AC Research
Accelerated Articles Anal. Chem. 1996, 68, 2721-2725
Detection of High Molecular Weight Narrow Polydisperse Polymers up to 1.5 Million Daltons by MALDI Mass Spectrometry David C. Schriemer and Liang Li*
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
The detection of very high molecular weight narrow polydisperse poly(styrene) samples by MALDI time-offlight mass spectrometry is reported. It is shown that accurate molecular weight determinations of samples up to 1 million can be achieved very rapidly from the singly charged polymeric species. For a poly(styrene) with a molecular weight of approximately 1.5 million, signals corresponding to the multiply charged ions of the principal distribution are observed. The molecular weights obtained by MALDI are in good agreement with classical molecular weight determination techniques. all-transRetinoic acid was used as the organic matrix for the laser desorption procedure, and the samples were analyzed as their silver cation adducts. This work demonstrates that, with proper matrix selection and sample preparation, MALDI can be a very useful tool for high molecular weight polymer sample analysis.
While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been widely used in biopolymer applications, this technique has also been rapidly developed as an analytical tool for the analysis of industrial synthetic polymers.1,2 Compared with the traditional methods of polymer analysis, MALDI MS has the potential to offer several advantages. It can provide accurate, fast, and direct molecular weight information. For relatively low molecular weight polymers where the instrumental resolution is sufficient to separate oligomer peaks, accurate mass analysis of the individual oligomers can be achieved to provide information on repeat unit, end group, and other modifica(1) Bahr, U.; Deppe, A.; Karas, M.; Hillenkamp, F.; Giessman, U. Anal. Chem. 1992, 64, 2866. (2) Danis, P. O.; Karr, D. E. Org. Mass Spectrom. 1993, 28, 923. S0003-2700(96)00442-8 CCC: $12.00
© 1996 American Chemical Society
tions of a polymer.3,4 A number of studies have been reported in the areas of oligomer characterization and new matrix and polymer/matrix preparation protocols, as well as comparative studies between MALDI and the more traditional molecular weight determination techniques.5-9 However, only a few cases of the detection of high molecular weight polymers over 100 000 by MALDI have been reported. Danis et al. reported the detection of water-soluble poly(styrenesulfonic acid) with a molecular weight just under 400 000.10 In addition, they also indicated the detection of nonpolar poly(styrene) with a molecular weight of approximately 120 000,11 and the detection of a poly(methyl methacrylate) sample with a molecular weight of approximately 256 000.2 Multiply charged ions from a starburst polyamidoamine dendrimer with a molecular weight as high as 1.2 million has been reported by Savickas.12 Electrospray ionization and detection of a similar dendrimer (approximately 1 MDa) has also been achieved.13 Electrospray ionization has proved capable of generating mass (3) Whittal, R. M.; Li, L.; Lee, S.; Winnik, M. A. Macromol. Rapid Commun. 1996, 17, 59. (4) Lee, S.; Winnik, M. A.; Whittal, R. M.; Li, L. Macromolecules 1996, 29, 3060. (5) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Rapid Commun. Mass Spectrom. 1994, 8, 1011. (6) Montaudo, G.; Montaudo, M. S.; Puglisi, C.; Samperi, F. Rapid Commun. Mass Spectrom. 1995, 9, 453. (7) Lloyd, P. M.; Suddaby, K. G.; Varney, J. E.; Scrivener, E.; Derrick, P. J.; Haddleton, D. M. Eur. Mass Spectrom. 1995, 1, 293. (8) Belu, A. M.; DeSimone, J. M.; Linton, R. W.; Lange, G. W.; Friedman, R. M. J. Am. Soc. Mass Spectrom. 1996, 7, 11. (9) Jackson, C.; Larsen, B.; McEwen, C. Anal. Chem. 1996, 68, 1303. (10) Danis, P. O.; Karr, D. E.; Holle, A.; Mayer-Posner, F.; Watson, C. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, CA; 1993; p 1093. (11) Xiong, Y.; Owens, K. G.; Danis, P. O.; Karr, D. E. Proceedings of the 42nd ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL; 1994; p 980. (12) Savickas, P. Poster presentation at the Sanibel Conference on Mass Spectrometry, Chicago, IL, 1994. (13) Schwartz, B. L.; Rockwood, A. L.; Smith, R.; Tomalia, D. A.; Spindler, R. Rapid Commun. Mass Spectrom. 1995, 9, 1552.
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spectra of poly(ethylene glycol)s with molecular weights up to 7 MDa; the lack of resolution between charged states prohibits its usefulness however.14,15 The difficulty in analyzing even higher mass polymers by MALDI could be attributed to instrumental limitations, fundamental limitations of the MALDI process, or both. With currently available detector designs (e.g., microchannel plate detectors, electron multipliers, or a combination of these two types), there can be a reduced instrumental detection sensitivity for high-mass ions.16 Furthermore, the MALDI process itself may have an upper mass limit conditional upon the nature of the analyte. This limit could be affected by the chemistry/photochemistry of the analyte, the availability of suitable matrices and ionization reagents, and suitable solvents. For example, a matrix that performs very well for a certain class of polymers might not be applicable to other types of polymers due to unfavorable chemical or photochemically induced reactions between the matrix and the analyte (during preparation or ionization). It is also possible that, for a particular analyte, no matrix is known with which it can cocrystallize. This could be due to a mismatch between the chemical nature of the analyte and the proposed matrix or simply that a common solvent does not exist. In addition to all of this, an ionization reagent must be introduced that can effectively ionize the analyte but not disturb favorable crystallization behavior. It is the combination of these factors that determines the overall efficiency of a preparation2 and, hence, the upper mass limit. It is felt by the authors that the nature of the MALDI sample preparation is the principal impediment to the analysis of high molecular weight polymers, rather than instrumental limitations. In this work, the detection of singly charged ions for narrow polydisperse linear poly(styrene) samples ranging to 1 million Da by MALDI is reported. For even higher molecular weight narrow polydisperse linear poly(styrene) (approximately 1.5 million), signals corresponding to multiply charged ions of the intact poly(styrene) can be observed. A novel mass calibration scheme for this high-mass range is also presented. Finally, the results obtained by MALDI are compared with those obtained by traditional molecular weight determination methods such as gel permeation chromatography (GPC), intrinsic viscosity (IV), membrane osmometry (MO), and laser light scattering (LLS). EXPERIMENTAL SECTION Instrumentation and Data Processing. Mass spectral data were collected on a Model G2025A MALDI time-of-flight system (Hewlett-Packard, Reno, NV), equipped with a pulsed nitrogen laser (337 nm radiation, 3 ns pulse width). This instrument was operated in positive mode at high voltage (28 kV). In general, 100 laser shots (2.5-6 µJ pulse energy) were averaged to produce a mass spectrum for samples demonstrating high detection sensitivity. With extremely high molecular weight samples that tend to have a lower detection sensitivity, the maximum number of shots averaged for a given sample was in the range of 200300 shots. Spectra were acquired and processed with HewlettPackard supporting software and reprocessed with the Igor Pro software package (WaveMetrics, Inc., Lake Oswego, OR). Macros (14) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37. (15) Fuerstenau, S. D.; Benner, W. H. Rapid Commun. Mass Spectrom. 1995, 9, 1528. (16) McCloskey, J. A., Ed. Methods in enzymology, Vol. 193; Academic Press: San Diego, 1990; Vol. 193, p 61.
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were written for this software platform that allow for the calculation of Mn and Mw. The equations used for molecular weight determination are
Mn ) Mw )
∑(N M )/∑M i
i
i
∑(N M )/∑(N M ) 2
i
i
i
i
where Ni and Mi represent signal intensity and mass at point i. Numerical integration of the polymer signal was performed in the mass domain, after the correction of the peak intensities required when converting from a time domain to a mass domain. This correction factor takes the form of 1/(dm/dt) and will be discussed in a future publication. Samples and Reagents. The polymers employed in this study were poly(styrene) standards purchased from Showa Denko KK (Tokyo, Japan), Polymer Laboratories (Amherst, MA), and Aldrich (Milwaukee, WI), ranging in molecular weight from approximately 90 000 to 1.5 million. The molecular weights of these samples were provided by the suppliers and are summarized in Table 1 (see Results and Discussion). all-trans-Retinoic acid was purchased from Aldrich, and silver nitrate from Terochem Laboratories Ltd. (Edmonton, Alberta, Canada). The proteins (carbonic anhydrase, bovine serum albumin, lactase) used in the calibration procedure were purchased from Sigma (Milwaukee, WI). Analytical-grade tetrahydrofuran (THF) was purchased from VWR (Toronto, Canada), treated with potassium hydroxide and then distilled in the presence of sodium metal. Sample Preparation. Polymer samples for MALDI analysis were prepared by combining the analyte, matrix, and cationizing agent in a common solvent, THF. The polymer samples were dissolved in dry THF to prepare stock solutions with concentrations ranging between 5 and 50 mg/mL. all-trans-Retinoic acid was used as the matrix, which was prepared to a 0.15 M concentration in THF. An aliquot of the analyte solution was added to the matrix, followed by the addition of a small amount of a saturated ethanolic solution of AgNO3 (1 µL to 100 µL of matrix/analyte solution). The concentration of analyte in the final solution ranged from 0.5 nM (higher molecular weight samples) to 50 µM (lower molecular weight samples). The concentration of matrix in the final solution remained virtually unchanged at 0.15 M. After brief mixing, 0.3-0.5 µL of the mixture was added to the MALDI probe tip and allowed to air-dry. It should be noted that very thin layer depositions on the probe tip favor the analysis of the very high mass polymers. For the initial mass calibration, a solution of the proteins (carbonic anhydrase, bovine serum albumin, lactase) was analyzed by MALDI using sinapinic acid (SA) as the matrix. An aliquot of a solution of SA in acetone (20 mg/mL) was first applied to the probe tip and air-dried, followed by an aliquot of the protein sample dissolved in a saturated SA solution (33% acetonitrile in water). This was allowed to air-dry as well. RESULTS AND DISCUSSION Figure 1 shows the mass spectra of three different poly(styrene)s with nominal masses of 330 000 (PS 330, Figure 1A), 600 000 (PS 600, Figure 1B), and 900 000 (PS 900, Figure 1C). At this high mass, the adjacent oligomer peaks with a mass difference of 104 Da are unresolved, and consequently, the entire oligomer distribution appears as a broad peak. The salient features of these
Figure 1. Mass spectra of three poly(styrene) samples with nominal molecular weights of (A) 330 000, (B) 600 000, and (C) 900 000.
spectra are the appearance of the singly charged molecular ion peaks as well as peaks from multiply charged ions. For Figure 1B, the peak at m/z ∼400 000 is from the triply charged dimer of PS 600. Likewise, the peak at m/z ∼620 000 in Figure 1C is from the triply charged dimer of PS 900. As the mass increases, it becomes clear that the multiply charged distributions begin to dominate the spectra. The relative decrease in the intensity of the principal distribution is likely due in part to a steady drop in detector efficiency. Nevertheless, it is remarkable that ions with a mass-to-charge ratio of 916 000 (Mn, Figure 1C) can be easily detected with a microchannel plate (MCP) detector. The HP Model G2025A system is equipped with a proprietary MCP detector system, which has a demonstrated high efficiency at high mass.17 It appears that the only other study demonstrating the ability of MALDI for the direct analysis of species approaching 1 million Da is the report of the detection of human IgM.18 The ability to detect such massive polymeric species is due in part to careful sample preparation. In the analysis of poly(styrene), all-trans-retinoic acid was used as the matrix. This matrix was previously reported by Wilkins and his co-workers for the analysis of dendrimers;19 however it was claimed that the cis form was more efficient for their application. Nevertheless, the all-trans (17) Chakel, L. Am. Lab. 1994, (Sept), 32C. (18) Nelson, R. W.; Dogruel, D.; Williams, P. Rapid Commun. Mass Spectrom. 1995, 9, 625. (19) Walker, K. L.; Kahr, M. S.; Wilkins, C. L.; Xu, Z.; Moore, J. S. J. Am. Soc. Mass Spectrom. 1994, 5, 731.
form appears to be extremely efficient in the analysis of both polar and nonpolar polymers, subject to the availability of suitable solvents. When this matrix is used, it is very important that the solvent is free of impurities such as excess water, peroxides and/ or acids, and antioxidants. For example, using analytical-grade THF without further purification results in complete suppression of even low molecular weight poly(styrene) signals, although it is unclear which contaminant(s) cause this suppression. In the current application, the THF was purified prior to use, as described in the Experimental Section, to remove the above-mentioned impurities. When prepared in this fashion, the matrix solution can be stored for over 1 week without loss in matrix efficiency. A small aliquot of a saturated ethanolic solution of AgNO3 was added to the matrix/analyte mixture immediately prior to analysis and proved to be an easy and effective means of introducing the cationization agent. It is felt that this procedure is more practical than preparing a silver salt of the matrix, as has been done with other matrices.11 A carboxylate salt of this matrix would be very difficult to prepare in the pure form, since such preparations would always contain Ag+ ions complexed to the polyalkenyl chain of the matrix.20 As the matrix slowly oxidizes in the presence of Ag+, it is preferable to introduce the cationizing agent immediately prior to analysis. In the preparation of samples for MALDI, a general rule of thumb is to use a matrix to analyte molar ratio ranging between 500:1 and 10 000:1. In the MALDI analysis of high molecular weight poly(styrene), this does not hold. When the experimenter experiences poor detection sensitivity, it is intuitively sensible that an increase in the amount of analyte would increase the signal strength. However, just the opposite is the case for the analysis of high molecular weight poly(styrene). For example, the analysis of PS 900 was achieved from 10 fmol of total polymer loaded on the probe tip, or a matrix to analyte ratio of ∼8 million:1. With these high molecular weight species, use of the molar matrix to analyte ratios in the range between 500:1 and 10 000:1 yielded no ion signals at all. It has been observed that with increasing molecular weight of the analyte, progressively lower molar amounts must be added to the probe tip in order to achieve highest sensitivity. Optimum ratios for this broad molecular weight range vary between 3000:1 (lower molecular weight) and 10 million:1 (higher molecular weight). This optimum ratio is defined as the composition at which a further increase in the amount of polymer added does not lead to an increase in signal strength (all other parameters being held the same). The 3300fold molar increase in the amount of matrix required in going from a 86 kDa to 1.391 MDa poly(styrene) is more than is indicated by a simple proportionality with polymer molecular weight. The reason underlying this observation is unknown; however, it seems likely that the amount of matrix present should be sufficiently high so as to preclude polymer entanglement and/ or the formation of regions of microcrystallinity. Furthermore, such massive species probably require more energy for a phase change in desorption, which would require a greater number of matrix molecules per analyte molecule being involved in the process. Because it is necessary to decrease the molar amount of polymer loading as the molecular weight increases, this would suggest a practical limit to the mass range for polymer analysis (20) Cotton, F. A.; Wilkinson, G. Advanced Inorganic Chemistry, 5th ed.; John Wiley and Sons: New York, 1988; p 945.
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Table 1. Results of Molecular Weight Measurements for Poly(styrene)s molecular weight polymer standarda PS 90 (A)
Mv ) 96 000 (IV)
PS 156 (SD)
Mn ) 154 000 (MO) Mv ) 159 000 (IV) Mw ) 162 000 (LLS) Mn ) 152 000 (GPC) Mw ) 156 000 (GPC) Mp ) 150 000 (GPC) Mw ) 216 000 (LLS) Mv ) 184 000 (IV) Mw ) 208 000 (GPC) Mn ) 198 000 (GPC) Mp ) 198 000 (GPC) Mn ) 302 000 (MO) Mv ) 306 000 (IV) Mw ) 322 000 (LLS) Mn ) 316 000 (GPC) Mw ) 327 000 (GPC) Mp ) 330 000 (GPC) Mw ) 382 000 (LLS) Mn ) 335 000 (MO) Mw ) 393 000 (GPC) Mn ) 340 000 (GPC) Mp ) 357 000 (GPC) Mn ) 502 000 (MO) Mv ) 498 000 (IV) Mw ) 505 000 (LLS) Mn ) 481 000 (GPC) Mw ) 505 000 (GPC) Mp ) 514 000 (GPC) Mw ) 670 000 (LLS) Mn ) 663 000 (LLS) Mw ) 649 000 (GPC) Mn ) 618 000 (GPC) Mp ) 649 000 (GPC) Mv ) 735 000 (IV) Mn ) 765 000 (LLS) Mn ) 728 000 (GPC) Mw ) 756 000 (GPC) Mp ) 771 000 (GPC) Mw ) 929 000 (LLS) Mw ) 942 000 (MO) Mn ) 892 000 (MO) Mp ) 949 000 (MO) Mv ) 932 000 (IV) Mv ) 1 507 000 (IV) Mw ) 1 420 000 (LLS) Mn ) 1 378 000 (GPC) Mw ) 1 460 000 (GPC) Mp ) 1 459 000 (GPC)
PS 200 (A)
Figure 2. Mass spectrum of poly(styrene) with a nominal molecular weight of 1.5 million.
PS 330 (PL)
PS 400 (A)
PS 514 (SD)
PS 600 (A)
PS 770 (PL)
Figure 3. Mass spectrum of poly(styrene) with a nominal molecular weight of 90 000.
by MALDI. As molecular weight increases, the sensitivity of the instrument is challenged on two fronts: decreased sensitivity due to loss in detector efficiency and decreased sensitivity from the requirement of lower (molar) sample loading. In the analysis of poly(styrene) with the current procedure, this limit appears to occur at ∼1.5 million Da. Figure 2 shows the mass spectrum of poly(styrene) with a nominal mass of 1.5 million Da. This spectrum was obtained from 5 fmol of total polymer loaded on the probe tip. Even a slight increase of the amount loaded to 15 fmol resulted in a total, reproducible signal suppression, and loading less than 5 fmol proved to be beyond the sensitivity of the instrument. The spectrum does not display any signal from the principal distribution, suggesting an insufficient detector sensitivity at this mass/charge ratio. Figure 2 shows a peak at m/z 700 000 corresponding to the doubly charged ion distribution and a peak at m/z 476 000 corresponding to the triply charged ion distribution. A challenge in determining the molecular weight of any species in the high-mass range is mass calibration. In the mass range of 100 000 Da and above, protein standards are rare, and ambiguity can exist as to their exact mass. The goal of obtaining extremely high mass accuracy can be relaxed in the present application (and indeed in all MALDI polymer analyses where oligomeric resolution is lost). A 1% relative mass accuracy would be sufficient in 2724 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
by classical methodsb,c
PS 900 (A)
PS 1460 (PL)
by MALDI Mn ) 86 800 Mw ) 87 000 Mn ) 153 700 Mw ) 153 900
Mn ) 202 500 Mw ) 202 800
Mn ) 308 900 Mw ) 309 100
Mn ) 402 400 Mw ) 402 900
Mn ) 469 800 Mw ) 470 500
Mn ) 589 100 Mw ) 589 400
Mn ) 738 400 Mw ) 741 100
Mn ) 916 200 Mw ) 917 000
Mn ) 1 391 300 Mw ) 1 393 400
a A, Aldrich Chemical Co.; SD, Showa Denko K. K.; PL, Polymer Laboratories. b These results are provided by the suppliers. c IV, intrinsic viscosity; LLS, laser light scattering; GPC, gel permeation chromatography; MO, membrane osmometry.
many cases. This allows for a novel calibration procedure, where the high-mass spectra are calibrated with a lower molecular weight poly(styrene) sample that demonstrates significant multimer formation. Figure 3 shows one of these relatively low mass narrow polydisperse poly(styrene) samples (PS 90), which exhibits extensive multimer formation. Through careful calibration of even lower molecular weight narrow polydisperse poly(styrene)s with protein standards, it was observed that the multimers are integer multiples of the principal distribution. The peaks in these spectra can be fit with Gaussian functions extremely well, justifying the selection of the Mp (most probable molecular weight) values of the peaks as calibration points. The Mp value of the principal
distribution, being in this case ∼86 000 Da, could be determined through a normal method of calibration by protein standards (using carbonic anhydrase, bovine serum albumin and lactase). The Mp’s of the multimer peaks were then simply integer multiples of the Mp of the principal distribution, corrected for the mass of the silver cation. This allows calibration of the instrument up to m/z ∼600 000. The above-described indirect calibration procedure does have an inherently higher degree of error associated with it. Based on this procedure, replicate experiments show that an average relative standard deviation (RSD) of 0.23% for mass determination can be expected. A more direct mass calibration procedure would be ideal, but at present, accurate mass calibrants are not available. A possible critique of this calibration procedure involves the issue of mass discrimination by the ion detection system. However, for narrowly distributed poly(styrene)s, the peaks span a limited mass range and retain their expected Gaussian symmetry. For such samples, the mass discrimination factor can be ignored. The examination of the mass discrimination over a broad mass range will be reported in a future publication. All the high molecular weight poly(styrene)s examined display multiply charged ion distributions. The presence of multiply charged distributions can be analytically useful, as inferred from the mass spectra of lower molecular weight poly(styrene)s. It is found that doubly charged distributions appear to be consistently overestimated by 0.9%, and triply charged distributions by 2.2%. Application of these correction factors to the 2+ (m/z 700 000) and 3+ (m/z 476 000) distributions in the spectrum of Figure 2 generates values for the principal distribution of 1 386 000 and 1 396 000 Da, respectively. A reasonably accurate estimate of the number-average molecular weight would then be 1 391 000 Da. Replicate determinations and factoring in the error from the calibration procedure would suggest an average RSD of 0.43% for this molecular weight determination. It should be noted that the accuracy of this MALDI technique and the precision of the m/z determinations for the two peaks in the spectrum are sufficient to identify them as corresponding to the 2+ and 3+ charge states. Also note that this correction procedure was applied to the determination of the principal distributions of PS 600, PS 770, and
PS 900, rather than reporting the value of these distributions directly. This procedure was followed in these cases since the principal distributions were outside of the calibrated mass range. Table 1 summarizes the data collected for a range of high molecular weight poly(styrene) standards. The molecular weights as determined by the classical methods were provided by the suppliers. It is readily evident that MALDI is capable of generating data for these poly(styrene) standards that follow the trend of the classical techniques of GPC, LLS, IV, and MO. While not explicitly shown in the table, it is to be observed that the polydispersity (Mw/Mn) as determined by MALDI is consistently lower than that reported by the other techniques. This has been observed by others6,8 for low molecular weight poly(styrene) samples and warrants further investigation. In conclusion, it would seem possible to determine the molecular weight of narrow polydisperse polymers by MALDI, with molecular weights approaching 1.5 million, by careful formulation of the matrix/analyte preparation and by using a sensitive MALDI detection system. The rapid analytical method described in this work produces results that are consistent with those obtained using traditional methods. Future work in this area will be focused on extending the capability of this MALDI technique to other types of polymers with extremely high molecular weights. ACKNOWLEDGMENT This work was supported by the Natural Sciences and Engineering Research Council of Canada through its Industrially Oriented Research Grant Program and by the Polymer Structure and Property Research Program of the Environmental Science and Technology Alliance Canada. D.C.S thanks the Killam Trust for a predoctoral scholarship.
Received for review May 6, 1996. Accepted June 12, 1996.X AC960442M X
Abstract published in Advance ACS Abstracts, July 1, 1996.
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