Organic Geochemistry 38 (2007) 1061–1072

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OrganicGeochemistry

Characterisation of the backbone structures of severalfulvic acids using a novel selective chemical reduction method

Rama D. Nimmagadda, Christopher McRae *

Department of Chemistry and Biomolecular Sciences, Division of Environmental and Life Sciences,

Macquarie University, Sydney, NSW 2109, Australia

Received 6 June 2006; received in revised form 2 February 2007; accepted 19 February 2007Available online 15 March 2007

Abstract

Fulvic acid ‘backbone’ structures were studied by subjecting fulvic acids isolated from Elliott soil, Suwannee River andWaskish peat to a selective reduction method. The products were subsequently analysed using gas chromatography andgas chromatography-mass spectrometry. The reducing agent was n-butylsilane in the presence of tris(pentafluorophe-nyl)borane, the latter serving as a Lewis acid catalyst. The method is the first chemical degradation method able to reducethe polar groups of fulvic acids without destroying any ‘core’ structural information. The degradation method also enablesreduction of polycarboxylic acids, alcohols, aldehydes, ketones and methyl esters to alkanes, with phenols being convertedto silyl ethers. Ester linkages are reduced to ethers and ethers are unaffected.� 2007 Elsevier Ltd. All rights reserved.

1. Introduction

Humic substances (HSs) are described as beinga mixture of naturally occurring precursors derivedfrom degraded organic matter in soil and naturalwaters (Hayes and Swift, 1990). They generallyconsist of a wide range of compounds, includingamino acids, sugars and polysaccharides. HSs aretypically classified into three main groups compris-ing humin, humic acid and fulvic acid. Humin isclassified as a material which is insoluble in aque-ous acids or bases, humic acid is a substance solu-ble in solutions with pH > 2 and fulvic acid soluble

0146-6380/$ - see front matter � 2007 Elsevier Ltd. All rights reserveddoi:10.1016/j.orggeochem.2007.02.016

* Corresponding author. Tel.: +61 2 9850 8288; fax: +61 2 98508313.

E-mail address: christopher.mcrae@mq.edu.au (C. McRae).

at any pH value. HSs are yellow to black in col-our, refractory and are considered to be of rela-tively high molecular weight ranging from 600 to500,000 Da (Malcolm, 1990).

The role of HSs in the environment is extremelyimportant. They hold micronutrients in the soilessential for plants and their root systems and playa crucial role in maintaining the acid-base bufferingability of soils. They are also able to bind mineralparticles together in soil environments, thereby con-tributing to the soil structure and helping to main-tain the moisture content. HSs can also dissolvecertain minerals, bind small organic compounds,reduce metal ions and act as mediating electronshunts in microbial and abiotic redox reactionsand are capable of forming and solubilising toxiccompounds in the environment (Hayes and Clapp,2001; Pena-Mendez et al., 2005).

.

1062 R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072

The structure of HSs is poorly understooddespite their important role in natural ecosystems.As a result of their chemical heterogeneity they arenot only complex but their composition is a func-tion of the different properties (vegetation, climateand topography) of the ecosystems in which theyare formed. Their chemical complexity is a resultof the presence and combination of numerous poly-carboxylic acid, phenol, alcohol, ketone, aldehyde,ester and quinone functional groups. Various0characterisation techniques have been used tostudy the bulk properties, including vapour pressureosmometry (Aiken and Malcolm, 1987), viscosity(Nikonova and Nikonov, 1982), laser light scatter-ing (Wagoner and Christman, 1998), gel permeationchromatography (Guel et al., 2003), field desorptionmass spectrometry (Schulten, 1987), laser desorp-tion mass spectrometry (Pena-Mendez et al., 2005)and size exclusion chromatography (Piccolo et al.,2002; These et al., 2004). These methods haveproduced quite varied molecular weight rangesfor HSs, attributed mainly to the limitationsof the methods when applied to polydispersesystems.

Recently, electrospray ionisation (ESI) mass spec-trometry (McIntyre et al., 1997, 2001; McIntyre andMcRae, 2005; These et al., 2004) become a populartechnique for studying the water soluble componentsof HSs. Although several limitations of ESI whenapplied to HSs have been identified (McIntyreet al., 2002; These and Reemtsma, 2003) the couplingof ESI to Fourier Transform Ion cyclotron Reso-nance (FTICR) mass spectrometry has, however,enabled the exact masses of a homologous series offulvic acids to be obtained (Stenson et al., 2003).Analysis of functional groups and structural elucida-tion of HSs has been performed using numerousspectroscopic methods, such as nuclear magnetic res-onance (Simpson, 2006), infrared (Gossart et al.,2003), UV–Vis (Barreto et al., 2004) and fluorescencespectroscopy (Sierra et al., 2005) together with wetchemical techniques such as potentiometric titration(Ghatak et al., 2004). Despite the advances made inunderstanding the structure of HSs that all thesetechniques have brought, definitive structural infor-mation, particularly at the molecular level, has yetto be obtained.

In an attempt to obtain structural information atthe molecular level, several chemical degradationtechniques, such as oxidation (Cuypers et al.,2002; Lehtonen et al., 2004), reduction (Egliteet al., 2003), pyrolysis (Ikeya et al., 2006) and ther-

mochemolysis (Ikeya et al., 2006) in combinationwith mass spectrometry have been applied to HSs.The major drawbacks with the degradation tech-niques used to date include molecular rearrange-ments, low yield and cleavage of ether and esterlinkages, thereby resulting in loss of the backboneinformation about HSs. Summaries of the progressmade on the structural characterisation can befound in the following four reviews: Abbt-Braunet al. (2004), Cardoza et al. (2004), Janos (2003),McDonald et al. (2004).

The approach taken in the present studyinvolves reducing the complexity of fulvic acidsby examining the backbone structures obtainedfrom a selective and quantitative reduction reac-tion (Nimmagadda and McRae, 2006a, 2006b)capable of converting polycarboxylic acids,ketones, aldehydes and alcohols to alkanes withoutlosing important backbone information. The con-version of the polar functional groups of fulvicacids to alkanes yields analytes that are muchmore amenable to analysis using gas chromatogra-phy (GC) and gas chromatography–mass spec-trometry (GC–MS). The chemical reduction offulvic acids would usually be a multi-step process;first reducing carboxylic acids to alcohols followedby reduction of the alcohols to alkanes. Similarly,the conversion of aldehydes and ketones to alk-anes requires a multi-step process. The need to iso-late products at each stage of the reduction,incompatibility of fulvic acids with the solvent sys-tems used, loss of structural information due tolow yield and harsh reducing reagents/conditionsmeans that the reduction of fulvic acids has metwith only limited success. In addition, there hasbeen no reduction method reported which is capa-ble of reducing polycarboxylic acids and polyalco-hols to alkanes. The method carried out in thepresent study enables the ‘one pot’ conversion ofpolycarboxylic acids, primary, secondary and ter-tiary alcohols, aldehydes, ketones and quinonesto their corresponding alkanes in high yield usingvery mild conditions. Another distinct advantageof this reduction method compared to other degra-dation reactions is that double bonds and ethersare unaffected. Further, esters are not cleavedand are instead reduced to ethers. As a conse-quence, fulvic acids can now be reduced to alkanesor ethers without loss of important backboneinformation, allowing additional structural infor-mation to be obtained compared with existing deg-radation techniques.

Table 1

Entry Substrate Product GC Yield (%) Identificationa

I OH

O

72% a,b,c

II OO

O 87% a,b,c

III

O

OH

HO

OH

OH

OH

O

28% a,c

IV OH

OH

HO

OO

HOO

36% a,c

V

OO O

83% a,c

VI

O OHO

HO

O

O

O

79% a,b,c

VII

O

OHO

O

OH

OHO

O

HO

O

62% a,c

VIII

COOH

O

HO

O

O

O

O

SiH 2Bu

73% a,c

IX

HOOC

O

O

O

O 84% a,b,c

(continued on next page)

R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072 1063

Table 1 (continued)

Entry Substrate Product GC Yield(%)

Identificationa

X O OH82% a,c

XI

HO

OH

OH

O

79% a,c

XII

OH

OOH

HO O

HO OH

O

O O

O OH2Si

H2Si SiH2

SiH 2

BuBu

Bu

Bu

67% a,c

XIII

OHO COOH

OH

O

71% a,c

XIV

OH

OH

O

COOHHO

OH

HO

OO

O

O

SiH2

SiH 2

Bu

Bu

91% a,c

XV

OHCOOH

OHO

O

OHO

OO

O

SiH2Bu

83% a,c

XVI

OO

OHO

O

HOOC

OHO OH

OHOH

HOOCHOOC

OO

O

O

32% a,c

a identified by molecular weight and fragmentation pattern; b identified by co-injection of standard; c structures for reduced modelcompounds; based on MS interpretation; structures were confirmed using NIST MS Interpreter where the quality indexes reported by theprogram for structure versus spectra were >90.

1064 R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072

R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072 1065

The reduction reaction was studied using a set ofmodel compounds, some proposed by variousauthors as fulvic acid components (Cappielloet al., 2003; Leenheer et al., 2001). Fulvic acids fromdifferent sources were also selected: fulvic acids iso-lated from Suwannee River, Elliott Soil and Was-kish peat were examined. The elementalcomposition, functional group analysis and struc-tural elucidation of these standard samples has beenreported by the International Humic SubstancesSociety (IHSS, St. Paul, MN, USA).

2. Experimental

2.1. Samples and chemicals

Fulvic acids from Suwannee River (2S101F),Waskish peat (1R107F) and Elliott soil (2S102F)were obtained from the International Humic Sub-stances Society (IHSS, St. Paul, MN, USA) andused as supplied [i.e. de-salted (H+-saturated)].Diethylsilane (DES), n-butylsilane (n-BS), tris(pen-tafluorophenyl)borane and nanograde CH2Cl2were obtained from Sigma–Aldrich. CH2Cl2 wasdried over CaCl2 and distilled from CaH2 toremove any trace of water prior to use. Ifrequired, the silane reagents were further purifiedby distillation over LiAlH4. The model com-pounds (Table 1) were obtained from Sigma–Aldrich and were purified using C18 solid phaseextraction.

2.2. Reduction procedure

An oven-dried 25 mL round bottom flask waspurged with N2 and substrate (5 mg fulvic acidor model compound) was added; the flask wasclosed with a septum and purged with Ar. Catalyst[B(C6F5)3, 0.5–1 mg] was introduced via a syringeas a freshly prepared solution in 0.5 mL anhydrousCH2Cl2. Stirring was continued (10 min) beforeaddition, via a syringe, of n-butylsilane. For thereaction involving model compounds, 2 equiv. n-butylsilane (99% pure) were added for each car-bonyl group or 4 equiv. for each carboxylic acidgroup present in the substrate. For reactionsinvolving fulvic acid 500 lL (3.9 mmol) of n-butylsilane (99% pure) were injected. A static Aratmosphere was established and the reaction mix-ture stirred for ca. 20 h. Blank reactions were per-formed without substrate, using an identicalamount of catalyst, silane and reaction time used

if the substrate were present. In all cases, the com-pleted reaction mixture was filtered using glassfibre filter paper before analysis using GC or GCMS.

GC yields reported for the model compoundswere calculated on the basis of introduction of aknown mass (weighed to 0.01 mg) of model com-pound and an internal alkane standard to the reac-tion vessel. The mass of reduced product wasdetermined using the known amount of internalstandard and assuming a response factor of 1 forthe analyte vs. the internal standard. The reportedyield is the molar percent conversion based on themolecular weights of the model compound and thereported product.

2.3. Analysis

A Shimadzu Gas Chromatograph 17A coupledto a Shimadzu QP5000 mass spectrometer wasused for qualitative analysis. A similar chromato-graph with flame ionisation detector (FID) wasused for quantitative analysis. In both cases anSGE BPX-5 column (30 m · 0.25 mm id, 0.5 lmfilm thickness) was used. Splitless injection modewith 1 lL of analyte was used, with a samplingtime of 1 min. Injector temperature was 270 �C,detector temperature 310 �C for the FID and theMS interface temperature was 290 �C. The tem-perature programme was 30 �C for 4 min, rampingto 350 �C at 4 �C/min, holding at 350 �C at for5 min. Column flow was 1 mL/min with a headpressure of 50 kPa. The mass spectrometer wasrun with 70 eV electron ionisation, and scanningfrom m/z 50–720 in 0.5 s. The solvent cut off timewas 4 min. Identification of reduction products(Table 1) was undertaken by examination of massspectral data. Identification was based on thepresence of a molecular ion and interpretation ofmass fragmentation patterns. Identification wasadditionally performed in some cases by co-injec-tion with known standards, together with massspectral matching using either the National Insti-tute of Standards and Technology (NIST) MassSpectral Standard Reference Database (June2002 Release) or the Palisade 600 k mass library,with a match factor of greater than 90%. Confir-mation was also performed using NIST MS Inter-preter V2.0. In all cases where the latter was usedfor confirmation, the quality indexe valuesreported by the program for structure vs. spectrawere > 90.

1066 R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072

3. Results and discussion

The mechanism for the reduction of a carboxylgroup by n-BS is shown in Fig. 1. The ability of n-BS and DES to reduce the carboxyl groups of arange of model compounds, including polycarboxy-lic acids such as cyclohexane 1,2,3,4,5,6-hexacarb-oxylic acid and benzene 1,2,3,4,5,6-hexacarboxylicacid, to methyl groups was demonstrated in earlierstudies (Nimmagadda and McRae, 2006a). In thelatter case it was found that DES was not as effec-tive as n-BS when applied to sterically hindered sub-strates. For this reason, n-BS was the primaryreducing agent used in this study. To illustrate thereagent’s usefulness when applied to fulvic acids,additional model compounds, containing variousfunctional groups and chosen from a range of pro-posed (Cappiello et al., 2003; Leenheer et al.,2001) fulvic acid structures were studied (Table 1).The reduction of a simple hydroxy ketone, 2-hydroxy-1,2-diphenylethanone (benzoin) I to 1,2-diphenylethane was achieved in high yield withoutreductive cleavage, thereby keeping the backboneinformation intact. The reduction of esters was

ROH

O

B(C6F5)3

HSi

B(C6F5)3H H

SiH

H

R COH

O

SiH

SiH2

ROH

O

H

B(C6F5)3SiH

H

B(C6 F5 )3SiH

H

ROH

H

H

RHO

H

SiR

OH

O

H

SiH2

CH2Cl2 ,r t+

H

H

H

H

1 eq 3 eq

Fig. 1. Proposed reduction mechanism (

demonstrated with benzyl benzoate II. Ester reduc-tion occurs at the carbonyl carbon, which is reducedto a methylene, resulting in an ether. The ether, as afunctional group, is unaffected, thus leaving thebackbone intact. Although the reduction needs tobe carried out in CH2Cl2, even water-soluble sub-strates that are insoluble in CH2Cl2, such as glucoseIII and ascorbic acid IV, were reduced withoutcleavage of the hemi-acetal and lactone, respec-tively. This point is particularly relevant for applica-tion of the reaction to fulvic acids, as solventsolubility is a major limitation with traditionalreduction procedures. The yield of reduced productfrom both these substrates was, however, quite lowcompared to the other substrates tested. Severalreasons for the low yield were identified. First, incommon with glycyrhizzic acid XVI, anotherlow-yielding compound, these compounds havenumerous functional groups in close proximity,resulting in steric hindrance preventing access tothe intermediate silyl ethers by the relatively bulkyhydride-carrying catalyst B(C6F5)3. Thus, themajority of the products formed from these sub-strates were silyl ethers. Secondly, the reduced prod-

SiH2

B(C6F5)3H

H

ROH

O

H2Si

B(C6F5)3H

ROH

O

H2Si

H

B(C6F5)3H

RH

H

SiH2

HO

H2

RH

HH

I

II

III

RH

HH

Nimmagadda and McRae, 2006a).

R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072 1067

ucts from III and IV are quite volatile and mighthave been lost through volatilisation, resulting inan underestimation of the overall yield. The possi-bility of the loss of products by volatilisation meansthat certain classes of compounds found in fulvicacids, such as some amino acids, pentoses or smallcarboxylic acids containing 5 or fewer carbons,may not be characterised using this procedure. Thisis not, however, seen as a problem, as these types ofcompounds can be readily analysed using standardderivatisation techniques.

The remainder of the model compounds listed inTable 1 illustrate the effectiveness of the reactionwith increasingly complex substrates. It is interest-ing to note that the carbon–carbon double bondsin substrates IX, X, XIII and XIV are stable to thereduction. Conjugated carbon–carbon doublebonds, such as those of a,b unsaturated ketones(XVI), do not appear to be stable and are reducedto alkyl moieties. Alcohols, be they primary, sec-ondary or tertiary, are readily reduced as illustratedby the reduction of III, IV, XI, XIII–XVI. Phenolsare not, however, reduced. Instead, they are eitherconverted to silyl ethers (VIII, XII, XIV and XV)or, if the phenol is sterically- hindered, are leftunchanged. The mild reaction conditions can be

18 25 277 8

9 10

12 13 1517 19 21 22

24

2328

2930

* * * *

41 2 3

Tota

lIon

Cur

rent

c 2S102F

10.00 20.00 30.00Time (min)

6 7 910 19

1415 1817 21

2224

2328 29 3

**

1 2 34To

talI

onC

urre

nt

8

b 2S101F

10.00 20.00 30.00Time (min)

9 1011 12

7

1415 16

17 18 1920

2223

24 26

28

3029

*

*

51 23

4

Tota

lIon

Curr

ent

8

a 1R107F

10.00 20.00 30.00Time (min)

Fig. 2. Total ion current of products from (a) Waskish peat fulvic acid (fulvic acid (2S102F) upon reduction with n-butylsilane(n-BS); * indicat

illustrated by the preservation of the glycosidic link-ages in glycyrhizzic acid XVI.

It should be noted that n-BS’s ability to reducecarboxyl groups requires the carboxyl group to bein protonated form. Carboxylates are either notreduced, or are reduced in very low yield. Asidefrom issues of steric hindrance, the low yield ofproduct from glycyrhizzic acid XVI could also bea result of one or more of the carboxylic acid groupsbeing in deprotonated form and thus not amenableto reduction.

In summary, the model compound study indi-cates that the backbone structures of even complexmolecules not readily amenable to GC (e.g. glyc-yrhizzic acid XVI) can be readily obtained throughreduction by n-BS.

To examine the applicability of n-BS as an effec-tive reducing agent for fulvic acids, reduction wasperformed on the three IHSS standard fulvic acids.In all three cases, reduction with n-BS was able tocompletely solubilise the fulvic acids in CHCl2.The total ion current (TIC) chromatograms of thereduced fulvic acids are shown in Fig. 2 and a sum-mary of the compounds identified is listed in Table2, the latter comprising ca. 10% of the total numberof resolvable compounds in the TIC and represent-

40

41

31 3334

3237 38 39

36

*** *

*

* * *

*

40.00 50.00 60.00

1 33 38 3935

* * * *

40.00 50.00 60.00

3133

36 39

* ** *

*

* * * * *

*

*

40.00 50.00 60.00

51

1R107F); (b) Suwannee River fulvic acid (2S101F); (c) Elliott soiles siloxane peaks.

Table 2Peak identifications for Fig. 2

Peaka Reduced fulvic acids Method of identificationb 1R107Fc 2S101Fc 2S102Fc

1 Methylcyclopentane a,b + + +2 1,3-Dimethylcyclopentane a,b + + +3 Methylcyclohexane a,b +++ ++ ++4 Toluene a,b ++ ++ ++5 3,5-Dimethylpentene a +6 Octane a,b ++7 1,4-Dimethylcyclohexane a,b ++ ++ ++8 1,2,3-trimethylcyclopentane a,b + + +9 1,3,5-Trimethylcyclohexane a,b + + +

10 1,3-Dimethylbenzene a,b + + +11 1,4-Dimethylbenzene a,b + +12 2-Ethyl-4-methyl-tetrahydrofuran a,c ++ ++13 3-Methyl-3-octane a,b +14 Nonane a,b ++ +++15 1,2,4-Trimethylbenzene + ++ ++16 Decane a,b ++17 2-Isopropyl-3,4,5-trimethylfuran a,b,c +++ + ++18 1,3-Dimethoxy-5-methylbenzene a,b,c ++ + ++19 2-Methylbutylbenzene a,b,c + + +20 4-Methylpropiophenone a,c ++21 4-Methylundecane a,b,c + ++22 Dodecane a,b ++ +++ ++23 3-Ethylpentylbenzene a,b,c ++ + +24 4-Ethyl-5,6,7-trimethylbenzofuran a,c ++ + ++25 1,4-diethyl-4-methylcyclohexa-2,5-dienol a,c +26 1,1,2,3,3-Pentamethyl-2,3-dihydroindene a,c +27 5-Methyl-7-phenylheptan-3-one a,c +28 1,3-Dimethoxy-5-(prop-1-enyl)benzene a,b,c + + +29 2-Chloronapthalene a,c ++ ++ ++30 1,2,3,5-Tetramethyl-4-(3-methylbutan-2-yl)benzene a,c + +31 2,6-Di-tert-butyl-4-methylphenol a,b,c + + ++32 1,5,9-Trimethyl-cyclododecatriene a,c +33 Hexadecane a,b + + +34 2,6,10-Trimethylpentadecane a,b,c +35 Octadecane a,b ++36 Tridecan-4-ylcyclohexane a,c + +37 (4-Methylpent-2-ene-2,4-diyl)dibenzene a,c ++38 Eicosane a,b ++ +39 n-Heptadecylcyclohexane a,c + + +40 2,6-Di-tert-butyl-4-(2-phenylpropan-2-yl)phenol a,c ++41 Bis(4-octylphenyl)amine a,c +++

a Numbers correspond to peaks represented in Fig. 2.b a identified using NIST 02 and/or Palisade libraries; b identified by co-injection; c confirmed using NIST MS Interpreter V2.0, where

the quality indexes reported by the program for structure vs. spectra were > 90.c The presence of a compound is indicated by + sign and the relative concentrations in the order + < ++ < +++.

1068 R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072

ing ca. 30% of the total yield of GC amenable mate-rial (excluding siloxanes), assuming a response fac-tor of 1 for all compounds. What is immediatelyevident is that complete reduction of fulvic acidswas not achieved (peaks 20, 25, 27, 31, 40), as com-pounds are readily identifiable in which the reduc-tion of carboxylic acids, alcohols and ketones toalkanes did not take place. This was to be expectedas a result of both the complexity of fulvic acids and

the presence of deprotonated carboxylic acids, giventhat the model compound work had indicated typi-cal yields of fully reduced products to be around80%. Interestingly, the reduction products from soiland peat fulvic acids show a greater abundance ofhigher molecular weight compounds, observed atlong relative retention times, than those from theSuwannee River. Interestingly these higher molecu-lar weight components have similar mass spectra

50 100 150 200 250 300 350 400 450 500 550 600 650 7000

100

50

379

351365323

246

253232 295

267 30928142355

218 337

479395 44971 535

4634371469783 561203 505112 137 70251 176 59408

m/z

Rel

ativ

e A

bund

ance

(%)

Fig. 3. Averaged and background subtracted mass spectrum of an unidentified component in the reduction products of all the three fulvicacids (compound is indicated by . in Fig. 2).

R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072 1069

(Fig. 3), although resolution or identification of thecomponents was not attempted.

As shown in the model compound work, car-boxyl groups are reduced to methyl groups by n-butylsilane, so the prevalence in all samples ofmethyl substituted compounds is indicative of thepossible location of carboxyl groups in the originalfulvic acids. Methyl substituted cycloalkanes, suchas methylcyclopentane, dimethylcyclopentane, trim-ethylcyclopentane, dimethylcyclohexane, trimethyl-cyclopentane, tetramethylcyclopentane and methylsubstituted aromatic hydrocarbons such as toluene,1,3-dimethylbenzene, 1,4-dimethylbenzene, 1,2,4-trimethylbenzene and 2-methylbutylbenzene arefound in the reduction products of all three fulvicacids, indicating the presence of small cyclic poly-carboxylic acids in the fulvic acids; 1,3-dimethoxy-5-(prop-1-enyl)benzene (peak 28) is likely thereduced product of 3,5-dimethoxycinnamic acid, aproposed fulvic acid component and the presenceof silyl ethers indicates free phenols. The presenceof the reduced analogues of various phenolic andaromatic carboxylic acids is consistent withreported fulvic acid characteristics (Haiber et al.,2001; Leenheer et al., 2003). Further, many of theseanalogues, as shown in Table 3, match with com-pounds previously proposed as model compoundsbut not detected using other degradation methodssuch as tetramethyl ammonium hydroxide (TMAH)

chemolysis and pyrolysis (Ikeya et al., 2006). Thepresence of straight chain aliphatic compounds suchas peaks 6, 14, 16, 22, 33, 35 and 38 is likely theresult of the reduction of straight chain diacids.However, as hydroxyl or carbonyl groups will alsobe reduced to methylenes, the straight chain aliphat-ics may equally be derived from long chain hydroxy-or keto- carboxylic acids or even polyalcohols in thefulvic acids. Straight chain diacids have beereported in HSs, where their presence was attributedto microbial degradation of aromatic compounds(Nanny Mark and Ratasuk, 2002) and simplerstraight chain aliphatics may be due to the oxidativemicrobial degradation of longer chain material(Spitzy and Ittekkot, 1991). The reduction productsof Suwannee River fulvic acid appear richer instraight chain aliphatic compounds than those fromthe soil and peat fulvic acids. This matches withIHSS 13C NMR data, which indicate a greater ali-phatic carbon content in Suwannee River fulvic acidthan in the soil and peat fulvic acids. The presenceof chloronaphthalene (peak 29) in all the samplesis intriguing as its presence could be an indicationof the chlorination of fulvic acids during isolation.Similar chlorinated compounds have also been iden-tified in the study of other HSs (Eglite et al., 2003).Alternatively, it could be an indication of the forma-tion of anomalous byproducts from the photolytic,or other, degradation of the CH2Cl2 solvent.

Table 3Compounds identified which are similar to the reduced analogues of compounds proposed by various authors as present in HS

Model compound Proposed bya Reduced product Peak no.

COOH

COOH

a,b,c 2

COOH

COOHHOOC

a,c 9

COOH

c 11

O

O

b

O

12

COOH

HOOC

COOH

c 15

O

COOHHOOC

HOOCCOOH

a,b

O

17

OO

COOHb

OO

18

COOH

O

O

HO

b

O

O

28

a Leenheer et al., 2001;b Cappiello et al., 2003;c McIntyre et al., 2002.

1070 R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072

Although it cannot be discounted, photolytic degra-dation of CH2Cl2 is considered unlikely as light wasexcluded from the reaction vessel during the reduc-tion. The number of methoxy-bearing compoundsin the products from all three fulvic acids was rela-tively low compared to that typically identified in

other degradation methods such as TMAHchemolysis. Methoxy groups in TMAH chemolysisproducts are considered to be formed from themethylation of phenols and so are generally notconsidered to be native. With n-butyl silane reduc-tion, however, phenols are either converted to silyl

R.D. Nimmagadda, C. McRae / Organic Geochemistry 38 (2007) 1061–1072 1071

ethers or left unchanged and alkyl ethers are stableto the reduction. Thus, any methoxy functionsfound are likely to be native or are the result ofthe reduction of formates. Alkylated furans (peaks12, 17, 24) are likely the reduced products of saccha-rides and appear in greater abundance in the soiland peat fulvic acids than in the Suwannee Riverfulvic acid. This correlates with the reported carbo-hydrate composition of the IHSS samples, whichindicate higher carbohydrate levels in the soil fulvicacids (355.3 lmol/g) than in the Suwannee Riverfulvic acids (43.2 lmol/g). The reduction productsfrom soil and peat fulvic acids contained noticeablygreater amounts of higher molecular weight andheterocyclic compounds than those obtained fromSuwannee River fulvic acids.

4. Conclusion

Our preliminary results, demonstrated with threestandard fulvic acids, clearly indicate the potentialof this selective chemical reduction reaction toreduce the complexity of fulvic acids and yieldimportant structural information. Although onlyca. 10% of the number of compounds producedwere identified, these were consistent with thereduced analogues of many of the compounds thathave been reported using other prominent degrada-tion methods such as TMAH hydrolysis (NannyMark and Ratasuk, 2002), pyrolysis-GC (Ikeyaet al., 2006) and reduction (Eglite et al., 2003).Additionally, reduced analogues of many proposed,but as yet unreported, compounds were found, suchas furan-derived compounds and even some simplearomatic and cyclic polycarboxylic acids, highlight-ing the advantage of this reduction methodology instudying the unfragmented backbone structures offulvic acids and supporting the theory of supramo-lecular structures for HSs (Piccolo, 2002; Janos,2003).

Acknowledgements

We sincerely thank Professor Barry Batts andDr. Cameron McIntyre for their contributions. Pro-fessor Kliti Grice and the anonymous reviewers arethanked for their critical review of the manuscriptand for constructive suggestions.

Associate Editor—Kliti Grice

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