LC-MALDI-TOF Imaging MS: A New Approach in Combining

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LC-MALDI-TOF Imaging MS: A New Approach in Combining Chromatography and Mass Spectrometry of Copolymers Steffen M. Weidner* and Jana Falkenhagen Federal Institute for Materials Research and Testing (BAM), D-12489 Berlin, Richard-Willstaetter-Strasse 11, Germany ABSTRACT: A new approach that utilizes MALDI-TOF imaging mass spectrometry as a new detector for polymer chromatography is presented. For the first time, the individual retention behavior of single structural units of polyethylene oxide (PEO)/polypropylene oxide (PPO) copolymers and changes of the copolymer composition could be monitored. Composition specific calibration curves could be easily obtained by displaying the copolymer ion intensity data. This approach provides completely new insights in the chromatographic principle of copolymer separation and could be used to easily modify and adapt conditions for separation. In combination with electrospray deposition, homogeneous sample/matrix traces of surprisingly high spatial resolution could be obtained.

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ALDI-TOF MS has been established as an important tool in polymer analysis. Ideally, information on molecular masses, mass distributions, and chemical heterogeneities (e.g., end groups) could be obtained in one single analysis. However, most synthetic polymers exhibit a broad polydispersity with regard to molecular masses and heterogeneity as well, which requires an additional chromatographic separation prior to MALDI-TOF MS. The situation becomes much more complicated when copolymers having an additional compositional distribution are matter of investigation. In a previous paper, we could show that each structural block of randomly composed polyethylene oxide (PEO)/ polypropylene oxide (PPO) copolymers can independently elute in a different chromatographic mode.1 Even a slight variation of chromatographic parameters (e.g., solvent composition) was shown to have a strong influence on the separation and could cause either PEO or PPO units to change their individual elution mode from adsorption to size exclusion mode (or vice versa). These experiments have demonstrated that a deeper understanding of separation processes of copolymers could be beneficial for a better separation. In contrast to many other liquid chromatography mass spectrometry (LC-MS) techniques, a LC-MALDI MS coupling cannot be done online. Although many attempts have been done to overcome this drawback, e.g., by means of rotating ball interfaces or using frits, suitable online coupling devices for commercially available instruments have not yet been developed.2 A comprehensive overview of existing coupling techniques for the analysis of synthetic and biopolymers can be taken from references.3 The most important off-line devices are based on the principle of fractionation (either manually or by means of fraction collectors) or utilizing sample/matrix spraying devices. Spray methods can be roughly divided in air spray and electrospray techniques. The air spray method is based on the fast evaporation of the chromatographic eluents through a heated capillary accompanied by the use of a heated sheet gas. Various applications have been r 2011 American Chemical Society

published demonstrating the applicability of this method, especially for the determination of the chemical heterogeneity distribution of complex polymers by means of liquid chromatography at/near critical conditions (LACCC). 4 In a recent paper, the homogeneity of such deposited sample/matrix traces was demonstrated by means of MALDI imaging mass spectrometry (MALDI IMS).5 However, the use of air spraying for the evaporation of eluent mixtures having a higher amount of hardly vaporizable solvent requires higher evaporation temperatures. This can cause degradation of thermally labile samples and, moreover, a blocking of the tip of the capillary by crystallization of the matrix. The use of electrospray deposition for synthetic polymers analysis has been reported by several groups.6 It was shown that this technique, on the one hand, increases the repeatability of both the MALDI signal intensity and the measured molecular mass distribution (MMD) but, on the other hand, may also influence the apparent MMD of a synthetic polymer, especially at higher voltages due to increased fragmentation.6c In contrast to air spray techniques, the electrospray deposition (ESD) requires much lower flow rates of the chromatographic system. Typically, 5 20 μL min 1 could be evaporated, whereas flow rates of up to 1 mL min 1 using air spray devices could be achieved. In a previous paper, it was proved that MALDI-TOF MS is sensitive enough to detect even such low polymer amounts that are available using micro-HPLC instruments.7 For our investigations, a custom-build ESD transfer device was applied. Since its introduction in the midnineties, MALDI-TOF IMS has become an important tool to monitor the 2D and 3D distribution of components in biological tissues.8 In contrast to that, the application of MS imaging techniques for investigating synthetic polymers so far has been limited mainly to secondary ion mass Received: September 8, 2011 Accepted: October 20, 2011 Published: October 22, 2011 9153

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Figure 1. Scheme of the home build electrospray deposition (ESD) interface used for continuous transfer of chromatographic samples onto the MALDI target.

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Figure 2. MALDI-TOF mass spectrum of the original PPO/PEO copolymer (2 mg/mL polymer, 10 mg/mL DCTB, 5 mg/mL KTFA [v/v/v = 1/10/1]).

’ EXPERIMENTAL SECTION

generated by a DC power supplier (FuG Elektronik GmbH, Rosenheim, Germany) and applied to the capillary. Additionally, heated gas could be applied through a series of concentric holes around the capillary to enable a better evaporation of solvents. The deposition flow could be varied from 5 to 20 μL min 1 by means of an adjustable flow splitter ASI-QuickSplit (Analytic Scientific Instruments, Richmond, CA). The matrix solution (5 10 μL min 1) was added via a t-piece to the eluent line using a microsyringe pump (Harvard Apparatus, Holliston, MA). Sodium trifluoroacetate (NaTFA, 2 mg/mL in THF) was added for a selective ionization (matrix/salt, 10/1, v/v). MALDI-TOF Imaging MS. An Autoflex III MALDI mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) equipped with a 200 Hz Smartbeam laser (355 nm) was used. For the imaging experiments, regions of 0.25  8 cm were selected. The image pixel size was 350  350 μm. Since the laser spot diameter is around 50 μm, 500 single spectra were randomly accumulated at each pixel. The evaluation of recorded spectra was performed using instrument specific FlexImaging software. For the generation of imaging plots, normalized ion intensities were taken.

Sample. The polyethylene oxide (PEO)/polypropylene oxide (PPO) copolymer having an average molecular mass of 2000 g/mol was obtained from Clariant AG, Germany. Liquid Chromatography. For chromatographic separations, a HP 1090 instrument (Hewlett-Packard, USA) running with a mixture of tetrahydrofurane (THF)/water (70/30 v/v) was used. The system was equipped with a reverse phase ODS column (250  4.5 mm, 120 Å, YMC). The temperature was kept at 45 °C. The flow rate was 0.5 mL min 1. The sample concentration was 5 mg/mL. Typically, 20 μL of sample solution was injected. An evaporative light scattering detector Sedex 45 (Sedere, France) was used for detection. Nitrogen gas was used as detector evaporation gas. ElectroSpray Deposition (ESD) Interface. A scheme of the interface is given in Figure 1. The interface consists of a Teflon x y table, which was adapted to the size of conventional 384 MALDI target plates. A small contact was connected to enable a contacting of the target plate to ground potential. The spray capillary (stainless steel, 0.1 mm inner diameter) was fixed in a Teflon block. The distance between target plate and capillary could be varied from 0.5 to 3 cm. A high voltage (3 5 kV) was

’ RESULTS AND DISCUSSION The MALDI-TOF mass spectrum of the original EO-PO copolymer sample is shown in Figure 2. A typical copolymer spectrum characterized by peak-to-peak distances of 14 Da was found. These series are overlaid by an intensive distribution having a peak-to-peak distance of 58 Da. Since potassium trifluoroacetate (KTFA) was added to the matrix, the peak at mass m/z 1799.5 could be attributed to a PPO potassium adduct ion that consists of 30 repeat units (Mtheo = 1799.7 g/mol). Obviously, the copolymer contains a considerable amount of PPO homopolymer probably formed as side product during the polymerization. Since the ionization yield of homo- and copolymers are unknown, quantitative data could not be given. The chromatogram of the EO-PO copolymer is presented in Figure 3. It is characterized by a nonspecific broad peak revealing no significant information neither on the chromatographic behavior nor on the sample composition. From previous experiments, the critical separation conditions of PEO and PPO for the chosen experimental setup (mobile and stationary phases) were known [PPO: THF/water 85/15 (v/v) and PEO: 19/81 (v/v)]. Thus, from our experience at the

spectrometry (SIMS)-TOF.9 The use of MALDI-TOF IMS for monitoring of segregation phenomena in dried droplet MALDI sample spots was reported first by Weidner et al.5,10 Jokinen et al. applied surface assisted laser desorption/ionization (SALDI) imaging MS for generating engineered droplet shapes.11 They observed that not all analytes behaved identically during the deposition process and reported similar to our findings that analyte separation can occur while drying. Since a deeper understanding of mechanisms in copolymer chromatography could be beneficial for a better separation, our aim was to gain deeper insights into separation processes by monitoring the elution of individual copolymer units. Thus, MALDI-TOF imaging MS was applied as a detector to characterize deposited chromatographic runs. The chromatographic separation was combined with electrospray deposition for a homogeneous transfer of samples onto the MALDI target.

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Analytical Chemistry chosen chromatographic conditions, the system was supposed to separate PEO’s in a SEC mode and PPO in adsorption mode. This chromatographic run was transferred onto the MALDI sample plate using the ESD transfer device. The matrix solution (containing NaTFA) was added simultaneously using a syringe pump to the eluents flow. The optical image of the sprayed sample trace (9  83 mm) is shown in Figure 4. As revealed by this picture, a thin and homogeneous sample trace could be obtained. For the imaging experiments, a region of 2.5  80 mm was selected (see Figure 4). PPO Homopolymer. Since PPO homopolymers were detected in the MALDI spectra of the original sample, the initial imaging experiment was focused on monitoring these compounds first. Beginning from the lowest detectable PPO homologue (PPO19),

Figure 3. LAC chromatogram of the PPO/PEO copolymer (250  4.5 mm, 120 Å column, THF/water (70/30 v/v), 20 μL injection volume, evaporative light scattering detector [ELSD]).

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the ion intensities of all PPO species (up to PPO47) were monitored. The result is shown in Figure 5a, which consists of a stack of single images. With an increase in the number of repeat units, the spots are continuously shifted from left to right, which can be related to higher retention times in the chromatogram. By a correlation of retention times to target positions, these data could be easily transformed into a conventional calibration curve, which is presented in Figure 5b. This result confirmed our assumption that at the chosen chromatographic separation conditions PPO homopolymers are predominantly separated according to adsorption-based interactions. In contrast to that, the mechanism of copolymer separation is much more complicated since two structural components (PEO and PPO) are combined in polymer chains having different building block sequences and molecular masses. However, similar to the previous experiment, MALDI-TOF imaging MS can be applied to monitor the ion intensity of a single copolymer as well. In order to change only one parameter, for the following experiments, either the number of PPO or PEO were kept constant. Copolymers with Constant PEO Numbers. Copolymers with a constant number of PEO units were chosen for these investigations. Surprisingly, our first ion intensity plots showed the presence of two or even more spots. This can be attributed to the large variety of different copolymer compositions having contiguous masses and, therefore, overlapping isotope patterns. A detailed explanation is given by Figure 6. In this figure, the ion intensities of four different ions each differing by ∼2 Da (or 3 PO and +4 EO units) are displayed. It shows that depending on the mass of the selected ion different spot patterns were obtained. For example, PPO40PEO2Na+ with a monoisotopic mass of 2450.73 g/mol shows only one spot. An overlapping with another copolymer isotope distribution was not observed. This could be explained by the fact that interfering with copolymers with negative

Figure 4. Optical image (composite of eight single photographs) of the sprayed sample trace overlaid by a frame indicating the imaged region (80 mm  2.5 mm, spot size 350  350 μm, ca. 2.800 spectra each of 500 shots).

Figure 5. (a) Set of ion intensity plots of PPO homopolymers (from homologue No 19 to 47) and (b) transformed data used for PPO calibration. 9155

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Figure 6. Overlay of isotope patterns of four copolymers with different composition (each differing by two dalton) and its effects on measured ion intensity plots.

Figure 7. (a) Set of ion intensity plots of PPO/PEO copolymers (having constant PEO and varying PPO numbers) and (b) transformed data used for specific calibration.

numbers of PEO units (in this case, PPO43PEO 2Na+) is simply not possible. The mass of its third isotope (2452.73 g/mol) completely overlaps with the monoisotopic mass of PPO37PEO6Na+ which is 2452.71 g/mol. Thus, monitoring of PPO37PEO6Na+ results in two spots, since PPO40PEO2Na+ (its third isotope) is also detected. The situation is even more complicated for PPO34PEO10Na+ having a monoisotopic mass of 2454.68 g/mol. It is monitoring results in three spots that are characteristic for PPO34PEO10Na+ (first isotope), PPO37PEO6Na+ (third isotope), and PPO40PEO2Na+ (fifth isotope). As expressed by the figures, measured ion intensities do reflect the intensities of the corresponding isotopes very well. This, by the first view confusing information, turned out to be very helpful because now different copolymer series could be monitored simultaneously. It also becomes clear that imaging of particular copolymer series can only be used for determining their position in the corresponding

sample trace, since the majority of copolymer ion plots represents an overlay of ion intensities of different compositions (shown in Figure 6). This is in contrast to homopolymers, where an imaging of ion intensities of homologue’s results in only one characteristic spot. Since our intention, however, was to get some information on the retention behavior in order to establish compound specific calibration curves, intensity values were not needed. As one of the most abundant PEO/PPO copolymers, a series with 10 PEO units was found in the mass spectra. The ion intensity plots of these copolymers (PEO10/PPOx, with x from 20 to 36) are arranged in Figure 7a. As mentioned before, series of copolymers having masses of plus/minus 2 Da could also become visible. These series are characterized by the homologue’s PEO6/PPOx+3 ( 2 Da) and PEO14/PPOx 3 (+2 Da). Thus, monitoring of copolymer series with 10 PEO units automatically 9156

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Figure 8. (a) Set of ion intensity plots of PPO/PEO copolymers (having constant PPO and varying PEO numbers) and (b) transformed data used for specific calibration.

Figure 9. (a) Set of ion intensity plots of PPO/PEO copolymers (having a constant number of PPO and PEO but varying PPO/PEO ratio) and (b) transformed data used for specific calibration.

results in displaying of PEO 6 and PEO 14 copolymer series as well. Figure 7a also reveals that all spot series slightly differ from each other. With an increase in the number of PPO units, the distances between PEO 6, PEO 10, and PEO 14 series increase. This becomes more obvious after transforming the ion intensities in retention times, which were used for generating the calibration plot shown in Figure 7b. As shown in Figure 5b, the elution of pure PPO homopolymers (PEO 0) proceeds in strong adsorption mode. With an increase in the PEO amount (from PEO 6 to PEO 10), the calibration curves reveal a less pronounced adsorption behavior of the copolymers. Copolymers having 14 PEO units elute independently of the number of PPO units at one retention time. From theory, this can be regarded as the so-called “critical” point of adsorption for PPO having an “end group” of 14 PEO units.12 A similar data evaluation was performed for copolymers having a constant number of PPO units. Copolymers with Constant PPO Numbers. For a better overview, an abundant series of copolymers having a constant number of 33 PPO units was chosen. Again, two additional series differing by 2 Da (PPO 30) and 2 Da (PPO 36) became additionally visible. This can be seen in Figure 8a. The characteristics

of these three curves are completely different from those with constant numbers of PEO units previously shown in Figure 7a. Consequently, the calibration curves shown in Figure 8b also reveal a different shape. Starting from PPO homopolymers (PPO30PEO0, PPO33PEO0, and PPO36PEO0), an increasing amount of PEO leads to calibration curves that clearly reveal a SEC behavior. Copolymers with higher mass elute first, whereas smaller molecules with lower molecular masses elute at higher retention times. Interestingly, the retention times for those spots that could be attributed to PPO homopolymers (PPO30PEO0, PPO33PEO0, and PPO36PEO0) perfectly fit to retention times obtained for the pure PPO, shown in Figure 5a. This confirms our finding that multiple spots representing copolymers with different compositions can be found in one ion intensity plot. Copolymers with Constant Numbers of Monomer Units. In contrast to the previously shown data, where the number of monomer units of the copolymers had increased successively by one unit, this data treatment was performed by keeping the number of structural (PEO and PPO) units constant. A successive replacement of PPO by PEO units changes the molecular masses of the copolymers by only 14 Da. Nevertheless, this substitution should be chromatographically visible by its “chemical shift”. 9157

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Analytical Chemistry Ion intensity plots of different series of these copolymers are presented in Figure 9a. The corresponding calibration curves are shown in Figure 9b. As expressed by these plots, a slight variation of the composition of copolymers (having nearly constant masses, expressed by nearly horizontal calibration curves) can, however, drastically change the retention time of the copolymers. Thus, PPO38 (∼2245 g/mol) elutes at approximately 10.4 mL, whereas PPO32PEO6 (∼2162 g/mol, i.e., 3.7%) reveals a retention time at 6.4 mL.

’ CONCLUSIONS For the first time, MALDI-TOF imaging mass spectrometry has been applied as a detector for copolymer chromatography. Using this technique, the simultaneous detection of discrete copolymer series in a chromatographic run was achieved. Moreover, the individual retention behavior of single structural units and changes of the copolymer composition could be easily monitored. Our results could be important for a better understanding of complex chromatographic separation processes and enable a “fine-tuning” of copolymer chromatography. Combined with electrospray deposition, a prerequisite for homogeneous sample traces, MALDI-TOF spectra with excellent spatial and peak resolution could be recorded.

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18, 1139–1146. (d) Lou, X. W.; van Dongen, J. L. J. J. Mass Spectrom. 2000, 35, 1308–1312. (7) Falkenhagen, J.; Weidner, S. M. Rapid Commun. Mass Spectrom. 2005, 19, 3724–3730. (8) Seeley, E. H.; Caprioli, R. M. Trends Biotechnol 2011, 29, 136–143. (9) (a) Hanton, S. D.; Clark, P. A. C.; Owens, K. G. J. Am. Soc. Mass Spectrom. 1999, 10, 104–111. (b) McDonnell, L. A.; Mize, T. H.; Luxembourg, S. L.; Koster, S.; Eijkel, G. B.; Verpoorte, E.; de Rooij, N. F.; Heeren, R. M. A. Anal. Chem. 2003, 75, 4373–4381. (c) Quirk, R. A.; Briggs, D.; Davies, M. C.; Tendler, S. J. B.; Shakesheff, K. M. Surf. Interface Anal. 2001, 31, 46–50. (d) Miyasaka, T.; Ikemoto, T.; Kohno, T. Appl. Surf. Sci. 2008, 255, 1576–1579. (e) Artyushkova, K.; Fulghum, J. E. J. Electron Spectrosc. 2005, 149, 51–60. (f) Belu, A. M.; Graham, D. J.; Castner, D. G. Biomaterials 2003, 24, 3635–3653. (10) Weidner, S.; Knappe, P.; Panne, U. Anal. Bioanal. Chem. 2011, 401, 127–134. (11) Jokinen, V.; Franssila, S.; Baumann, M. Microfluid. Nanofluid. 2011, 11, 145–156. (12) Gorshkov, A. V.; Much, H.; Becker, H.; Pasch, H.; Evreinov, V. V.; Entelis, S. G. J. Chromatogr. 1990, 523, 91–102.

’ AUTHOR INFORMATION Corresponding Author

*Address: BAM 1.35, D-12489 Berlin, Richard-Willstaetter-Strasse 11, Germany. Phone: +493081041633. Fax: +493081041137. E-mail: steff[email protected].

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