A modified thermal deposition unit for gel-permeation ... - SAGE Journals

E. Peter Maziarz and X. Michael Liu, Eur. J. Mass Spectrom. 8, 397–401 (2002)

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A Modified Thermal Deposition Unit for MS Analysis E. Peter Maziarz and X. Michael Liu, Eur. J. Mass Spectrom. 8, 397–401 (2002)

A modified thermal deposition unit for gel-permeation chromatography with matrix-assisted laser desorption/ionization and electrospray ionization time-of-flight analysis E. Peter Maziarz* and X. Michael Liu* Bausch & Lomb Inc., 1400 North Goodman Street, PO Box 450, Rochester, New York 14603, USA. E-mail: [email protected] and [email protected]

We describe a modified commercially available thermal deposition unit that incorporates a post-column mixing cell for gel-permeation chromatography (GPC) effluent and matrix solution prior to continuous deposition onto a matrix-assisted laser desorption/ionization (MALDI) sample target. This modification overcomes the shortcomings of using a prefabricated matrix foil. This GPC MALDI method with time-of-flight (ToF) mass spectrometry is demonstrated by the analysis of a poly(dimethylsiloxane) (PDMS) sample used in the production of implantable device materials. Keywords: automated GPC-MALDI MS, polymer characterization, thermal co-deposition, automation, post-column mixing

Introduction Due to the narrow limits of specification required for implantable device polymers, it is necessary to utilize analytical techniques that provide detailed information of the oligomers within the molecular-weight distribution. The combination of gel-permeation chromatography (GPC) and matrix-assisted laser desorption/ionization (MALDI) timeof-flight (ToF) mass spectrometry provides detailed information of this kind, including absolute average mass values, repeat unit sequence, end group chemistry, and level of impurities for polymer samples.1 During the polymer development process it is often necessary to evaluate samples from iterated polymer synthesis rapidly. For this reason, it is beneficial to use time-saving techniques such as an automated thermal deposition unit that places the GPC effluent directly on to a MALDI sample plate for subsequent mass spectrometric analysis.2-7 This automated fraction deposition is much less labor-intensive than the traditional manual fraction collection process. For the commercially available deposition units it is recommended that a prefabricated metal foil, containing embedded matrix, be fixed to the MALDI sample target. One major shortcoming of this experimental method is the limited number of different matrix foils available for use. Prefabricated foils also preclude the possibility of tuning the

DOI: 10.1255/ejms.500

matrix composition for optimal signal performance. Additionally there is no possibility for using custom matrix cocktails that may include new candidate matrix molecules, charging agents or mixtures thereof. These limitations are especially a concern when evaluating a polymer material that requires an uncommon matrix or charge agent. Discussion For the analysis reported here, we modified a commercially available thermal deposition instrument by incorporating a post-column mixing cell for GPC effluent and matrix solution prior to deposition onto the MALDI sample target [Figure 1(a)]. This modification precludes the need for a prefabricated matrix foil and thus overcomes its associated limitations. Figure 1(b) illustrates a GPC chromatogram of a custom poly(dimethylsiloxane) (PDMS) material used as a prepolymer for production of oxygen-permeable implantable devices. Figure 1(c) illustrates an image of the MALDI sample target with the continuously deposited GPC effluent/matrix mixture. Deposition starts with the highest mass oligomers near sample well 32 followed by increasingly lower mass oligomers to sample well 79 on the MALDI sample target. Figure 2 illustrates MALDI-ToF mass spectra from several positions along the MALDI sample target.

ISSN 1356-1049

© IM Publications 2002

398

A Modified Thermal Deposition Unit for MS Analysis

Figure 1. A modified thermal deposition unit for GPC MALDI-ToF and ESI-ToF analysis: (a) diagram of the thermal spray nozzle and MALDI sample target of the LC-Transform; (b) differential refractive index chromatogram of the PDMS sample; (c) digital image of the GPC-fractionated sample/matrix co-deposited on a MALDI target.

Figure 2. MALDI-ToF mass spectra (a)-(j) recorded at different GPC elution times for the PDMS sample.


From the track speed of the x-y motor [Figure 1(a)] it is calculated that each sample well (2 mm diameter) on the MALDI target represents approximately 11 seconds of elution time. Although it should be noted that we are not limited to the analysis of only a sample well as the matrix/sample deposit is continuous. The oligomeric peaks from each sampled position are narrowly dispersed so that average-mass values (Mn and Mw) can be determined, without mass discrimination effects, for selected GPC elution times. With this data, the original GPC trace can be calibrated to obtain absolute average-mass values for the polymer material.3 With this approach, absolute average-mass values of Mn and Mw were calculated as 5080 and 7210, respectively, for the PDMS polymer evaluated in this report. Qualitatively, sodiated PDMS oligomer ions are observed within the mass range of 1800 to 14000 Da. This is in contrast to the direct MALDI-ToF analysis of this material, which reported oligomers up to only 4000 Da (data not shown). Thus, incorporation of GPC allows us to examine the high-mass range of the molecular-weight distribution (MWD). This is important for us because we have observed for some PDMS polymers that the intensities of impurity peaks, with respect to one another, change as a function of mass throughout the molecular weight distribution.7,8 The insets in Figure 2 illustrate several expanded views through-

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out the MWD. Within each inset there is an intense distribution of peaks (labeled with a filled circle) differing by 74 Da. This confirms the repeat unit as dimethylsiloxane (C2H6SiO). From evaluation of the peaks in the filled-circle distribution it was determined that these represent the target polymer containing the expected end groups (proprietary information). A second, very minor distribution of peaks (labeled with a filled diamond) is observed in the inset from Figure 2(a). This second distribution of peaks also has a repeat unit mass of 74 Da. It was determined that these peaks represent PDMS oligomers containing one of the expected end groups together with another end group believed to originate from an unexpected side reaction during synthesis. This very minor impurity is observed throughout the mass distribution. However, due to a decrease in resolution with increasing mass, this minor distribution of peaks blends in with the major distribution of peaks [Figure 2(f) inset]. We do not observe any high-mass cyclized or branched oligomers in this sample which potentially occur in many polysiloxane-based products.9 We have realized an added advantage to preparing the fractionated polymer as illustrated in Figure 1(c). After MALDI analysis, most of the fractionated polymer sample remains unconsumed. Therefore it is possible, as we illustrate in Figure 3, to dissolve excised sample spots in a suit-

Figure 3. ESI-ToF mass spectra of excised samples from the sample/matrix co-deposited on the MALDI target.

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able solvent (in this case isopropanol) and analyze them by an alternative ionization technique, namely electrospray ionization (ESI). This advantage at least approaches equality with the one and only advantage of manual fraction collection, (i.e. the availability of plenty of fractionated sample for multiple technique evaluations). Comparatively, the mass of oligomer peaks observed by ESI-ToF mass spectrometry from both excised sample spots in Figure 3 complement those observed by MALDI-ToF MS. Conclusion In conclusion, we feel that, with this rapid and information-rich analytical technique in place, the advantages for implantable-device polymer development are two-fold. First, the rapidly obtained information from the iterated synthesis of a particular polymer material allows us to construct a database of comparative information that can be used to develop the most efficient synthetic route towards achieving the target polymer product. Second, we can use these capabilities to create quality control protocols that establish more detailed molecular specifications for a polymer material. Overall, the motivation here comes from the notion that a more detailed understanding of the product chemistry, and control thereof, results in a reduction of end-product failure rate. Experimental Sample preparation

For GPC experiments, the PDMS sample was dissolved in HPLC-grade tetrahydrofuran (THF) at a concentration of 0.15% (w/v). For MALDI-MS experiments a matrix solution was prepared from 1.5% (w/v) dithranol (Aldrich, St. Louis, MO, USA) doped with sodium nitrate (Aldrich) in THF. For the ESI-MS experiments, the fractionated PDMS/matrix deposit was scraped from the MALDI sample target with a scalpel. The residue was dissolved in 2.0 mL of HPLC-grade isopropanol (IPA) and directly infused into the ESI-ToF MS instrument.

A Modified Thermal Deposition Unit for MS Analysis

Modified LC-Transform

The LC-Transform 500 Series was purchased from Lab Connections, Inc. (Northboro, MA, USA). A separate Waters 515 HPLC pump was used to deliver the dithranol -1 matrix solution at a rate of 0.50 mL min . The GPC effluent and matrix solution were mixed in a Valco Tee prior to the thermal spray nozzle of the LC-Transform. The nozzle temperature was set at 183°C and the sheath gas (house nitrogen) pressure was adjusted to 25 psi. The nozzle height was set at 20 mm above the MALDI sample target. While evaporating most of the solvent, the analyte and matrix were codeposited onto the moving MALDI sample target. Upon finishing the GPC run, the MALDI target was introduced into the MALDI-ToF MS instrument. MALDI-ToF

The MALDI-ToF MS data were obtained with an Applied Biosystems DE-STR ToF mass spectrometer, operating in the linear mode. Ions were formed by laser desorption at 337 nm (N2 laser, 3 ns pulse width, 106 W cm-2, 100 µm diameter spot), acceleration to 25 kV and detection as positive ions. During the ionization process a delay time of 150 ns was applied before acceleration. Additionally, the grid and guide wire voltages were set at 88 % and 0.10 % of the applied acceleration voltage, respectively, to focus the beam of ions. Typically, 256 laser shots were averaged for each spectrum. ESI-ToF

The ESI-ToF MS data were acquired on a Mariner Biospectrometry Workstation (Applied Biosystems) using the turbo-ion source. The spray tip and nozzle potentials were set at 5000 and 125 volts, respectively. Nitrogen drying gas (250°C) was used for desolvation. Ions were accumulated for 1 second before being accelerated into the ToF mass analyzer with 4000 volts. The mass (m/z) range was set at 300 to 6000. All data acquisition and manipulation were controlled by Mariner Instrument Control software (Applied Biosystems). The sample was introduced into the ESI MS instrument using the built-in syringe pump at a flow rate of 35 µL min–1. The mass spectrum reported was the average of 50 scans.

GPC

The GPC system used a Waters Alliance 2690 Separation Module (Waters Corporation, Milford, MA, USA). The separation was performed on a set of two Plgel mixed-E columns (Polymer Laboratories, Amherst, MA, USA). The column dimensions were 300 mm x 7.8 mm and the particle size of the packing material was 3 µm diameter. HPLC-grade THF was used as the mobile phase at a flow rate of 1.0 mL min-1. The column temperature was maintained at 35°C. A Waters 2410 differential refractive index detector was used to monitor the GPC effluent. The internal temperature of the refractive index detector was set at 35°C. Fifty microliters of sample solution was injected into the GPC system.

References 1. 2.

3. 4. 5.

S.D. Hanton, Chem. Rev. 101, 527 (2001). C.E. Kassis, J.M. DeSimone, R.W. Linton, E.E. Remsen, G.W. Lange and R.M. Friedman, Rapid Commun. Mass Spectrom. 11, 1134 (1997). S.D. Hanton and X.M. Liu, Anal. Chem. 72, 4550 (2000). X. Lou and J.L.J. van Dongen, J. Mass Spectrom. 35, 1308 (2000). J. Dwyer and D. Botten, Am. Lab. 28, 51 (1996).


6. 7.

8.

M.W.F. Nielen Anal. Chem. 70, 1563 (1998). E.P. Maziarz, X.M. Liu, G.L. Grobe and J Bonafini, “Mass Spectrometry Analysis of Implantable Devices: Contact Lens Polymers and their Interaction with Proteins”. Proc. 50th ASMS Conf. On Mass Spectrometry & Allied Topics, Orlando, FL, USA, June 2–6, CD Edition (2002). E.P. Maziarz, X.M. Liu, E.T. Quinn, Y.C. Lai, D.M. Ammon and G.L. Grobe, J. Am. Soc. Mass Spectrom. 13, 170 (2002).

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X.M. Liu, E.P. Maziarz, F. Price, D.J. Heiler and G.L. Grobe, Eur. J. Mass Spectrom. 7, 473 (2001). Received: 24 June 2002 Accepted: 25 June 2002 Web Publication: 11 July 2002