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Journal ArticleDOI

Identification of Bound Alcohols in Soil Humic Acids by Gas Chromatography-Mass Spectrometry

20 Dec 2000-European Journal of Mass Spectrometry (SAGE PublicationsSage UK: London, England)-Vol. 6, Iss: 5, pp 439-441

Abstract: Humic acids are complex, partly macromolecular, yellow-brownish substances occurring in soils, waters and sediments. In order to shed some light on their molecular structure, crop humic acids were cleaved by alkaline hydrolysis (KOH). The products were fractionated by thin layer chromatography to give mono-alcohols which were analysed as acetate derivatives by gas chromatography coupled to mass spectrometry. Linear alcohols, sterols, stanols and plant-derived triterpenoid alcohols were identified by co-injection of pure standards and by comparison with literature data. These findings imply that alcohols could have been incorporated into the humic matrix by esterification with carboxylic acids. Furthermore, the presence of stanols as hydrogenated counterparts of sterols suggests that a process of hydrogenation is operating in soils.
Topics: Fatty alcohol (55%), Gas chromatography (52%), Thin-layer chromatography (52%), Gas chromatography–mass spectrometry (51%), Humin (50%)

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Identication of bound alcohols in soil humic acids by
gas chromatography-mass spectrometry
Gersende Berthier, Sen Dou, Torren Peakman, Eric Lichtfouse
To cite this version:
Gersende Berthier, Sen Dou, Torren Peakman, Eric Lichtfouse. Identication of bound alcohols in soil
humic acids by gas chromatography-mass spectrometry. European Journal of Mass Spectrometry, IM
Publications, 2000, 6 (5), pp.439-441. �10.1255/ejms.368�. �hal-00263408�

1
Revised version
European Journal of Mass Spectrometry 6, 439-441, 2000. doi: 10.1255/ejms.368
Correspondence: Dr. Eric Lichtfouse, INRA-CMSE-PME, 17, rue Sully, 21000 Dijon, France
Eric.Lichtfouse@dijon.inra.fr
Identification of bound alcohols in soil humic acids by GC-MS
Gersende BERTHIER
1
, Sen DOU
2
, Torren PEAKMAN
3
and Eric LICHTFOUSE
4
*
1. Collège Louisa Paulin, F-81120 REALMONT, France. 2. Depatrment of Soil Science, Jilin Agricultural University,
Changchun, P.R. China. 3. School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK. 4.
INRA/ENSAIA-INPL, BP 172, 54505 Vandoeuvre-lès-Nancy, France.
Abstract
Humic acids are complex, partly macromolecular, yellow-brownish substances occurring in soils, waters and
sediments. In order to bring some light on their molecular structure, crop humic acids were cleaved by alkaline
hydrolysis (KOH). The products were fractionated by thin layer chromatography to give mono-alcohols which were
analysed as acetate derivatives by gas chromatography coupled to mass spectrometry. Linear alcohols, sterols,
stanols and plant-derived triterpenoid alcohols were identified by co-injection of pure standards and by comparison
with literature data. These findings imply that alcohols could have been incorporated into the humic matrix by
esterification with carboxylic acids. Furtehrmore, the presence of stanols as hydrogenated counterparts of sterols
suggests that a process of hydrogenation is operating in soils.
INTRODUCTION
Humic substances represent a major pool of organic carbon occurring in soil, sediment and waters
1-2
. It is
generally accepted that humic acids are characterised as polydispersed, acidic, amorphous substances
ranging in molecular mass from a few hundred to several thousand daltons. However, the pathways of
formation of humic acids remains an enigma mainly because of the lack of structural identification of its
network at the molecular level. To unravel the molecular structure of complex, high molecular weight
natural organic substances such as kerogen, asphaltene, coal and humic substances, various chemical
and physical methods have been applied
3-5
. In particular, the breakdown of humic substances by chemical
and enzymatic degradation can release substantial amounts of compounds amenable by gas
chromatography coupled to mass spectrometry
5-7
. In a recent investigations, we identified alkane and
alkene biomarkers released by pyrolysis of humin
8-12
. Here, we report the identification of
n
-alkanols,
stanols, sterols and plant-derived triterpenoid alcohols released by mild alkaline degradation of soil humic
acids from crop soils.
EXPERIMENTAL
Labware decontamination procedures are described elsewhere
13
. Soils cultivated for 23 years with maize
(
Zea mays
) or wheat (
Triticum aestivum
) were sampled at Boigneville, France, then dried and sieved to 2
mm. 2 g of humic acids were extracted with 0.1 M sodium hydroxide under nitrogen to prevent oxidation of
organic matter, precipitated at pH 1.5 with HCl, dialysed against water to remove molecules smaller than
2000 daltons, freeze-dried, extracted with diethyl ether to remove free organic compounds then refluxed
16 h. in 5 M potassium hydroxide-methanol under nitrogen. After addition of water, alcohols were
extracted with diethyl ether (3 x), Na
2
SO
4
-dried then fractionated by thin layer chromatography (TLC) on
silica-gel eluting methylene chloride with cholesterol as reference (R
F
~ 0.13) to yield a mono-alcohol
fraction which was then derivatised with an excess of acetic anhydride and pyridine (50°C, 1 h). The

2
products were fractionated by TLC on silica-gel eluting methylene chloride with acetates of cholesterol,
ergosterol and
n
-hexadecanol as references (R
F
~ 0.70) to yield a mono-acetate fraction which was then
analysed by GC-MS. GC conditions: HP 5890 series II, on-column injector, 50 m x 0.32 mm i.d. capillary
column coated with 0.52 mm 100% polymethyl-siloxane phase (Ultra 1), Helium carrier gas: 0.66 b. head
pressure at 50°C (constant flow mode), oven temperature: 50-100°C (15°/min.), 100-310°C (3°/min.),
310°C (60 min.). MS conditions: HP 5989A quadrupole, electronic impact (70 eV), scanning from 50 to
700 amu.
RESULTS AND DISCUSSION
The GC-MS total ion current trace of mono-alcohols, as acetate derivatives, released by alkaline
hydrolysis of humic acids from a maize crop soil is presented on Figure 1. Three classes of alcohols were
identified in hydrolysis products of humic acids by mass spectrometry and by comparison with pure
standards: linear alkanols, sterols, stanols and triterpenoid alcohols (Figure 2). The spectra of all
compounds show low relative intensity of the molecular ion and high relative intensity of the M-60 peak
corresponding to the easy removal of acetic acid. Sterols give a high m/z 255 peak due to the cleavage of
the side-chain. The m/z 215 base peak of stanols is explained by the cleavage of the 5-carbon ring. β- and
α-amyrin are easily characterised by a m/z 218 base peak. Compounds 1,2,4,6-8,10 and 11 were
identified by co-injection of pure standards. The structure of the other compounds were assigned on MS
grounds and by comparison with literature data
14-19
.
Figure 1. GC-MS total ion current of alcools (as acetate derivatives) released by alkaline hydrolysis of humic acids
from a maize crop field. A similar fingerprint was obtained by analysis of humic acids from wheat crop soils. C
16
-C
30
refer to
n
-alkanols.
1 Cholest-5-en-3β-ol (cholesterol) , 2 5α-cholestan-3β-ol, 3 (3β, 22E)-ergosta-5,22-dien-3-ol
(brassicasterol), 4 (24R)-ergost-5-en-3β-ol (campesterol), 5 (5α, 24R)-ergostan-3β-ol, 6 (3β, 22E)-stigmasta-5,22-
dien-3-ol (stigmasterol), 7 (3β,5α,22E)-stigmast-22-en-3-ol, 8 stigmast-5-en-3β-ol (β-sitosterol), 9 olean-12-en-3β-ol
(β-amyrin), 10 5α-stigmastan-3β-ol 11 urs-12-en-3β-ol (α-amyrin).

3
The identification of bound alcohols in soil humic acids has several implications. First, a higher plant
source
20
can be inferred for most alcohols, e.g. odd-carbon number
n
-alkanols, campesterol, stigmasterol,
β-sitosterol, β-amyrin and α-amyrin. Whereas cholesterol is probably derived at least partly from other
organisms, as suggested by the higher β-sitosterol/cholesterol ratio of 8 in maize than in maize soils
(value: 2)
21
. Second, the most likely pathway of incorporation of plant alcohols into soil humic acids could
involve esterification of alcohols with carboxylic groups. This hypothesis is in agreement with the high
concentration of acidic groups in humic substances
1,5
. Third, we identified C
27
-C
29
stanols (2,5,9) as
hydrogenated counterparts of C
27
-C
29
sterols (1,4,8) in hydrolysis products of humic acids. Since stanols
are rare in living organisms, their occurrence in soil suggests thier derivation from sterols by
hydrogenation into the soil. This assumption is supported by an investigation showing the hydrogenation
of
14
C-cholesterol in sediments
22
.
Figure 2. Molecular structure of alcools released by alkaline hydrolysis of humic acids.
CONCLUSION
Several classes of alcohols have been identified by GC-MS as bound moieties of soil humic acids using
alkaline degradation. They are probably incorporated in the humic matrix by esterification with carboxylic
groups. Most alcohols are typical of plant substances whereas cholesterol and cholestanol are probably
inherited at least parlty from other organisms. Furthermore, the presence of stanols in humic substances
could be explained by hydrogenation of their sterols counterparts.

4
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3. J.A. Neyroud and M. Schnitzer,
Geoderma
13, 171-188 (1975).
4. J. Rullkötter and W. Michaelis,
Org. Geochem
. 16, 829-852 (1990).
5. A. Amblès, J.C. Jacquesy, P. Jambu, J. Joffre and R. Maggi-Churin,
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___________________________________________________________________________
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