Anal. Chem. 2000, 72, 1707-1710
A MALDI Sample Preparation Method Suitable for Insoluble Polymers Randall Skelton,† Fre´de´ric Dubois, and Renato Zenobi*
Department of Chemistry, Swiss Federal Institute of Technology, CH-8092 Zu¨rich, Switzerland
Polyamides are insoluble or poorly soluble in common organic solvents, which makes normal sample preparation for matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry very difficult. An new analytical protocol for MALDI analysis of polyamides or other insoluble samples is described. It consists of pressing a pellet from a solid mixture of the polymer and a matrix, both in the form of finely ground powder. This sample preparation is compared with the common dried droplet sample preparation method and found to perform much better, both in terms of robustness against variation of experimental parameters and high-mass capability. Insoluble or poorly soluble polymers pose particular challenges to analysis by MALDI MS because they do not readily form mixed polymer/matrix crystals, a requirement for preparing good MALDI samples. In addition, quantitative information is often required in mass spectrometric studies of synthetic polymers, to answer questions about their molecular weight distribution (MWD) or end group distribution.1-6 Obtaining reliable quantitative information is to a large extent dependent on homogeneous, high-quality samples but is difficult by MALDI MS of poorly soluble materials. As noted by Yalcin et al.,7 even slight differences in the solubilities of matrix and polymer can lead to grave errors in the measured polymer molecular weight. We describe a new analytical protocol for MALDI analysis of polyamides which circumvents solubility problems. It should be easily applicable to other insoluble samples as well. The polymers investigated here (Figure 1) were polyamides, produced either as condensates between 1,6-diaminohexane and 1,4-benzenedicarboxylic acid (resulting in oligomers with a low molecular mass, up to 1000 Da) or as oligomers of laurin lactame regulated by either 1,6-diaminohexane or by 1,10-decanedicarboxylic acid, with a somewhat higher molecular mass (2500-4000 † Present address: Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada. (1) Montaudo, G.; Garozzo, D.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Macromolecules 1995, 28, 7983-7989. (2) Lehrle, R. S.; Sarson, D. S. Rapid Commun. Mass Spectrom. 1995, 9, 9192. (3) Axelsson, J.; Scrivener, E.; Haddleton, D. M.; Derrick, P. J. Macromolecules 1996, 29, 8875-8882. (4) Martin, K.; Spickermann, J.; Ra¨der, H. J.; Mu ¨ llen, K. Rapid Commun. Mass Spectrom. 1996, 10, 1471-1474. (5) Jackson, C.; Larsen, B.; McEwen, C. Anal. Chem. 1996, 68, 1303-1308. (6) Kassis, C. M.; Desimone, J. M.; Linton, E. W.; Remsen, E. E.; Lange, G. W.; Friedman, R. M. Rapid Commun. Mass Spectrom. 1997, 11, 11341138. (7) Yalcin, T.; Dai, Y.; Li, L. J. Am. Soc. Mass Spectrom. 1998, 9, 1303-1310.
10.1021/ac991181u CCC: $19.00 Published on Web 02/05/2000
© 2000 American Chemical Society
Figure 1. General structures of compounds investigated.
Da). The production method had an important influence on the end groups of the polymers, which included diamine-terminated (N-N), mixed-terminus (N-H), and diacid-terminated (H-H) polymers. The samples were partially soluble in trifluoroethanol, concentrated sulfuric acid, and hot m-cresol; the best known solvent is hexafluoro-2-propanol. All of these solvents are incompatible with common MALDI matrixes. Only one study has appeared in the literature8 where the end groups of a polyamide, nylon-6, were analyzed by MALDI MS. 2-((4-Hydroxyphenyl)azo)benzoic acid was used as the matrix, and it was possible to dissolve the sample in trifluoroethanol. It is important to stress that these polyamides were real industrial products, not polymer standards that are often used in (8) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. J. Polym. Sci., Part A 1996, 34, 439-447.
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MALDI research. They had previously been characterized by other methods. Titration of the carboxyl and amine groups9 had been used for some of them to obtain a quantitative end group analysis. If no solubility problems occur, the accuracy of the end group distribution can reach about 5% with the titration method. A rough estimation of the average molecular weight can be obtained from these data, by using the relationship 2/Mn ) n(COOH) + n(NH2), where n(COOH) is the concentration of the acid end groups in mol/g, n(NH2) is the concentration of the amine end groups in mol/g, and Mn is the number-average molecular weight. Molecular weight distributions were also available from gel permeation chromatography (GPC) data. For the higher polyamide oligomers, GPC was only possible after chemical derivatization,10 which leaves some doubt as to the accuracy of the GPC data. Proton NMR spectroscopy has also been proposed for molecular weight and quantitative end group determination,11 by making use of the slight difference in chemical shift of terminal hexanamine and acid groups compared to those inside an extended chain. However, this method does not yield very accurate data. Solubility problems can hamper analysis both by GPC and by NMR spectroscopy. MALDI mass spectrometry has been shown to yield very useful information for polymer analysis,5,12-17 provided that a good sample can be prepared. We investigated experimental protocols for the detection and quantitative analysis of polyamide oligomers by MALDI MS. Efforts to circumvent problems associated with the poor solubility of these samples included work with liquid and slurry samples and ultimately led to the discovery of a new solid/ solid sample preparation for polymers that are insoluble in MALDIcompatible solvents. To judge the usefulness of a given analytical protocol, we observed how sensitive it was to changes in composition (e.g., matrix-to-analyte ratio) and experimental parameters (e.g., desorption laser power). EXPERIMENTAL SECTION Materials. All polyamide samples were donated by EMS CHEMIE (Domat-Ems, Switzerland). Solid MALDI matrixes included 4-hydroxy-R-cyanocinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 3-aminoquinoline (all from Fluka), 3,5dimethoxy-4-hydroxycinnamic acid, and dithranol (from Aldrich). They were used without further purification. Instrumentation. MALDI time-of-flight (TOF) experiments were performed on a home-built 2 m linear TOF mass spectrometer.18 The total acceleration potential was +25 kV. Delayed extraction was generally used, with delay times of 150-700 ns, (9) Roerdink, E.; Warnier, J. M. M. Polymer 1985, 26, 1582. (10) Jacobi, E.; Schuttenberg, H.; Schulz, R. C. Macromol. Chem. Rapid Commun. 1980, 1, 397-402. (11) Shit, S. C.; Maiti, S. Eur. Polym. J. 1986, 22, 1001-1008. (12) Bahr, U.; Deppe, A.; Karas, M.; Hillenkamp, F.; Giessmann, U. Anal. Chem. 1992, 64, 2866-2869. (13) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Macromolecules 1995, 28, 4562-4569. (14) Montaudo, G.; Scamporrino, E.; Vitalini, D.; Mineo, P. Rapid Commun. Mass Spectrom. 1996, 10, 1551-1559. (15) Chaudhary, A. K.; Critchley, G.; Diaf, A.; Beckmann, E. J.; Russell, A. J. Macromolecules 1996, 29, 2213-2221. (16) Belu, A. M.; De Simone, J. M.; Linton, R. W.; Lange, G. W.; Friedman, R. M. J. Am. Soc. Mass Spectrom. 1996, 7, 11-24. (17) Ra¨der, H. J.; Schrepp, W. Acta Polym. 1998, 49, 272-293. (18) Dubois, F.; Knochenmuss, R.; Steenvoorden, R. J. J. M.; Breuker, K.; Zenobi, R. Eur. Mass Spectrom. 1996, 2/3, 167-172.
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depending on the mass range investigated. Desorption was performed using a nitrogen laser (model VSL-337ND-T, Laser Science Inc.), incident at ca. 60° to the surface normal. The energy was generally slightly above threshold, in the range 10-20 µJ, as measured with a pyroelectric detector (model ED 100, Gentec, Quebec, Canada). The irradiance at the sample surface was estimated to be in the 106-107 W/cm2 range. Spectra were obtained in the positive mode, by averaging 50-100 single shots. Preparations. (a) Crystalline MALDI Samples. Initially, samples were prepared with crystalline MALDI matrixes. A layer of matrix solution was applied to the sample target, dried in air or under vacuum, and covered by a second layer of sample solution. The layered sample preparation technique was necessary because the matrix and the sample solutions were generally immiscible. Often, the addition of the sample solution onto the dried matrix was observed to disrupt the matrix microcrystals. (b) Solid/Solid Pressed Samples. The new solid/solid sample preparation is analogous to making a crystalline KBr sample for infrared analysis. The polymer samples were ground with a mortar and pestle to fine powders. Solid samples were then mixed with one of the common MALDI matrixes, for example, DHB, 3-aminoquinoline (Fluka), or dithranol (Aldrich), and the mixtures were ground together in the mortar. Other materials such as KBr, cobalt powder, and graphite were also tried, but without success. A variety of matrix-to-polymer mixing ratios were used, ranging from 1:10 to 10:1 (by weight). Empirically it was observed that mixing ratios between 1:1 and 1:5 provided optimal conditions for desorption/ionization of both the low molecular weight samples and the higher molecular weight polyamide oligomers. These are unusually low mixing ratios compared to those of standard MALDI. Increasing the matrix content only served to increase the intensity of the matrix peaks, which obscured the peaks of the low-mass oligomers present in both samples. Finally, a few milligrams of the finely ground mixture was pressed with 1.3 × 105 N/cm2 (10 tons) for approximately 2 min using a hydraulic press to yield a flat, thin (
