Anal. Chem. 2003, 75, 6531-6535
Quantitative Analysis of Synthetic Polymers Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Hui Chen, Meiyu He,* Jian Pei, and Haifeng He
Department of Chemistry, Peking University, Beijing 100871, China
Quantitative analyses of synthetic polymers were accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Many factors have hindered the development of quantitative measurement of polymers via MALDI TOF MS, e.g., laser power, matrix, cation salt, and cocrystallization. By probing the optimal conditions, two sets of polymers were studied. Fair repeatability of the samples ensures acceptable results. In set 1, two poly(ethylene glycols) with different end groups showed equal desorption/ionization efficiencies. Two synthetic polymers in set 2 with different chemical properties resulted in different MALDI responses. Good linearity was achieved by plotting the relationship between the sample concentration ratio and the total signal intensity ratio in both sets. Since being introduced in 1988,1,2 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been widely used in qualitative analysis. This soft ionization method convincingly proved its value in the study of biomacromolecules and synthetic polymers. For instance, MALDI-TOF MS has been applied to the determination of the molecular weights of proteins, peptides, and synthetic polymers;3,4 the mapping and sequencing of peptides;5 and the characterization of synthetic polymers.6,7 However, all of the studies mentioned above were related to qualitative analysis. Few achievements were presented demonstrating the capability of quantitative analysis by the use of MALDI TOF MS. In 1994, Hercules and co-workers first illustrated the concept of quantitative measurement on cyclosporine A by MALDI TOF MS.8 Subsequently, increasing efforts have been made on the quantitative measurement of biomolecules.9-12 * Corresponding author. Fax: 8610-62751708, e-mail:
[email protected]. (1) Tanaka, K.; Waki, H.; Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2, 151-153. (2) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (3) Busch, K. L. J. Mass Spectrom. 1995, 30, 233-237. (4) Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 2721-2725. (5) Fenselau, C. Anal. Chem. 1997, 69, 661A-665A. (6) Jackson, A. T.; Yates, H. T.; Lindsay, C. I.; Didier, Y.; Segal, J. A.; Scrivens, J. H.; Critchley, G.; Brown, J. Rapid Commun. Mass Spectrom. 1997, 11, 520-526. (7) Gooden, J. K.; Gross, M. L.; Mueller, A.; Stefanescu, A. D.; Wooley, K. L. J. Am. Chem. Soc. 1998, 120, 10180-10186. (8) Muddiman, D. C.; Gusev, A. I.; Proctor, A.; Hercules, D. M. Anal. Chem. 1994, 66, 2362-2368. (9) Nicola, A. J.; Gusev, A. I.; Hercules, D. M. Appl. Spectrosc. 1996, 50, 14791482. 10.1021/ac0344034 CCC: $25.00 Published on Web 10/30/2003
© 2003 American Chemical Society
In comparison with the development of biomolecules, the quantitative analysis of macromolecules has proceeded much more slowly, with a later start and fewer reports in the literature. The first report on the quantitative analysis of polymers was presented by Gardella and co-workers in 2002.13 In their study, quantitative measurements were completed for poly(dimethylsiloxane) (PDMS) of two different molecular weights. The measured results were in good agreement with predictions based on stoiciometry. This development indicates that MALDI TOF MS is a potentially useful tool for quantitative analysis, especially for synthetic polymers, which is considered a brand-new area of application for this technique. In addition, the application of MALDI TOF MS to the quantification of synthetic polymers is extraordinarily relevant. The changing properties of a polymer material induced by the varied percentages of its different components present investigators with a particular challenge to accurately measure the relative quantities of the polymers. There is a difference between quantitative measurements on biomolecules and on polymers. An internal standard has been employed for most biomolecule measurements. For example, quantification of the intact proteins in biological fluids and tissues requires measurements of the absolute quantities of proteins. In contrast, no internal standard was used in Gardella et al.’s study, because they finished the polymer quantitative analysis using the relative signal intensities of the two analytes in each set. In this contribution, two sets of experiments were conducted on the quantitative analysis of synthetic polymers using MALDI TOF MS. The first set included two synthetic polymers, P1 and P2, which were poly(ethylene glycols) (PEGs) with different end groups. Another included synthetic polymers P2 and P3, which were greatly distinct from each other in structure and desorption properties. Chart 1 shows the structures of the three polymers studied in the present paper. Table 1 shows the ratios of the polymer mixtures of sets 1 and 2. A plot of signal intensity ratio (y axis) vs sample concentration ratio (x axis) was linear for both sets of mixtures. (10) Muddiman, D. C.; Gusev, A. I.; Martin, L. B.; Hercules, D. M. Fresenius’ J. Anal. Chem. 1996, 354, 455-463. (11) Mehl, J. T.; Nicola, A. J.; Isbell, D. T.; Gusev, A. I.; Hercules, D. M. Am. Lab. 1998, 30, 30-38. (12) Gusev, A. I.; Wilkinson, W. R.; Proctor, A.; Hercules, D. M. Fresenius’ J. Anal. Chem. 1996, 354, 455-463. (13) Yan, W.; Joseph, A.; Gardella, J.; Wood, T. D. J. Am. Soc. Mass Spectrom. 2002, 13, 914-920.
Analytical Chemistry, Vol. 75, No. 23, December 1, 2003 6531
Chart 1. Structures of the Three Polymers P1-P3
Table 2. Measured Mw and Mn Values of Polymers P1-3
P1 P2 P3
Mw
Mn
PD (Mw/Mn)
865 950 2350
830 906 2280
1.04 1.03 1.03
method to prepare the sample target: 1.5 µL of sample mixture was applied to the sample target and air-dried for 5 min.2
Table 1. Ratios of the Polymer Mixtures in Sets 1 and 2 set 1 P1a
b
set 2 DIb
sample
(µL)
P2 (µL)
(µL)
sample
P3 (µL)
P2 (µL)
DI (µL)
1a 1b 1c 1d 1e 1f 1g 1h
1 1.5 2 3 4 4.5 4.8 5
5 4.5 4 3 2 1.5 1.2 1
30 30 30 30 30 30 30 30
2a 2b 2c 2d 2e 2f 2g 2h
1 1.5 2 3 4 4.5 4.8 5
5 4.5 4 3 2 1.5 1.2 1
30 30 30 30 30 30 30 30
a Concentrations of P1, P2, and P3 are all 5 × 10-5 mol/L. Concentration of DI is 40 g/L.
EXPERIMENTAL SECTION General Instrumentation and Conditions. MALDI experiments were carried out using a Bruker BIFLEX III time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Billerica, MA). The instrument is equipped with a N2 laser emitting at 337 nm (Laser Sciences Inc., Cambridge, MA), a 1-GHz sampling rate digitizer, a pulsed ion extraction source, and an electrostatic reflectron. The laser pulse width is 3 ns, and the maximum power is 200 µJ. Spectra were acquired in the linear positive-ion mode. The acceleration voltage was 19 kV. Typically, 200 single-shot mass spectra were summed to give a composite spectrum. All data were reprocessed using Bruker XTOF software. The mass scale was calibrated externally, using the peptides angiotensin II and bovine insulin b-chain (Sigma, St. Louis, MO) as mass standards. MALDI analysis of the polymers utilized Dithranol (DI) (Aldrich, Milwaukee, WI) as the matrix. Samples and Reagents. The solvent, inhibitor-free tetrahydrofunan (Aldrich, Milwaukee, WI), stored in a Sure/Seal bottle, was used as received. Polymer P1 was bought from Acros (Fairlawn, NJ). Polymers P2 and P3 were synthesized previously.14,15 We obtained the number-average molecular weight (Mn) and weight-average molecular weight (Mw) of each of the three polymers by MALDI (see Table 2). Mw was used to calculate the molar concentration for each polymer. The polymers were dissolved in THF at a concentration of 5 × 10-5 mol/L, and the matrix DI had a concentration in THF of 40 g/L. We used the dried droplet 6532
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RESULTS AND DISCUSSIONS The matrix is of considerable importance to the quality of a MALDI analysis.16 The function of the matrix is to absorb the laser energy and make the polymer molecules separate from each other into a single-molecule state. Then, the molecules are desorbed and ionized. The quality of the cocrystals depends on the properties of the chosen matrix. Generally, it is recommended that the polarity of the matrix be matched with that of the polymer under investigation. Nevertheless, matrix selection and optimization for polymer analysis is still often a trial-and-error process. Our previous studies17,18 indicated that dithranol (DI) was the best choice for the three polymers P1-3. The relative ratio of matrix to sample is as crucial to the formation of homogeneous cocrystals as the properties of the matrix. Less matrix will not absorb enough laser energy to induce desorption and ionization, whereas more matrix will also dilute the sample concentration. Both cause low signal intensities. The ideal mole ratio ranges from 100 to 50 000 (matrix vs sample).19 Herein, the mole ratio of 1800 was selected as the optimal choice for which the two analytes in each set showed relatively high signal intensities. Normally, a synthetic polymer is composed of several series of polymers with different end groups caused by different chain terminators and the side reactions. Not only the structures of these series of polymers but also the percentages of the component polymers can affect the physical and chemical properties of the synthetic polymer, e.g., surface activity, phase behavior, and light emission.20-22 Therefore, end-group structure characterization and component quantitative analysis are of great value to understand more about the mechanism of polycondensation and to gain a deeper insight into the limiting side reactions. In set 1, to investigate the feasibility of component quantitative analysis of different oligomers with different end groups, we chose P1 and P2 as the objects for their identical repeat units (PEG) but different end groups. The end-group analysis of P2
was conducted previously.17 Using the same method, we obtained (14) Liu, B.; Wang, Y.; Yee, L.; Wei, H. Chem. Mater. 2001, 13, 1984-1991. (15) Yang, L. F.; He, H. F.; Wan, X. H. Chin. J. Polym. Sci. 2002, 20, 401-406. (16) Nielen, M. W. F. Mass Spectrom. Rev. 1999, 18, 309-344. (17) Chen, H.; He, M. Y.; Wan, X. H.; Yang, L. F.; He, H. F. Rapid Commun. Mass Spectrom. 2003, 17, 177-182. (18) Chen, H.; He, M. Y.; Pei, J.; Liu, B. Anal. Chem. 2002, 74, 6252-6258. (19) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63, 1193A-1202A. (20) Sheila, J.; Motekaitis, R. J.; Martell, A. E. Inorg. Chim. Acta 1998, 278(2), 170-177.
Figure 1. Mass spectra of eight samples in set 1.
the end-group information on P1 (-H and -OCH3). The mass spectra of eight samples in set 1 are shown in Figure 1. Sodium adduct [M + Na]+ and potassium adduct [M + K]+ were found in each spectrum of the eight samples. We ascribed this result to the trace Na+ and K+ present as impurities in glassware, solvent, reagents, etc., given that we did not add any cationization salts during the experiment. To investigate the effect of the amount of sodium impurities varying from run to run, we conducted a contrast experiment by comparing the results of two runs using the same sample but with the addition of NaCl specifically to one while no cationization agent was added to the other. The absolute signal intensities of the two samples were the same, which means the impurity sodium in the system is relatively sufficient for the cationization of the sample, but its absolute amount is small. Therefore, we decided not to add cationization salts to simplify the operation. The absolute intensity of P1 or P2 changed as its amount varied. The greater the volume used used, the stronger the intensity observed. Furthermore, the relative intensity between P1 and P2 increased with increasing mole ratio. To obtain accurate results, we tried to calculate the total signal intensity of (21) Stelian, G.; Linda, D. K. Macromolecules 1996, 29, 1260-1265. (22) Tao, Y.; Donat-Bouillud, A.; D’Iorio, M.; Lam, J.; Gorjanc, T. C.; Py, C.; Wong, M. S.; Li, Z. H. Thin Solid Films 2000, 363, 298-301.
Table 3. Measured Signal Intensity Results for Sets 1 and 2 set 1
set 2
sample
MP1/MP2
ItP1/ItP2
sample
MP3/MP2a
ItP3/ItP2
1a 1b 1c 1d 1e 1f 1g 1h
0.20 0.33 0.50 1.0 2.0 3.0 4.0 5.0
0.24 ( 0.03 0.33 ( 0.04 0.53 ( 0.06 0.96 ( 0.09 2.0 ( 0.3 3.2 ( 0.4 3.8 ( 0.6 4.9 ( 0.7
2a 2b 2c 2d 2e 2f 2g 2h
0.20 0.33 0.50 1.0 2.0 3.0 4.0 5.0
0.013 ( 0.003 0.037 ( 0.004 0.049 ( 0.004 0.087 ( 0.006 0.142 ( 0.009 0.242 ( 0.016 0.321 ( 0.027 0.432 ( 0.048
a
MP3/MP2 ) molar ratio of polymer P3 to polymer P2.
each component polymer by the following equation13
It )
∑I
p
×
Mp - ME1 - ME2 - MC MR
(1)
where It represents the total signal intensity; Ip represents the intensity of each oligomer peak; and Mp, ME1, ME2, MR, and MC represent the mass of each oligomer peak, the mass of the two sides of the end groups, the mass of the repeat unit, and the mass of cation, respectively. In this study, we chose [M + Na]+ peaks for the calculations. Analytical Chemistry, Vol. 75, No. 23, December 1, 2003
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Figure 2. Plot of relative ion intensity ratio (ItP1/ItP2) vs mole ratio (MP1/MP2) for set 1 (y ) 0.98x, R2 ) 0.998). Error bars represent the standard deviation.
In the MALDI of synthetic polymers, rather inhomogeneous sample preparations are typically obtained, and one often has to search not only for the sweet spots showing good resolution and signal intensity but, in some cases, even for spots giving any polymer signal. Thus, a homogeneous sample is the basis for a successful MALDI analysis, which limits the use of MALDI for quantitative measurements. To decrease the error caused by sample inhomogeneities, we chose five different spots on the same
Figure 3. Mass spectra of eight samples in set 2. 6534
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sample target. The calculated results for set 1 are reported in Table 3. Also, a plot of the relative ion intensity ratio (ItP1/ItP2, as the y axis) as a function of the mole ratio of the two polymers (MP1/MP2, as the x axis) is shown in Figure 2. A line passing through the origin was obtained (slope 0.98, correlation coefficient, R2 ) 0.998). The results fit well with the expected ratio of stoichiometry, which indicates that polymers with identical polymer chains exhibit equal desorption/ionization efficiencies even when their end groups are different. This is crucial for component quantitative analysis of the synthetic polymer. The results of set 1 provided a successful quantitative measurement on polymers with identical polymer chains and different end groups. Then, we addressed the question of the capability of MALDI to be used for quantitative analysis of polymers with a great variety in structure, end groups, and chemical properties. To probe whether MALDI could be widely used for quantitative analysis, experiment set 2 was performed. Of the polymers used for this set of experiments, P2 is a type of PEG, and P3 is a conjugated polymer synthesized by the Suzuki coupling reaction.15 These polymers are completely different, from structure to chemical properties. Eight samples were prepared and propelled into the vacuum system for MALDI analysis (listed in Table 1). Symmetric Gaussian distributions of both analytes were observed in the spectra (see Figure 3). Considering that P3 only forms a radical ion,18 rather than an alkali adduct, even when extra alkali salts are added, and that the systemic sodium impurity can full ionize the sample, we did not use a special cationization agent in
Figure 4. Plot of relative ion intensity ratio (ItP3/ItP2) vs mole ratio (MP3/MP2) for set 2 (y ) 0.083x, R2 ) 0.996). Error bars represent the standard deviation.
these experiments. Both sodium and potassium adduct peaks were observed for polymer P2, whereas polymer P3 formed only radical ions. The change in the relative intensity of P3 vs P2 with changing mole ratio was obvious. Equation 1 was used to measure the total signal intensity for each polymer in each of the eight samples. The results are shown in Table 3. The line drawn through the data passes through the origin with a slope of 0.083 and a correlation coefficient of 0.996 [see Figure 4, which is a plot of the relative intensity ratio (ItP3/ItP2, as the y axis) as a function of the molar ratio (MP3/MP2, as the x axis)]. The ratios of total signal intensity were not in agreement with those expected on the basis of stoichiometry. We ascribed this deviation to the different desorption/ionization probabilities caused by the different structures and chemical properties of the two polymers. Fortunately, the good linearity of the plot implies tha promise of MALDI for quantitative analysis. Regarding this plot as a standard calibration curve, we might calculate the component percentage of a mixed sample composed of P2 and P3 by finding the value (on the x axis) corresponding to the ratio of total signal intensity (on the y axis) obtained from a MALDI spectrum. CONCLUSION Many problems intrinsic in MALDI have restricted its practical application to the quantitative measurement of synthetic polymers.
For example, inhomogeneity in the sample, matrix, and cationization salts is considered to be the biggest obstacle to obtaining sample-to-sample or spot-to-spot repeatability. Moreover, the relatively low detector saturation limits the dynamic range of MALDI. Laser power also contributes to the variable signal intensities of analytes. Despite these problems, we were still able to find the optimal conditions for our experiments by repeated trials. Actually, we feared that repeatability might be the fatal threat to successful results at the beginning of our study. We made several parallel trials to study the sample-to-sample repeatability by comparing results for different samples with the same analyte concentration prepared by different methods and for different times. The results exhibited pleasing reproducibility. Furthermore, the signals of five different spots on the same target were acquired. The small standard deviation verified the good spot-to-spot repeatability of these samples. In set 1, two PEGs with different end groups displayed equal desorption properties, and the ion intensity ratios were in good agreement with the predicted stoichiometric ratios. Successful quantitative results can help us understand more about the mechanism of polycondensation and gain a deeper insight into side reactions, which is of practical value in improving the properties of polymer materials. Although the results of set 2 were not as desirable as expected, the linear curve implied the feasibility of MALDI for the quantitative measurement of synthetic polymers with different chemical properties. The satisfying results of our study provide something fresh and new in the area of polymer quantitative analysis. It is believed that, with further development of the technique and instrumentation, MALDI TOF MS will be a practical tool for synthetic polymer quantitative analysis, as well as qualitative analysis, in the future. ACKNOWLEDGMENT Financial support from the National Natural Science Foundation of China is gratefully acknowledged. All experiments were carried out with the MALDI-TOF MS at the Beijing Mass Spectrometry Center.
Received for review April 17, 2003. Accepted September 19, 2003. AC0344034
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