Anal. Chem. 1998, 70, 707-715
A Graphite-Assisted Laser Desorption/Ionization Study of Light-Induced Aging in Triterpene Dammar and Mastic Varnishes Stefan Zumbu 1 hl,† Richard Knochenmuss,‡ Stefan Wu 1 lfert,† Fre´de´ric Dubois,‡ Michael J. Dale,§ and ,‡ Renato Zenobi*
Technologisches Labor, Schweizerisches Institut fu¨ r Kunstwissenschaft, Zollikerstrasse 32, CH-8032 Zu¨ rich, Switzerland, Laboratorium fu¨ r Organische Chemie, Universita¨ tsstrasse 16, ETH Zentrum, CH-8092 Zu¨ rich Switzerland, and Port Sunlight Laboratory, Unilever Research, Quarry Road East, Bebington, Wirral, L63 3JW, U.K.
The light-induced aging of natural triterpenes used in varnishes was studied using graphite-assisted laser desorption/ionization mass spectrometry. This method was found to be superior to conventional matrix-assisted laser desorption/ionization for these materials because of higher signal levels and less chemical noise from the matrix. The dammar and mastic raw materials were found to be partially oxidized compared to their nominal composition, with up to six oxygens incorporated. The light-induced aging process leads initially to polymerization, up to trimers. Further aging leads to a decrease in average molecular weight. These observations are supplemented by FTIR and 13C NMR spectra which show complete reaction of keto groups to acids and peroxy species. A general mechanism for the process is proposed, and the consequences for properties of varnish films are investigated. Naturally occurring triterpenoid resins are widely used as varnishes on artwork.1 Depending on the type of resin, the state of oxidation, and application parameters, films of these resins exhibit a variety of degradation phenomena. Crack or craze formation and yellowing are among the most apparent. Other authors2-4 have made large strides in establishing the connections between the mechanical properties of and chemical changes in natural resin films, yet varnish aging remains incompletely understood. In this study, we attempt to progress further toward such an understanding for two natural triterpene resin mixtures frequently used in the field of conservation and restoration of art, dammar and mastic (traditionally “gum” mastic, although it is a resin and not a gum). A need for such †
Schweizerisches Institut fu ¨ r Kunstwissenschaft. ‡ Laboratorium fu ¨ r Organische Chemie. § Unilever Research. (1) De la Rie, R. Anal. Chem. 1989, 61, 1228A. (2) De la Rie, R. Stable Varnishes for Old Master Paintings; Academisch Proefschrift, University of Amsterdam, Amsterdam 1988. (3) De la Rie, R. Stud. Conserv. 1988, 33, 53. (4) Feller, R. L. Am. Assoc. Museums 1952, 7. Feller, R..L. Stud. Conserv. 1957, 162. De Witte, E.; Goessens-Landrie, M. Bull. IRPA 1979, 17, 106. Michels, J. Tru ¨ bungen transparenter U ¨ berzuge und Farbschichten an Staffeleigema¨lden-Pha¨nomenologie, Terminologie und mo¨gliche Ursachen. Diplomarbeit, Fachklasse fu ¨ r Konservierung und Restaurierung, Ho¨here Fachschule fu ¨ r Gestaltung, Bern, 1995. S0003-2700(97)00574-X CCC: $15.00 Published on Web 01/21/1998
© 1998 American Chemical Society
Figure 1. Photomicrographs of mastic and dammar films (20× magnification). The broad structure in the mastic film (upper) extending from left to right is a craze. Broad crazes normally result when long-chain polymeric materials are forced into plastic flow. In contrast, brittle fracture occurs with dammar films (lower).
understanding is apparent from observations of resin films. While brittle fracture (cracking) occurred in certain films we prepared, crazes (related to plastic flow) were observed in other similarly produced films. Example micrographs are shown in Figure 1. The dammar film cracked while the mastic film exhibited only crazing at equivalent times after preparation. This difference could not be explained by prevailing models, such as the elastoplastic behavior predicted for a short-chain polymer5 or the embrittlement theory popular with art conservators.2,6 The different mechanical properties found seemed to have their origins in the degree of prior aging of the dammar and mastic Analytical Chemistry, Vol. 70, No. 4, February 15, 1998 707
Scheme 1
Scheme 2
materials. This study applies several modern spectrometric methods to the determination of the chemical basis of these aging effects. Using continuous exposure to high light fluxes, we focus on photoinduced processes, since these are believed to be the major intitiation and degradation pathways when the material has been exposed to short-wavelength light. Thermal aging also occurs in the absence of light1,3 but is dramatically enhanced if photoaging has occurred also. The initiation of photoaging is believed to occur in these resins at the ubiquitous aliphatic keto groups. Light of 275-295-nm wavelength excites the n-π* transition of these groups followed by bond homolysis and ring opening (Norrish type I reaction); see reaction Scheme 1.3,7 Reaction of the ensuing radicals leads to unsaturation and oxidation to aldehydes and acids. Secondary propagating autoxidation reactions are more complex. Attack of a radical on its neighbor can lead to dimerization and an increase in average molecular weight. On the other hand, oxidation proceeding via peroxy radicals can cause further ring opening or chain cleavage and generation of new ketones, as well as promoting further degradation by production of radicals; see Scheme 2. These reactions seem to be adequate to describe the bulk of our results but certainly do not represent the full range of chemistry that can occur in resins. Further discussion of possible degradation pathways can be found in refs 1-8 and references therein.
Mass-resolved information on the oxidation and the degree of polymerization was obtained using a variant of matrix-assisted laser desorption/ionization (MALDI). These results were complemented by Fourier transform infrared (FT-IR) and carbon nuclear magnetic resonance measurements to add qualitative structural information. MALDI is becoming one of the most important mass spectrometric methods for rapid analysis of a wide range of compounds. However, the method relies on the availability of a suitable matrix material for each class of analyte. These matrixes have been developed or discovered in an empirical fashion, and there is currently no method of systematically selecting an appropriate matrix for a new class of analytes. Some analytes have thus been found unsuitable for classical MALDI analysis or have only been found suitable after substantial effort was invested in finding a good matrix and/or sample preparation method. We encountered this difficulty in our efforts to study the aging of triterpene resins. Efforts to use standard MALDI met with limited success. The signals obtained were weak and matrix adduct bands greatly complicated spectral interpretation. We found that application of graphite-assisted, two-phase LDI substantially improved our ability to study these compounds, as has been demonstrated for a wide range of other small- and mediumsized molecules.9
(5) Chen, C. C.; Chheda, N.; Sauer, A. J. Macromol. Sci. Phys. 1981, 565. Hayashi, S. J. Macromol. Sci. Phys. 1981, p 623. Matsuoka, S. J. Macromol. Sci. Phys. 1981, p 715. Ishikawa, M.; Ogawa, H.; Narisawa, I. J. Macromol. Sci. Phys. 1981, p 441. Huang, W.; Aklonis, J. J. Chemistry and Properties of Cross-linked Polymers; Academic Press: New York 1977; p 453. Kramer, E. J. Adv. Polym. Sci., 1983, 52/53, 1. Rossmanith, H.-P.; et al. Grundlagen der Bruchmechanik; Springer: Vienna, 1982. Sauer, J. A.; Hara, M. Adv. Polym. Sci. 1990, 91/92, 69. Takemori, M. T. Adv. Polym. Sci. 1990, 91/ 92, p 263. Kramer, E. J.; Berger, L. L. Adv. Polym. Sci. 1990, 91/92, p 1. Michler, G. H. Kunstsoff-Mikromechanik-Morphologie, Deformations- und Bruchmechanismen; Hanser: Munich, 1992. (6) De la Rie, R. ICOM 8th Triennal Meeting Sidney Australia, Working Group 16, Vol. II, 1987; p 791. (7) DePuy, C. H.; Chapman, O. L. Moleku ¨ l-Reaktionen und Photochemie; Weinheim: New York, 1977. March, J. Advanced Organic Chemistry, 3rd ed.; Wiley: London, 1985. Von Bu ¨ nau, G.; Wolff, T. Photochemie-Grundlagen, Methoden, Anwendungen; Weinheim: New York, 1987; Suppan, P. Chemistry and Light; Cambridge University Press: Cambridge, U.K., 1994.
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EXPERIMENTAL SECTION “Batavia” dammar was obtained from Farbmu¨hle Kremer (Aichstetten, Germany). The “Chios” mastic was obtained from A. Grogg Chemie (Bern, Switzerland). The dipterocarpol reference triterpene was obtained from Fluka (No. 43610, >97%) and used with no further purification. For photoaging, the resins were dissolved in turpentine or commercial hydrocarbon solvents such as Dottisol D40 and then (8) Scott, G. Atmospheric Oxidation and Antioxidants; Elsevier: Amsterdam, 1965. (9) Dale, M.; Knochenmuss, R.; Zenobi, R. Anal. Chem. 1996, 68, 3321. Dale, M.; Knochenmuss, R.; Zenobi, R. Rapid Commun. Mass Spectrom. 1997, 11, 136.
spread on glass microscopic slides to a thickness of 50 µm, using a model 360 13-mm drawing knife from Erichsen GmbH (Hemer, Germany). Accelerated aging was carried out in a Heraeus Xenontest 150S (Heraeus GmbH, Hanau, Germany). The lamp power was 1.3 kW; the temperature was held at 26-28 °C. The relative humidity was held at 30% to minimize water-assisted homolysis aging. Initial matrix-assisted laser desorption mass spectrometry experiments used 2,5-dihydroxybenzoic acid (DHB) matrix purified by sublimation. The sample and matrix were dissolved in tetrahydrofuran (THF) and applied sequentially to the sample tip. Other matrixes tested were prepared similarly. A variety of matrix molecules were tested, including DHB, nicotinic acid, sinapic acid, R-cyano-4-hydroxycinnamic acid, and dithranol. Of these DHB was superior. Graphite-assisted spectra were, however, always stronger and less complicated by chemical noise. Particle-assisted laser desorption/ionization was first demonstrated by Tanaka et al. using cobalt particles.10 Sunner et al. introduced the use of graphite as a substrate,11 but other materials have also been found to be highly suitable.9,12 For this study, the graphite-supported samples were prepared in a two-step process. A 30 vol % suspension of 2-µm graphite particles (Aldrich) in methanol was placed on the sample tip and allowed to dry. In contrast to earlier graphite-assisted laser desorption studies,9 the addition of a liquid matrix was not found to be especially advantageous for these resins. As a result, the spectra reported here were mostly obtained from a dry graphite surface. After the graphite was dry, a THF solution of the resin was pipetted onto the graphite and also allowed to dry. The sample quantity was varied empirically for best signal and resolution. It was found that poor spectra were obtained if so much resin was used that the sample had a glossy appearance. Using both DHB and graphite laser desorption/ionization methods, these tripterpenes are observed largely as alkali metal ion adducts. To avoid spectral confusion resulting from the occurrence of both Na+ and K+ adducts, we enhanced Na+ adduct formation by addition of a small amount of NaCl to the graphite/ methanol slurry. This led to near complete disappearance of the K+ adducts. Although external calibrants were regularly used, the absolute mass accuracy is limited by the stability of the highvoltage supplies and by thickness variations in the graphite film. All m/z values reported here are estimated to be accurate to within 2 units. The Fourier transform infrared spectra were taken using a Perkin-Elmer System 2000 equipped with an i-Series IR/VIS microscope. The 13C nuclear magnetic resonance spectra were taken with a Bruker Spectrospin Avance DPX 200 instrument. The samples were dissolved in deuterated THF and measured for up to 10 240 sweeps. RESULTS AND DISCUSSION Triterpenes in natural dammar and mastic resins are tetra- and pentacyclic, largely saturated molecules. Before aging they (10) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151. (11) Sunner, J.; Dratz, E.; Chen, Y. C. Anal. Chem. 1995, 67, 4335. (12) Schu ¨ renberg, M. Doctoral Thesis, Westfa¨lische Wilhelms-Universita¨t Mu ¨ nster, Germany, 1996. Hillenkamp, F.; Schu ¨ renberg, M.; Schulz, T.; Dreisewerd, K. Proceedings of 46th ASMS Conference, 1997; p 1096.
Figure 2. Laser desorption/ionization mass spectra of dipterocarpol (hydroxydammarenone-II). In the upper spectrum, the dipterocarpol was desorbed from a graphite surface; in the lower, it was incorporated in and desorbed from DHB. The peaks annotated in parentheses were determined to be impurities in the graphite.
contain one to three oxygen atoms in the form of ketone, hydroxyl, or carboxylic acid groups. Molecular weights are in the range of 410-470. Natural resins contain 10-20 components, although 3-5 are typically dominant.1-3,13-16 A dammar component commonly present in large quantities is dipterocarpol, or hydroxydammarenone-II, MW ) 442.7. A mass spectrum of pure dipterocarpol is shown in Figure 2, using both matrix- and graphite-assisted laser desorption/ionization. Use of DHB as matrix leads to a strong band from the matrix at m/z ) 377, a strong unassigned adduct at m/z ) 553, and numerous weaker matrix adduct interferences up to m/z ) 900. The latter are in the range where we wish to observe oxidation and polymerization products of the resins and are therefore particularly problematic. These interferences are stronger than commonly observed with facile samples, largely due to the relatively high laser intensity needed to observe the resins. Signal strength from aged samples using DHB as matrix was not sufficient for acceptable signal/noise ratios in the high-mass region of interest. In contrast, the graphite-assisted spectrum shows a better signal/noise ratio and has only two significant interferences at m/z ) 412 and 507. Blanks showed that these are due to impurities in the graphite. The higher mass region is almost (13) Mills, J. S.; White, R. The Organic Chemistry of Museum Objects; Butterworth & Co.: London, 1987. (14) Dev, S.; Nagasampagi, B. A. In CRC Handbook of Terpenoids-Triterpenoids; Dev, S., Ed.; CRC Press: Boca Raton, FL, 1989; Vol. II. (15) Dev, S.; Gupta, S. A.; Patwardhan, S. A. In CRC Handbook of TerpenoidsTriterpenoids. Vol. II, Dev, S. , Ed.; CRC Press: Boca Raton, FL, 1989. (16) Karrer, W. Konstitution und Vorkommen der Organischen Pflanzenstoffe, 2nd ed.; Birkhauser: Basel, 1976.
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Figure 3. Laser desorption/ionization mass spectra of dammar on graphite, dammar in DHB, and DHB alone. The graphite substrate leads to much stronger dammar signals, without excessive interferences. In contrast, DHB is an inefficient matrix for dammar, and a number of interfering matrix bands appear with substantial intensity in the relevant m/z region.
entirely free of chemical noise. The small peak at m/z ) 482 is either the M + K+ adduct or, more likely, the Na+ adduct of dammarenolic acid, the first oxidation product of dipterocarpol, via the Norrish mechanism discussed below. The advantages of the graphite-assisted method are even more apparent when comparing spectra of natural dammar, Figure 3, in which many more components are present. The low intensities of the individual terpenes result in larger relative contributions of weak matrix-related bands from DHB to the spectrum, making assignments difficult and unreliable. The m/z ) 377 matrix peak is much stronger than any of the terpene signals. Other matrix bands are of comparable strength to the terpenes. The difficulty of differentiating analyte from matrix signal becomes even worse when trying to assign products of resin oxidation at higher mass. Unaged Dammar. De la Rie investigated unaged dammar resins similar to those studied here using gas chromatography followed by electron impact mass spectrometry.2,3 He found three major components in his natural dammar: dipterocarpol (MW ) 442.7), dammarenolic acid (MW ) 458.7), and ursonic acid (MW ) 454.7). Oleaonic acid differs from ursonic acid only in the location of one methyl group and can also be present in significant amounts. Including minority components, up to 10 compounds may be found in dammars, but the graphite-assisted mass spectrum of Figure 4 can be interpreted as largely due to the sodium adducts of the three main components, their fragments, and their oxidation products. Comparing with Figure 2, we assign m/z ) 466 as the sodiated dipterocarpol molecular ion. This was confirmed by addition of dipterocarpol to a dammar sample, which enhanced this signal proportionately. Singly oxidized and sodiated dipterocarpol is at m/z ) 482. This is almost certainly dammarenolic acid, as it is an expected product of photolytic oxidation and a known major component of dammar.2,3 This component was also found to be 710 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998
Figure 4. Laser desorption/ionization mass spectra of dammar (upper) and mastic (lower) on graphite. The lines are spaced by 16 Da and connect series of oxidized species. See text for further discussion. The inset shows the structure of isomasticadienoic acid, one of the major components of gum mastic, and closely related to the other main components.
in greater abundance near the surface of dammar nodules versus the interior, as expected for an oxidation product. The peak at m/z ) 494 in Figure 4 is assigned as sodium adducts of singly oxidized oleaonic and/or ursonic acids. Very little of the sodiated (m/z ) 478) or free (m/z ) 455) molecules are observed. These apparently oxidize much more readily than the other main dammar components. Further oxidation products readily explain the series of peaks indicated to higher mass (expected masses: m/z ) 494, 510, 526, 542, 558, and 574). Decarboxylation products are not observed (m/z ) 409), probably since the acid group is in a tertiary position. Finally, we note the weak band at m/z ) 448 is probably due to sodiated dammardienone (MW ) 424.7) and is the only peak that seems clearly assignable to a minority component of the dammar. Unaged Mastic. Major components of the mastic resin are expected to be tirucallol, isomasticadienonic acid, masticadienonic acid, and oleanonic acid.13,16,18 Tirucallol has a MW ) 426.7, the others all have MW ) 454.7. Since only two molecular weights are expected, the mastic mass spectrum of Figure 4 is surprisingly complex, at first glance. Tirucallol is not observed as the molecular ion but appears sodiated at m/z ) 449 (expected m/z ) 450). The other components do show a molecular ion at m/z ) 455, but the sodiated peak is much stronger, at m/z ) 477 (expected m/z ) 478). (17) Mathieu, J.-P.; Ourisson, G. Triterpenoids; Pergamon Press: London, 1958. (18) Wilhelm Sandermann, Naturharze-Terpentino¨l-Tallo¨l; Springer: Berlin, 1960.
The spectrum is dominated by the strong series of structured bands with average spacing of 16, indicating substantial oxidation. The right-hand edge of each peak cluster corresponds rather well to the series of sodiated oxidation products based on tirucallol: m/z ) 450, 466, 482, 498, 514, 530, and 546. The left edges correspond slightly less well to the oxidation products of the other components: m/z ) 478, 494, 510, 526, and 542. Up to six oxygens have been added to each of the presumed initial components of the mastic. This oxidation was not detected by de la Rie in dammar,2 even though both mastic and dammar can be significantly oxidized even as raw materials. This is due to the method of collecting them as exudates of plants of the Anacardiaceae and Diptercarpaceae families. The exudates are exposed to air for considerable time before processing and reaching the final user. Aged Samples. The effects of artificial or accelerated aging have been recently and extensively reviewed by Feller.19 High, controlled doses of light of a controlled spectrum and at elevated temperature or humidity are used to induce chemical changes in a short period which are similar to those induced by normal aging in a much longer time. However, natural aging also includes thermal processes that proceed in the dark and that modify the products of photolytic aging. We have deliberately used continuous exposure to high light fluxes at low temperature to enhance production of photoproducts, as these must be understood before the additional effects of dark reactions can be addressed. The results presented are therefore not to be understood as completely representative of natural aging. Nevertheless, since natural aging is believed often to be initiated via photolytic reactions, these results are representative of key natural processes. Aged Dipterocarpol. Figure 5 shows the mass spectrum of 300-h-photoaged dipterocarpol. There is relatively little oxygen addition to the molecule itself, as seen in the inset. At most two oxygens are added. Although not dominant in absolute terms, numerous reaction products are seen in the main figure at higher molecular weight. In the m/z region above 800, there is a substantial, slowly decreasing baseline of unspecific polymerization products. On top of this are numerous resolved peaks, some of which can be interpreted. At m/z ) 911 is the sodiated dipterocarpol dimer. The expected mass is 2 (dipterocarpol) (m/z ) 443) + Na + H ) 910. The hydrogen is abstracted from neighboring molecules (presumably at relatively labile tertiary positions) to terminate the radicals remaining from the initial ring cleavage. The peak at m/z ) 828 could correspond to dipterocarpol dimer which has lost the alkene tail group (see structure in Figure 2). The equally strong m/z ) 843 peak is assigned as the same fragment with one oxygen added. An oxygen addition peak is also observed above the unfragmented dimer, but with lower intensity. The peaks at m/z ) 1138 and 1154 are in the mass range between dipterocarpol dimers and trimers. They are therefore presumably of the composition 2(dipterocarpol) + fragment + Na. This is relevant for the natural resins discussed below, where it is also observed that the regions between the nominal oligomers are well filled in. (19) Feller, R. L. Accelerated Aging, Research in Conservation Series 4, The Getty Conservation Institute, Marina del Rey, CA, 1994.
Figure 5. Laser desorption/ionization mass spectra of photoaged dipterocarpol on graphite. The inset shows the pseudomolecular ion region, where few simple oxidation products are found. In the highmass region, the dipterocarpol dimer is observed, as well as numerous other radical addition products. Aging time 300 h.
Most intriguing is the series of peaks above m/z ) 1200 with average spacing of 74. These are also presumably due to addition of fragments onto dipterocarpol oligomers. The spacing of 74 corresponds to propionic acid, which is one of the fragments that can be cleaved from dipterocarpol after Norrish ring opening, but this assignment is tentative. It is remarkable that this pattern appears only in the upper mass range. Why these fragments do not also add strongly to dipterocarpol or the dimer is unclear, but may well be associated with the unsaturation that also accompanies aging. The fact that the progression is so welldefined indicates that one or a few photoproducts are the base material for fragment addition. Aged Dammar. As seen in Figure 6, photoaging of dammar leads to substantial changes in the high-mass region of the mass spectrum. After 150-300 h of photoaging, there has been polymerization of up to three triterpene units (m/z ≈ 1500). However, there has also been substantial cleavage of smaller units off the polymers since the regions between the monomers, dimers, and trimers are well filled in. Free fragments are also evident from the rising baseline below the original triterpenes. In the m/z ) 400-600 triterpene region the changes are not significant, as could be expected from the dipterocarpol results. The degree of oxidation of these molecules is not seen to increase significantly over that of the unaged samples. Further oxidation leads instead to reaction with neighboring triterpenes to form oligomers. At 300 h of aging, some structure appears in the dimer and trimer region, resembling the aged dipterocarpol spectrum. The spacing is, however, ∼24 Da, rather than 74 observed for dipterocarpol. This suggests that cleavage reactions lead to a few base structures and/or fragments differing in molecular weight Analytical Chemistry, Vol. 70, No. 4, February 15, 1998
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Figure 6. Laser desorption/ionization mass spectra of dammar on graphite, showing the effect of photoaging. The degree of polymerization increases up to 300 h, and then decreases with further aging. Polymerization of triterpene units is accompanied by nonspecific cleavage, leading to a general filling of the m/z range up to triterpene trimers. The original components do not undergo substantial further oxidation. The peak at m/z ) 412 is an impurity in the graphite; see Figure 2.
by two carbons. These then form the fragment addition series as seen with dipterocarpol. Pure dipterocarpol did not react as extensively as the natural dammar. In the 150-h spectrum of Figure 6, dipterocarpol accordingly remains less affected by aging than the other components. At longer aging times it is also consumed, as could be expected from the buildup of reactive species from the other dammar components. Aged Mastic. As seen in Figure 7, mastic undergoes a similar photoaging process. The original triterpenes do not become significantly further oxidized but rather react to form larger units, again up to trimers, and to lose fragments. In the 150-h-aged sample, the dimers are particularly prominent at m/z ) 900-1000, as are the fragmented dimer products at m/z ) ∼800. The structure at higher masses again has a typical spacing of 24 Da and is much more prominent than for dammar. Tirucallol and isomasticadienonic acid both have fully substituted double bonds between two of the central rings; see the inset in Figure 4. This structural element is not present in the main dammar components. Retro-Diels-Alder cleavage at this position leads to unsaturated fragments which fragment further.2 It seems a reasonable possibility that fragments differing by this C2 unit are the basis for the structure observed and the reason it is more pronounced in mastic than dammar. 712 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998
Figure 7. Laser desorption/ionization mass spectra of mastic on graphite, showing the effect of photoaging. The trends are similar to those seen for dammar in Figure 5. The peak at m/z ) 412 is an impurity in the graphite; see Figure 2.
For both mastic and dammar we find a somewhat smaller degree of maximum polymerization on aging than inferred by de la Rie from gel permeation chromatography of dammar.3 While interpretation of permeation chromatograms in terms of molecular weights is less certain than the mass spectral results presented here, we nevertheless hesitate to claim that the graphite-assisted LDI mass spectra are definitive. MALDI in general, and graphiteassisted LDI in particular, are known to suffer from decreasing sensitivity with increasing analyte molecular mass. It is possible that the small amount of 3000-20 000-Da polymeric material inferred by de la Rie is not efficiently desorbed or ionized in our instrument. On the other hand, problematic mass discrimination in graphite-assisted LDI is not expected until several thousand mass units above the highest molecular weights observed here.9 In addition, it should be emphasized that de la Rie also found the large bulk of his material in the mass range
