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Comprehensive Characterization of Grafted Expanded Poly(tetrafluoroethylene) for Medical Applications )
Adrienne F. Chandler-Temple,†, Edeline Wentrup-Byrne,‡ Hans J. Griesser,§ Marek Jasieniak,§ Andrew K. Whittaker, and Lisbeth Grøndahl*,† School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia Q 4072, Australia, ‡ Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Avenue, Kelvin Grove, QLD 4059, Australia, § Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia, and Centre for Advanced Imaging and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia Q 4072, Australia )
†
Received March 16, 2010. Revised Manuscript Received June 22, 2010 Successful implantation of any biomaterial depends on its mechanical, architectural, and surface properties. Materials with good bulk properties seldom possess the appropriate surface characteristics required for good biointegration. The present study investigates the results of surface modification of a highly porous, fully fluorinated polymeric substrate, expanded poly(tetrafluoroethylene) (ePTFE), with a view to improving the surface bioactivity and hence ultimately its biointegration. Modification involved gamma irradiation-induced graft copolymerization with the monomers monoacryloxyethyl phosphate (MAEP) and methacryloxyethyl phosphate (MOEP) in various solvent systems (water, methanol, methyl ethyl ketone, and mixtures thereof). In order to determine the penetration depth of the graft copolymer into the pores and/or the bulk of the ePTFE membranes, angle-dependent X-ray photoelectron spectroscopy (XPS) and magnetic resonance imaging (MRI) were used. It was found that the penetration depth was critically affected by the choice of monomer and solvent as well as by the technique used to remove dissolved oxygen from the grafting mixture: nitrogen degassing versus vacuum. Difficulties due to the porous nature of the membranes in establishing the lateral position of the graft copolymers were largely overcome by combining data from microattenuated total reflectance Fourier transfer infrared (μ-ATR-FTIR) mapping and time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. Results show that the large variation in graft heterogeneity found between different samples is largely an effect of the underlying substrate and choice of monomer. The results from this study provide the necessary knowledge and experimental data to control both the graft copolymer lateral position and depth of penetration in these porous ePTFE membranes.
Introduction The successful biointegration of implant materials is governed by their surface properties. One problem that can occur if surface properties are not addressed is the formation of a fibrous connective tissue layer around the implant which prevents effective integration and can lead not only to pain but also to loosening of the implant, damage to the local tissue, and the expense and trauma of revision surgery. Hence, researchers are continually investigating surface modification of many classes of materials using a variety of techniques. One technique that has attracted attention is radiation-induced graft polymerization, since it can be used to introduce a variety of functional groups and does not require chemical initiators or catalysts which may cause adverse in vivo responses.1 Many implant materials including tissue engineering scaffolds and facial augmentation membranes are porous, and this creates some specific challenges compared to solid materials. Our research into improving the bone-bonding ability of ePTFE facial membranes initially utilized graft copolymerization of the phosphate-containing monomers MAEP and MOEP onto a thin (1) Ikada, Y. Biomaterials 1994, 15, 725–736. (2) Grøndahl, L.; Cardona, F.; Chiem, K.; Wentrup-Byrne, E. J. Appl. Polym. Sci. 2002, 86, 2550–2556. (3) Grøndahl, L.; Bostrum, T.; Cardona, F.; Chiem, K.; Wentrup-Byrne, E. J. Mater. Sci.: Mater. Med. 2003, 14, 503–510. (4) Suzuki, S.; Grøndahl, L.; Leavesley, D.; Wentrup-Byrne, E. Biomaterials 2005, 26, 5303–5312.
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implant analogue.2-5 More recently, however, we have extended this work to a clinically relevant thicker ePTFE membrane.6 Earlier studies demonstrated that grafting of these phosphatecontaining monomers onto the ePTFE substrate does in fact improve the bioactivity of the membrane. In one study, the controlling experimental parameters were evaluated and the results used to formulate a grafting mechanism.5 In addition, we demonstrated that grafting outcomes can be controlled by judicious choice of the solvent in the graft polymerization process.5 Since our overall goal is to exclusively change the surface properties of these soft tissue-replacement membranes, ideally graft copolymerization should only occur at the outermost surface. It is therefore important to determine the depth of penetration of the copolymer into the pores and/or into the bulk of the substrate. Methods commonly employed to determine depth of penetration include scanning electron microscopy (SEM),7 pseudo confocal Fourier transform infrared spectroscopy (FTIR),8,9 (5) Wentrup-Byrne, E.; Grøndahl, L.; Suzuki, S. Polym. Int. 2005, 54, 1581–1588. (6) Chandler-Temple, A.; Wentup-Byrne, E.; Whittaker, A. K.; Grøndahl, L. J. Appl. Polym. Sci. 2010, 117, 3331–3339. (7) Hegazy, E.-S. A.; Ishigaki, I.; Rabie, A.; Dessouki, A. M.; Okamoto, J. J. Appl. Polym. Sci. 1981, 26, 3871–3883. (8) Cardona, F.; George, G. A.; Hill, D. J. T.; Rasoul, F.; Maeji, J. Macromolecules 2002, 35, 355–364. (9) Yang, P.; Meng, Z.; Zhang, Z.; Jing, B.; Yuan, J.; Yang, W. Anal. Chem. 2005, 77, 1068–1074.
Published on Web 09/02/2010
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Chandler-Temple et al. Table 1. Experimental Parameters and Characterization of Grafted ePTFE Membranes
sample
[monomer] (w/v %)
solvent
graft yield (%) ((1)
grafting extent (%)
ePTFE 0 20 ( 3a MAEP-1-N 10 H2O MAEP-2-N 30 H2O 3 84 ( 10b MAEP-3-N 30 MeOH 6 16 ( 1c MOEP-1-N 10 MEK 12 40 ( 13c MOEP-2-V 10 MEK 8 60 ( 20c MOEP-3-N 20 MEK 26 77 ( 3b MOEP-4-V 20 MEK 19 n.d.d MOEP-5-N 20 MeOH 10 10 ( 9b MOEP-6-V 30 MEK 34 63 ( 3b MOEP-7-N 30 MEK 33 88 ( 10c MOEP-8-N 30 MeOH/MEK 17 33 ( 10c a Number of samples: n = 4. b Number of samples: n = 2. c Number of samples: n = 3. d n.d.: not determined.
Raman spectroscopy,10,11 dye assays,12,13 angle-dependent X-ray photoelectron spectroscopy (XPS),14-16 and confocal laser scanning microscopy.17 SEM, Raman, FTIR, and dye assays all require destructive sample preparation, such as resin embedding or cross-sectioning, which can cause the introduction of artifacts. Cross-sectioning of highly porous substrates, particularly when soft in nature, is also a challenge because this can cause deformation of the sample. It has been previously reported that the polymer crystallinity of semicrystalline polymers can affect the lateral distribution of the graft copolymer and prevent full surface coverage.6,18,19 Since our ultimate goal is to produce a material that is capable of integrating with the hard tissue, this raises the issue of the lateral distribution of the graft-copolymer across the surface. This means that the “patchiness” is just as important as the amount of the graft copolymer. The lateral distribution is commonly assessed by analyzing several positions across the surface and using the data to generate a surface map or image of the distribution of a specific chemical functionality. Both Raman and microattenuated total reflectance FTIR (μ-ATR-FTIR) mapping have been previously used.20,21 In the present study, in addition to traditional characterization methods, both the lateral distribution and penetration depth of the graft copolymer in ePTFE membranes modified by γ-radiationinduced grafting with the phosphate-containing monomers MAEP and MOEP under a range of grafting conditions were comprehensively characterized. The techniques selected all require minimal sample preparation. Angle dependent XPS was used to investigate the outermost sample surfaces, while magnetic resonance imaging (MRI) proved very useful for examining the (10) Cardona, F.; George, G. A.; Hill, D. J. T.; Perera, S. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 3191–3199. (11) Hietala, S.; Paronen, M.; Holmberg, S.; Nasman, J.; Juhanoja, J.; Karjalainene, M.; Serimaa, R.; Toivola, M.; Lehtinen, T.; Parovuori, K.; Sundholm, G.; Ericson, H.; Mattsson, B.; Torell, L.; Sundholm, F. J. Polym. Sci., Part A: Polym. Chem. 1999, 37, 1741–1753. (12) Bisson, I.; Kosinski, M.; Ruault, S.; Gupta, B.; Hilborn, J.; Wurm, F.; Frey, P. Biomaterials 2002, 23, 3149–3158. (13) Grøndahl, L.; Chandler-Temple, A.; Trau, M. Biomacromolecules 2005, 6, 2197–2203. (14) Feng, D.; Chandekar, A.; Whitten, J. E.; Faust, R. J. Macromol. Sci., Part A: Pure Appl. Chem. 2007, 44, 1141–1150. (15) Paynter, R. W.; Roy-Guay, D.; Parent, G.; Menard, M. Surf. Interface Anal. 2007, 39, 445–451. (16) Costello, C. A.; McCarthy, T. J. Macromolecules 1987, 20, 2819–2828. (17) Schmidt, C.; T€opfer, O.; Langhoff, A.; Oppermann, W.; Schmidt-Naake, G. Chem. Mater. 2007, 19, 4277–4282. (18) Gupta, B.; B€uchi, F. N.; Scherer, G. G.; Chapiro, A. Polym. Adv. Technol. 1994, 5, 493–498. (19) Dargaville, T. R.; George, G. A.; Hill, D. J. T.; Whittaker, A. K. Prog. Polym. Sci. 2003, 28, 1355–1376. (20) Keen, I.; Rintoul, L.; Fredericks, P. M. Macromol. Symp. 2002, 184, 287–298. (21) Keen, I.; Rintoul, L.; Fredericks, P. M. Appl. Spectrosc. 2001, 55, 984–991.
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θAdv (deg) 135 ( 6 81 ( 21 136 ( 7 100 ( 14 105 ( 12 81 ( 14 87 ( 7 129 ( 10 72 ( 14 63 ( 4 120 ( 5
degree of grafting for the complete depth of the substrate, albeit at a lower resolution. μ-ATR-FTIR mapping and time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging were employed to investigate the gross lateral distribution across a large sample area as well as the more detailed distribution over 100100 μm areas, respectively.
Experimental Section Materials. Expanded poly(tetrafluorethylene) (ePTFE) was obtained from W. L. Gore & Associates, Inc. (Newark, DE) under the trade name Gore-Tex (thickness 1.0 mm). The membrane porosity is reported to be greater than 70%.22 The crystallinity for the membrane has previously been found to be approximately 59%.6 Methoxyacrylethyl phosphate (MOEP) was supplied by Sigma-Aldrich, and monoacryloxyethyl phosphate (MAEP) was supplied by Polysciences. Grafted samples were prepared as described in the Supporting Information by a method similar to that previously reported.6 The samples are named using monomer and graft condition terms such as “N” and “V” for grafting under nitrogen and vacuum, respectively. For example, the designation MAEP-1-N refers to the sample grafted with a solution of MAEP under nitrogen. Samples as well as the exact solvent and monomer concentrations used are listed in Table 1. Characterization. The overall degree of grafting was obtained gravimetrically as the percentage weight increase of the ePTFE membrane using the following equation: graft yield ð%Þ ¼
wg - wo 100 wo
where wg and wo are the weights of grafted and original ePTFE membranes, respectively. X-ray photoelectron spectroscopy (XPS) analysis were performed on a Kratos Axis Ultra X-ray photoelectron spectrometer using monochromated Al KR radiation (1486.6 eV) at 15 kV and 10 mA (150 W). Sampling depth was up to 10 nm with 63% of the signal being from the top 3 nm. Data was collected in a fixed analyzer transmission mode (FAT): survey scans at 1200-0 eV with 1.0 eV steps at an analyzer pass energy of 160 eV; narrow scans at 0.1 eV steps at an analyzer pass energy of 20 eV. Vision 2 software was used for data acquisition and processing including curve fitting. All binding energies were referenced by setting the highest component of the C 1s peak to 292.48 eV; this component corresponds to carbon in a fluorocarbon environment.23 Component energies, number of peaks, and peak widths (fwhm of 1.0 for all C 1s peaks) were fixed initially, and refinement was carried out only for peak heights. In a final refinement cycle, component energies and peak widths were also refined and these changed by (22) W. L. Gore & Associates. www.goremedical.com (Products - Head and Neck). Website, 2002-2006; accessed, 2006. (23) Beamson, G.; Briggs, D. High Resolution XPS of Organic Polymers. In The Scienta ESCA300 Database; John Wiley & Sons Ltd: West Sussex, 1992.
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less than 1.0%. The XPS grafting extent of the graft copolymer was determined from curve-fitted high resolution narrow scan spectra of the carbon region and calculated using grafting extent ð%Þ ¼
carbonAll - carbonC- F 100% carbonAll
where carbonAll is the atom percent of all C 1s and carbonC-F is the carbon-fluorine component of the C 1s region. Angledependent XPS was performed using stage orientations of φ = 0°, 30°, and 60° from normal, which allow analysis to depths of approximately 10, 6, and 3 nm, respectively. μ-ATR-FTIR mapping was performed using a Nicolet 870 Nexus FTIR spectrometer (64 scans, 4 cm-1 resolution, wavenumber range 400-4000 cm-1) with a Continuμm microscope incorporating an MCT detector and a Nicolet ATR objective equipped with a silicon crystal (refractive index of 3.49 (589 nm)). Spectra were measured from a 2000 2000 μm2 map area with a step size of 100 μm and an aperture size of 100 100 μm2, resulting in areas of approximately 30 30 μm2 being analyzed.24 The dp value varied from 0.205 μm (at 400 cm-1) to 2.098 μm (at 4000 cm-1) for an estimated refractive index of polymer/graft copolymer (ns) of 1.5. Background measurements were taken every 3 min, and all spectra were recorded at ambient temperature. The microscope stage was computer controlled in the x, y, and z directions, and contact between the crystal and sample was automated and monitored via a pressure gauge. Data was collected using OMNIC professional suite software (Thermo Electron Corporation, Waltham, Massachusetts) and processed using GRAMS/32 software (GRAMS/32, Galactic Industries Corporation, Salem, NH). Time-of-flight secondary ion mass spectrometry (ToF-SIMS) analyses were performed using a PHI model TRIFT 2100 (PHI Electronic Ltd.) spectrometer equipped with a 69Ga liquid metal ion gun (LMIG). A 15 kV pulsed primary ion beam was used to desorb and ionize species from the sample surface. Pulsed, low energy electrons were used for charge compensation. Mass axis calibration was done with CH3þ, C2H5þ, and C3H7þ in positive mode and with CH-, C2H-, and Cl- in negative mode of operation. A mass resolution of m/Δm of ∼4500 at nominal m/z = 27 amu (C2H3þ) was typically achieved. Damage to the uppermost monolayer was minimized by applying low primary ion fluxes to ensure that data was obtained from a virtually intact surface; the primary ion fluxes used were between 3 1011 and 6 1011 ions per cm2, meeting the static conditions regime.25 This means that less than 0.1% of the surface atomic sites (1 in 1000) are struck in the time of the measurement (4-5 min). Images were produced from single ions fragments over an analyzed area of 100 100 μm2. Topographical effects were minimized by normalization to total ion emission. Magnetic resonance imaging (MRI), using a Bruker AMX300 spectrometer, was used to visualize the location of the sorbed water in saturated samples. This made it possible to confirm the depth of grafting, since water is only taken up by the graft copolymer and not the hydrophobic ePTFE substrate. Acquisition utilized the standard Bruker spin-echo three-dimensional, SE3D, pulse sequence using a Hahn sequence to generate an echo. This was combined with the phase and read gradients to spatially encode the signal. The three-dimensional images were generated by repeating the experimental slice gradient in increments. Images were acquired using a 90° pulse, duration, 12.5 μs, and echo and repetition times of 4.4 ms and 1.0 s, respectively. The strength of the read gradient was 1.5 Tm-1, and the images consisted of 128 128 8 voxels. All the images have an in-plane resolution of 62.5 62.5 μm and a slice thickness of 2.5 (or 3) mm in a field of (24) Chan, K. L. A.; Kazarian, S. G. Appl. Spectrosc. 2003, 57, 381–389. (25) Briggs, D. Surface Analysis of Polymers by XPS and Static SIMS; Cambridge University Press: Cambridge, U.K., 1998.
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view of 0.8 0.8 cm2. Two averages were coadded, giving a total acquisition time of 35 min. Samples were prepared by cutting to enable them to fit easily into the NMR tube (4 mm 10 mm). The samples were then placed in Milli-Q water overnight to ensure saturation. Excess surface water was removed, and the saturated samples were wrapped in low density polyethylene (LDPE) to ensure that the samples did not dry out and that they fitted securely in the NMR tube. Scanning electron microscopy (SEM) analyses of platinumcoated samples were performed using a Jeol JSM-6460 LA scanning electron microscope. Imaging parameters depended on the age of the filament, on the detector, and on the nature of the sample. Samples with high graft copolymer coverage tended to show less outgassing and appeared less susceptible to beam damage, thus allowing greater optimization of the image. Sessile contact angle measurements were performed using a custom-built apparatus comprising a combined stage and lens assembly fitted with a CCD camera. Images were processed using Scion Image software. Measurements were performed by placing the sample on the backlit Teflon stage. Five microliter aliquots of Milli-Q water were syringed onto the sample surface up to a volume of 20 μL using a 50 μL glass syringe fitted with a stainless steel needle. The needle tip remained in the droplet during the experiment to eliminate vibrations caused by removal of the needle. The effect of the surface free energy of the needle was calculated and found to lie within the standard deviation. The advancing contact angle was calculated using the following equation: tan
θ 2h ¼ 2 d
where h is the height and d is the diameter of the drop. Six areas on each sample were examined (three positions on each side of the sample), and the average of the 5, 10, and 15 μL measurements used. Errors were calculated as the standard deviation of the mean. There are specific challenges associated with contact angle measurements of highly porous materials. See discussion below.
Results and Discussion Characterization of Untreated and Grafted ePTFE Membranes. The importance the surface morphology of implant materials plays in their behavior in vivo has long been recognized and studied.26-28 The highly porous ePTFE membranes have a gross morphology consisting of islands or “nodes” interconnected by fibrils; the nodal diameter and internodal distance averages 10-20 μm, as illustrated in the SEM image shown in Figure 1A. This morphology is generally uniform. However, regions with nodal or fibril congestion occur (Figure 1B), and some islands are only a few micrometers in size. A range of surface morphologies was created as a result of modification involving judicious selection of solvent and using the monomers of choice (Supporting Information Figure S1). Other important characteristics of the grafted membranes are the graft yield, the grafting extent as determined using XPS6, and contact angles (Table 1). Contact angle measurements probe the outermost molecular layer and therefore give a relative evaluation of the surface coverage, while XPS analyses to a depth of ∼10 nm. The graft yield, on the other hand, reflects bulk changes to the substrate. In the current study, there appears to be no benefit to using a mixed solvent system (Table 1). This strongly suggests that methanol has an inhibitory effect on the grafting outcome, as has previously been reported for other grafting systems.29,30 (26) (27) (28) (29) (30)
Schwartz, Z.; Boyan, B. D. J. Cell. Biochem. 1994, 56, 340–347. Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573–1583. Wong, J. Y.; Leach, J. B.; Brown, X. Q. Surf. Sci. 2004, 570, 119–133. Mukherjee, A. K.; Gupta, B. D. J. Appl. Polym. Sci. 1985, 30, 265–2661. Gupta, B.; Saxena, S.; Ray, A. J. Appl. Polym. Sci. 2008, 107, 324–330.
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Figure 1. Scanning electron micrographs of untreated ePTFE showing (A) a normal and (B) an island congested area.
By comparing the information from the techniques listed in Table 1, it is possible to gain useful information, albeit limited, regarding the relative location of the graft copolymer (see the Supporting Information). However, when the differences in the grafting outcomes are subtle (e.g., samples MOEP-1-N vs MOEP-2-V and samples MOEP-6-V vs MOEP-7-N), these techniques prove inadequate for the analyses of the different graft copolymer distributions. In the present study, where comprehensive characterization of the grafted membranes is the goal, we extended the suite of analytical techniques employed to include those additional techniques capable of probing both the depth and lateral distribution of the graft copolymers. Penetration Depth of Graft Copolymer. As well as the influence of solvent, monomer, and monomer concentration on the penetration depth of the graft copolymer, the effect of method of degassing as part of the polymerization process was also investigated. We found that cross-sectioning the soft porous samples was very difficult, and because artifacts were sometimes introduced, we chose techniques which did not require sample preparation, namely, angle-dependent XPS and MRI. In samples with relatively low grafting extent and graft yield, the penetration depth of the graft copolymer was evaluated using angle-dependent XPS because it allows sampling to different depths within the outermost layers of the substrate. However, since quantitative information requires a solid, flat surface, only a comparative measure of the depth of the grafted copolymer was possible where these membranes are both porous and rough. In order to compare the graft penetration between samples where penetration was greater than ∼10 molecular layers, MRI was successfully used to visualize the water bound to the graft copolymer. None of the techniques used here can distinguish between graft copolymer inside the bulk of the substrate or inside the pores. It is, however, possible that the so-called “grafting front mechanism” is promoting grafting into the bulk of the substrate.31 In addition, a grafting-front-like mechanism could be operating by which grafting into the pores in a poorly wetting solvent most likely proceeds (31) Hegazy, E. S. A.; Dessouki, A. M.; Elassy, N. B.; Elsawy, N. M.; Elghaffar, M. A. A. J. Polym. Sci., Part A: Polym. Chem. 1992, 30, 1969–1976.
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Figure 2. Angle-dependent XPS multiplex spectra of samples (A) MOEP-1-N and (B) MOEP-2-V at three different angles relative to the normal angle of analysis: (a) 0°, (b) 30°, and (c) 60°.
by swelling of the initially grafted regions, thereby aiding monomer solution diffusion into the pores as grafting proceeds. It is generally found that the solvent has a large effect on the penetration depth of a graft copolymer into the bulk of a substrate, with solvents capable of swelling the bulk substrate promoting this effect.32-34 In addition, monomer concentration is likely to affect the grafting process into the porous substrate, and it is inferred that higher monomer concentration will penetrate further into a porous substrate during the grafting process, since the graftingfront-like mechanism will be more pronounced unless other factors, as discussed below, contribute. Angle-Dependent XPS Characterization. A comparison was made between two samples, one grafted under vacuum and the other under nitrogen, MOEP-1-N and MOEP-2-V, both of which were grafted in a solution of 10% MOEP in MEK. The multiplex scans between 600 and 100 eV (Figure 2) show the O 1s (532 eV), C 1s (285 eV), and P 2p (131 eV) signals. Spectra labeled (a), (b), (32) Irwan, G. S.; Kuroda, S.; Kubota, H.; Kondo, T. J. Appl. Polym. Sci. 2003, 87, 458–463. (33) van Os, M. T.; Menges, B.; Forch, R.; Knoll, W.; Timmons, R. B.; Vancso, G. J. Thin film plasma deposition of allylamine: Effects of solvent treatment. In Plasma Deposition and Treatment of Polymers; Materials Research Society: Warrendale, PA, 1999. (34) Gupta, B.; Jain, R.; Anjum, N.; Singh, H. Radiat. Phys. Chem. 2006, 75, 161–167.
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Figure 3. MRI contour images of grafted ePTFE samples (A) MOEP-6-N, (B) MOEP-7-N, (C) MAEP-1-N, and (D) MAEP-3-N. The field of view is 0.8 0.8 cm2. The contours represent relative image intensity which is proportional to water content.
and (c) were collected using takeoff angles of 90°, 60°, and 30°, corresponding to sampling depths of approximately 10, 6, and 3 nm, respectively. The carbon regions show two peaks corresponding to carbon in a fluorine environment at high binding energy (C-F) and carbon in an oxygen or hydrogen environment at low binding energy (C-other) corresponding to the ePTFE substrate and the graft copolymer, respectively. For the sample prepared under nitrogen, the multiplex scan indicates some grafting within the top 5-10 molecular layers (Figure 2A(a)) as determined by comparing the C-other and C-F peaks (grafting extent = 40%) and the oxygen and phosphorus peaks. However, as the angle increases and the analysis depth becomes shallower (Figure 2A(b)), it can be seen that there is a higher concentration of C-other relative to C-F (grafting extent = 50%). This increase is also seen in the oxygen and phosphorus signals. With even shallower probing (Figure 2A(c)), it is clear that the C-other peak dominates relative to the C-F peak (grafting extent = 60%). These results suggest that the nitrogen degassing process produces a graft copolymer that is more concentrated on and near the surface (predominant C-other found in the top 5 nm) than within the substrate (predominantly C-F found when monitored to a depth of ∼10 nm). In contrast, for the sample prepared under vacuum (Figure 2B), the graft copolymer (grafting extent = 60%) is uniformly distributed throughout the ∼10 nm depth probed by XPS, as evidenced by the similarity of the multiplex scans at all three emission angles. This suggests that, under vacuum, the monomer solution is drawn into the substrate, allowing monomer to react with substrate radicals within the pores. This results in uniform grafting throughout the sample to a depth of at least 10 nm. Hence, the preparation protocol clearly influences the distribution of the graft copolymer within the top 10 nm of the sample at this relatively low monomer concentration. Substantial grafting to >10 nm is only achieved under vacuum, which enhances Langmuir 2010, 26(19), 15409–15417
monomer diffusion under the relatively poor wetting conditions of the ePTFE membranes by the 10% MOEP/MEK solution. MRI Characterization. Due to the significantly different spin-spin relaxation times, T2, of the protons in the graft copolymer and the penetrant water molecules, the proton signal from the penetrant is selectively measured by MRI. Since PTFE is extremely hydrophobic, water will only penetrate the porous substrate when interacting with the graft copolymer. This provides a method of visualizing the graft copolymer distribution within the ePTFE membrane. The MRI experiments involved spin-echo 3D imaging resulting in contour plots showing the proton density across a slice cross section of the 3-D image. These contour maps are compiled from water concentration profiles of saturated samples taken in increments in the y-direction, the x-direction is the width of the sample (∼1 mm), and the z-direction the water concentration. The samples are positioned such that the outer surface is along the y-axis. The different preparation methods (vacuum and nitrogen) were compared by analyzing the samples MOEP-6-V and MOEP-7-N (Figure 3A and B). The concentration profiles show that the bound water is evenly distributed throughout the thickness of sample MOEP-6-V (Figure 3A). This strongly suggests an even distribution of the graft copolymer throughout the membrane. On the other hand, in sample MOEP-7-N, the water concentration is highest on the exterior surfaces of the membrane and decreases in concentration toward the center (Figure 3B). However, there appears to be some water signal above noise level throughout the membrane. This indicates that although grafting has occurred throughout the membrane, it is more concentrated at the surface. Thus, for both samples, some diffusion of the monomer solution (30% MOEP/MEK) into the ePTFE membrane during the grafting process is inferred. However, it is only when grafting is carried out under vacuum, which further assists in diffusion of the monomer solution into the pores, that an even DOI: 10.1021/la1010677
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distribution of the graft copolymer is seen. Comparing concentrations of 10% (samples examined by angle-dependent XPS) versus 30% for MOEP in MEK, we found that the more concentrated solution gave a greater penetration depth. This can be understood in terms of the grafting-front-like mechanism which will be more pronounced for the 30% solution as described above. Samples MAEP-1-N and MAEP-3-N were both grafted with the acrylate monomer under nitrogen, the first in water and the second in methanol. The contour images for these samples (Figure 3C and D) indicate that water is associated only with the outermost surface and hence this is where grafting exclusively occurs. In contrast, the profile image of the sample grafted with MOEP in methanol under nitrogen (MOEP-5-N) indicates uneven distribution of bound water throughout the membrane. This suggests that grafting has occurred heterogeneously throughout the sample. It can be concluded, therefore, that the nature of the monomer strongly influences the penetration depth of the graft copolymer. In order to rationalize the experimental observations regarding graft copolymer penetration depths for samples prepared in different solvents and using different monomers, a number of aspects of the grafting system need to be taken into account. The diffusion of the monomer solution into the porous substrate depends on the time allowed, the ability of the monomer solution to wet the ePTFE membrane, which in turn depends on both the monomer and the solvent, and the viscosity of the solution. In order to minimize variability, the same amount of time was allowed between sample preparation and irradiation in all experiments. It can be safely predicted that wetting of the ePTFE substrate is likely to be higher for the methacrylate monomer as it is less hydrophilic. In addition, we found that wetting of PTFE by the solvents used in this study follow the order water , methanol < MEK. The relative density of the solvents is water > methanol ∼ MEK. Based on these physical parameters, it is possible to propose the following order for depth of penetration: MAEP(water) < MAEP(MeOH) < MOEP(MeOH) < MOEP(MEK), which agrees well with our experimental observations. Thus, comparing the MAEP-3-N and MOEP-5-N samples grafted in methanol, we found that, in the case of the former, grafting was confined to the outermost surface whereas when MOEP was used grafting penetrated further into the membrane. This can be explained by the higher hydophobicity of MOEP and the lower viscosity of the MOEP/methanol solution. In conclusion, complementary MRI and angle-dependent XPS data enabled us to estimate the depth of penetration of the graft copolymer either at the surface or into the bulk of the sample. This information makes it possible to optimize the grafting conditions in order to fulfill the aim of exclusive surface grafting where desired. This can now be achieved by grafting using MAEP or low MOEP concentrations, and using nitrogen degassing. Surface Coverage of the Graft Copolymer. Previously, we have used the grafting extent to evaluate the surface coverage of the graft copolymer.2,4-6 However, as we recently pointed out, this is not always the best measure of this parameter since XPS probes to a depth of ∼10 nm. The “apparent” contact angle of a porous membrane cannot be interpreted as the exact contact angle the liquid would make with a single fiber of the material, and hence a thermodynamic interpretation of such “apparent” contact angles is futile.35 However, the contact angles obtained do (35) Spelt, J. K.; Vargha-Butler, E. I. Contact Angle and Liquid Surface Tension Measurements: General Procedures and Techniques. In Apparent and microscopic contact angles; Drelich, J., Laskowski, J. S., Mittal, K. L., Eds.; Utrecht, Boston, 2000.
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give a relative measure of the surface coverage of the top molecular layer.6 Indeed, a comparison of samples MOEP-1-N and MOEP-2-V reveals that, although they have similar apparent contact angle values, the grafting extent is significantly different (40 and 60%, respectively). A comparison of the angle-dependent XPS data obtained at an angle of 60° to normal, which probes to a depth of ∼3 nm, shows comparable values for the grafting extent (Figure 2). Thus, if the probing angle in XPS is shallow, we can confidently state that the grafting extent is a good measure of the surface coverage of the graft copolymer. However, neither contact angle measurements nor grafting extent gives any information regarding the lateral distribution of the graft copolymer. In order to assess this, it was necessary to use imaging techniques, and hence, ToF-SIMS and μ-ATR-FTIR mapping were used. Lateral Distribution of the Graft Copolymers. MicroATR-FTIR is a technique often used to assess the lateral distribution of chemical functionalities. It probes at a resolution of 30 30 μm to a depth of approximately 1-2 μm and therefore gives information regarding the gross heterogeneity. ToF-SIMS in the imaging mode allows a 100 100 μm area to be imaged at a resolution of ∼120 nm with a probe depth of 1 nm. This method is therefore capable of giving information about the lateral distribution of the graft copolymer at the outermost molecular layers of the membrane and at very high resolution. The combined data from these techniques, therefore, gives a comprehensive view of the lateral distribution of the graft copolymers. Micro-ATR-FTIR Mapping. The highly porous nature of these membranes creates characterization challenges with respect to several common techniques including μ-ATR-FTIR. Maps of 2000 2000 μm were compiled from 20 20 FTIR spectra for samples MOEP-3-N and MOEP-5-N and a 2000 1500 μm map from 20 15 FTIR spectra for MAEP-3-N. In order to determine the areas of surface maps that represent good contact with the ATR crystal, the average intensity of the 1800-750 cm-1 spectral region was plotted (Figure 4A, D, and G). These maps of the whole spectral region show intensities of both the ePTFE substrate and the graft copolymer vibrations (Supporting Information Figure S2). From consideration of the low and high intensity regions (blue and red, respectively), it can be clearly seen that both MOEP-3-N and MOEP-5-N have large areas of poor or no contact with the ATR crystal, mainly but not confined to the edge of the samples. For MAEP-3-N, poor crystal contact throughout the sample is very pronounced. The presence of such areas of little or no contact with the ATR crystal can arise due to the soft and porous nature of the ePTFE membrane and clearly complicates the use of μ-ATR-FTIR mapping for such materials. It is, however, possible, by comparing maps of specific vibrations with the total spectral region maps, to assess to some degree the gross lateral graft copolymer distribution. It should be noted that, when mapping the intensity of one of the strong C-F vibrations36 (1146 cm-1, Figure 4B, E, and H) for the same total area as the total spectral region maps, there is some overlap between this C-F stretching vibration and the P-O-C and C-O-C stretching vibrations. Thus, in areas of contact with the ATR crystal, some intensity is expected irrespective of the graft density. Mapping the intensity of the CdO vibration (1721 cm-1,37 Figure 4C, F, and I) for the exact same area as the previous two maps identifies regions of high graft density as the CdO band arises from the graft copolymer. (36) Liang, C. Y.; Krimm, S. J. Chem. Phys. 1956, 25, 563–571. (37) Lin-Vien, D.; Colthup, N. B.; Fateley, W.; Grasselli, J. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press Limited: London, 1991.
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Figure 4. μ-ATR-FTIR map of samples MOEP-3-N (A-C), MOEP-5-N (D-F), and MAEP-3-N (G-I). Maps were produced using the
1800-750 cm-1 spectral region (A, D, G), the C-F vibrational mode at 1146 cm-1 (B, E, H), and the CdO vibrational mode at 1721 cm-1 (C, F, I). Red and blue signify high and low relative intensities, respectively.
For MOEP-3-N, there is clearly a region (central in x-direction, lower half in y-direction) of generally much higher CdO intensity than total intensity in the total region map, yet this area correlates well to areas with good crystal contact. Even within this general region, some 100 100 μm areas have higher C-F than CdO intensities. Outside this region, there is a reasonably good correlation between areas of good crystal contact (Figure 4A) and high C-F intensities (Figure 4B). In the MOEP-5-N and MAEP-3-N CdO maps (Figures 4F and I), only very few isolated areas show high intensities while the C-F maps (Figures 4E and H) have more areas of high intensity correlating with good contact. Thus, on the whole for these two samples, relatively higher intensities are observed in the C-F maps, indicating little or no grafting for most of the regions with good crystal contact. Combining information for all three samples, this strongly suggests that sample MOEP-3-N has a higher graft density than either sample MOEP-5-N or MAEP-3-N, in good agreement with both the graft yields (a bulk measure) and the grafting extent (Table 1). An interesting feature of the grafted membranes, as seen in the μ-ATR-FTIR maps, is the fact that on a gross level at least grafting is heterogeneous. Thus, in MOEP-3-N, there are some areas where high crystal contact corresponds to high C-F vibrations while others correspond to CdO vibrations. Similarly, for MOEP-5-N and MAEP-3-N, there is clear evidence that Langmuir 2010, 26(19), 15409–15417
grafting is heterogeneous when examining the distribution of the high intensity areas of the CdO and C-F maps. A similar heterogeneous grafting effect was reported previously for Sumitomo ePTFE membranes grafted with MAEP in water.3 Taking into account the microstructure of the ePTFE membranes of the current study and the size of the island regions (10 μm, Figure 1A), any effect of the substrate morphology cannot be resolved using FTIR maps because the resolution is only 30 30 μm. Hence, a technique with much higher resolution is required in order to resolve the graft distribution in terms of these structural features. ToF-SIMS Imaging. ToF-SIMS analysis of both untreated and grafted ePTFE membranes gave distinct spectra in both the positive and negative ion mode (Table 2). In the case of the untreated membranes, in addition to ions corresponding to the C-F backbone, minimal levels of OH- and O- were observed. Oxidation of the chemically inert ePTFE substrate is known to be minimal, and trace oxygen is sometimes also evident in XPS spectra. The negative ion mass spectrum of MOEP-3-N (Table 2) contains ions not only relating to the ePTFE substrate, but also significant intensities for oxygen, hydrocarbon, and phosphorus species, all originating from the graft copolymer. ToF-SIMS imaging with a spatial resolution of ∼120 nm was used to examine a surface area of 200 μm2. Individual ions were imaged across a surface. The F- and PO3- fragments were chosen DOI: 10.1021/la1010677
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Chandler-Temple et al. Table 2. Main Positive and Negative Ion Fragments Observed from Samples ePTFE and MOEP-3-N
sample
positive fragments
negative fragments
ePFTE MOEP-3-N
Cþ, CFþ, CF3þ, C3F3þ, C2F4þ, C3F5þ Cþ, C2H3þ, Pþ/CFþ, C3H5þ, CF3þ, C3F3þ, C2F4þ, C3F5þ
C-, FC-, O-, OH-, F-, C2H-, PO2-, PO3-
Figure 5. Selected negative ion ToF-SIMS images; bright field regions show dominant elemental species present on surface. Scale bar is 10 μm
on all images. (A) F- fragment of untreated ePTFE; (B) F- fragment of sample MOEP-3-N; (C) F- fragment of a different area of sample MOEP-3-N; (D) OH- fragment of untreated ePTFE; (E) PO3- fragment of sample MOEP-3-N, same area as (B); (F) PO3- fragment of sample MOEP-3-N, same area as (C); (G) F- fragment of sample MOEP-4-V; (H) F- fragment of sample MOEP-5-N; (I) F- fragment of samples MAEP-3-N; (J) PO3- fragment of sample MOEP-4-V; (K) PO3- fragment of sample MOEP-5-N; (L) PO3- fragment of samples MAEP-3-N.
for mapping due to their high intensities. The F- ion was used to image the presence of the exposed fluoropolymer substrate, while the PO3- fragment was used to image the graft copolymer. 15416 DOI: 10.1021/la1010677
As expected, the F- ion ToF-SIMS image of the ePTFE membrane (Figure 5A) shows a uniform distribution across the surface. For sample MOEP-3-N (Figure 5B and E), in contrast, Langmuir 2010, 26(19), 15409–15417
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the F- and PO3- ion images reveal confined areas with exclusive emission of F- ions, surrounded by areas exclusively emitting PO3- ions. This grafting topography is also observed for different parts of the same sample (Figure 5C and F). The confined areas range in size from 5 to 20 μm in diameter identical to the island regions observed in the SEM images (Figure 1). This strongly suggests that the F- ion is associated with the island/nodal regions whereas the graft copolymer is residing on the interdispersed fibrillar areas. Whether this effect is due to differences in the crystallinity of the island/nodal and fibrillar regions or is in fact due to the much higher surface area of the fibrillar regions is not clear. Upon closer inspection of the two PO3- MOEP-3-N maps (Figure 5E and F), it can be seen that the latter has a higher area covered by the graft copolymer; that is, the graft density is higher. These variations in graft density between different areas of the same sample can thus be related to the morphological inhomogeneity of the underlying ePTFE substrate (Figure 1). Combining this data with the μ-ATR-FTIR mapping data (Figure 4A-C), where heterogeneity was observed on a much larger scale (mapping areas of 3030 μm), it is now possible to explain our results in terms of the underlying substrate morphology and the observed preference for grafting in the fibrillar region. Comparing the ToF-SIMS images for samples MOEP-4-V (Figure 5G and J) and MOEP-3-N, fluorine-rich islands surrounded by phosphate-rich nodules are also evident. The only difference between these samples is vacuum versus nitrogen degassing in the grafting procedure. Although the MOEP-4-V images represent a more congested area of the underlying substrate, which makes it somewhat less easy to distinguish between island and fibril areas, it is still clear that the PO3- ions are associated with the fibril area. This data indicates that, regardless of the preparation technique, MOEP samples grafted in MEK produce a heterogeneous graft copolymer selectively on the fibril regions of the substrate. For the sample grafted with MOEP in MeOH (MOEP-5-N), the PO3- ion signals are again distributed around the islands (Figure 5K). However, in contrast to the corresponding samples grafted in MEK, sample MOEP-5-N reveals F- ion emission covering the entire sampling area (Figure 5H). This is evidence of a much lower graft density (surface coverage), since signals from the underlying substrate are observed even in regions where (a low amount of) grafting has occurred. From the ToF-SIMS image for the MAEP-3-N sample grafted in MeOH (Figure 5I and L), it is difficult to determine the morphology of the underlying substrate, which appears to be a fibril-congested region with ridges of islands. Even though the darkest regions of the phosphate image (Figure 5L) correspond well to the brightest regions of the F- ion image (Figure 5I), F- and PO3- ions are visible across the entire surface, indicating that grafting has occurred on both on islands and fibril areas, albeit predominantly in the fibril area as noted previously. It can be concluded that although the nature of the grafting on MAEP-3-N is more homogeneous than that observed for MOEP-5-N (mainly fibrillar), the graft densities in both cases are low. Throughout this discussion, we have used the distribution and intensity of PO3- ions as an indication of the distribution of the graft copolymer and were able to correlate this to the μ-ATRFTIR mapping. In addition, we used the distribution of the Fion signal as an indication of the graft density or surface coverage of the outermost layer of the substrate. The latter should therefore
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correlate with the contact angle values for these samples and indeed this is what we find. Samples MOEP-3-N and MOEP-4-V displayed (relatively) high graft densities; their “apparent” contact angle values were also very similar (81° and 87°, respectively). On the other hand, those samples with (relatively) low graft densities, that is, samples MOEP-5-N and MAEP-3-N, also gave similar, but significantly higher, contact angle values of 129° and 136°. Thus, the judicious choice of characterization techniques made it possible to obtain the relevant data and overcome the issue of a highly porous material.
Conclusion Comprehensive characterization of grafted ePTFE membranes was achieved using a suite of techniques. Of particular importance was the penetration depth of the graft copolymer, which was assessed using angle-dependent XPS and MRI as well as the lateral distribution using μ-ATR-FTIR mapping and ToF-SIMS imaging. It was established that the penetration depth of the graft copolymer into the porous substrate was affected by the monomer, monomer concentration, and solvent (correlating with solution wettabilities) as well as by the sample preparation technique (nitrogen degassing versus vacuum). Using these results, it is now possible to optimize the grafting conditions to produce grafting exclusively on the surface of the membrane (i.e., by using the monomer MAEP in either water or methanol and using nitrogen degassing). Thus, the aims of the present study were achieved. Using ToF-SIMS imaging, it was found that, for all grafting conditions explored, the graft copolymer formed preferentially in the fibrillar regions of the membrane. For samples grafted using MOEP in MEK, this was most pronounced with distinct regions of either the graft copolymer or the ePTFE substrate being detected. Samples prepared under other grafting conditions showed less pronounced segregation of the chemical groups, and for samples grafted using MAEP in methanol some graft copolymer was detected in all regions of the membrane. Since the underlying ePTFE substrate is heterogeneous on the micrometer scale, this leads to a heterogeneous graft distribution on a much larger scale than imaged by ToF-SIMS, and this was observed using μ-ATR-FTIR mapping. In conclusion, this study provides the necessary knowledge and experimental data to control the lateral distribution as well as the penetration depth of the phosphate containing graft copolymer and hence opens up the commercial possibility of controlled production of bioactive ePTFE to be used as a medical implant material. Acknowledgment. Part of this work was funded by a School of Molecular and Microbial Sciences (UQ) Chemistry Alumni Fellowship (A.F.C.-T.). The authors thank Dr. Llew Rintoul, Coordinator of Vibrational Spectroscopy at Queensland University of Technology, and Dr. Barry Wood from the Centre for Microscopy and Microanalysis at the University of Queensland for their assistance with infrared and X-ray photoelectron spectroscopy, respectively. Supporting Information Available: Graft copolymer description and two figures. Figure S1: SEM images of grafted membranes. Figure S2: ATR-FTIR spectra of a selection of the graft copolymers. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la1010677
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