Technical Notes Anal. Chem. 1994, 66,4366-4369
Determination of Absolute Mass Values in MALDl-TOF of Polymeric Materials by a Method of Self-Calibration of the Spectra Giorgio Montaudo*and Maurizio S. Montaudo Dipartimento di Scienze Chimiche, Universita di Catania, Viale A. Doria, 6-95125Catania, Italy
Concetto Puglisi and Filippo Samperi lsfituto per la Chimica e la Tecnologia dei Materiali Polimerici, Consiglio Nazionale delle Ricerche, Viale A. Doria, 6-95125Catania, Italy
A novel calibration procedure is introduced that allows one to obtain absolute mass values in MALDI-TOFspectra of polymeric materials. The masses of the oligomers are written as the sum of two masses: pn = an 8, where 8 is the mass of the fist oligomer and an is a multiple of the polymer repeat unit and represents the distance in daltons from the first oligomer. The new calibration procedure considers 8 as a variable parameter and performs a best-fit minimization. Two examples of a p plication of the new procedure are given. Two synthetic polymers, poly(capro1actone) and poly(Bispheno1A carbonate), were analyzed by MALDI-TOF, and complex spectra showing one or more mass series up to 20 kDa were obtained. Application of this procedure allowed the interpretation of each single peak in the mass spectra and established that none of the peaks in the spectrum corresponds to fragments. It was found that the poly(caprolactone) sample consists of a series of oligomers with H and OH as terminal groups, which form sodiumcationized ions in the MALDI process. The composition of the poly(Bispheno1 A carbonate) sample was more complex, since it contains a series of cyclic oligomers desorbing as sodium-cationizedions, plus two series of oligomers bearing Wphenol and phenyVpheno1 as terminal groups, respectively, which form ions by capturing a proton in the MALDI process.
+
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a recently introd~cedl-~ soft ionization technique that allows desorption and ionization of very (1) b a s , M.; Hillenkamp, F. Anal. Chem. 1988,60,2299. (2)Hillenkamp, F.; Karas, M.; Beavis R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A (3) Bahr, U.; Deppe, A; Karas, M.; Hillenkamp, F.; Giessman, U. Anal. Chem. 1992,64, 2866. (4) Cotter, R A Anal. Chem., 1992,64, 1027A (5) Bruker, User Software Manual, Reflex MALDI-TOF. (6) Danis, P. 0.; Karr, D.E.; Mayer, F.; Holle, A; Watson, C. H. O q . Mass Spectrom. 1992,27, 843. Danis, P. 0.; Karr,D.E. Org. Mass Spectrom. 1993,28, 923.
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large molecules even if in complex mixtures, such as the wide molecular weight distributions present in synthetic polymer~~.~,7 and also in some natural macrom~lecules.~~~ Since TOF measures only differences of masses, the determination of absolute masses appears a d a c u l t task, and calibration procedures using external and internal standards of known mass are currently used.4~~ However, the use of external standards may lead to ambigous results in the case of polydisperse polymeric materials constituted of one or more series of oligomers extending over a large mass range. Furthermore, internal standards are difficult to find since each synthetic polymer class would need structurally identical species, possibly monodisperse, with mass values such to avoid isochrony with the mass series displayed by the polymer sample. The task of obtaining absolute mass values in MALDI-TOF spectra of synthetic polymeric materials is of outmost importance, since it would make possible the direct determination of the species contained in each polymer sample and also of d ~ e r e nend t groups present in the oligomer series. End group identification and determination of the actual composition of a polymer sample are so crucial in polymer analysis that their importance cannot be overemphasized. At the present time, calibration problems in MALDI-TOF analysis of polymers are such that often workers are able to obtain good spectra of polymeric samples but are unable to interpret them because of the difficulties in making the correct mass assignments and in discerning whether the polymer molecules desorb intact or not. In the present paper, we report an attempt to solve this problem by introducing a new self-calibration method for the MALDI-TOF spectra of polymers. To this purpose, two samples of polydisperse (7) Danis, P. 0.; Karr, D.E.; Westmoreland, D.; Piton, M. C.; Christie, D. I.; Clay, P. A; Kable, S. H.; Gilbert, R G. Macromolecules 1993,26, 6684.
(8) Burger, H.M.; Muller, H.M.; Seebach, D.; Bomsen, K. 0.; Schar, M.; Widmer, H. M. Macromolecules 1993,26. 4783. (9) Abate, R; Ballistreri, A; Montaudo, G.; Garozzo, D.; Impallomeni, G.; Critchley, G.; Tanaka, K Rapid Commun. Mass Spectrom. 1993,7, 1033. Garozzo, D.; Spina, E.; Shuiale, L.; Montaudo, G.; Rizzo, R Rapid Commun. Mass Spectrom. 1994,8,358.
0003-2700/94/0366-4366$04.50/0 0 1994 American Chemical Society
1000
1070
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,
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5010
6000
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Figure 1. MALDI-TOF mass spectrum of poly(caprolactone).
synthetic polymers, poly(capro1actone) and poly(Bispheno1 A carbonate), were analyzed by MALDI-TOF, and spectra up to 20 kDa were obtained. To our best knowledge, MALDI-TOF spectra of the two polymers are unreported. Application of the new calibration procedure allowed us to establish that the poly(caprolactone) sample consists of oligomers having H and OH as terminal groups, which form sodium-cationized ions in the MALDI process. The composition of the poly(Bispheno1 A carbonate) sample was more complex, since it contains a series of cyclic oligomers desorbing as sodium-cationized ions, plus two series of oligomers bearing H/phenol and phenyl/phenol as terminal groups, respectively. EXPERIMENTAL SECTION Materials. 2-(CHydroxyphenylazo)benzoic acid (HABA) , poly(caprolactone), and poly(Bispheno1 A carbonate) were purchased from Aldrich and used without further puritication. Sample Preparation. Probe tips were loaded with 100 pmol of polymer sample and 300 nmol of matrix. MALDI-TOF Mass Spectra. A Bruker Reflex mass spectrometer was used to obtain the matrix-assisted laser desorption/ ionization time-of-flight mass spectra. The spectrometer was equipped with a nitrogen laser (337 nm, 5 ns), a double-plate microchannel detector, and a flash ADC (time base 10 ns). Accelerating voltage was 30 kV. The laser irradiance was not measured. However, it was slightly above threshold (-lo6- lo7 W/cm2>. The detection was in the reflected mode. Ions below m/z 350 were removed with pulsed deflection, and 100 transients were summed. The resolution was -300 at 5000 Da. FAB Mass Spectra. A double-focusing Kratos MS 50s equipped with the standard FAB source and a DS90 data system was used. The FAB gun was operated with a 7 keV Xenon beam. The instrument was scanned from m/z 3000 to 100 with a scan rate of 10 s/decade. The accelerating voltage was 8 kV. FAB
MS spectra were obtained in positive and negative modes using 3-nitrobenzyl alchol as matrix. Cesium iodide was used to calibrate the instrument. RESULTS AND DISCUSSION The correspondence between the time needed for an ion of mass p to fly through the tube and the mass of the ion itself is a quadratic relation~hip,48~ as shown in eq 1, where a and 4, are p, = a(t
+ IO-~/I)'
(1)
two calibration constants. The knowledge of at least two absolute mass values is therefore necessary to determine the two constants. In order to yield correct results, this procedure requires that the absolute mass values used are exact. Unfortunately, even in the most favorable case (i.e., using an internal standard of known mass), the mass value is uncertain because of the effect of the desorption ionization in MALDI. In fact, a particular species can be desorbed as a cation associated with H+, Lit,Na+, Kt,or Cs+, or it can form a matrix adduct. The ambiguity in the mass value used reflects itself on the calibration constants, yielding erroneous mass assignments and complicating the interpretation of the TOF spectra. To illustrate the pitfalls of the conventional calibration procedure, some calibration attempts using eq 1will be illustrated below. Figure 1 reports the MALDI-TOF mass spectrum of poly(caprolactone). The spectrum is constituted of a single mass series (see inset), with no evidence for ion fragmentation. TINS, the peaks correspond to caprolactone oligomers, but their exact structure is unknown. The spectrum was initially obtained as a temporal spectrum, which reports the number of ions arriving at each time. The temporal spectrum displays a first peak at 0.033 58 ms, followed by a second one at 0.035 52 ms, a third one at 0.037 39 ms, and so on. The total number of peaks is 97; the last peak is at 0.109 11 ms. Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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In a first calibration attempt, it was assumed that the peak at 0.033 58 ms corresponds to the oligomer H[O(CH2)&0190HLi+ (Le., to a poly(capro1actone) nonamer bearing H and OH as end groups and cationized with lithium), making in fact a largely wrong assumption, since the peak actually corresponds to an octamer (see below). Similarly, it was assumed that the peak at 0.035 52 ms corresponds to the compound HIO (CH2)sCOlloOHLi+, that the peak at 0.037 39 ms corresponds to the compound H[O(CH2)5CO111OHLit, and so on, up to the compound H[O(CH2)5COIlosOHLi+. These assumptions were used to calibrate the MALDI-TOF spectrum according to eq 1,as follows: (i) A set of data was built containing the square roots of the mass numbers and the corresponding times of flight. The vector p,, contained the following mass numbers: 1051.9, 1166.0, 1280.1, ...., 12 005.8. It was assumed that the oligomer mass numbers are equally spaced and they were computed using the formula p,, = n114.1f 25; (ii) The above set of data was fitted with a straight line by a linear regression procedure. The calibration contants turned out to be a = 825899 and ,B = 1691.7 with a good coefficient of regression 12 = 0.999 985. It is apparent that, even in the case of large errors in the assignment, the conventional calibration procedure may be misleading. In a series of successive attempts, other more reasonable assignments were made. Each calibration attempt yielded different sets of calibration constants, each with a good regression value, leading to ambiguous mass values for the 97 caprolactone oligomers detected in the MALDI spectrum in Figure 1. In order to overcome these difkulties, and to obtain the absolute mass values of the oligomers, a novel calibration procedure was adopted, which takes advantage of a unique property of polymers. Considering that the mass of a homopolymer results from the sum of identical units, eq 1can be rewritten imposing the condition that the total mass of each oligomer results from the sum of the two masses:
where 0 is the mass of the first oligomer and on is a multiple of the polymer repeat unit and represents the distance in daltons from the first oligomer. Using the above formula, eq 1becomes
This equation is more useful than eq 1. In fact, the set of parameters has been enlarged to include 8. If one fures the value of 8, one can minimize over a and /?and calibrate the spectrum. However, when 8 is held constant, eq 3 is of very limited utility. In fact, it reproduces exactly the results obtained with conventional calibration procedures. An alternative consists in letting 8 vary. As a matter of fact, the novel calibration procedure consists of considering 8 as a variable parameter. A new set of data for poly(capro1actone) was built, in which the nth time of flight corresponds to the distance on= (n - 1) 114.1 Da. In this way, time-of-flight 0.033 58 corresponds to the distance 0 Da, time-of-flight 0.035 52 corresponds to the distance 114.1 Da, time-of-flight 0.037 39 corresponds to the distance 228.2 Da, and so on. The data set was given, along with eq 3, as input 4368 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
to a computer program which performs a least-squares minimization. The iterative process converged quickly toward a sharp minimum. The parameters at the minimum were a = 843 745.6, ,B = 44.8, and 8 = 953.8 with a coefficient of regression 12 = 0.999 998 5. This allowed us to calculate the absolute mass values associated with the experimental times of flight. Thus, it was found that the absolute mass of the peak at 0.033 552 is 953.8 Da and corresponds to the ion H[O(CH2)5COlsOHNa+. This analysis therefore ascertained that the poly(caprolactone) sample consists of a series of oligomers with H and OH as terminal groups, which form sodium-cationized ions in the MALDI process. Figure 2 shows the MALDI TOF mass spectrum of poly(Bisphenol A carbonate). The spectrum is constituted of three mass series (see inset)." The first (SI) ranges from about m/z 3000 to 13 000, the second (SZ) ranges from m/z 2000 to 20 000, the third (&) ranges from m/z 2000 to 8000, (the repeat unit of poly(Bispheno1 A carbonate) is 254.2 Da). The complexity of this MALDI spectrum does not allow unambiguous interpretation of the structure corresponding to the peaks observed, and furthermore, ion fragmentation cannot be a priori excluded. An inspection of the temporal spectrum associated with the mass spectrun showed that the first peak of the mass series S2 is at 0.043 74 ms, the second peak is at 0.047 08 ms, and the third one is at 0.050 19 ms. The total number of peaks in the mass series is 74; the last peak is at 0.154 55 ms. Assuming that the calibration constants found for poly(caprolactone) (a = 843 745.6, ,B = 44.8) were also valid for the polycarbonate spectrum, it was found that the peak at 0.043 74 ms corresponds to mass 1617.6, which is meaningless in terms of molecular structure. Therefore, the TOF calibration constants cannot be transferred.1° This is likely due the geometry of MALDI-TOF instruments, since the time of flight of desorbing species varies with the distance between the point of desorption from the crystal matrix and the accelerating plate. In fact, the point of desorption varies from a probe tip to another and also from spot to spot within the same tip. For the above reasons, a new set of calibration constants, specific for the spectrum in Figure 2, were sought. A set of data for poly(Bispheno1 A carbonate) was built, in which the nth time of flight corresponds to mass number on= (n - 1) 254.2. The data set was given, along with eq 3, as input to the least-squares minimization program. The iterative process converged toward the parameters a = 844 736.4, ,B = 6.8, and 8 = 1620.2 with a coefficient of regression 12 = 0.999 997 7. These values were used to compute absolute mass values associated with the experimental times of flight. Thus, it was found that the absolute mass of the peak at 0.043 70 is 1620.2 Da, which corresponds to the ion HO[BPAOCOO]sP.H+ (BPA = Bisphenol A; Ph = phenyl). This allows us to establish that peaks belonging to mass series S2 are poly(Bispheno1 A carbonate) oligomers having OH and (10) However, this is not strictly true in the case of MALDLFT mass spectra. See, for example: Castoro, J. A; Wilkins, C. L. Anal. Chem. 1993,65, 2621.
(11) FAE3-MS spectra of the polycarbonate were recorded and there were no peaks present up to 2000 Da. The same was found for poly(capro1actone). Manifacturers do eliminate the low molecular weight tail of the distribution curve in order to obtain useful materials.
3653 a
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1 1
2000
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Figure 2. MALDI-TOF mass spectrum of poly(Bispheno1A carbonate).
phenyl terminal groups, which form ions by capturing a proton in the MALDI process. The mass calibration also allowed the determination that peaks belonging to mass series SI consist of ions having the general formula PhO[BPA-OCOO],Ph.H+ and that peaks belonging to mass series SBconsist of carbonate cyclic oligomers which form sodium-cationized ions in the MALDI process. Since none of the three mass series corresponds to ion fragments, it was concluded that the experimental conditions allowed the desorption of intact polymer molecules. Summarizing our results, we note that synthetic polymers are not monodispersed as proteins and therefore ambigous peak assignments are the rule in MALDI-TOF spectra, when the conventional calibration procedure is used. One should use our method when the former has led to ambigous results.
Our calibration procedure can be applied, as such, to mixtures of polymers and to copolymers. The procedure can be applied only when the repeat unit of at least one component in the polymeric material analyzed is known. ACKNOWLEDGMENT Partial hancial support from the Italian Ministry for University and for Scientific and Technological Research (MURST) and from the National Council of Research (CNR, Rome) is gratefully acknowledged. Received for review April 5, 1994. Accepted August 11, 1994.m Abstract published in Advance ACS Abstracts, September 15, 1994.
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