AND MASS SPECTROMETRY 5
9. ORGANIC POLYMER e BASED LAYERS
It should be taken into consideration that monoliths in layer format were the initial shape when the organic polymerebased monoliths emerged (vide supra). The advantage brought to the field of MS through the use of organic polymerebased monolithic structures lies, among some other positive features, in the ease of the layer preparation and its availability in a variety of chemistries attained via selection of monomers used for their preparation. Also, the actual formation of these layers is simple.
Once prepared, these layers can be applied just to simply enhance desorption and ionization prior to mass spectrometric detection; they can be used for separation using one dimensional thin-layer chromatography (1D TLC) followed by MALDI MS, and also for two-dimensional thin-layer chro- matography (2D TLC) separation and MALDI MS.
FIGURE 5.6
Matrix enhanced surface-assisted laser desorption/ionization time-of-flight mass spectrum of angiotensin I using the poly(vinyl alcohol) substrate. An amount of 800 amol of angiotensin I was applied, and the CHCA matrix concentration was 0.1 mg/mL. The spectrum is the sum of 100 laser shots.
Adapted with permission from Lu, T., Olesik, S.V., 2013a. Electrospun nanofibers as substrates for surface-assisted laser desorption/ionization and matrix-enhanced surface-assisted laser desorption/ionization mass spectrometry. Anal. Chem. 85, 4384e4391. Copyright American Chemical Society, 2013.
9.1 PREPARATION OF MONOLITHIC LAYERS
9.1.1 Monolithic SpotsThe first paper concerning preparation of a monolithic thin layer for surface enhanced laser desorption- ionization (SELDI) TOF MS of small molecules was published in the early 2000s (Peterson et al., 2004). The 10mm “tall” monolithic cylinders were prepared from butyl methacrylate or benzyl methacrylate cross-linked with ethylene dimethacrylate in the presence of porogenic solvents, 1-dodecanol and cyclohexanol, and a photoinitiator, 2,2-dimethoxy-2-phenylacetophenone, using UV initiated polymerization. The mask shown inFig. 5.7, featuring 100 circular spots with a diameter of 3 mm, was designed using Corel Draw software and printed on a transparency film using a standard office laser printer. This mask was attached on top of a glass wafer. A small volume of the poly- merization mixture was placed on the top of a MALDI stainless steel plate, covered with the wafer- mask assembly, and polymerization carried out under UV lamp irradiation. The mask assembly was then carefully removed from the plate and the plate surface, with the attached monolithic spots shown inFig. 5.7, was rinsed with methanol. An alternative procedure had to be used for polymerizations of mixtures containing styrene and divinylbenzene monomers that are not UV transparent. The mono- lithic spots were prepared via thermally initiated polymerization using 2,2ʹ-azobisisobutyronitrile as the initiator. This polymerization mixture was spotted on a MALDI plate covered with a 10-mm-thick polyethylene film perforated with 3 mm holes that also acted as a sealing gasket on the top of which an
FIGURE 5.7
Mask used to prepare monoliths with a circular shape via photopolymerization of butyl methacrylate and ethylene dimethacrylate on the top face of a matrix-assisted laser desorption/ionization (MALDI) plate (A), top view of the MALDI plate with monoliths (B), optical micrograph of two adjacent monolithic spots (C), and scanning electron microscope micrograph of macroporous structure of monolith C (D).
aluminum plate was placed. The polymerization reaction was allowed to proceed at 80C for 24 h.
After the preparations and washing, the plates were dried and ready for use.Fig. 5.7also shows the respective optical and scanning electron microscope (SEM) images of the monolithic spots and their morphology.
9.1.2 Continuous Monolithic Layers
Continuous layers were most often prepared as attached to a glass plate.Fig. 5.8presents one of the implementations using a mold assembled from two glass plates separated by two Teflon strips placed along the longer sides of the plates, which define the thickness of the layer, and are held together with spring clamps. The bottom part of the figure then shows the morphology of the polymerized monolithic layer. In the typical preparation, the mold is filled with a polymerization mixture using capillary force, followed by photoinitiated or thermally initiated polymerization. The setup used for the preparation of monolithic layers is obviously very simple. However, some potential problems with the layer have to be taken into consideration.
Use of native glass for assembly of the mold might be the first choice. For example, Wouters et al. (2013)prepared a porous poly(butyl methacrylate-ethylene dimethacrylate) mono- lithic layer using photoinitiated polymerization of the monomer mixture placed between two untreated commercial microscopic glass slides. The problem was that the layer produced by
FIGURE 5.8
Mold consisting of two glass plates separated by Teflon strips located along the long side used for the preparation of monolithic layer (A), the mold containing the white monolithic layer (B), scanning electron microscope image of the cross section of the monolith (C), and detailed view of the morphology of the monolithic layer(D).
polymerization had a smooth surface “skin” with occasional cracks. So, properties of the skin differed from those of the bulk. This is well demonstrated with the SEM image inFig. 5.9A. The explanation for this has to be searched for in the basics of polymerization reactions. During the reaction, the monomer units are combined in a chain in which they are closer to each other compared FIGURE 5.9
Scanning electron micrographs of poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic layer prepared between two native glass plates (A) and a layer prepared using one plate (B), and both plates (C) functionalized with 3-(trimethoxysilyl)propyl methacrylate.
Adapted with permission from Wouters, B., Vanhoutte, D.J.D., Aarnoutse, P., Visser, A., Stassen, C., Devreese, B., Kok, W.T., Schoenmakers, P.J., Eeltink, S., 2013. Visualization procedures for proteins and peptides on flat-bed monoliths and their effects on matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection. J. Chromatogr. A 1286, 222e228.
Copyright Elsevier, 2013 and Han, Y., Levkin, P.A., Abarientos, I., Liu, H., Svec, F., Fre´chet, J.M.J., 2010. Monolithic superhydrophobic layer with photopatterned virtual channel for the separation of peptides using two-dimensional thin layer chromatographyedesorption electrospray ionization mass spectrometry. Anal. Chem. 82, 2520e2528. Copyright American Chemical Society, 2010.
with the distance between them in monomer solution. As a result, shrinkage in the volume occurs during polymerization. The UV light, which penetrates through the top plate, has the highest intensity closest to the underside of the top plate. The intensity of the UV light then gradually attenuates because of the self-screening action of the polymerization mixture. Thus, the initiation rate in the vicinity of the bottom plate is the slowest. As a result, the monolith adheres to the top slide while it is released from the bottom slide already during the preparation process. The space between the released monolith and the bottom glass plate is then filled with the air, the polymerization conditions are different from those in the bulk of the layer, and the nonpermeable thin “skin” is formed.
Although not explicitly mentioned in the paper (Wouters et al., 2013), it is likely that the adhesion of the monolith to the native glass surface may not be too strong, and the monolith may peel off. This is why it appeared useful to functionalize the top glass plate, targeted for support of the thin layer, with 3-(trimethoxysilyl)propyl methacrylate, whereas the bottom plate was not. The reason why the bottom glass plate should not be functionalized was to avoid adhesion of the layer to that plate and to facilitate disassembly of the mold. The polymer layer prepared under these conditions is shown inFig. 5.9B.
Not surprisingly, it exhibited a smooth surface “skin” assembled at the polymer-glass interface (Han et al., 2010).
The situation changed when the bottom plate was also silylated. As noted previously, the poly- merization rate is slower in the vicinity of the bottom plate leading to less firm attachment to that plate.
On disassembling the mold, most of the polymer then adhered to the top plate, whereas only a light coating of polymer remained attached to the bottom plate. As a result, the desired internal structure of the monolithic layer was revealed at the surface (Fig. 5.9C). The morphology of the surface was also reflected in its apparent hydrophobicity. Although for the smooth surface the water contact angle was only 77 degrees, as it lacked topographical features, the “open” surface then featured a dual scale roughness and the water contact angle increased to a superhydrophobic value of 154 degrees (Han et al., 2010).
So far, we have discussed situations occurring during photoinitiated polymerization, which is well suited for monomers that are UV transparent such as methacrylates. Switching to non-UV transparent monomers, such as styrene and its derivatives, and divinylbenzene as the cross-linker, requires use of thermal initiation (Lv et al., 2013). In contrast to photopolymerization with the gradient of UV light intensity across the polymerizing layer, thermally initiated polymerization occurs in all the bulk mixture homogeneously, and adhesion of the monolith to both functionalized glass plates is equal.
When the mold was disassembled, the polymer was torn into two parts with no priority for either of the glass plates. This resulted in a poorly defined thickness and a rough surface, as shown inFig. 5.10A.
The single glass plate vinylized approach did not work either. Certainly, the monolithic polymer layer did not adhere to the plain nonvinylized plate, whereas the entire monolith adhered to the vinylized plate. However once again, a smooth surface “skin” on the monolith was formed for the reason pre- sented above (Fig. 5.10B). This problem was eventually solved using a simple trick. A strip of a commercial Scotch tape was attached to the smooth surface, and on removal of the tape, the top polymer “skin” adhered to the tape and was homogeneously removed from the entire plate, whereas the desired well-developed globular structure from beneath attached to the glass slip was revealed as shown inFig. 5.10C. The change in surface morphology is again reflected in the increase in the water contact angle from 122 degrees for the smooth surface to 157 degrees observed for the globular counterpart (Lv et al., 2013).