Comparisons of Humic Substances Extracted from Contiguous Alfisols and Mollisolsof Southwestern Ohio

J. M. Novak* and N.E. Smeck

ABSTRACTHumic and fulvic acids extracted from contiguous Alfisols and

Mollisols were characterized to determine if any dissimilarities couldbe documented between humic substances extracted from soilsformed under nearly identical environmental conditions, but belong-ing to different taxonomic classes. Comparisons of humic substancesfrom two Alfisol-Mollisol pairs, one from the Wisconsinan and onefrom the Illinoian till plain of southwestern Ohio, were based on wetchemical, infrared, and "C cross-polarization magic angle spinningnuclear magnetic resonance (CPMAS NMR) spectroscopy studies.Using 0.1 M NaOH, more humic substances were extracted fromthe Mollisols than the Alfisols, with the majority being humic acids.Small amounts of fnlvic acids were recovered from all pedons. Datafrom infrared and 13C CPMAS NMR spectroscopy indicate thathumic acids from both the Alfisols and Mollisols are dominated byalkyl-substituted aromatic structures, followed by aliphatic, O-sub-stituted alkyl, carboxylic acid, and small amounts of carbonyl struc-tures. Whereas I3C NMR spectroscopy data suggest Mollisol humicacids contain higher aromatic and carboxylic acid contents thanthose of the Alfisols, Alfisol humic acid spectra show more evidencefor phenolic-OH groups and lignin and lignin-like products thanthose of the Mollisols. Fulvic acids from Alfisols and Mollisols con-tained mostly aliphatic, O-substituted alkyl, and carboxylic acid,followed by aromatic groups and small amounts of carbonyl groups.The proportion of nonaromatic and aromatic structures in the fulvicacids of the Alfisols and Mollisols was similar, but slight differenceswere noted in elemental ratios and "C NMR spectra. Although onlyminimal differences were noted among fulvic acids, differences wereevident in the chemical and structural properties of humic acids fromcontiguous Alfisols and Mollisols in southwestern Ohio.

PRIOR TO THE 1980s, reports indicated that humicsubstances in soils from widely differing climatic

zones exhibited similar chemical and compositionalproperties. Both Lowe (1969) and Khan (1971) re-ported that humic acids extracted from several majorsoil groups in Alberta were similar with respect to el-emental and functional-group composition. On a moreglobal scale, Schnitzer (1977), using wet chemical andoxidative-degradation methods, reported that humicsubstances extracted from soils formed in different cli-matic zones were also similar in composition.

Following the application of 13C CPMAS NMR inthe 1980s, however, compositional differences of hum-ic substances among soils were reported. In 1981, 13CCPMAS NMR spectra published by Hatcher et al.(1981) for humic acids extracted from soils of cooltemperate, subtropical, and temperate climates re-vealed different spectral patterns for Inceptisols, Mol-lisols, and Ultisols with wide ranges in aromaticity(43-92%). Nuclear magnetic resonance studies byJ.M. Novak, Savannah River Ecology Lab., Drawer E., Aiken, SC29801. N.E. Smeck, Dep. of Agronomy, Ohio State Univ., Colum-bus, OH 43210. Salaries and research support provided by state andfederal funds appropriated to the Ohio Agricultural Research andDevelopment Center, Ohio State Univ. Ohio State Journal no. 240-89. Received 26 Feb. 1990. Corresponding author.

Published in Soil Sci. Soc. Am. J. 55:96-102 (1991).

Skjemstad et al. (1983) and Saiz-Jimenez et al. (1986)both showed wide differences in the aromatic characterof humic acids extracted from Inceptisols and Molli-sols of Australia and Spain, respectively. Recently, Lo-bartini and Tan (1988) provided 13C NMR spectra ofhumic acids extracted from Entisols, Inceptisols, Mol-lisols, Spodpsols, and Ultisols of Indonesia, the USA,and Argentina and concluded that a distinctive spec-trum was produced from each humic acid studied.These studies indicate that 13C NMR spectra of humicacids extracted from soils belonging to various soilorders are different. These differences cannot, how-ever, be attributed solely to inherent differences amongsoil orders because the humic acids in these studieswere extracted from soils of widely different climaticzones and environmental settings.

In Ohio, Mollisols and Alfisols with evident differ-ences in organic-matter content often occur in closeproximity and in very similar environmental settings.Such a situation provides an opportunity to examinehumic substances from different soil orders withoutconfounding environmental differences. The soil dif-ferences are thought to be due to differences in nativevegetation — forest for the Alfisols and prairie grassesfor the Mollisols.

The hypothesis for this study is that humic sub-stances from Mollisols and Alfisols can be distin-guished even when the soils occur in very similarenvironmental settings. Therefore, the objective is tocharacterize and compare humic and fulvic acids ex-tracted from the A horizons of contiguous Alfisols andMollisols. The humic substances were examined bywet chemical, infrared (IR), and 13C NMR techniques.

MATERIALS AND METHODSSampling Sites

Contiguous well- or moderately well-drained Alfisols andMollisols are common in southwestern Ohio. Warren Coun-ty was selected for the site due to surficial parent-materialuniformity. The entire county is blanketed with a mantle ofPepria loess. Two sites were selected, with each containingadjoining Alfisol and Mollisol polypedons (Fig. 1). The siteswere carefully selected to ensure that the soil-forming factors,parent material, age, topography, and climate, were essen-tially identical for the adjoining polypedons. The site is inthe western portion of Warren County on a uniform 1%slope, and is underlain at a depth of 76 to 84 cm by Wis-consinan glacial till, whereas the site in the eastern half ofthe county occurs on a uniform 3% slope and is underlainat a depth of 86 to 102 cm with Illinoian glacial till. All fourpedons are moderately well drained and the Mollisol andAlfisol pedon at each site are < 175 m apart. Only nativevegetation differs between the Mollisol and Alfisol at eachsite. Information in the Warren County soil survey report(Garner et al., 1973) indicates that the Alfisols formed underhardwood forest, whereas the associated Mollisols formedunder mixed prairie vegetation and trees. Both sites havebeen cleared and cultivated for more than 50 yr, with maize(Zea mays L.) and soybean [Glycine max (L.) Merr.] beingthe dominant crops for the last 15 to 20 yr. The main dif-

96

NOVAK & SMECK: HUMIC SUBSTANCES EXTRACTED FROM SOILS OF SOUTHWESTERN OHIO 97

Table 1. Classification and properties of the horizon extracted for the four pedons used in this investigation.Particle-size distribution

Pedon Order Family Colorf Organic C pH Sand Silt Clay

Dana 1Dana 2XeniaRossmoyne

MollisolMollisolAlfisolAlfisol

Fine-silty,Fine-silty,Fine-silty,Fine-silty,

mixed,mixed,mixed,mixed,

mesic Typic Hapludollmesic Typic Hapludollmesic Aquic Hapludalfmesic Aquic Fragiudalf

10YR 3/210YR 3/110YR 4/210YR 4/2

24.921.014.811.0

5.86.65.97.5

6.44.39.6

10.0

68.761.974.773.5

24.933.812.216.5

t Munsell notations: 10YR 3/2 (very dark grayish brown), 10YR 3/1 (very dark gray), and 10YR 4/2 (dark grayish brown).

ference between the Mollisols and Alfisols is in surface colorand organic-matter content (Table 1). The Alfisols on theWisconsinan surface are mapped as Xenia, whereas those ofthe Illinoian surface are mapped as Rossmoyne; becauseboth Mollisols are mapped as Dana, they will be referred toas Dana 1 (Wisconsinan) and Dana 2 (Illinoian) (Fig. 1).

Pits were excavated for each pedon and morphologicaldescriptions were prepared using standard soil-survey pro-cedures and terminology (Soil Survey Staff, 1981). Bulk sam-ples were collected from the horizons with the darkest color(highest organic-matter content), which is the surface hori-zon (Ap) of all pedons except Dana 2, where the horizon (A)at 25 to 43 cm was selected. Samples were dried in a forced-draft oven at 60 °C and crushed to pass a 2-mm sieve.Soil Characterization

Particle-size distributions were determined using the pi-pette method of Kilmer and Alexander (1949) and Soil Sur-vey Staff (1972). Organic carbon (OC) contents weredetermined by dry combustion (950 °C) according to themethod of Nelson and Sommers (1982). Soil pH was meas-ured from a 1:1 soil/water suspension.Extraction ofHumic and Fulvic Acids

Humic substances were extracted and purified using pro-cedures outlined and distributed by the International HumicSubstances Society (Aiken et al., 1985). Briefly, 1.0 to 1.2 kgof 2-mm oven-dry soil were suspended in 0.1 M NaOH (so-lution/soil ratio of 10:1) in a 19-L plastic bucket under anN2 atmosphere for 24 h at room temperature. The extractwas acidified with 6 M HC1 to a pH of < 1. Humic and fulvicacids were then separated by centrifugation at 1350 g. Thehumic acid fraction was purified with 0.1 M HC1-0.3 M HF,and then dialyzed in deionized water using no. 20 dialysistubing (Union Carbide Corp. Chicago, IL) with a molecular-weight cutoff point of 12 000. Fulvic acids were purified withrepeated passages through Amberlite XAD-8 no. A-6525, 1-to 0.5-mm resin (Sigma Chemical Co., St. Louis, MO), fol-lowed by washings with 0.3 M HF, and dialyzed in the sameway as the humic acids. To ensure removal of salts, the fulvicacids were then passed four times in succession through Bio-Rad Ag MP-50, 0.5- to 0.2-mm resin (Bio-Rad Laboratories,Richmond, CA) in the H form. Both fractions were thenfreeze-dried and stored over P2O5.Characterization ofHumic and Fulvic Acids

Total acidity, carboxylic acid, and phenolic-OH contentwere measured on all humic acid samples using methodsdescribed by Schnitzer (1982). Sufficient quantities of fulvicacids were not available for these measurements. Ash con-tents and C, H, O, and N contents (oven-dry, ash-free basis)were determined for humic and fulvic acid samples by Gal-braith Labs of Knoxville, TN. Infrared absorption spectrafor all humic and fulvic acid samples were obtained usingthe KBr pellet technique described by Schnitzer (1982). Spec-tra were collected on a Beckman model 4250 diffuse double-beam IR spectrophotometer (Beckman Co., Los Angeles,CA).

Sample Siteso Xenia and Dana 1• Rossmoyne and Dana 2

Fig. 1. Location of sample sites and major glacial deposits in Ohio.

Solid-state 13C CPMAS NMR spectra for all humic acidswere obtained on a Bruker CXP-100 spectrometer (BrukerIndustries, Berlin, Germany) operating at 22.6 MHz. Sam-ples were spun at the magic angle (54.7 °C) at approximately3 to 3.5 kHz. Spectra based on 12 000 scans were obtainedusing a contact time of 1 ms and a recycle time of 1 s. Solid-state 13C CPMAS NMR spectra for all fulvic acids were ob-tained on a Nicolet NT- 150 spectrometer (General Electric,Los Angeles, CA) operating at 1 5 MHz at the Regional NMRCenter at Colorado State University, Ft. Collins, CO. Sam-ples were spun at the magic angle at 3.8 kHz. Spectra basedon 9000 scans were obtained using a contact of 1 ms and arecycle time of 1 s. Chemical shifts in the humic and fulvicacids were reported downfield from tetramethylsilane. Thespectra were evaluated in a manner similar to that of Hatcheret al. (1983), Saleh et al. (1983), and Skjemstad et al. (1983).This was accomplished by dividing each spectrum into fiveregions: aliphatic C (0-50 ppm), O-substituted alkyl C (50-110 ppm), aromatic C (110-160 ppm), carboxylic acid C(160-190 ppm), and carbonyl C (190-240 ppm). The areasfor the previously defined chemical-shift regions were delin-eated by dropping verticals to a baseline. Areas were esti-mated by tracing the spectra on plastic sheets, cutting outthe defined regions, weighing, and expressing as a percentageof the total OC content. Hatcher et al. (1983) reported thatthe error associated with estimating the area of well-definedpeaks in I3C NMR spectra is ± 5%.

RESULTS AND DISCUSSIONThe OC contents of the soil horizons selected for

this study range from 11.0 to 24.9 g kg-1 (Table 1).The Mollisols have OC contents that are 1 .5 to 2 timesgreater than those in the associated Alfisols. The min-

98 SOIL SCI. SOC. AM. J., VOL. 55, JANUARY-FEBRUARY 1991

eral fractions are dominated by silt, which is typicalfor soils derived from loess (Table 1). The pH valuesof the A horizons are mildly acidic, except Rossmoyne,which has an alkaline pH.

Chemical AnalysesYields of humic substances extracted from all four

pedons have been expressed as a percentage of the totalorganic carbon (TOC) content (Table 2). Twenty-fourto 40% of the TOC content of the soils was extracted.The values are similar to those reported by Lowe(1969) for an Orthic Gray Wooded and an OrthicBlack soil (23-38%). The Mollisols (Dana 1 and 2)yielded approximately 1.2 to 1.6 times more humicacids than the Alfisols (Xenia and Rossmoyne). Lowe(1969), Tan (1978), and Stevenson (1982) have all re-ported that grassland soils (Mollisols) yielded morehumic acids than forest soils (Alfisols). In contrast, theyields of fulvic acids were very similar for the Alfisolsand Mollisols, but much lower than those of humicacids. The low yields of fulvic acids were considerablylower than those reported by Lowe (1969) and Ste-venson (1982). The low yield of fulvic acids in thisstudy is attributed to a poor choice of dialysis tubing(molecular-weight cutoff point 12 000) and the stren-ous, multiple-step purification process used. The low-molecular-weight component of the fulvic acids wasprobably lost during purification. Consequently, thefulvic acids recovered in this study may not representthe entire range of fulvic acids present in the soils.

The elemental composition of the humic acids ex-tracted in this study ranged from 528 to 583 g C, 38to 49 g H, 338 to 380 g O, and 31 to 48 g N kg-1 ofoven-dry, ash-free humic acid (Table 3). Ash contentsranged from 7 to 20 g kg"1 humic acid. The compo-sition is similar to that of an ideal soil humic acidreported by Schnitzer (1977) and humic acids extract-ed from a Gray Solonetz and a Brown Solod reportedby Lowe (1969). Some trends, however, are apparent.The C content of humic acids from soils on the Illi-noian surface (Dana 2 and Rossmoyne) was lower andthe O content higher than that of humic acids fromsoils on the Wisconsinan surface (Dana 1 and Xenia)as illustrated by differences in the O/C ratios (Table3). This suggests that organic matter of soils on theWisconsinan surface is more humified than organicmatter of soils on the Illinoian surface. The perceiveddifferences in humification may be indicative of dif-ferences in the length of time the two sites have beencultivated. Some differences can also be noted betweenthe humic acids of the Alfisols and Mollisols. Thehumic acids extracted from the Alfisols had wider H/C ratios and higher H and N contents than the Mol-lisols. Generally, H and N contents increased with adecrease in OC content of the soil. Wide H/C ratiosare indicative of humic substances with less condensedor more open aromatic-type structures and a relativelyhigh content of aliphatic components (Visser, 1983).This implies that the humic acids extracted from theAlfisols were not as structurally condensed as the Mol-lisol humic acids.

Elemental composition of the fulvic acids rangedfrom 405 to 460 g C, 28 to 36 g H, 292 to 371 g O,and 20 to 28 g N kg"1 of oven-dry, ash-free fulvic acid

Table 2. Yield of humic and fulvic acids expressed as a percentageof the total organic carbon (TOC) content.

Yield of humic substances

Soil Soil OC Fulvic acids Humic acids Total

Dana 1Dana 2XeniaRossmoyne

g kg'1

24.921.014.811.0

1.20.61.11.2

—— % ———

38.828.523.224.2

40292425

Table 3. Elemental composition and ratios of humic and fulvicacids.f________________________________

Elemental ratios

Soils

Dana 1Dana 2XeniaRossmoyne

Dana 1Dana 2XeniaRossmoyne

C

583528565528

418405416460

H

— g

38414849

36322833

O N H/C O/C N/CK6

Humic acids338 31361 39350 40380 48

Fulvic acids351371292344

20282220

0.770.931.011.10

1.010.960.810.86

0.440.510.460.54

0.630.690.530.56

0.050.060.060.08

0.040.040.050.04

t Reported on an oven-dry, ash-free basis.

(Table 3). Ash contents ranged from 37 to 115 g kg"1

fulvic acid. Although the C and N contents are similarto those of the ideal fulvic acid (Schnitzer, 1977), theH and O contents are lower. Steelink (1985) reportedthat the H/C, O/C, and N/C ratios for fulvic acidstypically cluster around 1.0,0.7, and 0.05, respectively.Whereas the H/C, O/C, and N/C ratios for fulvic acidsfrom the Mollisols were similar to values reported bySteelink (1985), the H/C and O/C ratios for fulvicacids from the Alfisols were lower. The data generatedin this study suggest some slight compositional differ-ences between the fulvic acids of the Mollisols andAlfisols.

Total-acidity values of the humic acids ranged from5.18 to 7.36, carboxylic acid content from 3.94 to 5.13,and phenolic-OH content from 1.24 to 2.34 mol kg"1

of humic acids (Table 4). Functional-group analyseswere not performed on fulvic acids because of insuf-ficient sample. Humic acids extracted from the Mol-lisols contained higher quantities of total acidity,carboxylic acid, and phenolic-OH groups than thoseextracted from the Alfisols. Humic acids extractedfrom Mollisols contained similar contents of total ac-idity, higher contents of carboxylic acids, and lowercontents of phenolic-OH groups than reported byLowe (1969) for an Orthic Brown and an Orthic Blacksoil, and by Schnitzer (1977) for an ideal humic acid.Whereas the total acidity and phenolic-OH contentsfor Alfisol humic acids were lower than the ideal humicacid (Schnitzer, 1977), carboxylic acid groups werehigher and all contents were lower than those reportedby Lowe (1969) for an Orthic Gray Wooded soil.

Infrared SpectroscopyInfrared spectra of both the humic and fulvic acids

(Fig. 2 and 3) show a strong -OH absorption band at

NOVAK & SMECK: HUMIC SUBSTANCES EXTRACTED FROM SOILS OF SOUTHWESTERN OHIO 99

Table 4. Total acidity, carboxylic acid, and phenolic-OH content ofhumic acids as measured by chemical methods, t Mollisols

Soils

Dana 1Dana 2XeniaRossmoyne

Total acidity

6.797.365.185.74

Carboxylic acid

4.455.133.943.95

Phenolic-OHJ

2.342.231.241.79

f Means of three replicates.| Determined by difference between total acidity and carboxylic acid groups.

3430

4000 3200 2400 1700 1300

Wave No. cm-'Fig. 2. Infrared spectra (4000-900 cnr1) for (a) Dana 1, (b) Dana

2, (c) Xenia, and (d) Rossmoyne humic acids.

3430 to 3250 cm-1, a C-H absorption band at 2960 to2900 cm-1, a C=O stretch of COOH absorption bandat 1730 to 1710 cm-1, and an aromatic C=C absorp-tion band at 1640 to 1610 cm-1. All humic acid spectra,with the exception of the Dana 1 specimen, exhibit aweak band indicative of C=O stretching of amidesand quinones at 1650 cnr1. Other bands common toboth the humic and fulvic acid spectra are attributableto weak -OH deformation and C-O stretching of phe-nolic-OH or COO-antisymmetric stretching at 1420 to1390 cm'1, a C-O stretch and -OH deformation ofCOOH groups at 1230 to 1200 cm-1, and a C-Ostretching of pplysaccharide-like substances or Si-O ofsilicate impurities at 1110 to 1080 cm-1. All humicacids exhibit a =C-H out-of-plane bending of aro-matic and alkene structures at 900 cm-' (Pavia et al.,1979). Only the Dana 2 fulvic acid spectrum, however,exhibits this same peak. It is interesting to note thatthe spectra of fulvic acids exhibit a weaker band at1640 to 1610 cm-1 than those of the humic acids, whichindicates that there was less aromatic character in thefulvic acids than the humic acids. The IR spectral pat-terns of humic and fulvic acids in this study are similarto those of Schnitzer and Gupta (1964) and Stevensonand Goh( 1971).

No differences are evident by IR spectroscopyamong the humic and fulvic acids extracted from thefour soils in this study. Infrared analyses did revealthat humic and fulvic acids were composed of aro-matic, aliphatic, carbonyl (as carboxylic acids, ke-tpnes, and phenolic-OH) groups and polysaccharide-like components and that the fulvic acids were not asaromatic as the humic acids.

3400 2960

Alfisols VV29203420

1720 111010851640

1200 f (C)

3380 2960

4000 3200 2400 1700

Wave No. cnr11300 900

Fig. 3. Infrared spectra (4000-900 cm-') for (a) Dana 1, (b) Dana2, (c) Xenia, and (d) Rossmoyne fulvic acids.

SpectraSolid-state 13C CPMAS NMR spectra for the humic

and fulvic acids are presented in Fig. 4 and 5, respec-tively. Each spectrum was divided into regions of res-onance frequencies for major organic structures asdescribed earlier. To facilitate spectral interpretation,the most significant peaks in each resonance frequencyregion will be identified and the significance of eachregion discussed.

In the aliphatic region (0-50 ppm), spectra of humicacids from Dana 1 and Xenia exhibit a broad peak(18-32 ppm), whereas those of Dana 2 and Rossmoyneshow a relatively well-defined peak at 32 to 34 ppm(Fig. 4). This suggests that, although humic acids fromall soils contained methyl-C in long chains and alkyl-C bonded to aromatic ring structures (Saleh et al.,1973), Dana 1 and Xenia humic acids may have con-tained a more heterogenous mixture of aliphatic struc-tures than the Dana 2 and Rossmoyne humic acids.

In the O-alkyl region (50-110 ppm), peaks are ev-ident in the spectra of all humic acids at 57 to 58 andat 73 to 74 ppm. The peak at 58 ppm has been at-tributed to methoxy associated with lignin and lignin-like products (Hatcher et al., 1980; Piotrowski et al.,1984), whereas the 74-ppm peak has been attributedto carbohydrates (Voelter and Breitmaier, 1973; Pres-ton and Ripmeester, 1982). The Alfisol humic acidsexhibited more prominent methoxy signals than thoseof the Mollisols, which suggests that the Alfisol humicacids contained more lignin and lignin-like products.This supports the soil surveyors' contention (Garneret al., 1973) that trees are the native vegetation of theAlfisols, because trees contribute more organic debrishigh in lignin and lignin-like products to the pool ofsoil organic matter than do grasses.

The most intense peak in the humic acid spectra ofthis study occurred at 129 to 134 ppm, which Schnitzerand Preston (1986) have attributed to aromatic struc-tures substituted with alkyl groups. Minor peaks, in-dicative of phenolic-OH groups, also occurred at 153to 154 ppm in all humic acids. The phenolic-OH grouppeaks are somewhat more prominent in spectra of theAlfisol humic acids than in those of the Mollisol humicacids. In contrast, chemical analyses (Table 4) indi-

100 SOIL SCI. SOC. AM. J., VOL. 55, JANUARY-FEBRUARY 1991

134^-129 172

200 1005ppm

Fig. 4. Solid state I3C cross-polarization magic angle spinning nu-clear magnetic resonance spectra of (a) Dana 1, (b) Dana 2, (c)Xenia, and (d) Rossmoyne humic acids.

cated a larger concentration of phenolic-OH groups inthe Mollisol humic acids than in the Alfisol humicacids. Irrespective of the contrasting trends, slight dif-ferences in phenolic-OH contents are indicated byboth methods.

In the 160- to 190-ppm region, all humic acid spectraexhibited a prominent peak at 173 to 174 ppm, whichSaleh et al. (1983) reported to be indicative of car-boxylic acid, amides, and esters. Minor peaks in thecarbonyl region (190-240 ppm) of the spectra wereevident for all four humic acids (Fig. 4).

Solid-state 13C CPMAS NMR spectra are shown inFig. 5 for fulvic acids from all soils. In the aliphaticregion, spectra for all fulvic acids showed a broad,noisy peak between 30 and 41 ppm, but only those ofthe Alfisols exhibited a shoulder at 17 to 18 ppm. Salehet al. (1983) indicated that the Alfisol humic acid typ-ifies aliphatic-side-chain C and C from terminal meth-yl groups or aromatic rings, and the shoulder isindicative of terminal methyl groups. The occurrenceof the shoulder at 17 to 18 ppm in spectra of the Alfisolfulvic acids that is absent in those of the Mollisolssuggests that there are some differences between thefulvic acids of the Mollisols and Alfisols.

All fulvic acid spectra exhibited a weak signal at 56ppm that provided little evidence for lignin and lignin-like products. Strong absorption peaks at 70 to 72 ppmsuggested that carbohydrate and carbohydrate-likecomponents are major constituents. Preston and Rip-meester (1982), Hatcher et al. (1983), and Blondeau(1986) have reported that soil fulvic acids are domi-nated by carbohydrate and carbohydrate-like com-ponents. Unlike for the humic acids, the peaksindicative of aromaticity occurring between 130 and

40 37

(d)

200 1008 ppm

Fig. 5. Solid-state I3C cross-polarization magic angle spinning nu-clear magnetic resonance spectra of (a) Dana 1, (b) Dana 2, (c)Xenia, and (d) Rossmoyne fulvic acids.

137 ppm were not the most intense signals in the fulvicacid spectra. Saleh et al. (1983) suggested that this in-dicates that, although fulvic acids have some aromaticcharacter, they are mainly alkyl substituted. Very weakpeaks at 146 to 149 ppm indicated that phenolic-OHgroups were either not detectable with 13C NMR orwere only a minor constituent in the fulvic acids. Lowdetectability of phenolic-OH groups by 13C CPMASNMR has been reported previously by Hatcher et al.(1981) and Schnitzer and Preston (1986). The mostprominent peak in the fulvic acid spectra occurs be-tween 172 and 174 ppm and is due to carboxylic acids,amides, and esters. A dominance of carboxylic acidadsorption peaks has been noted previously by Saiz-Jimenez et al. (1986) in spectra of soil fulvic acids. Allfulvic acid spectra exhibited some minor peaks in thecarbonyl region, but the signals were weak and barelydiscernable above background noise.

Distribution of Carbon in Humic and Fulvic AcidsExamination of the C distribution in both the Mol-

lisol and Alfisol humic acids (Table 5) indicates that30 to 45% of the C occurred in aromatic structures.The percentages found in this study are similar tothose reported for humic acids from Mollisols bySchnitzer and Preston (1986) and Lobartini and Tan(1988), but slightly lower than values reported byHatcher et al. (1981). It should be noted, however, thataromatic C content was distinctly higher in the Mol-lisol humic acids than in those of the Alfisols. Thepercentage of C occurring as carboxylic acid structuresin humic acids was also noticeably higher in the Mol-lisols than in the Alfisols, which is consistent with therelative amounts of carboxylic acids measured by wet

NOVAK & SMECK: HUMIC SUBSTANCES EXTRACTED FROM SOILS OF SOUTHWESTERN OHIO IOI

Table 5. Distribution of C in humic and fulvic acids as determinedby 13C nuclear magnetic resonance (NMR) spectroscopy.

SoilsAliphatic O-alkyl

C CAromatic Carboxylic Carbonyl

C acidC C- % of total organic C -

Humic acidsDana 1Dana 2XeniaRossmoyne

Dana 1Dana 2fXeniaRossmoyne

18.116.726.522.9

24.625.223.827.3

19.4 42.623.7 41.622.3 30.129.6 33.2

Fulvic acids30.4 19.323.8 18.828.8 18.023.7 18.8

18.518.517.614.9

23.228.024.225.5

1.44.73.51.7

3.54.25.24.7

t Fulvic acids extracted from the Ap (0-25 cm) horizon.

chemical methods (Table 4). Conversely, the amountof C occurring as aliphatic structures was higher inhumic acids of the Alfisols than in those of the Mol-lisols. The C found in aliphatic structures in this studywas somewhat lower than that reported by Lobartiniand Tan (1988) for Mollisols. The percentage of Coccurring in O-alkyl and carbonyl structures was sim-ilar for humic acids of the Alfisols and Mollisols. Theamount of carbonyl groups was small and similar tothat reported by Hatcher et al. (1980).

In contrast to the humic acids, only 18 to 20% ofthe C in the fulvic acids occurred in aromatic struc-tures. This is consistent with previous reports by Wil-son and Goh (1977), Preston and Ripmeester (1982),Hatcher et al. (1983), and Blondeau (1986). The fulvicacids consisted of approximately equal contributionsof aliphatic, O-alkyl, and carboxylic acid structuresand somewhat smaller contributions of aromaticstructures. Small quantities of carbonyl groups werepresent in all fulvic acids. The only apparent differenceamong the fulvic acids was that those extracted fromthe soils on the Illinoian surface had slightly less O-alkyl character. An explanation for this is not appar-ent.

SUMMARYMore humic substances were extracted from the

Mollisols than the Alfisols, with the majority beinghumic acids. Although no differences were detectedbetween humic substances of contiguous Alfisols andMollisols by IR spectroscopy, some differences weredetected with chemical and 13C GPMAS NMR ex-aminations. Differences noted between the Alfisol andMollisol fulvic acids were minimal: Lower H/C andO/C ratios and the presence of a shoulder at 17 to 18ppm in the 13C NMR spectra for the Alfisols. Severalcharacteristics, however, were noted that distinguishthe humic acids extracted from the Alfisols and Mol-lisols. Data from elemental analyses and 13C NMRspectroscopy indicate that humic acids of the Mollisolswere distinctly more aromatic than those of the Alfi-sols. Likewise data of the functional group analysesand 13C NMR spectroscopy indicate that Mollisolhumic acids contained more carboxylic acid groupsthan those of the Alfisols. Conversely, aliphatic struc-tures comprised a larger portion of Alfisol humic acids

than those of Mollisols. Furthermore, 13C NMR spec-tra of Alfisol humic acids exhibited more prominentmethoxy and phenolic-OH signals than those of theMollisols. The former is associated with lignin andlignin-like components, which are more indicative oforganic matter derived from trees than grasses. Thissuggests that the native vegetation associated with theAlfisols and Mollisols may have differed and is con-sistent with the concept that Alfisols form under for-ests and Mollisols form under prairies. We concludethat humic substances, particularly humic acids, canbe distinguished between contiguous Alfisols andMollisols.

ACKNOWLEDGMENTSAcknowledgment is extended to the Colorado State Uni-

versity Regional NMR Center for the 13C NMR analysis.The NMR Center is funded by the National Science Foun-dation Grant no. CHE 8208821. Sincere thanks to Mr. LeeBurras and staff at the Ohio State University Soil Charac-terization Laboratory for soil analyses.

102 SOIL SCI. SOC. AM. J., VOL. 55, JANUARY-FEBRUARY 1991