained without mono or diprotonated carbons. Spectra of this type, obtained with a dipolar dephasing time of 40psec, are shown in Fig. 2. Comparison of Figs 1 and 2 indicates that much of the aromatic carbon is non-protonated. Moreover, the resonance at about 200 ppm also is present in the dipolar-dephased spectra; therefore, the resonance can be assigned to ketone rather than to aldehyde carbon. The aliphatic region of the dipolar-dephased spectra contains resonances from methyl and quaternary carbon. However, a comparison of the spectra in Figs 1 and 2 indicates that most of the aliphatic carbon is protonated. The residual resonance in the aliphatic region of the dipolar-dephased spectrum is centered at 27 ppm. This cannot result from methyl groups in linear alkanes because these resonate at 14-16 ppm (Stothers, 1972). Likewise, most methyl groups adjacent to aromatics resonate at about 21 ppm (Stothers, 1972), although some exceptions exist. It is noteworthy that the methyl group of methyl-phenyl ketones resonate at about 25-27ppm (Stothers, 1972); however, the observed resonance could also

275

result from methyl groups in branched-chain alkanes, which can produce a range of chemical shifts. Although integrating 13C spectra, such as those shown in Figs i and 2, to determine the functionalgroup content of humic substances is common, the quantitative nature of functional-group analysis by CP/MAS is, by no means, established. Total signal intensity is not always proportional to the quantity of carbon in the sample, possibly because localized regions of the samples are not observed, but the observed carbon still is representative of the whole. In contrast, carbon in different parts of a molecule or of selected molecules may not be seen, so that one functional group is observed preferentially to another. These matters have been fully discussed in Wilson et al. 0983); here, it suffices to say that checks on quantitation are best made by measuring Tt pH (proton rotating frame spin-lattice relaxation time) of the sample and comparing solid-state with solution measurements. The TmpH of the humic acid was determined to be 4.9 msec. The fulvic-acid value (4.8 msec) was similar. These values are within the ranges determined for other humic substances and soils. However, the T~pH of the oxygenated-aliphatic resonance appears to be slightly less than that of the resonances of other functional groups, so that data obtained at the 1-msec contact time may underestimate slightly the oxygenated-aliphatic resonance. The error is more serious at longer contact times. Estimates of functional-group content at a range of contact times are listed in Table I. Notwithstanding the unfortunate limitations for these samples, estimates of functional-group contents of these substances still can be made based on the constraints of the experimental data. Thus, we can conclude that about one ketone group per monocyclic aromatic ring exists for both humic and fulvic acids. Humic acid clearly is more aromatic than fuivic acid with the fraction aromatic carbon, f, = 0.410.52, compared with f, = 0.23--0.30. In contrast, the fulvic-acid fraction contains more carboxylic carbon (~0.22) than does the humic acid (~0.10). The liquid-state ~3C-NMR spectrum of the Suwannee River fulvic acid is shown in Fig. 3. The resolution of the aromatic, carboxylic, and ketone regions of the liquid-state spectrum is superior to these regions in the solid-state spectra of Figs 1 and 2. The acetone carbonyl peak at 206 ppm delineates aliphatic ketones, which appear at or downfield from acetone carbonyl, from aryl-alkyl and diaryl ketones, which appear upfield from acetone carbonyl. Quinones resonances are centered in the valley between the ketone and carboxyl peaks; thus ketone groups appear to be predominantly aryl-alkyl and diaryl ketones from the Suwannee River fulvic acid. Proton NMR spectra of humic, and fuivic-acid samples are shown in Fig. 4. The proton NMR spectra were determined on samples in the free-acid state because conversion of the samples to their sodium salts caused an upfield shift of 0.2-0.3 ppm of

276

J . A . LEENHEERet a/.

Table I. Relative integrated intensitm of resonances in '3C-NMR spectra of humic and fulvic acids from the Suwannee River, Georgia Parts per million <60 60-100 100-165 165-190 190-210 Contact P u l s e Fraction Fraction Fraction Fraction Fraction time delay alkyl O-alkyl aryl carboxyl ketone Sample Method (nude) (sec) carbon carbon carbon carbon carbon Humi¢ acid Solid.state I 0.25(0,25) 0.14(0.14) 0.41 (0.40) 0.14 (0,15) 0.06(0.06) Hunfi¢ acid Solid-state 2 0.28 0.11 0,50 0.10 0.02 Humic avid Solid.state 3 (0-100 ppm) 0.32 0.52 0.I2 0.04 Humic avid Sofid.state 4 (0-100 ppm) 0,34 0.50 0.13 0.03 Humic acid Solid-state 5 (0-100 ppm) 0.38 0.50 o. 12 0.04 Fulvic a c i d Solid-state 1 0.35 (0.32)" 0.17 (0.16) 0.24 (0.21) 0.22 (0.23) 0.05 (0.07) Fulvic acid Fulvic acid Fulvic acid

Solid-state Solid-state

Solid-state Solid-state Solid-state

Fulvic a c i d Fuivi¢ a c i d Fulvic a c i d

2 3 4

0.38 0.33 0.32

0.15 0.15 0.15

0.23 0.25 0.24

0.20 0.20 0.24

0.04 0.05 0.05

5

0.32

0.18

0.27

0.23

--

6 8 I0

0.32

0,17

0.30

0.21

--

0.32 0.14 0.26 0.28 Fulvi¢ acid 0.36 0.18 0.27 0.18 ~¢alucs in brackets obtained in Professor Maciels' laboratory, Colorado State University, Department of Chemistry. Solid-state Solid-~tate

the methyl protons of aromatic-acetyl groups. Peak assignments, based on comparison with published spectra, became ambiguous (because o f this shift). This proton N M R shift was discovered by obtaining spectra o f a series o f substituted acetyl-benzoic acids and hydroxy aceto-phcnones, in both neutral and sodium-salt forms. Both humic acid and fulvic acid (Fig. 4) have a broad major peak at 2.7 ppm, near where aromatic methyl ketones occur. Significant quantities o f methylene and methine hydrogens on carbons adjacent to the aromatic ketone also may be present at 2.8-3.0 ppm. Methyl groups o f aliphatic ketones occur at 2.1 ppm; however, aliphatic methylene hydrogen, adjacent to a carbonyl, carboxyl, or

---

ester group, also could occur in the 2.2-3.0 ppm range. To determine whether the broad 2.7-ppm peak in the proton NMR was the result of methylene protons adjacent to carbonyl groups of carboxyl or ester groups, sodium-borohydride reduction of the carbonyl group to the carbinol group was performed. Carboxyl and ester groups are not reduced by sodium borohydride. The proton NMR spectra of the reduced humic and fulvic acids are shown in Fig. 5. Both spectra have a substantial decrease in the broad 2.7-ppm peak, and a substantial increase in the 1.25-ppm peak, when compared to the unreduced spectra of Fig. 4.

i r T r i ~ z z

I

ll~

2(30

z i

j

i

i

i

i

150

[

i

1Q0 ~)

i

i

I

] 50

[

:

i

;

j 0

~ i

i

ii

,

JTI

-50

Io 4~ ( P P M )

(PPM}

Fig. 3. Liquid-state 13C-NMR spectrum of the Suwann~ River fu]vic acid.

Fig. 4. Proton NMR spectrum of Suwannee River standard humic and fuivic acids.

Aromatic-ketone groups in aquatic humic substances

277

Hum< Ac*d

l 1900

I 11100

I 1700

Wllvenumb~

,'o'

;

'

g

'

~

'

~

'

'

,,

, , , ~ ,

,,

4

0

(PPM)

Fig. 5. Proton NMR spectra of Suwannee River standard humic and fulvic acids reduced by sodium borohydride.

The valley at 1.7 ppm in the unreduced samples is much less pronounced in the reduced fulvic-acid sample; this difference indicates methylene hydrogen, adjacent to aromatic ketones, has been converted to methylene, adjacent to aromatic carbinols. The spectral shift of protons from 2.6 to 1.25 ppm and from 2.8 to 1.7 ppm after sodium-borohydride reduction is consistent with the following reactions:

a

l 1000

I

(CM")

Fig. 6. Infrared spectra of tetrabutylammonium salt (pH 10) of Suwannee River standard humic and fulvic acids.

Finally, infrared spectroscopy provided direct insight into the nature of the carbonyl group. The infrared spectra of the tetrabutylammonium salt of the humic and fulvic acids are shown in Fig. 6. Spectral interference of the free carboxyl-group absorbance, which absorbs at about 1700cm -~, is shifted to 1600 cm -~, and carbonyl-group absorhance can be viewed directly in the tetrahutylammonium salts. The humic acid gave a carbonybgroup peak at 1650 cm-~ and the fulvic acid gave a carbonyl-group peak at 1670 c m - ~.

OH

0 II

-- C - CHa 2.6 ppm

O

NaBH4 H20, 60°C

i

- C - CHa ] 1.25 ppm (x) H

O

OH

0 II -- C - CHaR

2.8 ppm

I

NaBH4 ""i

H20, 60°C

Significant quantities of unconjugated aliphatic ketones are ruled out by the results, because the chemical shift of methyl protons adjacent to a carbinol group is 1.1 ppm, and the chemical shift of methylene protons adjacent to a carbinol group is 1.4 ppm (Simons, 1978).

" CH2 :" R 1.7 ppm

C2)

Acetylation and methylation also were used to shift the interfering carboxyl group to 1740 cm -I, and to decrease the hydrogen-bonding effects on the carl> onyl group. The infrared spectra of the acetylated and methylated humic- and fulvic-acid samples are shown in Fig. 7. The humic-acid spectrum has only

278

J. A..Lr~NH~R et al.

one carbonyl peak at 1650 cm-~ and the fulvic-acid spectrum has two carbonyl peaks at 1670 and 1650 cm- ]. Both the tetrabutylammonium salt-form and the acetylated methylated derivative give similar infrared spectra for carbonyl-group absorbances at 1670 and 1650 cm-l. The absorbances can be assigned to either singly- or doubly-conjugated (quinones and aryl-aryl ketones) carbonyl groups. Examination of published infrared spectra (Pouchert, 1985) indicated that singlyconjugated, phenolic aromatic ketones (hydroxyl group methylated or acetylated) have carbonyl-group absorbances from 1670 to 1690 cm-l; however, aryl-

tannins, lignins, and steroids, do not have aromaticketone groups in their structure (Robinson, 1980). Flavones have aromatic kctones in their structure, but their carbonyl-group resonances in 13C NMR and carbonyl-group stretches in IR do not agree with measured data for the humic and fulvic acids. Two hypotheses for the formation of aromatic-ketone groups in aquatic humic substances are proposed: the first hypotheses is that a photo-Fries rearrangement (Anderson and Reese, 1960), of phenolic esters occurs [reaction (3) and (4)] to give aromatic ketones substituted ortho and para to phenols.

0

OH

0

(3) C -

R

H o

HO

0

0

0

H 2

C4)

•

HO

aryl ketones and quinones have carbonyl-group absorbances at about 1650 cm -~. The 13C-NMR spectra of humic acids (Figs 1 and 2) indicate many more aromatic C-linkages in the 145- to 165-ppm spectral region, than the fulvic acid [3C-NMR spectra do. DISCUSSION

A r o m a t i c c o m p o u n d s f o u n d in plants, such as

Phenolic esters are known to occur both as plant constituents (Robinson, 1980), and in soil humic and fulvic acids (Schnitzer, 1978). Esters, being relatively insoluble in water, are likely to occur in surface films, where photolytic rearrangements may occur. The second hypothesis is photo oxidation of aryialiphatic hydrocarbons shown in reaction (4) (Ranby and Rabek, 1975).

CH3

~

CH3 I

~

i

R + 02

hv

I

C-R

I

hv

o

I

o

I

H

0

CH3

11

I

-

o.

÷

• OH

.....>

CH3 +•R

(5)

Aromatic-ketone groups in aquatic humic substances

279

did not acetylate aromatic acetyl groups; however, acetylation catalyzed by boron trifluoride did acetylate the aromatic acetyl group, producing a beta diketone. Third, the aromatic-ketone group may cyclize with adjacent hydroxyl groups to form hemiketals [reaction (7)], or with carboxyl groups to form lactols [reaction (8)].

,J

o H ~ ' i

~'~

OH

?OR I

H- CH3

~C - R

(7)

I H

H l, i~o

,

Isoo

,

i 17oo

,

I

leoo

,

I ~WO

O

OH

Wavenumbm {CM "~}

Fig. 7. Infrared spectra of Suwannee River standard humic and fulvic acids acetylated with acetic anhydride and methylated with diazomethane.

Branched aliphatic-aromatic hydrocarbons, such as p-cymene and thymol, are commonly found in plants (Robinson, 1980); these hydrocarbons may be photooxidized to aromatic-keto groups, as shown in reaction (5). Aromatic-keto groups may be of significancein the following reactions and properties of aquatic humic substances. First, the acetyl group may chlorinate to form chloroform in reaction (6).

(s)

II o

v

These possible cyclic forms need to be considered when making structural assignments based on ]3C-NMR, ~H-NMR, and infrared spectra of humic substances. It is noteworthy that residual resonances at 100-110ppm in ~3C-NMR spectra commonly occur in dipolar-dephased spectra, including those reported here, and may be, therefore, assigned to hemiketal [reaction (7)] and lactol [reaction (8)], as well as to ketal previously reported (Wilson et al., 1983).

0 II

o 11 CH 3 + 3 C12 + OH-

>O

The haloform reaction [reaction (6)] generally has been discounted to occur with aliphatic methyl ketones in natural waters because of the strongly basic conditions needed to form carbanions that chlorinate; however, aromatic methyl ketones from carbanion and enol forms, that chlorinate under less basic conditions than unconjugated ketones (Gould, 1959). Second, the aliphatic protons on carbons adjacent to aromatic ketones are a source of active hydrogen that will methylate, acetylate, and exchange with deuterium under the acidic or basic conditions commonly used in derivatization procedures (March, 1968). These active hydrogens usually can lead to determinations of large hydroxyl contents in aquatic humic substances, when addition of a derivative, such as an acetyl group, is used to estimate hydroxyl content. The acetylation procedure used in this study

II o

c "

OH + 3 CI- + 3H20 + CHCI a

(6)

Finally, the determining of aromatic ketones attached to aliphatic methylene groups indicates that aromatic ketones may provide a point of attachment between afiphatic and aromatic moieties in the structure of aquatic humic substances. Acknowledgements--We gratefully acknowledge Kevin

Thorn and Robert Wershaw, U.S. Geological Survey, Denver, for their advice and assistance in obtaining the liquid-state J3C-NMR spectrum.

REFERENCES

Anderson J. C. and Reese C. B. (1960) Photo-induced Fries rearrangements. Pro¢. Chem. Soc. (June issue) 217. Gould E. S. (1959) Carbanions and enolization. In Mechanism and Structure in Organic Chemistry (E~ted by Gould E. S.), pp. 365-399. Holt, Rinehart, & Winston, New York.

280

J . A . LEENrmERet al.

gations on the chemistry of fungal humic acids. Soil Biol. Biochem. 7, 365-371. Schnitzer M. and Ortiz de Scrra M. I. (1973) The sodiumamalgam reduction of soil and funsal humic substances. Geoderma 9, 119-128. Simons W. W. (Ed.) (1978) The Sadder Handbook of Proton NMR Spectra, 1254 pp. Sadtler Research Laboratories, Philadelphia. Stothers J. B. (1972) Carbon-13 NMR Spectroscopy. Academic Press, New York. Thurman E. M. and Malcolm R. L. (1981) Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15, 463-466. oxidation, and Photostabilization of Polymers: Principles Thorn K. A. (1984) NMR structural investigations of aquatic humic substances. Ph.D. thesis, School of agriculand Applications, 573 pp. Wiley, New York. tural Biochemistry and Nutrition, University of Arizona, Robinson T. (1980) The Organic Constituents of Higher 193 pp. Plants, 352 pp. Cordus Press, North Amherst, Mass. Wilson M. A., Pugmire R. J. and Grant D. M. (1983) Schnitzer M. (1978) Humic substance: Chemistry and Nuclear magnetic resonance spectroscopy of soils and reaction. In Soil Organic Matter (Edited by Schnitzer M. related materials: relaxation of t3C nuclei in cross polarand Khan S. U.), pp. 1-64. Elsevier, Amsterdam. Schnitzer M. and Neyroud J. A. (1975) Further invest_i-... ization nuclear magnetic resonance experiments. Org. Geochem. 5, 121-129. Hayes M. H. B. and Swift R. S. (1978) The chemistry of soil organic colloids. In The Chemistry of Soil Constituents (Edited by Greenland P. J. and Hayes M. H. B.), pp. 179-320. Wiley, New York. Leenheer J. A. and Noyes T. L (1986) Derivatization of Humic Substances for Structural Studies. Wiley (in press). March J. (1968) Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 1095 pp. McGntw-HiU, New York. Pouchert C. J. (1985) The Aldrich Library of FT-IR Spectra, 1st edn, "Col. 2, pp. 1-99. Aldrich Chemical Company, Milwaukee, Wis. Ranby B. and Rabek J. F. (1975) Photodegradation, Photo-