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 Malcolmet 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 13Cand 15NRMN, 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 Biberet 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 Hartzerset 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 (Bruchetet 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 770C 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-Jimenezet 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-Jimenezet 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 ; Martinet al., 1995), fulvic acid of water (Saiz-Jimenezet 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 (McKinneyet al., 1996). To better understanding the mechanism of the TMAH, the compounds such as aldehydes (Tanczoset al., 1997) and fatty acids (Hartgerset 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 Bruchetet 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. Alcanizet 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-Jimmenezet 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 200C (1 s) to 700C (10 s) at the rate of 20C/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 220C at the rate of 3C/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 Bruchetet 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), Reckhowet 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 Malcolmet al. (1993).
The fractions and the waters were concentrated by evaporation at 40C and the fulvic acids could be drying.
Biopolymers
The Chitin, Cellulose acetate, Dextran and Serum albumin bovine were used by Bruchetet 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-(14)--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 Hartgerset al. (1995) on hexadecanoic and 12-hydroxyoctadecanoic 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 Tanczoset 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. McKinneyet al. (1996), Del Rio and Hatcher, (1996) and Saiz-Jimenez (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.