COMPARISON BETWEEN THE PYROLYSIS AND THE
PYROLYSIS DERIVATISATION WITH
TETRAMETHYLAMMONIUM HYDROXIDE TO CHARACTERIZE
SOME BIOPOLYMERS AND SOME ORGANIC MATTER
FRACTIONS OF WATER
MOUSSET B. and D. RECKHOW
Department of Civil Engineering, University of Massachusetts, at Amherst
Amherst, MA 01003
Abstract
The organic matter was fractionated to use together the procedure of LEENHEER (1981)
and MALCOLM et al. (1993). The fractions were isolated from a Wachusett reservoir and
on the end of Andover treatment plant.
C:\DAR\RES\Epa2\Mousset\pap2\PyrolysisTMAHmethod.doc
Keywords
Fulvic, humic and weak hydrophobic acids of surface and treated waters, biopolymers,
pyrolysis and pyrolysis/methylation with the tetramethylammonium hydroxide.
Introduction
The natural organic matter (NOM) was characterized by non-specific parameters such as
COT, DOC, and UV absorbance, by reactivity with coagulant and oxidant and most
recently by biodegradable organic matter (BDOC), by sophistical analytical such as 13C
and 15N RMN, fluorescence and pyrolysis. However, those sophistical techniques are
easily applicable to pure substances. Their applicability to NOM is more uncertain,
because the salts could be disturbed the results and need a high quantity of sample. So it’s
necessary to extract, to concentrate and to purify the NOM. Different isolation procedures
have been used during the last decades, including adsorption into XAD, anionic and
cationic resins, filtration with membranes (ultrafiltration, nanofiltration and reverse
osmosis), and rotary evaporation. But the basis of the most widely used procedures is
proposed by THURMAN and MALCOLM (1981) or by LEENHEER (1981). The organic
natural matter separated based on charge and hydrophobic properties. The fractionation
scheme divided the organic matter into two major categories : hydrophobic and
hydrophilic and at least three subcategories : acids, bases and neutrals.
The pyrolysis is a very promising method that can already be used to estimate the overall
composition of the organic matrix of waters. This method is a thermal degradation
method and fragments the organic matter in reproducible and significant products, which
analyzed by gas chromatography. Those fragments could be compared with model
compounds and can be related to the structure of the undegraded products such as
polysaccharides, proteins, lignin, and aromatic and polyhydroxyaromatic compounds.
The pyrolysis is a semi-quantitative method. However the fragments could be shown
correlation between the results of the pyrolysis and the results obtained with other
quantitative methods (MARTIN, 1995 and LABOUYRIE-ROUILLER, 1997). Examples, the
relation between the aminoacids and the structure derived by proteins, the
polysaccharides and the sugars or the polyhydroxyaromatic structures and the UV
absorbance characterization of the aromatic groups. So the pyrolysis is an interesting
method. And more, the fragments obtained by pyrolysis show information on the origin
of the water, aquagenic or pedogenic organic mater than unlined BRUCHET (1990) and
BIBER et al. (1996).
But this technique presents some limitations. The first limitation is the aromatic
hydrocarbons (such as benzene, styrene, naphthalene biphenyl and their alkylated
derivatises) might derive from highly aromatic nucleus or from cylclisation reactions or
defunctionalised fatty acids (GOBBELS and PUTTMANN, 1997 and HARTZERS et al., 1995).
The second one is the sample was burnt and a significant percentage of carbon is
transformed into small molecules such as CO2, CH4 (BRUCHET et al. 1990). So a lot of
carbon is unknown origin. The third one is the benzonitriles and N containing
heterocyclic compounds are known to be generated by pyrolysis of proteins. However
GOBBELS and PUTTMANN (1997) have found that the pyrolysis of acidic and alkaline
hydrolyzed fulvic and humic acids at 770C generated also such compounds. Therefore,
in the present case the compound can not originate from proteins. The four one is the
problem of polar pyrolysates that often show peak, tailing characteristics, poor
reproducibility, long elution times or in some cases no chromatographic peaks are
obtained (CHALLINOR, 1989 ; SAIZ-JIMENEZ et al., 1994).
To resolve those limitations, a main way is to achieve simultaneous pyrolysis and
alkylation by incorporating the derivatizing reagent that could be the
tetrabutylammonium hydroxide (TBAH) or the tetramethylammonium hydroxide
(TMAH). In general the mechanism of pyrolysis of polyester involves the scission of the
RCOO-R bond and is considered to progress via a cyclic transition state. Pyrolysis results
in the formation of respective carboxylic acid and alkene. In contrast the pyrolysis with
the TMAH results in the formation of the methyl esters of the carboxylic acids and
methyl esters of alcohol (CHALLINOR, 1989). With the TMAH, DEL RIO and HATCHER
(1996) show that the methyl carboxylic groups and hydroxyl groups are more amenable
to chromatographic separation. They found that this technique provides excellent
preservation of the original structures containing carboxyl and hydroxyl groups in lignin
monomers owing to protection of the functional groups from thermal reactions. They
compared the pyrolysis and the pyrolysis/methylation and they observed that the humic
substances reveal the presence of a series of benzene carboxylic acid methyl esters as
well as long-chain fatty acid methyl esters and dimethyl esters. The methylated structures
produced by TMAH differ dramatically from those obtained by conventional pyrolysis,
calling into question the recently proposed structure of humic acids that are bases mostly
on conventional pyrolysis.
SAIZ-JIMENEZ et al. (1994) compared the pyrolysis/methylation and the solvent
extraction. They found that the data obtained by solvent extraction are similar to those
obtained by pyrolysis/methylation. The similitude proved that the pyrolysis methylation
is an analytical procedure of great sensitivity for investigating organic compounds in
inorganic matrices.
Previous papers demonstrated the feasibility of the pyrolysis methylation method for
analysis of soil humic acid (SAIZ-JIMENEZ, 1994-a and b ; MARTIN et al., 1995), fulvic
acid of water (SAIZ-JIMENEZ et al., 1993), synthetic polymers (CHALLINOR, 1989), tomato
and natural polyester cutin (DE LEEUW and BASS, 1993), humic acid isolated from a
volcanic soil (DEL RIO and HATCHER, 1996) and aliphatic biopolymer cutan (MCKINNEY
et al., 1996). To better understanding the mechanism of the TMAH, the compounds such
as aldehydes (TANCZOS et al., 1997) and fatty acids (HARTGERS et al., 1995) were
analyzed.
The aim of the present work was :
-
to compare pyrolysis and pyrolysis/methylation on biopolymers,
-
to compare pyrolysis and pyrolysis/methylation on aquatic organic matter,
-
to characterize isolated aquatic organic matter.
The focus was not on the mechanism occurred during the conventional pyrolysis or
pyrolysis/methylation, but only on the comparison.
Materials and Methods
Pyrolysis gas chromatography/mass spectrometry GC/MS
The method of pyrolysis alone used was similar to the one published by BRUCHET et al.
(1990). The fractions were concentrated by different methods reported upstairs. The
sample must be have got a DOC more than 100 mg C/l. A few milliliters were
transformed, under a nitrogen stream, to a solid fraction. Then the pyrolysis GC/MS
experiments were run on around 50 mg of sample deposited into quartz tube, which was
inserted into a filament pyrolyzer. The salts were lost during the evaporation on the wall
of the tube. ALCANIZ et al. (1989) were demonstrated that the usual salts, that found in
the water, could be reduced the intensity of response without changed the pyrogram.
For the pyrolysis/methylation, one step of methylation was added before pyrolysis. A
derivatizing reagent used was the tetramethylammonium hydroxide (25 wt % solution in
water) noted TMAH. This method is described in detail elsewhere (CHALLINOR, 1989,
SAIZ-JIMMENEZ et al., 1993 ; De LEEUW and BAAS, 1993 ; DEL RIO and HATCHER, 1996)
and is briefly explained here. Approximately 5 l of the TMAH was placed with the
sample in the quartz tube. The all was inserted into a filament pyrolyzer.
The temperature programmation of pyrolysis was 200C (1 s) to 700C (10 s) at the rate
of 20C/ms with a CDS 1500 pyroprobe 2000. At this temperature, the organic matter
produced different fragments separated on a 30 cm length DB WAX capillary column by
Hewlett Packard 5890A gas chromatography flushed with helium gas. The oven
programmation was from 25 to 220C at the rate of 3C/min and the identification was
made by a Hewlett Packard 5988A mass spectrometer operated at 70 ev and scanned
from 20 to 400 amu at 1 scan/s.
The semi-quantitative interpretation of pyrograms of the fractions was followed as
BRUCHET et al. (1990) except that the sums were not multiplied by a corrective factor. An
other fragment : the aromatics were added at this list as LABOUYRIE-ROUILLER (1997).
And the carboxylic acid methyl ester comes from fatty acids what represents an other
group.
Some standards (benzene, acetone, 2,3 dimethyl naphthalene, 2 methyl naphthalene, 1
methyl naphthalene, naphthalene, phenol, pyridine, toluene, 2,3,5 trimethyl naphthalene,
o xylene, m xylene and p xylene) were diluted in hexane and injected directly in the
GC/MS to confirm the retention time.
DOC measurement
Dissolved organic carbon (DOC) was measured using a Shimadzu Model TOC-500A
with ASI-5000A autosampler, calibrated with a potassium hydrogen phthalate standard
(C8H5O4K) solution containing 2, 5 and 10 mg C l-1. For each sample, a minimum of
triplicate measurements was made.
Extraction and fractionation of NOM
NOM fractions, used in this study, were according to the procedure developed by
LEENHEER and NOYES (1984), RECKHOW et al. (1993) and MALCOLM (1981).
The NOM was filtered on tow filters : the first is type DH rated the retain 98% of
particles 25 m in diameter and the second filter unit contains filter tube (type AAH)
rated to retain 98% of particles 0.3 m in diameter.
The extraction system consisted of two steps. The first one, around 200 liters of water
was filtered at pH neutral through the three resin columns of 2 liters (XAD-8, MSC-1 and
A-7) connected in series at the rate of 6 liters per hour.
The second step corresponds of an elution at 0.85 l/h of fractions the weak hydrophobic
acids with NaOH 0.1 N in XAD-8. On resin A-7 were eluted together, fulvic acids and
humic acids (corresponded of humic substances), hydrophilic acids and the ultra
hydrophilic acid. The fractions, that have a DOC more than 200 mg C/l, were acidified at
pH 1 for the separation of humic acids that precipitated. The humic acids were spited by
centrifugation (30 min, 500 rpm).
The procedure consisted by the separation of fulvic and hydrophilic acids at pH 2 on 0.5
liter of resin XAD-8 at the rate of filtration 0.8 l/h on 0.5 liter of resin XAD-4 at the rate
of filtration 0.4 l/h. Then the resins were rinsed with Milli-Q water. And the elution was
made with NaOH 0.1M. On XAD-8, the fulvic acid was eluted and on XAD-4, the
hydrophilic acid was recovered. Finely the fractions were filtered thought the MSC-1
(cationic exchanger) to eliminate the salts. The volume of fulvic, hydrophilic and ultra
hydrophilic acids is 12 liters with a DOC < 10 mg C/l in accordance with the procedure
of MALCOLM et al. (1993).
The fractions and the waters were concentrated by evaporation at 40C and the fulvic
acids could be drying.
Biopolymers
The Chitin, Cellulose acetate, Dextran and Serum albumin bovine were used by BRUCHET
et al. (1990) and represented after pyrolysis a lot of fragments which could be found in
the organic matter. The Tetracosanoic acid was added to this list because the pyrolysis
with TMAH reveals methyl ester carboxylic and alcohol groups, represented the fatty
acid fragments.
Chitin (Sigma) (C8H13NO5)n cam from crab shells. This biopolymer consisting
predominantly as unbranched chains of poly-(14)--N-acetyl-D-glucose. It found in
fungi, yeasts, marine invertebrates and arthropods, where it is a principal component in
the exoskeletons.
Cellulose acetate (Fluka) was obtained by treating cellulose with acetic anhydride at
various temperatures for different lengths of time to produce amorphous white solid
material in granular, flake or powder form. So it became directly of the cellulose,
polysaccharide with glucose units. It’s chief constituent of the fiber of plants.
Dextran (Sigma) mol wt 37 500 a term applied to polysaccharides produced by bacteria
(Leuconostoc mesenteroides) growing on a sucrose substrate. It’s composed exclusively
of –D-glucopyranosyl units.
Serum albumin bovine contained a group of proteins characterized by heat coagulability
and solubility in dilute salt solution. It’s found in nearly all living body tissues.
Tetracosanoic acid (Aldrich Chem, Co) C24H48O2 was obtained from beechwood tar or by
the distillation of rootten oak wood, represented a long chain of fatty acid.
Results and discussions
The biopolymers are the chitin, cellulose acetate, dextran, serum albumin bovine and the
tetracosanoic acid were used to compare the conventional pyrolysis/pyrolysis
methylation. A other group of compounds was used is the fractions of organic matter that
were extracted from reservoir Wachusett October 23, 1997 (Wachusett 1) and May 26,
1998 (Wachusett 2), from chlorinated reservoir Wachusett May 26, 1998 (Wachusett 2 T)
and from the treated water of Andover plant (March 16, 1998).
1- Comparison pyrolysis and pyrolysis/methylation of biopolymers
The cellulose acetate and the dextran come from polysaccharides. The chitin comes from
polysaccharides and N-acetyl. The albumin serum contained a mixture of proteins and the
tetracosanoic acid was a fatty acid.
Chitin
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 1 and 2.
With the conventional pyrolysis, the main fragments obtained are acetic acid, furfural,
toluene, derived pyridine, derived pyrazole, derived acetamide, butanamide and
pyridinone. Almost the same compounds were found by MARBOT (1997). The chitin
mainly cam from polysaccharides and derived proteins. Any aromatic compound except
the small peak of phenol was presented, that mean that the fatty acids, found with the
pyrolysis/methylation didn’t produced aromatic fragments. This remark was in
accordance with the results of HARTGERS et al. (1995) on hexadecanoic and 12hydroxyoctadecanoic acids.
With the pyrolysis/methylation, the same nucleus was found : pyridine, acetamide,
pyrrole and pyrazole.
The pyridine carboxaldehyde disappeared and could be transformed into pyridine
carboxylic acid and pyridine methanol acetate ester. Those results are almost in
accordance with TANCZOS et al. (1997) that shown that the aldehydes with the TMAH
could form aldehydes alcohol, methoxy aldehydes or carboxylic acid aldehydes in
different proportions depending on the kind of the aldehydes.
The pyridine carboxylic acid was not transfer into corresponded methyl ester that can
explain by the competition reactions with the TMAH in accordance with CHALLINOR
(1989). The same was observed for the 5-acetyl 3,4 dimethyl 3 pentanoic acid, but maybe
the environment of the carboxylic function prevents the reaction with the TMAH.
The peaks of tetradecanoic acid methyl ester, hexanoic acid methyl ester, 9 octadecenoic
methyl ester, butanoic acid methyl ester and acetic acid methoxy methyl ester cam from
the fatty acids. MCKINNEY et al. (1996), DEL RIO and HATCHER, (1996) and SAIZJIMENEZ (1994-a) shown that the fatty acids give the carboxylic acid fragments, but not
only. SAIZ-JIMENEZ (1994-a) found that in the conventional pyrolysis, the pyrograms of
fulvic and humic acids presented no carboxylic groups other than those of the few fatty
acids (mainly the C16 and C18 members). With in pyrolysis/methylation, the range of the
free fatty acids was C4-C30 for the methyl ester and C6-C26 for the dicarboxylic acids.
The TMAH preserved the function attached to the phenol, in the fingerprint with the
conventional pyrolysis only the phenol was found and with the pyrolysis/methylation, the
peak of phenol disappeared and replaced by the phenol 4 amino 3 methyl.
The toluene was disappeared and maybe it was co-eluted with the acetic acid methoxy
methyl ester which com from the reaction of TMAH and the acetic acid.
Note the desperation of the furfural in the fingerprint of pyrolysis/methylation that can
came from the acetamidofuran after scission C-N that was presented in the fingerprint of
pyrolysis/methylation and not in the pyrochromatogram of the conventional pyrolysis.
The acetamide reacts with the TMAH and gives acetamide N methyl or acetamide N, N
dimethyl.
Cellulose acetate
The fingerprints of cellulose acetate were in the figures 3 and 4.
The main products of conventional pyrolysis of cellulose acetate include acetic acid,
derived furan, butanone, methyl furfural, phenol, derived benzene diol and
cyclopentenone.
The fingerprints of the conventional pyrolysis and the pyrolysis/methylation were very
similar, except the first peak, pentenyne, butadiene and propenal in the conventional
pyrolysis that in the pyrolysis/methylation co-eluted with the TMAH.
In each pyrochromatogram was found a lot of ketones, derived furan represented the
polysaccharides. Any fatty acid was presented. So in this case the pyrolysis/methylation
was not necessary.
A great peak of acetic acid and some derived were presented in the two chromatograms.
Note the presence of derived benzene (benzene diol diacetate, benzene triol triacetate and
benzene diol).
But, the cellulose acetate became mainly from polysaccharides attached with acetyl
groups.
Dextran
The pyrochromatograms of the pyrolysis and pyrolysis/methylation are presented on the
figures 5 and 6.
The main products of conventional pyrolysis of dextran include butanone, derived of
acetic acid, ethanamide N N diethyl, thiophene, furfural and cyclopentanedione.
The pyrochromatograms of the two pyrolysis have some mutual peaks : propanal 3
ethoxy, acetic acid methyl ester, thiophene, derived furanone and derived
cyclopentanone. The propanoic acid methyl, pentanoic acid 4 oxo, trimethoxy benzene,
octanoic and nonanoic acid and decanoic acid methyl esters represent the fatty acids.
Maybe those fatty acids for the conventional pyrolysis produced the 3 hexyn 1 ol and the
trimethyl hexene. The formation of the alkenes from fatty acids has been shown by
HARTZERS et al. (1995).
In pyrolysis/methylation, the dimethoxy benzene was appeared. It could come from the
hydroquinone. The mechanism of the methylation of the function alcohol to form
methoxy has been observed by CHALLINOR (1989).
So the dextran used was not contain only polysaccharides but some fatty acids and
aromatic fragments.
Serum Albumin bovine
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of serum Albumin
bovine are presented on the figures 7 and 8.
The main fragments were derived of proteins (indol, toluene and derived nitriles).
Note the presence of polyhydroxy aromatic, phenol, cresol, phenol ethyl thio and phenol
3 ethyl. They transformed with the TMAH into methoxy fragments in accordance with
CHALLINOR (1989).
With the pyrolysis methylation it found the trisulfide dimethyl shown a present of salts.
The TMAH reacts with the salts that can co-eluted with other fragments.
The benzene was not found in the pyrolysis/methylation, maybe because it has the same
retention time than the 2 butanoic acid 2 methyl ester.
Tetracosanoic acid
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of tetracosanoic
acid are presented on the figures 9 and 10.
The products of conventional pyrolysis of fatty acid include alkenes and small peak of
alkanes and some aromatic compounds (cyclopropane and cyclohexane). The mechanism
of the formation of those fragments was explain by HARTZERS et al. (1995).
2- Comparison pyrolysis and pyrolysis/methylation of some fractions
Fulvic acid of Wachusett 1
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of the fulvic acid
of Wachusett 1 are presented on the figures 11 and 12.
The conventional pyrolysis conducted only of benzene, toluene, phenol and propanol.
Principally the aromatic compounds represent this fulvic acid. The source of the aromatic
signature was probably decomposition of the detritus materiel produced by leaves of
plants and algae death in the beginning of the fall. But the phenol and the toluene were
the fragments typically associated with biological signature, which is certainly reduced
because the peaks such as acetonitrile, acetic acid and acetamide were not found.
The fingerprint with the pyrolysis/methylation was very different with a great number of
different fragments. The similitude was the presence of the phenol methyl that can
became from the phenol in the pyrolysis/methylation and the 3 ethyl alpha 4 dimethoxy
toluene that can correspond to the toluene.
The most fragments obtained with the pyrolysis/methylation were aliphatic carboxylic
acid methyl ester and derived benzoic methyl ester. The same results than DEL RIO and
HATCHER (1996) with a soil fulvic acid.
In the pyrolysis/methylation, a peaks of phenol and methoxy phenol could be observed
that can explain due to competing side reactions that may cause the incomplete
conversion to the respective ethers.
Note the presence of methoxy fragments, butane 2 methoxy 3 methyl, propane 2
methoxy, benzene 1 methoxy 4 methyl, trimethoxy hexane and 3 ethyl-alpha 4 dimethoxy
toluene and methoxycarbonyl 1 1 diethyl 2 butene.
The pyrolysis/methylation permits to observe a lot fragments that were not existe and
transformed into small molecules in conventional pyrolysis.
Humic acid of Wachusett 1
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 13 and 14.
The phenol and the derived phenol were transformed to methoxy benzene except the
trimethyl phenol. The toluene and the derived nitrile were presented in the two
pyrograms. But globally the fingerprint with pyrolysis/methylation was very different
than the fingerprint with conventional pyrolysis.
Note that the program of temperature of the column was not exactly the same for the
humic acid with conventional pyrolysis.
Weak hydrophobic acid of Wachusett 1
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 15 and 16.
The figure 15 shows that most of the identified compounds from pyrolysis of weak
hydrophobic acid are toluene, acetone, and 1,3 pentadiene 2,3 dimethyl derived by
polysaccharides and proteins. This important proportion of those classes could be
explained that this fraction is the most biodegradable fraction. The fingerprint was
represented by small fragments with a retention time smaller than 4 min.
With pyrolysis/methylation, all the peaks co-elute with the TMAH and the toluene with
the butanoic acid methyl methyl ester.
In this case, it is difficult to interpret except that the weak hydrophobic acid correspond to
a agencement of small molecular of protein origin or small fatty acids. It’s important to
have the results of the both pyrolysis.
Fulvic acid of Wachusett 2
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 17 and 18.
The fingerprint of the fulvic acid with the conventional pyrolysis presented a lot of
fragments that can showed the complexity of the organic matter and the humification.
The main fragments were methyl or methoxy forms that can explain the stability with the
conventional pyrolysis.
The presence of benzoic acid 4 hydroxy methyl ester, benzoic acid dimethoxy methyl
ester and benzoic acid methoxy methyl ester underline the humification of the organic
matter. SAIZ-JIMENEZ (1994-a) showed that the 3,4,5 trimethoxy benzoic acid and the
benzene carboxylic acid represent final steps in the oxidation of side chains in the lignin
units though microbial degradation.
The fingerprint of the two pyrolysis were very similar except the fatty acid fragments.
Some fragments found with the conventional pyrolysis, were not found with the
pyrolysis/methylation. Maybe it could be occurred co-elution with those fragments such
as the carboxylic acid methyl esters or methoxy derived.
The derived butanoic acid was present in the conventional pyrolysis, it’s very unusual.
Maybe other groups protect the function or this acid was in big proportion.
Note the presence with the pyrolysis/methylation of bisulfide dimethyl.
Hydrophilic acid of Wachusett 2
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 19 and 20.
As the Wachusett 2fulvic acid, the hydrophilic acid present a lot of fragments and the
presence of 1 3 benzene dicarboxylic acid methyl ester underline the humification of the
organic matter.
The main products of conventional pyrolysis of hydrophilic acid include benzene,
toluene, acetic acid, p+m xylene, derived furan, derived ethanone, derived naphthalene
and cyclopentenone.
Note again the problem with the disulfide with the pyrolysis/methylation.
Fulvic acid of Wachusett 2 chlorinated water
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 21 and 22.
Any chlorinated compounds were found in the chlorinated water. When the compounds
were burnt, the chlorine was released.
As the fulvic acid of Wachusett 2, the same remark could be made with the chlorinated
fulvic acid.
Fulvic acid of Andover
The conventional pyrolysis/pyrolysis methylation pyrochromatograms of chitin are
presented on the figures 23 and 24.
The main products of conventional pyrolysis of fulvic acid include toluene, derived
benzene, derived cyclopentenone, derived phenol, indene and acetic acid.
As the fulvic acid of Wachusett 2, the presence of the benzoic acid dimethoxy or
methoxy methyl ester underline the humification of the organic matter.
The Andover fulvic acid was sampling in the end of the winter.
3- Characterization of some fractions
The figures 1 to 22 were compared each other.
The major part of the fragments was the first peak with CO2, H2S, CO. It represents
between 80% and 40% of the fulvic acid of the chlorinated water.
The main fragments were found in the fulvic acids except the fulvic acid of wachusett 1 :
benzene, p, m and o xylene, phenol, toluene, acetic acid, derived benzene, derived
naphthalene and derived phenol. With conventional pyrolysis or with
pyrolysis/methylation the aromatic compounds represent the main fragments of those
fulvic acids. The source of the aromatic signature was more important for the fulvic acid
of Wachusett 2 than Wachusett 1, greater than Andover that correlated with the SUVA
(results not showed). This aromatic signature of Wachusett 2 was due to an input of
aromatic compounds with the snow melting. The presence of benzene carboxylic acid or
benzoic acids where found in the lignin fragments. And SAIZ-JIMENEZ (1994-a) shown
that for a soil humic acid, the most significant fatty acid was identification of furan
carboxylic acids, benzene carboxylic acids and aliphatic dicarboxylic acids as their
respective methyl ester. The fulvic acid origin was more pedogenic (compounds
represented by derived benzene and naphthalene) than aquagenic.
The acetic acid, the phenol and the toluene were the fragments typically associated with
biological signature.
Those fulvic acids contain a little proportion of polysaccharides with the peaks of derived
cyclopentenone, of furan and of indene to compare to the results of (LABOUYRIEROUILLER, 1997). The small proportion of polysaccharides is in accordance with the
small activity algae in beginning of the spring and in the fall. And, any amino sugar was
included in the structure. Maybe, the activity of microorganisms was just started and the
production of the amino sugar is to small to detect them in GC/MS in the case of the
sampling Wachusett 2 and Andover and it was just finished for Wachusett 1.
Note a gig difference on the polyhydroxyaromatic and aromatic fragments to compare
other authors (LABOUYRIE-ROUILLER, 1997).
With the pyrolysis/methylation, the fulvic acid of the Wachusett 2 presents a butanoic
acid methyl ester peak that was not in the fulvic acid of the Wachusett 2 chlorinated
water. And, in the fulvic acid of the chlorinated water, the fatty acids were represented
with a lot of derived benzoic acid methyl ester that can be explain the 73.5% of the sum
of the aromatic + polyhydroxy aromatic fragments greater than the sum of the other
fulvic acids. In the fulvic acid of the chlorinated water, the methoxy phenol, benzene and
pentane were absented.
With the conventional pyrolysis, the pyrochromatograms of fulvic acids of Andover,
Wachusett 2 chlorinated water and of Wachusett 2 present a lot of fragments to compare
with the fulvic acid of Wachusett 1.
In the case of fulvic acid of Andover or of Wachusett 2, the pyrochromatograms present a
lot of methyl fragments. The methylation could be appended during the winter.
Note that after the treatment, the fingerprint of Andover fulvic acid is not very different
than the one of Wachusett 2. They present together a pronounced biological signature
based on peaks of acid acetic, toluene, methyl or ethyl indene and pyrrole (the last one
only in fulvic acid of Andover).
It could be understand that after different treatments the fulvic acid conserved it global
structure or resulted of the released compounds of the CAG or of the biomass.
It is not suppressing that for the fulvic acid of Andover a fragments represented the
biological signature are present, because the organic matter become of filtered water on
CAG where it is existing a biological activity. So it’s mean that the second hypothese is
more probable.
In the case of fulvic acid of Wachusett 2, the sampling was conducted during Mai
corresponding at the beginning of spring. So the microorganisms and algae activity was
starting.
In the hydrophilic acid, the peaks of toluene and benzonitrile were height than in the
fulvic acid represented the fragments derived of protein. The fingerprint of hydrophilic
acid presents a lot of ramified indene that represents the polysaccharide. The benzofuran
2 methyl and the benzofuran 4,7 dimethyl that represent the polysaccharide and the 1 H
pyrrole 2,5 dione 3 ethyl 4 methyl and piperidine that represent the protein were
presented in the hydrophilic acid and not in the fulvic acid.
With the pyrolysis/methylation, the pyrochromatograms of each fulvic, hydrophilic and
humic acids (except the fulvic acid of Wachusett 1) were not very different of the
fingerprints of each fraction with the conventional pyrolysis except the apparition of the
fatty acid fragments.
Note that the humic acid present a big diversity for it fragments that have been underline
by MARTIN (1995), results taken again by KRASNER et al. (1996).
In the case of the pyrolysis, the first peak represented the small molecules was ignored. In
the case of the pyrolysis/methylation, this peak was co-eluted with the TMAH. Seven
classes of compounds (polysaccharides, proteins, amino sugars, polyhydroxy aromatic
and aromatic non-substituted fragments, fatty acids and unknown origin) were regrouped
in the table 1.
Table 1 : Different classes of pyrolysis fragments of the organic matter fractions
Origin
Wachusett 1
Sample
fulvic acid
Wachusett 1
humic acid
No
TMAH
Wachusett 2
fulvic acid
Wach 2 T
Methylation PHA %
no
44.9
TMAH
0.7
Ar %
4.9
1.7
Pr %
29.6
2.5
Ps %
20
2
Fatty A AS %
0
0
92.9
0
u%
1.7
0.2
54.4
50.3
15.4
7.9
12
12.9
14
12.9
0
12.9
0
0
4.2
3
No
TMAH
14
26.5
35.4
21.4
15.5
4.6
12.8
7.1
4.3
34.9
0
0
18
5.5
hydrophilic acid
No
TMAH
5.9
6.6
38.4
29.3
27.1
33.8
27.6
14.9
0
11.3
0
0
1
4.1
fulvic acid
No
34
39.2
13
11.2
0
0
2.6
Andover
fulvic acid
TMAH
2
48.4
20.2
7.4
19.1
0
2.9
No
TMAH
15.8
17.1
27.3
21.8
17.1
16.7
14
20.2
0
11.8
0
0
25.7
12.4
Ps : polysaccharides, Pr : proteins, As : amino sugars, PHA : polyhydroxy aromatic fragments, Ar :
aromatic non substituted fragments, fatty A : fatty acids, u : unknown origin
The humic acid was presented a richer aromatic and polyhydroxy aromatic structure than
the fulvic acids, greater than hydrophilic acid.
Note that the Wachusett 2 and Andover have a lot of unknown to compare to Wachusett
1. A low level of the proteins and the polysaccharides was found for Wachusett 2 and
Andover. Maybe, the microorganisms consumed the proteins and the polysaccharides
first.
They are more aromatic fragments for Andover and Wachusett 2 to compare at
Wachusett 1 and reverse they are more polyhydroxy aromatic fragments for Wachusett 1
to compare at Wachusett 2 and Andover, that could be explain by the origins. GOBBELS
and PUTTMANN (1997) have shown that the aromatic fragments come from the soil humic
matter and the polyhydroxy aromatic fragments are originating from lignin. So the fulvic
acids of Wachusett 1 was more issue to lignin than the soil humic matter and inverse for
Wachusett 2 and Andover. This result could be in accordance with the fact that after the
winter the leaves were degradable and transform into soil humic matter and the fulvic
acids of Andover and Wachusett 2 contain a lot of methylated fragments than the fulvic
acid of Wachusett 1 and in contrary during the fall the lignin was entire.
A comparison between the fulvic and the hydrophilic acids of Wachusett 2 could be
done. The proportion of polyhydroxy aromatic fragments plus aromatic in hydrophilic
acid was less than fulvic acid and inversely, the proportion of protein plus polysaccharide
in the hydrophilic acid was greater than the fulvic acid, that was in accordance with the
knowledge on the two fractions.
The comparison of fulvic acids of Wachusett 2 and Wachusett 2 chlorinated waters were
presented a rich aromatic and polyhydroxy aromatic structure in accordance with the
important relative UV absorbance, 49.4% of aromatic and polyhydroxy aromatic
fragments for the fulvic acid of the raw water and 73.2% for the fulvic acid of the
chlorinated water. For the fulvic acid of the chlorinated water, maybe it better to consider
the aromatic and polyhydroxy aromatic fragments of the pyrolysis/methylation : 50.2%.
GOBBELS et al. (1990) showed that the defonctionalised fatty acids could be conduct to
the formation of aromatic fragments.
Note that the fulvic acid of raw water have more unknown fragments than the fulvic acid
of the chlorinated water. That could explain by the greater proportion of fatty acid of the
fulvic acid of the raw water. With the pyrolysis alone, the fatty acids produced some
alkanes and alkenes (HARTGERS et al., 1995) that were considered as unknown
fragments.
A low level of the proteins and the polysaccharides was found for those fulvic acids of
the two waters.
Conclusions
To characterize the organic matter completely, it’s necessary to have the results of two
pyrolysis. Nevertheless, the pyrolysis with TMAH is more useful than the other one
except for the weak hydrophobic acid because the conventional pyrolysis data are
incomplete and must be revised to confirm the results. The pyrolysis/methylation was
characterized by more different fragments than the conventional pyrolysis.
For the pyrolysis/methylation, the technique gives additional information about the
composition of carboxylic acids and alcohol and substituted phenolic compounds.
The advantage of the pyrolysis/methylation was the increasing sensitivity underline by
CHALLINOR (1989).
However, the pyrolysis methylation is inappropriate for the derivatization of low
molecular weight pyrolysis products, which elute at very low retention times in the same
times than the TMAH or whose methyl esters co-elute with other pyrolysis products and
it methylated some salts such as phosphoric and sulfuric acids that can co-elute with other
pyrolysis fragments.
For the peak of the toluene, BRUCHET et al. (1990) and Biber (1996) underline that the
toluene became from the proteins, but HARTZERS et al. (1995) shown that the fatty acids
such as 16(4’-methyl phenyl) hexadecanoic acid and 122(5’ methyl thienyl) dodecanoic
acid formed some toluene. And HARTZERS et al. (1995) and GOBBELS and PUTTMANN
(1997) found that the aromatic fragments might derive from cyclisation reactions of some
fatty acids. So the interpretation semi-quantitative was maybe more difficult than it
expect. But It’s seem that in the organic matter (fulvic, hydrophilic, weak hydrophobic
and humic acids) the pyrolysis/methylation shown that the most importance of the fatty
acids were small and if the comparison of the results between the pyrolysis and the
pyrolysis/methylation, except for the fulvic acid of Wachusett 1, were almost the same.
Pyrolysis/methylation drastically changed the fingerprints of fractions or biopolymers
when compared with the conventional pyrolysis, but globally the interpretations after
calculate the sum of the fragments was the same.
The method of the pyrolysis/methylation involves minimal manipulation, used the same
cost instrumentation and is much more sensitive than the conventional technique.
Thus for each case it’s necessary to realize the conventional pyrolysis and the
pyrolysis/methylation, the combination of the both pyrolysis appear to be good analytical
approaches for investigating the chemical nature of organic matter and oxidation
treatment.
Acknowledgements
This research was conducted in the Department of Civil Engineering, University of
Massachusetts at Amherst and supported by the EPA and MWRA projects.
References
Alcaniz J., J. Romera, L. Comellas, R. Munne and A. Puigbo, 1989, EFFECTS OF SOME MINERAL
MATRICES ON FLASH PYROLYSIS-GC OF SOIL HUMIC SUBSTANCES, Sci. Total Environ., 81/82,
81-90.
Biber M.V., F.O. Gulacar and J. Buffle, 1996, SEASONAL VARIATIONS IN PRINCIPAL GROUPS OF
ORGANIC MATTER IN A EUTROPHIC LAKE USING PYROLYSIS/GC/MS, Environ. Sci. Technol.,
30, 3501-3507.
Bruchet A., C. Rousseau and J. Mallevialle, 1990, PYROLYSIS-GC-MS FOR INVESTIGATING HIGHMOLECULAR-WEIGHT THM PRECURSORS AND OTHER REFRACTORY ORGANICS, J.AWWA.,
september, 66-74.
Challinor, J.M., 1989, PYROLYSIS-DERIVATISATION-GAS CHROMATOGRAPHY TECHNIQUE
FOR THE STRUCTURAL ELUCIDATION OF SOME SYNTHETIC POLYMERS, Journal of Analytical
and Applied Pyrolysis, 16, 4, 323-333.
De Leeuw J.W. and M. Baas, 1993, THE BEHAVIOUR OF ESTERS IN THE PRESENCE OF
TETRAMETHYLAMMONIUM SALTS AT ELEVATED TEMPERATURES; FLASH PYROLYSIS OR
FLASH CHEMOLYSIS ?, J. Analytical Applied Pyrolysis, 26, 175-184.
Del Rio J.C. and P.G. Hatcher, 1996, STRUCTURAL CHARACTERIZATION OF HUMIC
SUBSTANCES
USING
THERMOCHEMOLYSIS
WITH
TETRAMETHYLAMMONIUM
HYDROXIDE, “Humic and fulvic acids, Isolation, structure and environmental role”, Gaffney J.G., N.A.
Marley and S.B. Clark Editor, ACS Symposium series 651, American Chemical Society, Wachington DC,
Chapter 6, 78-95.
Gobbels F-J. and W. Puttmann, 1997, STRUCTURAL INVESTIGATION OF ISOLATED AQUATIC
FULVIC AND HUMIC ACIDS IN SEEPAGE WATER OF WASTE DEPOSITS BY PYROLYSIS-GAS
CHROMATOGRAPHY/MASS SPECTROMETRY, Wat. Res., 31, 7, 1609-1618.
Hartgers W.A., J.S. Sinninghe Damste and J.W. De Leeuw, 1995, CURIE-POINT PYROLYSIS OF
SODIUM SALTS OF FUNCTIONALIZED FATTY ACIDS, Journal Analytical Applied Pyrolysis, 34,
191-217.
Hatcher P.G. and R.D. Minard, 1996, COMPARISON OF DEHYDROGENASE POLYMER (DHP)
LIGNIN WITH NATIVE LIGNIN FROM GYMNOSPERM WOOD BY THERMOCHEMOLYSIS
USING TETRAMETHYLAMMONIUM HYDROXIDE (TMAH), Org. Geochem, 24, 593-600.
Labouyrie-Rouillier L.,1997, ETUDE COMPARATIVE DES TECHNIQUES DE FILTRATION
MEMBRANAIRE ET D'ADSORPTION SUR RESINES MACROPOREUSES NON IONIQUES EN VUE
DE L'EXTRACTION ET DE LA CARATERISATION DES MATIERE ORGANIQUES NATURELLES
DISSOUTES D'EAUX DE SURFACE, These de Doctorat, University of Poitiers.
Leenheer J.A., 1981, COMPREHENSIVE APPROACH TO PREPARATIVE ISOLATION AND
FRACTIONATION OF DISSOLVED ORGANIC CARBON FROM NATURAL WATERS AND
WASTEWATERS, Environmental Science and Technology, may, 578-587.
Malcolm R.L., J.P. Croue and B. Martin, 1993, Isolation of XAD-4 acids from natural waters and their
importance as precursors to TOX and THM upon chlorination, to presented at DELFT CONGRESS, Sept.
13-18, 1-34.
Martin B., 1995, LA MATIÈRE ORGANIQUE NATURELLE DISSOUTE DES EAUX DE SURFACE :
FRACTIONNEMENT, CARACTÉRISATION ET RÉACTIVITÉ, Thèse de Doctorat, Université de
Poitiers.
Martin F., J.C. Del Rio, F.J. Gonzalez-Vila and T. Verdejo, 1995, PYROLYSIS DERIVATIZATION OF
HUMIC SUBSTANCES 2. PYROLYSIS OF SOIL HUMIC ACIDS IN THE PRESENCE OF
TETRAMETHYLAMMONIUM HYDROXIDE, Journal Analytical Applied Pyrolysis, 31, 75-83.
Marbot R., 1997, THE SELECTION OF PYROLYSIS TEMPERATURES FOR THE ANALYSIS OF
HUMIC SUBSTANCES AND RELATED MATERIALS 1. CELLULOSE AND CHITIN, Journal
Analytical Applied Pyrolysis, 39, 97-104.
McKinney D.E., J.M. Bortiatynski, D.M. Carson, D.J. Clifford, J.W. De Leeuw and P.G. Hatcher,
TETRAMETHYLAMMONIUM HYDROXIDE (TMAH) THERMOLYSIS OF THE ALIPHATIC
BIOPOLYMER CUTAN : INSIGHTS INTO THE CHEMICAL STRUCTURE, Org. Geochem, 24, 641650.
Saiz-Jimenez C., 1994-a, ANALYTICAL PYROLYSIS OF HUMIC SUBSTANCES : PITFALLS,
LIMITATIONS AND POSSIBLE SOLUTIONS, Environ. Sci. Technol., 28, 11, 1773-1780.
Saiz-Jimenez C., 1994-b, PYROLYSIS/METHYLATION OF SOIL FULVIC ACIDS.
BENZENECARBOXYLIC ACIDS REVISITED, Environmental Science and Technology, 28, 1, 197.
Saiz-Jimenez, C., B. Hermosin and J.J. Ortega-Calvo, 1994, PYROLYSIS/METHYLATION : A
METHOD FOR STRUCTURAL ELUCIDATION OF THE CHEMICAL NATURE OF AQUATIC
HUMIC, Water Research, 27, 11, 1693-1696.
Saiz-Jimenez, C., B. Hermosin and J.J. Ortega-Calvo, 1993, PYROLYSIS/METHYLATION : A
MICROANALYTICAL METHOD FOR INVESTIGATING POLAR ORGANIC COMPOUNDS IN
CULTURAL PROPERTIES, Intern J. Environ. Anal. Chem., 56, 63-71.
Tanczos I., M. Schoflinger, H. Schmidt and J. Balla, 1997, CANNIZZARO REACTION OF
ALDEHYDES IN TMAH THERMOCHEMOLYSIS, Journal Analytical Applied Pyrolysis, 42, 21-31.
Thurman E.M. and R.L Malcolm, 1981, PREPARATIVE ISOLATION OF AQUATIC HUMIC
SUBSTANCES, Environ Sci. Technol., 15, 463-466.