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Journal of Analytical and Applied Pyrolysis, 26 ( 1993) 175- 184 175 Elsevier Science Publishers B.V., Amsterdam The behaviour of esters in the presence of tetramethylammonium salts at elevated temperatures; flash pyrolysis or flash chemolysis?lv2 J.W. de Leeuw * and M. Baas Netherlandr Institute for Sea Research (NIOZ), Division of Marine Biogeochemistry, P.O. Box 59, 1790 AB Den Burg, Texel (Netherlands) (Received November 2, 1992; accepted in final form January 26, 1993) ABSTRACT Flash-heating experiments with model compounds such as tomato cutin, octadecyl octadecanoate, hexadecanol, triacontanol and L-cc-lecithin with and without tetraalkylammonium salts, in particular tetramethylammonium hydroxide (TMAH), show that we have to discriminate between pyrolysis products sensu strict0 and products resulting from bond breaking induced by chemical reagents at elevated temperatures. This discrimination is required to improve the structural elucidation of the macromolecules to be analyzed. Esters; flash chemolysis; flash pyrolysis; pyrolysis; tetramethylammonium salts. INTRODUCTION To improve the gas chromatographic separation of relatively polar pyrolysis products, attempts have been made to perform so-called on-line derivatization of such pyrolysis products generated during flash pyrolysis-gas chromatography (Py-GC) or flash pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) analyses of macromolecular organic matter. Challinor [l] noticed that synthetic polymers subjected to pyrolysis conditions in the presence of tetraalkylammonium salts such as tetramethylammonium hydroxide (TMAH) yielded methyl esters and methyl ethers. He suggested that fatty acids and alcohols are produced as primary pyrolysis products and that these compounds were derivatized, i.e. methylated in this case. Other workers [2-61 have reported on the methylation of free acids * Corresponding author. ’ NIOZ Division of Marine Biogeochemistry Contribution 280. * Presented at: Pyrolysis ‘92-Proceedings of the 10th International Conference on Fundamental Aspects, Processes and Applications of Pyrolysis, Hamburg, Germany, September 28-October 2, 1992. 01652370/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved 176 J. W. de Leeuw and hf. Baas / J. Anal. Appl. Pyrolysis 26 (1993) IV- 184 with tetraalkylammonium hydroxides at elevated temperatures. It was shown that free carboxylic acids are quantitatively converted to the corresponding tetramethyl~monium salts which yield methyl esters and trimethylamine between about 250 and 350°C. This procedure was sometimes referred to as “pyrolytic methylation” and was applied more recently to improve the gas chromatographi~ resolution of mixtures of fatty acids. In a number of more recent papers [7-lo] natural esters were converted to their methyl esters by tetramethyl- or other tetraalkylammonium hydroxides under pyrolysis conditions. It was indicated that the methyl esters were the result of transeste~fi~ation reactions via intermediate tetramethyla~onium hydroxide salts and were not generated by methylation of pyrolytically formed fatty acids, although the genesis of free carboxylic acids from esters by pyrolysis is well known [ 1l]. Based on the above-mentioned observations it is not completely clear if tetraalkylammonium hydroxide salts under pyrolytic conditions act as chemical reagents inducing bond breaking within the macromolecular substances to be analysed (chemolysis) or if they derivatize, i.e. methylate, pyrolysis compounds which are generated from the macromolecular substances by bond breaking induced by heat only (pyrolysis). In the study presented here a number of selected model compounds were subjected to pyrolysis conditions with and without TMAH to determine whether the products obtained have to be considered as pyrolysis or chemolysis products. Such discrimination is important to enable a correct molecular characterization of the macromolecular substances based on the products formed. ~urthe~ore, if we are dealing with thermally assisted chemolysis, many new possibilities complementary to analytical pyrolysis will become available for characterizing complex mixtures of synthetic polymers and biomacromolecules using all kinds of specific chemical reagents. EXPERIMENTAL Curie point pyrolysis-gas chromatography (Curie point Py -GC) The samples were pressed onto flattened ferromagnetic wires at a Curie temperature of 358 and 610°C. Approximately 2 ~1 of a 25% w/w solution of tetraammonium hydroxide (TMAH) in water was used as the reagent for in situ methylation. A high frequency generator (Fischer 9425) was used to induce the magnetic field. The gas chromatograph (Hewlett-Packard HP5890) was equipped with a cryogenic unit and programmed from 0°C (5 min) to 300°C (10 min) at a rate of 3”C/min. Separation of the products was achieved by a 25 m fused silica capillary column coated with CP-Sil 5 (0.32 mm i.d.; film thickness 0.45 pm). Helium was used as carrier gas. The temperature of the flame ionization detector was 320°C. J. W. de Leeuw and M. Baas 1 J. Anal. Appl. Pyrolysis 26 (1993) 175- 184 Curie point pyrolysis-gas chromatography/mass (Curie point Py - GCIMS) 111 spectrometry Pyrolysis was performed as described above. The column was directly inserted into the EI ion source of a VG-70SE double focusing mass spectrometer (mass range m/z 40-800; cycle time 1.8 s; ionization energy 70 eV). RESULTS AND DISCUSSION Figure 1 shows the gas chromatogram of the pyrolysate of the natural polyester cutin isolated from the fruit of Lycopersicon esculentum, the tomato plant. This pyrolysate has already been analysed by Tegelaar et al. [ 111 and the pyrolysis products were explained by assuming six-membered rearrangement reactions occurring at the ester linkages, resulting in the formation of di-unsaturated fatty acids and mono-unsaturated mid-chain hydroxy fatty acids corresponding to the building blocks of cutin. For convenience the mechanisms are schematically indicated in Fig. 2 [I]. It is emphasized that the terminal ester-linked alcohol moieties yield terminal alkene moieties and that free alcohol groups at C9 or Cl0 are not dehydrated during pyrolysis. It has been speculated that the ratio of pyrolysis products with a mid-chain hydroxy group and those with a mid-chain double bond is an indicator for the degree of cross-linking in these natural TOMATO CUTIN Py-GCFID (610-c) 4 c, C,B C-C-yo-Co-C*- c=c I OH OH I I C,BC-c OH -c40 ‘OH 10 \ Fig. 1. Gas chromatogram of the pyrolysis cutin isolated from L. esculentum. products obtained from the natural polyester 178 J. W. de Leeuw and M. Baas / J. Anal. Appl. Pyrolysis \;/o-; - CtiO IO I'O-_c/ 16 O\ o#=m /o-- 10 1 16 OH Fig. 2. Proposed pyrolysis mechanism hexadecenoic acids from the polyester TOMATO 26 (1993) 175-184 CUTIN for the genesis of hexadecadienoic cutin. MeOC Py(Me)-GCFID (610°C) MeOC - C=$,y acids and hydroxy- $0 -c 16 OMe I ’ OMe C MeOC 40 ‘OMe \ / -C,, C 90 ‘OMe Y OH I c,,c C tieOC,,-C,: fro ‘OMe OMe 40 * HC=,6-C,oC, Fig. 3. Gas chromatogram of the products obtained from the natural polyester from L. esculentum in the presence of TMAH under pyrolysis conditions. OMe cutin isolated polyesters. p-Vinylphenol may originate from esterified p-coumaric acid moieties, which generate p-coumaric acid as a primary pyrolysis product that subsequently undergoes decarboxylation [ 111. Figure 3 shows the gas J. W. de Leeuw and M. Baas / J. Anal. Appl. Pyrolysis 26 (1993) 17% 184 119 chromatogram of the compounds obtained by flash heating the tomato cutin sample in the presence of TMAH under the same conditions as mentioned above for the pyrolysis experiment. The compounds indicated were identified by comparison of their mass spectra and GC retention times with those of standards. The major compounds are dimethoxymethyl esters and monohydroxymonomethoxymethyl esters. Products with mid-chain and/or terminal double bonds are minor compounds. The virtual absence of these latter compounds, which were the dominant products generated by pyrolysis (cf. Fig. l), and the abundant presence of methoxy and hydroxy compounds clearly shows that pyrolysis has occurred not at all or only to a minor extent. Most of the products can be explained by assuming a hydrolysis reaction followed by quantitative methylation of the fatty acid moieties as already suggested by Dworzanski et al. [9] and partial methylation of the hydroxyl groups. It is also noteworthy to indicate the presence of the dimethyl ester and the 16-methoxymethyl ester, products not encountered in the pyrolyzate obtained without TMAH. The assumption that p-vinylphenol in the pyrolyzate originated from p-coumaric acid moieties was substantiated by the relatively abundant presence of ~-methoxycinnamic acid methyl ester. To further investigate the mechanism of demacromolecularization in the presence of TMAH through a hydrolysis-methylation pathway, a C36 saturated wax ester, octadecyl octadecanoate, was subjected to pyrolysis/evaporation conditions with and without TMAH. Figure 4 shows the GC traces of reaction mixtures obtained from experiments performed with ferromagnetic wires at Curie temperatures of 358 and 610°C. The results of the experiments performed without TMAH are very similar and clearly indicate that the wax ester does not pyrolyze but simply evaporates. The peaks in the middle of the chromatogram correspond to impurities present in the wax ester sample. Their relative abundances are greatly overestimated because the high boiling point of the intact wax ester severely hampers a quantitative transfer from the pyrolysis chamber to the capillary column. Direct on-column injection of the wax ester sample substantiated the presence of small amounts of these impurities. The product mixture obtained in the presence of TMAH using a ferromagnetic wire at a Curie temperature of 358°C shows the abundant presence of octadecanoic acid methyl ester, octadecanol and 1-methoxyoctadecane next to the intact wax ester. The same results were obtained v+th a ferromagnetic wire at a Curie temperature of 61O”C, although the wax ester was barely recognizable. These results clearly show that no pyrolysis occurred in all ex~riments with the wax ester; in the absence of TMAH only evaporation takes place whereas in the presence of TMAH hydrolysis and partial methylation of the hydrolysis products occurs as indicated in Fig. 5. It should be noted that in the presence of TMAH using a 358°C wire competition between hydrolysis-methylation and evaporation takes place whereas hydrolysis-methylation is virtually the only process if more energy is available in the case of a 610°C wire. 180 J. W. de Leeuw and M. Baas / J. Anal. Appl. Pyrolysis 26 (1993) 175- 184 C36:O WAXESTER PY-GC (S5S/610%) C36:O WAXESTER Py(Me)-GC (35e%) ‘t i I t C3S:O WAXESTER tin Py(Me)-GC (610%) I t ; C , ,,-OMe . - t retention time Fig. 4. Gas chromatograms of the products from octadecyl octadecanoate pyrolysis conditions with and without TMAH. generated under J. W. de Leeuw and M. Baas 1 J. Anal. Appl. Pyrolysis 26 (1993) 175- 184 ‘I,~,- 181 $0 "-0-C 0- I H 18 37 (CH,),N+ OH- + (CH3)4N+ C,,H,-b-0-C,,H, I OH 1 ‘17~35- //O ‘\OH + //O C,7H35-C\o- -O - Cl 857 -4 Ho - 5 e.%7 _ W,)4N+ I C,7H,-C90 ‘O-N+(CH I //O C17H~-c’O-CH C,,H,,OH + (CHJ4N+OH- ) 34 n + 3 = (CH&N C,8H370-N+(CH3)4 + H,O n 5eH370Me + (CH,),N Fig. 5. Mechanisms of methyl ester and methyl ether formation. To investigate the methylation behaviour of alcohols, 1-hexadecanol and I-tricosanol were subjected to pyrolysis conditions with and without TMAH. Figure 6 shows the GC traces of the I-hexadecanol experiments. The alcohol evaporates with and without TMAH. Obviously, the energy required for evaporation is insutkient to methylate the alcohol. In the case of the I-triacontanol the GC data (Fig. 7) showed that the energy required for evaporation of this higher boiling alcohol is similar to the amount of energy needed to methylate the alcohol. As a result, the alcohol is partly methylated. In a separate experiment it was shown that the C30 alcohol also became partly methylated in the presence of methanol. Experiments with J. W. de Leeuw and M. Baas 1 J. Anal. Appl. Pyrolysis 26 (1993) 175-184 182 i3,CH2OH Cl 6 ALCOHOL Py-GC-FID (610%) I I Cl 6 ALCOHOL Py(Me)-GC-FID (610%) - retentiontime Fig. 6. Gas chromatograms obtained after heating I-hexadecanol with and without TMAH. TMAH and hexadecanoic acid showed, as expected, a quantitative conversion to the methyl ester. Because natural samples may contain compounds with tetraai~ylammonium moieties it was interesting to study the behaviour of a lecithin J. W. de Leeuw and M. Baas 1 J. Anal. Appl. Pyrolysis26 (1993) 115-184 183 C30 ACOHOL Py-GC-FIR (610%) Cz9H 59CH20Me ‘20 ALCOHOL Py(Me)-GC-FID (610°C) retentiontime - Fig. 7. Gas chromatograms obtained after heating I-triacontanol with and without TMAH. (L-a-phosphatidylcholine), a very common cell membrane component of many organisms. CC/MS analysis of the product mixture revealed the abundant presence of both the free carboxylic acids and their methyl esters 184 J. W. de L.eeuw and M. Baas / J. Anal. Appl. Pyrolysis 26 (1993) 175-184 indicating that the carboxylic acid moieties were partly transesterified by the trimethylammonium group. The data presented above clearly show that we have to discriminate between pyrolysis products sense strict0 and products generated through a reaction between the macromolecules and a specific chemical reagent at an elevated temperature. The latter process may be indicated by the term the~ally assisted chemolysis (TAC). 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