Assessing the Nanoscale Structure and Mechanical Properties of

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Assessing the Nanoscale Structure and Mechanical Properties of Polymer Monoliths used for Chromatography Martin Laher, Tim J Causon, Wolfgang Buchberger, Sabine Hild, and Ivo Nischang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac401303k • Publication Date (Web): 28 May 2013 Downloaded from http://pubs.acs.org on May 29, 2013

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Analytical Chemistry

Assessing the Nanoscale Structure and Mechanical Properties of Polymer Monoliths used for Chromatography Martin Lahera, Tim J. Causonb, Wolfgang Buchbergerb, Sabine Hilda, Ivo Nischangc* a

Institute of Polymer Science, Johannes Kepler University Linz, Altenberger Strasse 69, A-

4040 Linz, Austria b

Institute of Analytical Chemistry, Johannes Kepler University Linz, Altenberger Strasse 69,

A-4040 Linz, Austria c

Institute of Polymer Chemistry, Johannes Kepler University Linz, Welser Strasse 42, A-4060

Leonding, Austria *Contact: Institute of Polymer Chemistry, Johannes Kepler University Linz Email: [email protected]; Phone: +43 732 671547 66; Fax: +43 732 671547 62 --------------------------------------------------------------------------------------------------------------------------

Abstract Concerning polymeric monolithic materials utilized in separation science, the structural and mechanical characteristics from the nanoscopic to the macroscopic scale remain of great interest. Suitable analytical tools are urgently required to understand the polymer monolith’s constituent structure, particularly in the case of nanoscale polymer properties that tend to develop gel porosity in contact with a mobile phase ultimately affecting the chromatographic performance. Herein described are our first findings from a characterization of commercially available analytical polymer monoliths based on styrene/divinylbenzene and methacrylate chemistries utilizing confocal Raman spectroscopy imaging and atomic force microscopy (AFM). Confocal Raman spectroscopy can be used to generate a three-dimensional representation of monoliths in both dry state and in contact with solvent. AFM forceindentation measurements on individual cross-sectioned globular features permit detailed assessment of mechanical properties of the stationary phase. This approach allowed so far unprecedented insight and identification of a heterogeneous cross-link density distribution of polymer material within individual globular features on a sub-micrometer scale. Keywords:

atomic

force

microscopy

(AFM),

confocal

Raman

imaging,

chromatography, nanoscale heterogeneity, polymer monolith, stationary phase

1

ACS Paragon Plus Environment

liquid


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Introduction Pioneered in the late 1980s and early 1990s by several authors including Hjertén,1 Tennikova, Svec and Fréchet,2-5 porous polymer monoliths are now widely used and studied in the chromatographic community. Polymer monoliths differ from conventional packed columns in that the mobile phase flows through the pores of a single, interconnected porous (co)polymer network.6 Characterization of the micro- and nanoscale properties of monolithic materials remains a topic of great interest for improving the chromatographic performance of this stationary phase format.7-11 This is with the notion that more informative approaches are urgently required to resolve the nanoscale soft matter polymer structure in contact with a mobile phase on very small length scales.12 Considering the dry state morphology of polymer monoliths, scanning electron microscopy (SEM) continues to be widely used for qualitative characterization of monolithic structures.12, 13 The morphology of polymer monoliths has been probed using a range of techniques other than SEM with varying degrees of resolution. These techniques include transmission electron microscopy imaging (TEM) of monolithic material embedded in epoxy resin which allows microtoming and subsequent reconstruction for statistical macropore space analysis,7 an approach that works as well for serial block face SEM,14 small angle neutron scattering of smeared bulk samples,8 and atomic force microscopy (AFM) utilizing topography imaging of polymer monoliths in dry and solvated states.9 Related studies of materials used in separation science include studying the surface topography of coated capillaries,15 AFM imaging in combination with TEM three-dimensional pore structure reconstruction of beadbased media,16 and testing the smoothness of silica-based stationary phases.17 While the heterogeneities in the macroporous flow-through pore space are wellknown

11, 12, 14, 18

, our current interest concerns resolving the nanoscale structure of porous

polymer monoliths of and within individual globular features. The polymer develops gel porosity in contact with solvent irrespective of the existence of micro- or mesopores in the dry state.12 The overall aim is to resolve the physicochemical properties associated with nanoscale heterogeneities and distribution of suspected cross-linking density.12,

18-20

The

properties of individual globular features may underpin chromatographic performance of polymer monoliths under typical operating conditions.21 However, the previously described characterization

approaches

have

not

yet

provided

resolution

or

indication

of

physicochemical properties at the sub-micrometer scale. For this quest, we believe that AFM is currently the most suitable analytical tool as it has matured since its inception22 into a valuable technique for studying nanoscale properties 2


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of organic polymers in a wide range of samples.23-25 Our approach to this challenge is by using tailored AFM-based mechanical measurements within individual globular features in conjunction with non-invasive Raman imaging. Experimental Materials. Three commercially available analytical scale polymer monolithic columns with a diameter of 4.6 mm were used for this study. These were poly(styrene-codivinylbenzene) columns (Dionex ProSwiftTM RP-1S and RP-3U) having differently sized globular features and flow-through pores as well as one methacrylate-based column with weak cation-exchange properties (Dionex ProSwiftTM WCX-1S). These commercial columns were chosen since they allow an unbiased view for studying the potential of our techniques, are readily available to the scientific community and do not exhibit typical differences from varying experimental procedures used for monolith preparation. Furthermore, these columns have been studied chromatographically in detail.21 Sample Preparation. The sample preparation for confocal Raman spectroscopy and AFM measurements required extrusion of the polymer monoliths from the stainless steel column housing. After flushing with HPLC grade acetonitrile for 2 hours, the end fittings were removed and the columns placed in a vacuum desiccator to dry the monolithic rods. Once dried, the rods were gently pushed out from the column housing and smaller sections were cut with a razor blade to obtain suitable sample sizes. Obtained samples were directly used for confocal Raman imaging in the dry state and in contact with solvents. Further samples from the monolithic rods were prepared in plastic molds with epoxy resin that filled the structures (Figure S1). A detailed description of embedding the samples in epoxy, microtoming, and microscale imaging can be found in the supporting information. Raman and AFM Measurements. A complete description of confocal Raman and AFM instruments as well as additional measurement details and results can be found in the supporting information.

Results and Discussion Confocal Raman Spectroscopy In initial experiments, we pursued reconstruction of a sample of the RP-3U monolith with confocal Raman spectroscopy in the dry state and in contact with either acetonitrile or water. A stack of images calculated from discrete Raman peaks representing monolith, water, or acetonitrile (Figure 1a), allows independent volume reconstructions that show respective distribution of polymer and solvents. Overlaying these individual reconstructions 3



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was then used to compose a three-dimensional representation for polymer in the dry state and in contact with either acetonitrile or water (Figures 1b-d). Figure 1b clearly shows the three-dimensional globular morphology of the dry RP-3U polymer monolith. While water is seen predominantly inside the large micrometer-sized flow-through pores (Figure 1c), acetonitrile is present even within globular features (Figure 1d). This aspect is shown in more detail for an individual slice of the reconstructed volume and example confocal Raman spectra found in the supporting information (Figure S2). These results confirm that the crosslinked polymer contains solvent and becomes permeated to a varying degree by typical mobile phase components. This is a common situation in liquid chromatography in which the variably solvated, cross-linked polymer structure becomes permeated to a varying degree by solutes.21, 26 We suspect that the origin must be found in the cross-linked polymer properties on the nanoscale which was subsequently addressed by tailored AFM measurements. Atomic Force Microscopy To provide a benchmark comparison for AFM measurements, SEM (Figure S3, upper row) and AFM tapping mode phase images of monolithic samples embedded in epoxy resin and microtomed to a flat surface (Figure S3, lower row) of all materials used in this study were acquired at a scale of 20 × 20 µm. The SEM images show substantial differences in porous properties with respect to polymer globule and flow-through pore structure and the AFM phase images the clear distinction between individual globules and the macropore space filled with cured epoxy resin. Figure 2a shows an AFM phase image of the transition region of an individual globular feature of the RP-3U monolith to embedding epoxy resin. The shown area of 1 × 2 µm was subsequently probed by AFM force-indentation measurements to derive nanoscale mechanical properties. For this purpose, the cantilever was moved toward and into the material’s surface until a predetermined force of 500 nN was reached and was then retracted. A plot of the recorded force and indentation signal during approach and withdrawal is shown in Figure 2b. The observed hysteresis between the signal while approaching (bold lines) and withdrawing (thin lines) indicates indentation of the sample. This further provides information about local mechanical properties of polymer material at the probed position. For example, the contact stiffness is given by the slope of a linear fit to the approach curve (see dotted lines) and the indentation depth at the maximum. Lower stiffness and higher indentation depth on positions in the embedding epoxy compared to the cross-sectioned globule show a substantial difference and the consistently softer nature of the epoxy, an observation made for the other materials investigated in this study as well (Figure S4).

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An array of 16 × 32 force-indentation measurements over an area of 1 × 2 µm allows indentation imprints to be seen on globule regions in the phase image (Figure 2a). The stiffness image derived from curves (such as in Figure 2b) is shown in Figure 2c and effectively shows that the polymer monoliths are on average much stiffer than that of the embedding epoxy, together with a broad variation of stiffness on an individual globule scale. To highlight the increasing indentation depth and decreasing stiffness from inner regions of this globule to the surrounding epoxy, Figure 2d provides individual profiles of these properties along the shown diagonal line. Apparent from these figures is a smooth transition from deep regions of the polymer globule to embedding epoxy, demonstrating penetration of the polymer structure by epoxy prepolymer which becomes fixed by the curing process. Furthermore,

the

data

demonstrate

that

the

polymer material

in

its

preserved

physicochemical state has variable mechanical properties on length scales of approximately 1 µm (Figure 2d). For example, a stiffness of approximately 70 N/m was measured in the dense inner regions of the globule, while a stiffness of only 20 N/m was determined at its outer edge. This more than threefold decrease indicates a varying degree of cross-linking and softening of the globules toward its outer regions. Despite the heterogeneity with one material in itself, and the variety of globular feature sizes considering all materials presented in this paper (Figure S4), some generalization of this issue can be made. This is shown for the commercially available WCX1S methacrylate-based monolithic polymer which bears ion-exchange functionality. Focusing on a single microtomed globule cross-section (Figure 3a) shows with extremely high resolution, characterized by a 2 x 2 µm forcemap with 64 x 64 indentations, that variations of polymer properties for this material are apparent over a sub-micrometer length scale. Again, an increase in indentation depth (Figure 3b) and consequently a reduction in stiffness (Figure 3c) toward the outer regions of the globule can be seen. The plastic energy, derived from an individual force-indentation measurement with a typical hysteresis, is represented by the area within the curve. A derived image for plastic energy from all force-indentation curves with an example profile shown in Figure 3d shows the opposite trend to that of stiffness, confirming a possible lower degree of cross-linking at the outer regions of the globule. The generally observable heterogeneity of these materials on a globule scale and suggested cross-link density distribution for both styrene/divinylbenzene and methacrylate based related materials warrants further investigation. Such heterogeneities have been suggested based on the observation of the compositional drift of the polymerization precursor mixture during monolith formation.18, 19 Because of the importance of both partition and adsorption processes in liquid chromatography, this cross-link density distribution has been reasoned to have a significant influence on the chromatographic performance. The 5



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presented results and interpretation are based on a resolution below the radar screen of typically-used imaging techniques. Conclusions Results presented in this study demonstrate the importance of investigating mechanical properties of polymer monoliths at the nanometer-scale to determine variations in cross-linking and other potential heterogeneities. This is due to the wide range of possibilities to adjust properties of polymer monoliths such as the inclusion or surface decoration of nanoparticles,27 and variation in crosslinking chemistry, that all certainly result in different morphologies and chromatographic properties.12, 20 Utility of AFM to assess the structure and mechanical properties of polymer monoliths at the nanoscopic level permits unprecedented insight in studying their constituent material properties on the currently smallest experimentally accessible length scale. Despite the lower resolution afforded by confocal Raman imaging, it provides useful information about solvating properties of different mobile phase compositions, which relate to the performance of polymer monoliths. The demonstrated polymer solvation indicated on the microscale with confocal Raman spectroscopy imaging is recognized together with the indication of a heterogeneous crosslink density distribution. Thus, we believe that the combination of the demonstrated techniques can help to complete the picture on structural and mechanical properties of porous polymer monoliths and may provide guidance for design of novel materials with better understood properties. Acknowledgements The authors thank Dr. Kelly Flook (Thermo Fisher Scientific, Sunnyvale, California) for the provision of the columns used in this work.

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Figure Captions

Figure 1. Example confocal Raman spectra and x-y-z stack overlays of a 40 x 40 x 10 µm RP-3U polymer monolith sample volume in the dry state and in contact with different solvents. (a) Example confocal Raman spectra of a single slice from the image stack highlighting the peak(s) used to derive the structure of (b) dry monolith in greyscale; (c) monolith in contact with water; and (d) monolith solvated with acetonitrile. Images (c) and (d) are polymer (greyscale) superimposed with water or acetonitrile (each in red scale). Increased color intensity for each case corresponds to increased polymer or solvent concentration respectively.

Figure 2. Atomic force microscopy (AFM) data of a RP-3U monolithic sample at the transition region of inner globule polymer structure and cured epoxy resin. (a) AFM phase image of 1 x 2 µm in size showing indentation imprints of a force map and selected positions of individual force-indentation curves as shown in (b). The stiffness image given in (c) is calculated from linear fit data for each force-indentation curve as exemplified in (b) for an array of 16 x 32 measurements. Indentation depth and stiffness along the diagonal line in (c) is shown in (d). Figure 3. Atomic force microscopy data indicating crosslink density distribution within a single globule of the WCX-1S monolith with (a) topography image of an individual globule cross-section with 2 x 2 µm in size. Mechanical data calculated from a 2 x 2 µm force map of 64 x 64 measurements with images of (b) indentation depth, (c) stiffness from forceindentation curves, and (d) plastic energy. The cross-section profiles of stiffness and plastic energy are shown along the marked diagonal line for (c) and (d).

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Figure 1

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Figure 2

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Figure 3

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Assessing the Nanoscale Structure and Mechanical Properties of

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