Anal. Chem. 1995, 67,1575-1579
Determination of Molecular Weight Distributions of Polymers by MALDbFTMS Michael Dey, John A. Castoro, and Charles L. Wilkins* Universiv of Califomia, Department of Chemistv, Riverside, Califomia 9252 1
The advantages of matrix-assistedlaser desorptiodionization (MALDI) and Fourier transform mass spectrometry (FI'MS)are combined to create a powerful tool for the characterization of polymer samples. MALDI permits generation of intact, singly charged, high-mass(m/z lo3lo4) ions of polar and nonpolar analytes, while FlMS provides unique high resolution and mass accuracy. Use of a gated decelerationpotential allowsfor FTMS detection of MALDI-generatedions. However, when a single delay time is used between desorptiodionizationand restoration of normal trapping potentials, mass discrimination is apparent for broad polymer distributions. Here, an integral method for obtaining molecular weight distributions of polymers by MALDI-FI'MS is presented. By means of a simple procedure of averaging time domain transients obtained from a series of polymer sample spectra recorded at systematicallyvaried gated deceleration times, accurate polymer distributions are obtained. The technique is demonstrated with several poly(ethy1ene glycol) samples containingoligomers with masses covering a 10 kDa mass range. Since the introduction of matrix-assisted laser desorption/ ionization (MALDI),1-3its applicability to many types of analytes has been investigated. The interest of a number of research groups has focused on biomolecules, primarily proteins, nucleotides, and polymers. Most biomolecules examined by MALDI to date have a single defined sequence,which produces molecular ions of a single nominal mass. In contrast, polymers are mixtures of oligomers differing in mass by the mass of the repeating units. Therefore, in polymer mass spectra, distributions of molecular ions are detected, rather than a single type of molecular ion. MALDI timeof-flight mass spectrometry (TOFMS) has been used for characterization of polymers, since it allows for generation and detection of polymer ions4with minimal mass discrimination. Two features of MALDI are its main advantages for polymer analysis: (1) analyte polarity is not critical and (2) with most matrices, MALDI generates almost exclusively singly charged ions. Thus, in principle, MALDI mass spectra are interpretable even if a polymer sample consists of a large number of oligomers with different masses due to their chain lengths. However, drawback of TOFMS for polymer analysis are its relatively low (1) Karas, M.; Bachmann, D.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987,78,53-68. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988,60, 2299-2301. (3) Tanaka,K; Waki,H.; Ido, Y.; Akita, S.; Yoshida, Y Rapid Commun. Mass Spectrom. 1988,2, 151-153. (4) Bahr, U.; Deppe, A; Karas, M.; Hillenkamp, F.; Giessmann, U. Anal. Chem. 64,1992,2866-2869. 0003-2700/95/0367-1575$9.00/0 Q 1995 American Chemical Society
mass resolution and its limited mass accuracy (generally no better than 0.01%for masses below 20 000 Da). Fourier transform mass spectrometry (FTMS) offers high resolution and correspondingly improved mass measurement accuracy. With the introduction of a gated ion deceleration potential technique permitting trapping of MALDI-generated ions, it became possible to obtain MALDI-FT mass spectra of highmass molecules (mlz 103-104).5*6Since then, the available mass range for MALDI-FTMS has been increased and the method applied to a number of higher molecular weight biomolecules7t8 Thus, bovine insulin ions with m/z 5734 have been detected with resolving power of 90 OO09J0 and biomolecules have been detected with mass accuracy in the low partaper-million range7J1 Furthermore, MALDI-FTMS has been used to obtain polymer ~pectra.~ It seems obvious that MALDI-FTMS can be a powerful tool for polymer analysis. The high sensitivity of MALDI combined with the high resolution and mass accuracy of FTMS results in precise characterization of polymers, including determination of monomer units, end groups, and molecular weight distributions. Because polymer distributions may cover a wide mass range, the velocities of MALDI-generated ions must be considered in designing MALDI-ETMS measurements of such mixtures. MALDIgenerated ions leave the surface with an almost mass-independent mean velocity of approximately 750 m/s.12J3 Therefore, the mean kinetic energy of desorbed ions increases with increasing mass, making the trapping of high-mass ions (with correspondingly higher energies) in a FTMS dif6cult. Because the efficiency of trapping ions is related to their kinetic energy, use of a fured gated deceleration potential is an energy-selective method. The portion of desorbed analyte molecules trapped and observed depends on the decelerating potential applied at the rear trap plate. This potential acts as a kinetic energy band pass filter, where lower energy ions are decelerated while higher energy ions are not. Ions of a given kinetic energy have velocities related to their masses. When the gated deceleration potential is kept at a constant value, the optimum deceleration time for any analyte is a function of its mass. (5) Castoro, J. A; Koster, C.; Wilkins, C. Rapid Commun. Mass Spectrom. 1992, 6, 239-241. (6) Koster, C.; Castoro, J. A; Wilkins, C. L.J.Am. Chem. SOC.1992,14,75727574. (7) Castoro, J. A; Wilkins, C. L. Anal. Chem. 1993,65, 2621-2627. (8) Solouki, T.; Gillig, K. J.; Russell, D. H. Anal. Chem. 1994,66, 1583-1587. (9) McIver, R T., Jr.; Li,Y.; Hunter, R L. Int. J. Mass Spectrom. Ion Procwes 1994,132,Ll-L7. (10)Pastor, S.; Castoro, J. A; Willcins, C . L.Anal. Chem. 1995,67, 379-384. (11) Li, Y.; McIver, R T., Jr.; Hunter, R L.Anal. Chem. 1994,66, 2077-2083. (12) Beavis, R C.; Chait, B. T. Chem. Php. Lett. 1992,181, 479-484. (13) Pan, Y.; Cotter, R J. Ow. Mass Spectrom. 1992,27, 3-8.
Analytical Chemistty, Vol. 67, No. 9, May 1, 1995 1575
Table 1. Calculated Kinetic Energies and Velocitleo for MALDLQenerated Ions In a Mass Range from 1000 to l o o 0 0 Da m/z of ions
kinetic energy of ions with a velocity of 750 m/s (in ev) velocity of ions with 9.5 eV kinetic energy (in m/s)
lo00
2000
3000
4000
5000
6OOO
7000
8000
9000
10000
2.9 1355
5.8 957
8.7 782
11.7 677
14.6 606
17.5 553
20.4 512
23.3 479
26.2 452
29.1 428
Because MALDI-generated biomolecules usually contain only a single molecular ion species, the gated deceleration time can be optimized for this mass. However, for polymer samples containing oligomers with a wide range of masses, no single choice of deceleration time is optimal. The gated trapping time cannot be optimized for the complete polymer distribution simultaneously, and consequently, the measurement is inherently mass discriminating. Therefore, MALDI-FTMS measurements of polymers may result in misleading conclusions about molecular weight distributions, especially for broad distribution polymers, unless a procedure such as that described here is employed. To eliminate the discrimination introduced by using a single deceleration time, a systematic procedure using a series of deceleration times has been developed. The method is simple. MALDI-FT mass spectra collected using a range of gated deceleration times are integrated. This is accomplished by adding the corresponding series of time domain'transients. The resulting averaged composite time domain transient is then subjected to Fourier tranformation to produce the spectrum reflecting the true polymer distribution. The technique is demonstrated with several poly(ethy1ene glycol) samples containing oligomers with masses covering a 10 kDa mass range. EXPERIMENTAL SECTION The experimental details are described in greater detail el~ewhere.~ The experiments were performed using a 7 T ETMS 2000 instrument (Extrel-FTMS;Waters, Madison, WI). The 2-in. cubic source cell of the dual cell instrument was used for the experiments presented here. Samples were desorbed from a stainless steel probe tip of an automated solids probe. The 308 nm output of a XeCl excimer laser (EMG-201 MSC; Lambda Physik, Gottingen, Germany) was focused by a 12.5cm fused silica lens onto the probe tip. The laser beam was grossly attenuated by an iris diaphragm to an energy between 2 and 30 mJ. Laser energy was measured with a RLP-734 energy probe and a RJ7610 energy radiometer (Laser Precision Corp., Utica, NY). The spot size of the laser beam on the sample was adjusted to approximately 0.1 "2. 2,SDihydroxybenzoic acid (DHB) (Fluka, Buchs, Switzerland) was used as matrix for the poly(ethy1ene glycol) BEG) samples. The polymer samples PEG 1000 and PEG 8000 (Sigma, St. Louis, MO) and PEG 3000 and PEG 6000 (Fluka, Buchs, Switzerland) were used without further purification. Methanol solutions of the samples with a concentration of 1mmol/L were mixed with a 50 mmol/L matrix solution. The volumes were adjusted to yield a 1:5000 analyte-matrix molar ratio for single polymer experiments and a 1:l:l:l:lO000 molar ratio for the fourcomponent polymer experiments. A homogeneous layer of the analyte-matrix mixture was obtained by spraying the analyte-matrix solution as an 1576 Analytical Chemistry, Vol. 67, No. 9,May 1, 1995
aerosol onto a rotating stainless steel probe tip. This procedure improves spot-to-spot repeatability. However, in the present study, in order to eliminate that factor in comparisons, each set of experiments utilized data acquired from a single sample spot. Furthermore, several spectra were acquired for each measure ment to ensure reproducibility before the resulting data were stored. Gated ion deceleration was utilized for all MALDI-FTMS polymer experiments. The rear trap plate (the conductance limit) of the source cell is set to a 9.5 V potential, while the front trap plate (the plate -2 mm from sample probe) is grounded prior to the laser pulse. The laser is triggered, followed by a variable delay depending upon the mass of the analytes to obtain the most efficient deceleration and trapping of the desorbed ions, resulting in a maximum signal-to-noise ratio. The gated deceleration time was adjusted between 40 and 400 p s for the analytes used. Values outside of this range did not produce spectra. Following gated deceleration, the potentials of the front and rear trap plates were set to a 2 V trapping potential for a short delay prior to the excitation/detection events. A broad-band chirp excitation of 200 V peak-to-peak sweep from 50 to 200 kHz at a sweep rate of 240 H z / p was chosen to excite all ions into coherence simultaneously. All spectra were detected using an acquisition rate of 1000 kHz and collecting 65 534 data points. To obtain frequency domain mass spectra, each data set was augmented by an equal number of zeroes and baselinecorrected prior to magnitude mode Fourier transformation. No apodization was used. Sodiumationized PEG lo00 was used as an external calibrant, with the same trapping voltage, trapping cell, and excitation conditions as used for the analyte. Resolution in all cases is estimated from the ratio of peak position to peak width at half-height. RESULTS AND DISCUSSION Deceleration and trapping of MALDI-generated ions with high kinetic energies in a FTMS cell must be accomplished prior to the detection. To utilize MALDI-FTMS for the characterization of polymers, the deceleration parameters must be chosen carefully. Table 1 shows the calculated mean kinetic energies of MALDI-generated ions withii a mass range from 1000 to 10 000 Da. These values were calculated using the equation
Eldn= '/2mv2 assuming a mean velocity of 750 m/s for MALDI-generated ionsl1J2 The values indicate increasing kinetic energy with increasingmass of MALDI-generatedions. At 4000 Da, the mean kinetic energy of desorbed ions exceeds the 9.5 V trapping potential, which is the standard maximum voltage available in commercial FTMS instruments. The upper kinetic energy limit of trapped ions is therefore 9.5 eV.
h
goua
Ill,
120 us
Figure 1. MALDI-FT mass spectra of PEG 3000 recorded for (a) 90 and (b) 120 ps gated deceleration times.
The duration for which this potential is applied prior to trap ping is another experimental parameter directly related to the kinetic energy of the ions being trapped. Because the velocities of ions with a given kinetic energy are related to their masses, ions with higher masses require longer gated trapping times than lower mass ions. This is obvious when one considers Table 1, where the velocities of ions with masses ranging from lo00 to 10 000 Da at a kinetic energy of 9.5 eV are listed. Because heavier ions are slower than lighter ones with the same kinetic energy, they are more efficiently trapped at longer deceleration times. Ions with the same kinetic energy but different masses have different velocities (e.g., 9.5 eV mass 1000 ions have a velocity of 1355 m/s, while 10 000 Da ions with the same energy have a velocity of 428 m/s). Thus, choice of deceleration (gated trapping) time has a strong innuence on I T mass spectra observed. Therefore, a polymer distribution observed by MALDI-FTMS using a single deceleration time is dependent not only on the oligomer distribution of the polymer analyzed but also on the deceleration time. Figure 1 shows the MALDI-FT mass spectra of sodium-attached PEG 3000 measured using 90 (Figure la) and 120ps (Figure lb) gated deceleration times. Although the same sample and the same experimentalparameters were used in both experiments, with the exception of the gated deceleration time, the mass spectra are significantly different. As seen in Figure la, when 90 ps deceleration is used, the spectrum shows oligomer ions having masses between 2500 and 3600 Da, with a maximum abundance of ions with masses of about 3000 Da. For a 120 ps deceleration period, polymer ions with masses between 3000 and 3900 Da are observed (Figure lb), with a maximum abundance of ions with masses of about 3400 Da. The influence of the deceleration potential duration upon spectral appearance as shown in Figure 1is in accord with what might be predicted from Table 1. For shorter gated deceleration times, lower mass (and, therefore, faster) ions are preferentially detected, while for longer gated deceleration times, higher mass (slower) ions are preferentially detected. As a consequence, the observed distribution is strongly dependent upon the choice of gated deceleration time. However, except for its possible mass discrimination, MALDIFl?vlS with gated deceleration offers some major advantages for
Figure 2. High-resolution MALDI-FT mass spectrum of a sodiumattached PEG oligomer (n = 65) from PEG 3000. Mass measurement accuracy for this oligomer is 3.1 ppm, and resolving power is 30 000. Table 2. Observed Molecular Weight Distributions for Sodium-Attached PEQ 3000 Ions at Different Gated DecelerationTimes
gated trapping time (s)
obsd M,,
obsd M,
80 90 100 110 120 130 integral method, 80-130
2947 3028 3211 3331 3471 3545 3325
2963 3041 3218 3338 3478 3552 3337
characterization of polymers. High-resolution mass spectra allow for accurate determination of the monomer units and the isotopic peak pattern of the oligomers. Figure 2 displays the sodiumattached PEG 3000 oligomer, n = 65. The measured mass for this oligomer is 2902.714 Da, which compares favorably with a theoretically calculated mass of 2902.705 Da. The isotopic peaks of the oligomer are clearly resolved, because the resolving power in this spectrum exceeds 30 000. The mass accuracy, determined as the difference between measured and calculated mass for this oligomer, is about 3.1 ppm. The average mass accuracy for PEG 3000, n = 60 to n = 75, is 3.0 ppm. The calculated theoretical mass of the ethylene glycol repeating unit (-OCHzCHz-) in a PEG polymer is 44.0262 Da. The average measured repeating unit mass for PEG 3000 oligomers is 44.0266 Da. To characterize a polymer, the number average and weight average molecular weight values are calculated according to the following equations: number average
M, = &Mi/&j
weight average
M, = &M:/&Mj
where ni are the intensities of the signals and Mi are the corresponding masses. Table 2 lists the number and weight average molecular weights observed for sodium-attached PEG 3000 using gated trapping times ranging from 80 to 130ps. There is a significant increase in the observed M,values, from 2947 at 80 ys to 3545 at 130 ys, with a similar increase in the M, values. Table 2 shows the range of distributions that can be obtained for Analytical Chemistry, Vol. 67,No. 9,May 1, 1995
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this polymer using MALDI-FTMS and the indicated single deceleration times. Although high-resolution and high-mass-accuracy mass spectra of oligomers are obviously important, it is equally important to obtain accurate polymer distributions. As we have demonstrated, the actual distribution cannot be obtained by MALDIFTMS using a single laser shot experiment. However, with use of an integral method, accurate molecular weight distributions of polymers can be determined by MALDI-FTMS. The gated deceleration time determines the mass range of ions initially trapped in the cell, and, for observation, analytical trapping potential (2 V) controls the z-axis energies of the trapped ions. Different portions of a polymer distribution are detected using different gated deceleration times. Increasing the gated deceleration time by small increments, typically 10 ps, allows sytematic sampling of the entire mass range of a complete polymer distribution. Summation of the time domain data sets thus acquired results in an integral measurement. Fourier transformation of these summed data results in a spectrum representative of the entire polymer distribution. From this information, accurate molecular weight distributions of polymers are obtained by MALDI-FTMS. For example, the method was applied to the FT mass spectra obtained for PEG 3000 by adding the data at the various gated deceleration times from 80 to 130 ,us taken by 10 ps intervals. For deceleration times outside these limits, polymer ions were not detected. Adding the transients from these six experiments resulted in a FT mass spectrum of PEG 3000 representative of the complete distribution from 2500 to 3900 Da. The calculated Mn (from peak heights) for the sodium-attached PEG 3000 according to the integral method was 3325 (Table 2) ,and M, was calculated to be 3337. As a further example of the effectiveness of determining a polymer molecular weight distribution by the integral method, a polymer with a wide distribution was simulated with a mixture of four PEG samples. PEG 1000, PEG 3000, PEG 6000, and PEG 8000 were mixed in a 1:l:l:l molar ratio. The mixture contains analyte molecules with masses from
