Single Molecule Nanospectroscopy Visualizes Proton-Transfer Processes within a Zeolite Crystal

Visualizing proton-transfer processes at the nanoscale is essential for understanding the reactivity of zeolite-based catalyst materials. In this work, the Brønsted-acid-catalyzed oligomerization of styrene derivatives was used for the first time as a single molecule probe reaction to study the reactivity of individual zeolite H-ZSM-5 crystals in different zeolite framework, reactant and solvent environments. This was accomplished via the formation of distinct dimeric and trimeric fluorescent carbocations, characterized by their different photostability, as detected by single molecule fluorescence microscopy. The oligomerization kinetics turned out to be very sensitive to the reaction conditions and the presence of the local structural defects in zeolite H-ZSM-5 crystals. The remarkably photostable trimeric carbocations were found to be formed predominantly near defect-rich crystalline regions. This spectroscopic marker offers clear prospects for nanoscale quality control of zeolite-based materials. Interestingly, replacing n-heptane with 1-butanol as a solvent led to a reactivity decrease of several orders and shorter survival times of fluorescent products due to the strong chemisorption of 1-butanol onto the Brønsted acid sites. A similar effect was achieved by changing the electrophilic character of the para-substituent of the styrene moiety. Based on the measured turnover rates we have established a quantitative, single turnover approach to evaluate substituent and solvent effects on the reactivity of individual zeolite H-ZSM-5 crystals.

S2 UV-Vis micro-spectroscopy: UV-Vis absorption spectra were measured with an Olympus BX41 upright microscope working in reflectance mode, equipped with a 50× 0.5 NA high working-distance microscope objective lens. A 75 W tungsten lamp was used for illumination. Reflected light was directed to a CCD video camera (ColorView IIIu, Soft Imaging System GmbH) via a 50/50 double viewport tube, and to a UV-Vis spectrometer (AvaSpec-2048TEC, Avantes BV) via a 200 μm core fiber. Figure S1. UV-Vis absorption spectra of the studied oligomerization reactions under solvent-free conditions. The spectra were measured with an UV-Vis microscope at the center of zeolite crystals; top panels -parent H-ZSM-5, bottom panels -steamed H-ZSM-5 crystals. a,b) UV-Vis absorption spectra of 4-methoxystyrene oligomers; (a) was recorded for two different crystals. c, d) UV-Vis absorption spectra of 4-fluorostyrene oligomers at room temperature and 393 K. The numbers above the absorption maxima indicate the tentative assignment to the carbocationic species shown in Scheme 1 of the main text of the article.
A notable visual difference in reactivity between the 4-methoxy-and 4-fluorostyrenes is noticed with a UV-Vis microscope. The Brønsted acid-catalyzed oligomerization of 4-methoxystyrene readily proceeds at room temperature, coloring the individual zeolite H-ZSM-5 crystals due to the formation of the stable carbocationic species (Figure S1a,b). 1 In contrast to 4-methoxystyrene, 4fluorostyrene does not display any significant reactivity at room temperature (Figure S1c,d) due to the high electronegativity of the fluoro-substituent that hinders the proton transfer from the Brønsted acid sites of the zeolite material to the double bond of the styrene moiety. 2 The difference in reactivity of the tested molecules is visible in the comparison of UV-Vis spectra taken from the reaction products, as summarized in Figure S1. Two distinct absorption bands initially appear for both types of zeolite H-ZSM-5 crystals at ~ 565 nm and 590 nm. A similar absorption profile was reported previously; the absorption ~ 590 nm was attributed to the linear dimeric carbocation (5) in Scheme 1 of the main text of the article. 1,3,4 We attribute these absorption bands to the existence of two isomers of the allylic dimeric carbocation (5). The assignment of the band at ~ 530 nm has never been attributed before for 4-methoxystyrene as it appears as a satellite band to the observed linear dimeric species. Sprung and Weckhuysen have recently reported 20-40 nm shifts in the absorption bands of the cyclic carbocations in the sinusoidal pores of zeolite H-ZSM-5. 5 Additionally, as the formation of the absorption band follows similar trends as for the trimeric (6) and cyclic species (7), suggesting their bulkier nature, we tentatively attribute this band to the cyclic dimeric species. The absorption originating from the trimeric species (6) was previously proposed, 1,5 but to date their precise molecular structure has not been identified in the experiments.

S3
Confocal fluorescence microscopy: The fluorescence microscopy measurements were carried out using an upright Nikon Eclipse 90i confocal laser scanning microscope, equipped with a 100× 0.73 NA dry objective lens. Confocal fluorescence microscopy images were recorded using the excitation from two laser light sources (i.e., 488 nm and 561 nm) connected to a Nikon-Eclipse A1R scanning head equipped with corresponding dichroic mirrors to separate the excitation and emission light. The long axes of the zeolite crystals were aligned perpendicular to the polarization vector of the laser light in order to efficiently excite the fluorescent molecules, as explained in reference. 5 The light emission was detected in the range of 495-700 nm by using a spectral detection unit equipped with a diffraction grating and a 32 photomultiplier tube array. The fluorescence spectra obtained by 488 nm (from 490-600 nm) and 561 nm lasers (from 570-700 nm) were combined. The UV-Vis micro-spectroscopy data with confocal fluorescence microscopy results recorded for both probe reactions as well as for the parent and steamed zeolite H-ZSM-5 crystals are compared in Figure S2. Both emission bands overlap spatially for the top subunits of both the parent and steamed zeolite H-ZSM-5 crystals ( Figure S2a,b). At the edge, the 600 nm band seems to be higher in intensity, as reported in the literature. 1 The recorded fluorescence predominantly originates from the molecules aligned in the direction of the straight pores directed along b lattice vector ( Figure S2e). This was verified in a polarization-sensitive experiment by rotating the zeolite H-ZSM-5 crystal for 90°. 5 The emission band at 540 nm was not present for the majority of the crystals. Thus, based solely on confocal fluorescence microscopy measurements, we have concluded that the concentration of the bulkier trimeric and cyclic dimeric species depends on the inter-particle differences in chemical reactivity, with the formation of the trimeric species being more visible in fluorescence.

S4
Our experiments suggest that a more intense coloration from increased visible light absorption, of zeolite H-ZSM-5 crystals exposed to 4-methoxystyrene does not directly translate into higher fluorescence intensity. The absence of such dependency clearly indicates the presence of additional non-radiative relaxation pathways. This behavior was most pronounced for 4methoxystyrene and most probably relates to its high reactivity that leads to a locally high concentration of absorbing reaction products. The reported emission bands should be rather used as a guideline for the possible fluorescence species, as the oligomerization kinetics will largely depend on the reaction conditions in different concentration regimes UV-Vis spectra of H-ZSM-5 crystals: interparticle differences Figure S3. a) UV-Vis spectra of the reactive and non-reactive parent zeolite H-ZSM-5 crystals recorder after 1 h of a reaction with 4-methoxystyrene. b,c) A time evolution of UV-Vis spectra for two steamed zeolite H-ZSM-5 crystals indicating a difference in the distribution of the colored reaction products.
It is noteworthy that the extent of crystal coloration, time-dependent changes, as well as the number of absorption bands differed significantly from one zeolite H-ZSM-5 crystal to another ( Figure S3). The optical microscopy observations indicated large interparticle differences in reactivity that can be most probably attributed to the large surface diffusion resistance and inhomogeneous uptake of reactants. As a consequence, some parent zeolite H-ZSM-5 crystals reached dark-purple coloration, whereas other did not show visible absorption, even after 1 h of reaction in 4methoxystyrene.

S5
Localization of the individual fluorescent events: scatter plots

Optimization of the localization parameters with the emitter tracking algorithm
In our recent work we have described the procedure to determine the survival times of individual fluorescent emitters by using the emitter tracking algorithm. 6 The algorithm takes into account subsequent localizations in time (blinking gap parameter) and space (pixel jump parameter) and determines whether they originate from the same or different molecule. This is schematically illustrated in Figure S5. If the subsequent localized events (localizations ) appear within 57 nm of distance and within 2 s in time, they are counted as one emitter (one fluorescent product molecule). The determined numbers are a good physical approximation of the analyzed data set. This is due to the fact that the many photostable emitters appear highly localized (see Figure S4) and with shorter blinking times (mostly bellow 2 s). The purpose of this analysis is to clearly identify the "hotspots" of fluorescence activity that are attributed to the formation of the highly photostable emitters. The complexity of fluorescence blinking and survival time distributions cannot be quantitatively determined by our analysis. Instead, the applied algorithm relies on the statistically sound description of the large number of molecules. It should be noted that the localization parameters in this analysis differ from the ones determined previously for the oligomerization of furfuryl alcohol. 6,7 This is due to a different nature of fluorescent species and significantly short survival time distributions of individual emitters. The long survival times of the trimeric styrene products and their blinking shifts the distribution towards higher values of the blinking gap parameter.