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Single Molecule Nanospectroscopy Visualizes Proton-Transfer

Processes within a Zeolite Crystal
Zoran Ristanović,† Alexey V. Kubarev,‡ Johan Hofkens,§ Maarten B. J. Roeffaers,*,‡
and Bert M. Weckhuysen*,†
†
Inorganic Chemistry and Catalysis, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands
‡
Centre for Surface Chemistry and Catalysis and §Department of Chemistry, KU Leuven, Celestijnenlaan 200 F, 3001 Heverlee,
Belgium
*
S Supporting Information
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ABSTRACT: Visualizing proton-transfer processes at the

nanoscale is essential for understanding the reactivity of
zeolite-based catalyst materials. In this work, the Brønsted-
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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 environ-
ments. 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.

■ INTRODUCTION
Zeolites are microporous aluminosilicates that are heavily used
recent characterization studies at the single particle level
emphasize the importance of the underlying structural
as solid acid catalysts due to the unique combination of acidic differences between individual zeolite particles where surface
and shape-selective properties.1,2 In particular, zeolites are of diffusion barriers and crystalline defects may play an important
high importance in catalytic processes in the petrochemical role.11−17
industry. More recently, they are also considered to be It should be evident that the chemical nature of the above-
implemented in future biorefineries.3−8 A challenge in the described processes at the solid−liquid interface of zeolites is
rational design of zeolite-based catalysts is to control the poorly studied at the single particle level and relevant time and
number, distribution and nature of (Brønsted) acid sites. length scales, partially due to the insensitivity and ensemble
Significant progress has been made recently in the direction of averaging nature of traditionally used spectroscopic methods.
tailoring hierarchical zeolite catalysts with remarkably improved Consequently, spatially resolved information about the
catalytic activity, selectivity, and stability.9,10 accessibility of Brønsted acid sites and the real-time changes
Understanding the synergy between the numerous phys- in reactivity of a single zeolite particle cannot be visualized by
icochemical processes taking place in zeolite-based catalysts these bulk characterization methods.12,18 To overcome this
demands for fundamental insights into the host−guest limitation, several techniques based on optical microscopy
interactions, preferably at the single particle−single molecule methods have been developed in recent years for the study of
level. Porosity, crystalline defects of the catalyst, guest mobility, Brønsted acid-catalyzed reactions at the single particle
as well as solvent effects, can strongly interfere with Brønsted level.19−21 Hence, selective staining of zeolite acid sites22 is
acid-catalyzed processes taking place within zeolite catalysts. becoming a popular strategy to study Brønsted reactivity of
Nowadays, there is a tendency to perform catalytic reactions in zeolite particles where fluorescent species are generated after an
the liquid phase leading to an abundance of various organic
guest molecules that influence the proton-transfer processes Received: June 13, 2016
taking place within the cages and channels of zeolites. Finally, Published: October 6, 2016

© 2016 American Chemical Society 13586 DOI: 10.1021/jacs.6b06083

J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

acid catalyzed conversion of molecules, such as furfuryl Single Molecule Fluorescence Microscopy. An inverted
alcohol,23−26 thiophene,27 and styrene.28−30 epifluorescence wide-field microscope (Olympus IX-71) was used
After the great success in life sciences,31 single molecule for the single molecule experiments. Both a 532 nm excitation (with a
545 nm long-pass filter) and a 561 nm excitation (a 575 nm long-pass
fluorescence microscopy has become a powerful and sensitive
filter) were used. The excitation light was focused by a 100× oil
tool to study chemistry, physics and materials science at the immersion objective lens (1.4 NA) and the emitted fluorescence light
molecular scale. The localization of fluorescent emitters leads to was detected by a highly sensitive electron multiplying charge-coupled
the spatiotemporal resolutions in the orders of 10 nm and 10 device (EMCCD) camera (ImagEM Enhanced C9100-23B, Hama-
ms, provided that the fluorescent species possess sufficient matsu). The fluorescence microscopy movies were recorded with
brightness and photostability. Several excellent reviews high- acquisition times of 30 and 100 ms per frame.
light the high relevance of the single molecule fluorescence Data Analysis. The recorded single molecule fluorescence
approach in many fields of chemistry.19,32−38 The broad scope microscopy movies were analyzed with the Localizer software
of single molecule fluorescence microscopy includes studies of developed by Dedecker et al.62 for Igor Pro (Wavemetrics) and
Matlab (MathWorks). The localization of fluorescent events was done
kinetics and mechanisms of stoichiometric chemical reac- by independent segmentation of each frame into emissive spots and
tions,39,40 collisions of conjugated polymers in liquid phase,41 background as described by Sergé et al.63 A list of initial emitter
distribution and transport of charge carriers in semiconduc- positions was determined with subdiffraction limited resolution by
tors,42,43 diffusion in porous materials,44,45 localization of fitting a 2-D Gaussian using the Levenberg−Marquardt least-squares
plasmonic hotspots,46,47 and single-turnover kinetics of solid algorithm as implemented in the GNU Scientific Library. The emitter-
catalysts.24−26,48−53 tracking algorithm, as implemented in the Localizer, has been used for
Among the above-mentioned fluorogenic reactions, the analysis of the single emitter survival.
Brønsted-acid-catalyzed oligomerization of styrene-based de- Experiments. All single molecule fluorescence microscopy experi-
ments were performed at room temperature. H-ZSM-5 crystals were
rivatives has received high interest due to the existence of loaded on a cover glass that was previously attached in a reactor
several carbocationic species that are considered as frequently designed for liquid-phase single molecule microscopy experiments.
encountered reaction intermediates in zeolite chemistry.54 Typically, 1 mL of the solution containing 4-methoxystyrene or 4-
Importantly, styrene and its derivatives fit inside the pores of fluorostyrene dissolved in either heptane (spectroscopic grade) or
medium and large pore zeolites and oligomerize further to butanol (spectroscopic grade, vacuum distilled) was subsequently
generate carbocationic species that absorb and emit light in the added. The reaction was then monitored by focusing at the surface of
visible region. By changing the substituents on the styrene the bottom facet or by moving the focus to any provisional focal depth
moiety, it is possible to probe the acidic and shape-selective in the axial Z-direction up to Z = 20 ± 0.2 μm. Prior to measurements,
the zeolite crystals were activated at 773 K (1 K/min) for 24 h in static
properties of zeolites.29 The oligomerization reactions using 14
air to avoid residual fluorescence.

■
different styrene substituents were initially performed on large
zeolite H-ZSM-5 crystals as model systems28,29,55 and further
extended to industrially relevant zeolite-based catalysts, such as RESULTS AND DISCUSSION
fluid catalytic cracking (FCC) particles and catalyst bodies Brønsted Acid-Catalyzed Oligomerization of Styrene
based on H-ZSM-5 and H-Y.56−58 Derivatives as Single Molecule Probe Reaction. Previous
In this work, we apply for the first time the Brønsted-acid- studies of the styrene oligomerization reaction suggested a
catalyzed oligomerization of styrene derivatives to study the reaction mechanism for the formation of fluorescent
influence of solvent polarity and defect density on the reactivity carbocationic species,29,30,64−66 as outlined in Scheme 1.
of zeolite H-ZSM-5 at the single particle−single molecule level. Upon protonation of the neutral styrene molecule (1), the
Using 4-methoxystyrene and 4-fluorostyrene as selective and
versatile probes we showcase the importance of pore Scheme 1. Potential Reaction Pathways and Species
accessibility and defect chemistry for the selective formation Proposed for the Oligomerization of Styrene Derivatives, In
of different reaction intermediates. To accomplish this, we have Particular 4-Methoxystyrene and 4-Fluorostyrene, in Acidic
studied two types of zeolite crystals, namely, highly micro- Zeolites29,30,64,66a
porous parent zeolite H-ZSM-5 crystals and defect-rich, mildly
steamed zeolite H-ZSM-5 crystals with induced mesoporos-
ity.26,30,59−61 Based on this study, a spectroscopic marker for
nanoscale quality control of zeolite-based materials could be
identified. Furthermore, we show that the single molecule
oligomerization kinetics and stability of fluorescent carboca-
tions can be further controlled by the polarity of solvent
molecules.

■ EXPERIMENTAL SECTION
Zeolite Materials. Large zeolite H-ZSM-5 crystals (dimensions:
100 × 20 × 20 μm3) were used as provided by ExxonMobil
(Machelen, Belgium). The synthesis has been reported elsewhere.59,61
Tetrapropylammonium as template was removed by calcination at 823
K for 8 h (1 K/min). The crystals were subsequently converted into
their acidic form by a triple ion exchange with 10 wt % ammonium
nitrate (99+% Acros Organics) at 353 K, followed by 6 h calcination at
a
773 K. The steamed zeolite H-ZSM-5 was treated at 773 K in a water- The absorption maxima for the species (5−7) represent the
saturated N2 flow (150 mL/min) for 5 h. The resulting crystals were experimentally observed absorption bands shown in Supporting
previously characterized in detail by Aramburo et al.61 Information Figure S1.

13587 DOI: 10.1021/jacs.6b06083

J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

initial benzylic carbocation (2) is formed. Further dimerization UV−vis and fluorescence microscopy studies reported
leads to the formation of the linear dimeric 1,3-bis(phenyl)-1- previously.30,67
butylium cation (3), that can transform into neutral diphenyl Figure 1 summarizes the results for a parent zeolite H-ZSM-5
alkene upon proton transfer to the zeolite framework (4), or crystal recorded in solvent-free conditions and at two different
undergo cyclization to the cyclic dimeric 3-methyl-1,4-phenyl-
indanyl (indanyl) carbocation (7). The formation of the linear
allylic carbocation (5) has been the subject of particular interest
due to its photoproperties. According to Cozens et al.64 and
Buurmans et al.,66 molecules (3) and (4) can undergo
bimolecular hydride transfer to form an allylic carbocation
(5). The conjugated carbocations (5) and (7) are reported in
the literature as the stable carbocations that can absorb visible
light. The absorption originating from the trimeric species (6)
was previously proposed,28,30 although its precise molecular
structure is still unknown.
The absorption and emission properties of the 4-methox-
ystyrene and 4-fluorostyrene carbocations (5−7) under study
were determined in a series of UV−vis and confocal
fluorescence microscopy experiments, which are summarized
in the Supporting Information Figures S1 and S2. The
oligomerization of both probe molecules resulted in several
absorption bands indicated in Scheme 1. The fluorescence
spectra of H-ZSM-5 crystals exposed to 4-methoxystyrene
exhibit two main emission bands at ∼600 and 650 nm
(Supporting Information Figure S2) that are assigned to the
linear dimeric and trimeric species, respectively. Similar
conclusions can be made with respect to the origin of the Figure 1. High-resolution single molecule fluorescence microscopy
fluorescence bands observed for the 4-fluorostyrene oligomers. imaging of fluorescent reaction products recorded after 1 h during
In this case, the corresponding emission bands were observed ∼500 s of the solvent-free oligomerization of 4-methoxystyrene on a
at ∼580 and 640 nm. In what follows, we will use these parent zeolite H-ZSM-5 crystal; the excitation wavelength was 561 nm.
(a,b) Accumulated maps of the total number of detected fluorescent
spectroscopic assignments to explain the observations from our
events counted separately in each of 5000 frames (color bar: number
single molecule fluorescence microscopy measurements. of events per 200 × 200 nm2 presented on the logarithmic scale,
Single Molecule Fluorescence Microscopy Imaging of noncorrected for repeated detection of single product molecules), (a)
Crystalline Defects in Zeolite ZSM-5 Crystals. Under recorded in the near-surface region of the crystal and (b) recorded at Z
optimal conditions for single molecule imaging, the speed of = 8 μm from the surface and toward the middle of the crystal. The
fluorescent product disappearance due to photobleaching or edges are not fluorescent due to the orientation of the product
migration out of focus should match the formation of novel molecules with respect to the laser beam and light polarization. (c)
fluorescent oligomers. The room temperature reaction in pure Normalized occurrence of emitters with different survival times,
4-methoxystyrene resulted in a very fast buildup of the calculated for the surface (N1(t), red) and Z = 8 μm (N2(t), blue). (d)
fluorescence signal that could not be matched by the The difference in normalized occurrences for the distributions in (c).
photobleaching induced by the excitation laser. Under such
conditions, for the large majority of the crystals, the fast focal depths−at the zeolite surface (Figure 1a, Z = 0 ± 0.2 μm)
accumulation of overall fluorescence hampers the continuous and in the middle of the zeolite crystal (Figure 1b, Z = 8 μm).
detection of single fluorescent reaction products being formed. The reconstructed high-resolution maps in Figure 1a,b present
In this case, the reaction rate needs to be controlled by the total number of detected single emitting molecules,
introducing a solvent, which will be discussed below. Notably, accumulated independently over 5000 frames (≈ 500 s). At
single molecule sensitivity could be reached in solvent-free the surface, fluorescent reaction product molecules cluster in
conditions by studying H-ZSM-5 crystals with low Brønsted certain regions (Figure 1a), whereas at the middle of the crystal
acidity, making use of the large interparticle differences in (Z = 8 μm) an active region with more evenly distributed
reactivity (Supporting Information Figure S3). fluorescence events is present (Figure 1b). The scatter plots of
The localization of fluorescent reaction products that individual localizations support this conclusion (Supporting
appeared as the result of stochastic catalytic turnovers was Information Figure S4). The low surface reactivity of the
achieved by the Nanometer Accuracy by Stochastic Chemical studied parent H-ZSM-5 crystal (as compared to the more
reActions (NASCA) super-resolution fluorescence method. reactive crystals) points to the acid site inaccessibility due to the
The method enables following real-time catalytic events taking extremely high and inhomogeneous surface diffusion resistance
place within a catalytic solid.24 By using NASCA approach, the caused by zeolite pore blockage.13,15
4-methoxystyrene oligomerization was followed with a 30−100 An important observation from Figure 1a is that a high
ms temporal resolution and individual product molecules were number of fluorescent events can be detected in the yellow/red
localized using 2-D Gaussian fitting procedure with an color-coded crystalline domains. Inspection of the recorded
estimated lateral resolution of about 20 nm (Supporting fluorescent movie (Supporting Information Movie S1) reveals
Information Figure S4). This is more than a 10-fold high fluorescence movie activity in these regions. Highly
improvement of the diffraction-limited resolution and a photostable fluorescent emitters were found to contribute
remarkable improvement in sensitivity as compared to the about 25% to the observed fluorescence activity. To confirm
13588 DOI: 10.1021/jacs.6b06083
J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

this observation we have analyzed the photostability of the

individual fluorescent reaction products. A particle-tracking
algorithm was applied to account for reappearing individual
fluorescent molecules within certain spatial and temporal
constrains.26 Based on the analysis described in Supporting
Information Figure S5, we considered that the consecutive
localizations within ∼60 nm distance originate from the same
molecule; additionally, the blinking dark states (off times) in
duration shorter than 2 s were considered to originate from the
same molecule. These values were found to represent well the
reappearance of the highly photostable emitters.
By using determined localization values, we have examined
the survival times of fluorescent 4-methoxystyrene oligomers
for both studied cases in Figure 1a and b, respectively. Figure 1c
summarizes the normalized distributions of survival times for
the fluorescent molecules at the surface (N1(t)) and at Z = 8
μm (N2(t)). We detect a broad range of survival times for
fluorescent emitters, covering 3 orders of magnitude from 100
ms to 50 s. The difference (Δ) between the normalized
distributions N2(t) − N1(t) is shown in Figure 1d. The result
indicates that the fraction of molecules with the lower survival
times (≤700 ms) is higher for the movie recorded at Z = 8 μm,
Figure 2. High-resolution single molecule fluorescence microscopy
whereas the movie recorded at the surface of the parent zeolite maps of the short-lived (<0.7s; dimeric) and long-lived (>0.7s;
H-ZSM-5 crystal has a higher contribution of the highly trimeric) species, reconstructed based on the survival times of
photostable emitters (survival times > 700 ms), Supporting fluorescent molecules shown in Figure 1a,b. (a−d) Accumulated
Information Movie S1. maps of (a) dimeric products at the outer surface of the crystal, (b)
The measured differences in the survival times of fluorescent trimeric products at the outer surface of the crystal, (c) dimeric
products suggest the formation of fluorescent species with products at Z = 8 μm, and (d) trimeric products at Z = 8 μm. The
different photostability. The linear dimeric carbocations (5) white squares indicate the region of interest magnified in Figure 3a.
form easily in the zeolite micropores and under highly diluted Color scale bars denote a total number of dimeric and trimeric
conditions they should be the predominant species. The emitters per 200 × 200 nm2 detected during 500 s.
trimeric (6) and cyclic dimeric (7) carbocations are likely to be
formed at more accessible acid sites close to the zeolite outer whereas the trimeric species were predominantly detected close
surface. Defects in the crystalline structure may facilitate further to the outer surface of the zeolite H-ZSM-5 crystal (Figure
oligomerizations in larger voids where the local porous 2b,d).
environment does not induce severe shape constraints.30,66,68 Our interpretation can be further supported by inspecting
The formation of the trimeric carbocation species (6) seems to the nanoscopic regions of interest, such as the region
be kinetically controlled and follows the formation of the linear highlighted in Figure 2a,b and presented as a scatter plot of
dimeric carbocation species (5),28 as evidenced from Scheme 1 fluorescent events in Figure 3a. We represent the dimeric and
and the UV−vis spectroscopy experiments (Supporting trimeric fluorescent species by the blue and red circles,
Information Figure S3). In the single molecule fluorescence respectively. Qualitatively, the highly photostable events (in
microscopy experiments we have used 532 and 561 nm nm red) seem to appear in agglomerates and in close proximity to
laser excitation, meaning that we simultaneously excite both the each other. The less photostable dimeric species (in blue)
linear dimeric (5) (emission at 600 nm) and the trimeric appear more evenly distributed within the analyzed region. A
species (6) (emission at 650 nm). Therefore, the formation of similar conclusion can be made for the entire maps of dimeric
the photostable trimeric fluorescent products close to the and trimeric species presented in Figure 2a,b. This is a clear
surface could explain the observed differences in survival times indication that the two types of fluorescent reaction products
of individual emitters. The high stability of the trimeric species are formed in conditions of different accessibility of acid sites.
is most probably related to their low reactivity, as further Consequently, the local reactivity rates and the kinetics of
oligomerization is unlikely to happen. dimer/trimer formation are largely determined by the
After applying the correction for the reappearance of accessible Brønsted acid sites.
individual molecules, it was possible to construct super- The spatial arrangement of the dimeric and trimeric
resolution NASCA reactivity maps by binning individual fluorescent species can be quantified by calculating the nearest
catalytic turnovers in 200 × 200 nm2 areas. To distinguish neighbor distances for both types of the formed products. The
between dimeric and trimeric species, we have chosen a results are shown in Figure 3b for both short-lived (dimeric)
threshold value for survival times of 0.7 s. Note that this and long-lived (trimeric) species. A clear distinction can be
arbitrary threshold value, as determined from Figure 1d, will be made between the distributions of NN distances. Both
only used to illustrate the concept of distinguishing fluorescent distributions show a maximum number of counts for the very
species with different lifetimes. In Figure 2, we have spatially short NN distances (<25 nm). A close inspection of the scatter
resolved the appearance of trimeric fluorescent molecules plot in Figure 3a confirms that these localizations predom-
(survival times > 0.7 s) and dimeric fluorescent molecules inantly originate from clustered events, where the formation of
(survival times ≤ 0.7 s). The dimeric species are formed both at both dimeric and trimeric species is facilitated. It is not possible
the surface and in the middle of a zeolite crystal (Figure 2a,c), to make a clear distinction between both reaction products at
13589 DOI: 10.1021/jacs.6b06083
J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

evenly formed throughout the microporous crystalline domains,

as qualitatively presented in Figure 2.
The presented analytical approach offers a possibility to
study defect-induced chemistry of zeolite ZSM-5 by following
two distinct types of fluorescent species. The working
hypothesis was further tested by deliberately inducing structural
defects into the zeolite framework by dealumination of the
parent zeolite H-ZSM-5 crystals via mild steaming at 773 K. 4-
Methoxystyrene appeared to be very sensitive to the induced
crystalline defects. The reactivity of parent and steamed zeolite
H-ZSM-5 crystals were compared in a series of concentration
experiments with a 4-methoxystyrene solution in n-heptane.
As discussed earlier, the exposure of the parent H-ZSM-5
crystals to liquid (nondiluted) 4-methoxystyrene leads to the
fast accumulation of fluorescence where single fluorescent
reaction products cannot be resolved anymore, as illustrated in
Figure 4a. Significant dilution of 4-methoxystyrene in heptane
(7.4 mM) was necessary to observe well-separated and localized
Figure 3. Nearest neighbor (NN) analysis for the formation of the fluorescent reaction products in such active parent H-ZSM-5
dimeric (blue) and trimeric fluorescent species (red). (a) Scatter plot crystals (Figure 4b). Reducing the concentration of 4-
of the fluorescent products. (b) Frequency of the NN distances for the methoxystyrene also slows down the intracrystalline diffusion
dimeric (blue) and trimeric carbocations (red) calculated for the entire
with fluorescent oligomers predominantly detected near the
data set presented in Figure 2.
zeolite surface (∼0.5 μm). As expected, the reactivity decreased
for more than 1 order of magnitude when the crystals were
exposed to a 1.5 mM 4-methoxystyrene solution (Figure 4c). In
distances that are below the resolution limit as the optimized both cases, the individual single molecule products could be
parameters of localization are arbitrary and cannot exclude the clearly resolved by the localization algorithm (red circles in
blinking of individual molecules and statistical distribution in Figure 4). However, when the steamed H-ZSM-5 crystals were
survival times of fluorescent products. Therefore, within this exposed to a 1.5 mM 4-methoxystyrene solution, an over-
resolution limit, the measured numbers should be seen as a whelming amount of fluorescent carbocation products was
semiquantitative description of the NN distances. However, the detected making it impossible to efficiently resolve individual
dimeric species show also a considerable fraction of NN molecules (Figure 4e). Reducing the concentration 10-fold did
distances that are in the range of 100−300 nm (Figure 3b), not resolve this problem (Figure 4f); only further 50-fold
values that are well above the resolution limit. This is expected dilution of 4-methoxystyrene to 30 μM made it possible to
from the initial assumption that the dimeric species are more observe isolated fluorescent species (Figure 4g).

Figure 4. Probing the accessibility of parent (top) and steamed zeolite H-ZSM-5 crystals (bottom). Wide-field fluorescence micrographs of the
Brønsted acid-catalyzed oligomerization of 4-methoxystyrene at different molar concentrations in heptane (bottom right); images are recorded at the
surface of the parent (a−c) and steamed (e−g) zeolite H-ZSM-5 crystals by using a 532 nm excitation; (a) was measured in solvent-free conditions
(7.4 M). The red circles indicate the efficiency of the localization algorithm to detect individual fluorescent events. (d, h) Cartoons illustrating the
formation of dimeric and trimeric fluorescent species in parent (d) and steamed (h) zeolite H-ZSM-5 crystals. The light blue dots indicate the
formation of linear dimeric carbocations, while the red dots represent the bulkier trimeric carbocations, formed close to defects/mesopores.

13590 DOI: 10.1021/jacs.6b06083

J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

Figure 5. High-resolution fluorescence microscopy imaging of defect-induced reactivity of 4-methoxystyrene in a steamed H-ZSM-5 crystal, recorded
in 30 μM solution in heptane by using a 532 nm excitation with 30 ms acquisition time. (a) Accumulated images of photoactivity for different focal
depths denoted in the top left corner, recorded after ∼1 h from the start of the reaction. The color scale bar represents the number of detected
fluorescent styrene oligomers per 200 × 200 nm2 during 150 s of the recording time. (b) High resolution scatter plot indicating the location of
fluorescent products formed in a steamed zeolite H-ZSM-5 crystal at Z = 2 ± 0.3 μm. (c,d) Corresponding (c) scatter plot and (d) fluorescence
intensity trajectory of the single molecule indicated with the red arrow in (b). The background intensity in (d) is around 10k.

Our previous work has shown that the measured turnover bulkier oligomers that are apparently more photostable (red
frequencies of the parent and mildly steamed H-ZSM-5 crystals dots depicted in Figure 4h).
differ by a factor of 2−5.26 The variation in fluorescence The low concentrations of 4-methoxystyrene that are used in
response with styrene derivatives is hence probably related to the experiments (<1 mM) favor the predominant formation of
the formation of different, more photostable oligomers at the the linear dimeric carbocations in the micropores. Considering
crystal defects giving rise to a pronounced increase in the high density of Brønsted acid sites, the formation of the
background fluorescence, as postulated earlier in the text. highly photostable trimeric carbocationic species can be used to
Spectroscopic evidence for this conclusion can be found by selectively probe more accessible Brønsted acid sites at the
UV−vis microscopy. Two distinct absorption bands initially crystalline defects. A significant fraction of the parent zeolite
appear for both types of zeolite H-ZSM-5 crystals at ∼565 and crystals appeared to have intracrystalline defects (Supporting
590 nm (Supporting Information Figure S3). We attribute Information Figure S6), such as cracks and mesoporous defects,
these absorption bands to the existence of two isomers of the which are inherent to the sample preparation, such as synthesis,
allylic dimeric carbocation (5). The absorption bands at 490, activation and calcination. Notably, the important role of
530, and 640 nm appear later in the spectrum, but faster on crystalline defects in base-leaching of zeolite H-ZSM-5 was
steamed zeolite H-ZSM-5 crystals and parent crystals with reported recently on nanosized zeolite H-ZSM-5 crystals,
visible crystalline defects (Supporting Information Figure S3). making the single molecule visualization of the defects a very
Similar absorption profiles were reported previously.28,64,65 The relevant topic for zeolite chemistry.16,17 In a similar fashion, the
absorption band at ∼640 nm has been attributed to the trimeric developed method can be used to qualitatively visualize
carbocationic species (6).28,67 Despite the lack of evidence for interparticle differences in the uptake of probe molecules.
their precise molecular structure(s), the extent of conjugation These become obvious for the steamed zeolite H-ZSM-5
and late appearance of these species supports their assignment crystals where different crystals can have strikingly different
to the trimeric carbocationic species. The additional non- reactivity (Supporting Information Figure S6). We speculate
radiative relaxation pathways and energy transfer between the that the interparticle differences arise due to structural
different fluorescent species are expected to be minimized in heterogeneities, such as amorphous deposition or framework
highly diluted solutions of the reactant and large product-to- distortions that may strongly influence local surface mass
product distances−conditions that are met in our experiments. transport and reactivity.69
The observed behavior underlines the importance of pore The selective formation of trimeric carbocations can be
accessibility and the shape-selective role of framework in the maximized in the conditions of extremely low concentration of
formation of bulkier reaction products. For instance, the highly the selected styrene derivatives. The high-resolution maps of a
crystalline parent zeolite H-ZSM-5 crystals typically have a Si- steamed H-ZSM-5 crystal, measured at four different focal
rich layer of up to 100 nm in thickness, that is characterized by depths, are shown in Figure 5a. The majority of the photostable
a very low acid site density (Si/Al ratio of ∼160).59,61 This layer reaction products were formed in a 2−3 μm thick surface layer
also seems to act as a shape-selective surface diffusion barrier of the zeolite crystal (Supporting Information Movie S2).
hindering the formation of the bulkier fluorescent products at Previous high-resolution SEM studies of steamed zeolite H-
the very surface, as depicted in Figure 4d. The local diffusion ZSM-5 crystals detected near-surface mesopores with diameters
properties are drastically changed with the formation of larger of 5−50 nm. However, almost no damage of the crystalline
mesoporous defects,11 which can facilitate the formation of structure (i.e., mesoporosity) was observed in H-ZSM-5 crystals
13591 DOI: 10.1021/jacs.6b06083
J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

Figure 6. Oligomerization of 4-methoxystyrene (2.5 M in 1-butanol) at parent zeolite H-ZSM-5 crystals. (a−c) Wide-field fluorescence micrographs
recorded 30 min after start of the reaction by using a 561 nm excitation. The sequence illustrates the effect of photobleaching, (a) at the beginning of
recording, (b) after 20 s, and (c) after 130 s; (d) recorded after 24 h, at Z = 4 μm. (e,f) Accumulated high-resolution reactivity maps recorded at (e)
the middle of the zeolite crystal, (f) the edge of the zeolite crystal. (g) Integrated total fluorescence background intensity and the number of detected
fluorescent events, as measured for the crystal in (f). Color scale bars denote the total number of turnovers per 200 × 200 nm2 detected during 500 s.
Acquisition time in (a−d) was 100 ms. Scale bars are 5 μm.

further away from the outer surface.60,61 This observation is in The initial fluorescence signal, recorded 30 min after
accordance with the 3-D reactivity profiles of furfuryl alcohol in initiating the reaction, vanished after 130 s of continuous
the steamed zeolite H-ZSM-5.26 Therefore, it is reasonable to illumination due to fast photobleaching of individual
suggest that the photostable emitters in Figure 5 reveal the fluorescent molecules (Figure 6a−c). After 24 h, we observed
locations of the accessible mesopores. Even though the significant penetration and accumulation of the fluorescent
concentration of 4-methoxystyrene in this experiment (30 oligomers along the sinusoidal pores of the zeolite H-ZSM-5
μM) corresponds to only 17 molecules per 100 nm3 of solvent, crystal tips, also in the deeper regions of the crystals (Figure
the kinetics of the product formation is elevated in mesopores 6d). The observed reactivity and directionality of the
by specific local adsorption and diffusion pathways. At Z = 2 fluorescent products in 1-butanol is a result of anisotropic
μm below the zeolite surface, a higher number of photostable diffusivity and reactivity of 4-methoxystyrene along the straight
and highly localized fluorescent oligomers was detected (Figure and sinusoidal channels. Kox et al. reported higher accessibility
5b). The detected emitters did not show significant diffusion of the tip regions via the straight pores (running along the b
and consecutive localizations are within the localization lattice parameter) open to the surface, as illustrated in
precision of our method (∼20 nm), as shown in Figure 5c. Supporting Information Figure S2.28 The fluorescence response
The recorded fluorescent reaction products show high can be described in terms of low single molecule reactivity
photostability with occasional blinking, as evidenced from presented in Figure 6e and f; the analysis of the average
their fluorescence intensity trajectories (Figure 5d). By using fluorescence background signal and the number of detected
single molecule fluorescence microscopy with 30 ms acquisition fluorescent events showed that both quantities decayed fast in
time it is possible to track the temporal changes in fluorescence time (Figure 6g). We concluded that the rate of the
for every individual fluorescent product. Several representative oligomerization reaction in 1-butanol was very low and that
fluorescence intensity trajectories are presented in Supporting only the initially formed fluorescent products were photo-
Information Figure S7. bleached.
The Effect of Solvent Polarity on the Oligomerization The change in the reaction medium affects not only the
Reaction. In attempt to optimize the reaction rates for the reactivity, but also the survival times of fluorescent products.
highly reactive 4-methoxystyrene, we have tested the The analysis of the survival times of the 4-methoxystyrene-
oligomerization reaction in 1-butanol. Whereas 4-methoxystyr- derived carbocationic species in n-heptane and 1-butanol is
ene reacted readily at the Brønsted acid sites in a 1.5 mM (0.02 shown in Figure 7a. Although the total number of detected
vol %) solution in n-heptane, hardly any fluorescent events fluorescent events is 2 orders of magnitude higher for the
were observed in a highly concentrated 2.5 M (33.3 vol %) fluorescent reaction products in n-heptane, the measured
solution in 1-butanol. The spatial reactivity distribution was exponential decays of the survival time frequencies clearly
clearly different with respect to n-heptane, with extremely low indicate a lower photostability for the fluorescent reaction
reactivity mostly observed at the edges of the H-ZSM-5 crystals products in 1-butanol; all exponential time constants appeared
(Figure 6a). to be smaller in the latter case.
13592 DOI: 10.1021/jacs.6b06083
J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

molecules per acid site (measured for a Si/Al ratio of 72).70

The close packing of 1-butanol molecules is due to their strong
interaction with Brønsted acid sites, but also due to favorable
interactions of the hydrocarbon chain with the zeolite wall
(Figure 7e).71 As a result, the formation and thermodynamic
stability of the benzylic and dimeric carbocationic species is
severely inhibited by the presence of the polar solvent
molecules. Heptane, on the other hand, has a similar pore
filling as 1-butanol for the same reasons of C−H/zeolite wall
interactions,72 but does not interact strongly with the Brønsted
acid sites, which can additionally promote the formation of
carbocationic species (Figure 7d). Similar tests in 1,4-dioxane
did not yield any visible photoproducts. In line with our results,
Yoon et al. have reported in their bulk experiments a full
conversion of styrene in dichloromethane over zeolite H−Y
powder, and no evidence of reaction in tetra-hydrofuran or
acetonitrile.73
The Effect of the Para-Substituent on the Oligome-
rization Reaction. The presented single molecule fluores-
cence microscopy approach can be successfully extended to
other substituted styrene probe molecules. In this way, a broad
range of reaction conditions can be achieved considering the
large difference in reactivity between differently substituted
derivatives. The trends in reactivity, as determined by the
stabilization of the initial benzylic carbocation, can be predicted
by Hammett parameters.74−76 In a comparative study, Stavitski
et al. have shown that the presence of different functional
Figure 7. (a) Survival times of fluorescent reaction products during groups may affect the net stabilization of the benzylic species.29
the oligomerization of 4-methoxystyrene with parent zeolite ZSM-5
crystals in 1-butanol (2.5 M, red circles) and n-heptane (7.4 mM, blue
For example, fluoro- substituent has less stabilizing effect than
circles). Exponential time constants for the fits are 0.055 ± 0.001 s methoxy- group in the para position, resulting in the much
(27% contribution), 0.22 ± 0.01 s (45%), and 1.1 ± 0.1 s (28%) for lower reactivity of 4-fluorostyrene at room temperature.
the emitters in n-heptane (blue fit) and 0.015 ± 0.05 s (18%), 0.13 ± However, it should be noted that the reactivity trends in
0.01 s (64%) and 0.8 ± 0.2 s (18%) for the emitters in 1-butanol zeolites may strongly deviate from the Hammett equation, as
(black fit). Note the logarithmic scale on y-axes. (b,c) Examples of the the confinement effects may play important role.29 The
fluorescence intensity trajectories of individual fluorescent molecules fluorescent reaction products for 4-fluorostyrene can be
in (b) n-heptane and (c) 1-butanol. (d,e) Schematic of the styrene detected at room temperature by using single molecule
oligomerization reaction in (d) n-heptane and (e) 1-butanol; the fluorescence microscopy. Using this less-reactive probe
interaction of a solvent molecule with a Brønsted acid site is
molecule similar solvent effect is observed, whereas imaging
highlighted.
of the crystalline interior is readily possible in solvent-free
conditions (Supporting Information Figure S8).
By inspecting the fluorescence behavior of individual Quantitative Comparison of the Oligomerization
emitters, it is possible to give a more qualitative description Rates. Single molecule fluorescence microscopy can be
of their interactions with the zeolite framework. In a parent considered as a quantitative technique provided that conditions
zeolite crystal, the fluorescent reaction products are confined for efficient localization of fluorescent molecules are met. By
within the micropores and the measured survival times provide quantifying the individual fluorescent reaction products it was
information about framework-emitter interactions. Manual possible to estimate the averaged turnover frequencies recorded
inspection of a large number of individual fluorescence in the studied oligomerization reactions. For simplicity we have
trajectories confirmed the results presented in Figure 7a. The considered each fluorescent event to be one catalytic turnover.
fluorescent reaction products in n-heptane on average have The results are summarized in Figure 8. A concentration range
longer survival times, as illustrated for the fluorescence intensity for successful single molecule fluorescence imaging of zeolites
trajectory of an emitter in Figure 7b. In stark contrast, will depend on the reactivity of a probe molecule (i.e, the
fluorescent reaction products in 1-butanol predominantly show substituent effect) and the polarity of a solvent (i.e., the solvent
short survival times, often within the acquisition time of 100 effect). In both cases the oligomerization rate is determined by
ms. This is illustrated in Figure 7c, where one fluorescence the tendency of the reactant to form the initial benzylic and
trajectory consists of several short intensity bursts possibly dimeric carbocations. The polarity of a solvent and its
originating from one fluorescent molecule. interaction with Brønsted acid sites plays a crucial role in the
The experimentally observed results can be explained by the carbocation formation, as illustrated for the reactions in n-
competing role of solvent molecules as they may adsorb on the heptane and 1-butanol. The oligomerization of both reactants is
Brønsted acid sites, as depicted in Figure 7d,e. The significant significantly inhibited when n-heptane is replaced with 1-
drop in the reaction rates and the photostability of individual butanol (Figure 8a).
fluorescent reaction products are due to the strong The oligomerization of 4-methoxystyrene in zeolite H-ZSM-
chemisorption of 1-butanol at Brønsted acid sites. This solvent 5 proceeds much faster than in the case of 4-fluorostyrene, as
is known to occupy 96% of the zeolite volume with about 2 visible from the comparison of their reactivity in heptane
13593 DOI: 10.1021/jacs.6b06083
J. Am. Chem. Soc. 2016, 138, 13586−13596
Journal of the American Chemical Society Article

hand, we have established a probe concept that is very sensitive

to chemical changes in the nature of the zeolite host as well as
of the guest species and that is capable of resolving nanoscopic
details in the reactivity of zeolite crystals with the ultimate
single molecule sensitivity.

■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b06083.
Additional spectra and high-resolution images of intra-
and interparticle heterogeneities, and single molecule
fluorescence intensity trajectories (PDF)
Figure 8. (a) Reactivity of 4-methoxystyrene (in red) and 4-
fluorostyrene (in blue) as probe molecules for single molecule Photoactivity of a parent zeolite ZSM-5 crystal in 4-
chemical imaging in n-heptane (circles) and 1-butanol (rhombi). The methoxystyrene (AVI)
squares represent 99% pure 4-fluorostyrene. Averaged turnover Reactivity of a steamed zeolite ZSM-5 crystal in 30 μM
frequencies of the parent and steamed zeolite H-ZSM-5 crystals solution of 4-methoxystyrene in n-heptane (AVI)

■
(denoted as “S”) were recorded close to the outer surface of the single
crystals; x-axis denotes the concentration of the probe molecules in the
solvents. The blue and red lines indicate the lowest and highest
AUTHOR INFORMATION
turnover rates that can be imaged by the presented single molecule Corresponding Authors
approach. The linearity shown is provided only as a guidance for the *maarten.roeff[email protected]
observed trends in reactivity. (b) Frames indicating the density of the *[email protected]
individual emitters and an approximate level of reactivity for
corresponding turnover frequencies.
Notes
The authors declare no competing financial interest.
(Figure 8a). A successful single molecule fluorescence
microscopy imaging of zeolite H-ZSM-5 crystals reacting with
4-methoxystyrene can be performed at a high dilution of the
■ ACKNOWLEDGMENTS
B.M.W. acknowledges The Netherlands Organisation for
reactant (10−5−10−2 M in n-heptane). In contrast, the Scientific Research (NWO) Gravitation program (Netherlands
experiments with 4-fluorostyrene can routinely be performed Center for Multiscale Catalytic Energy Conversion, MCEC)
with the pure reagent (8.3 M) for at least 30 min, even for the and a European Research Council (ERC) Advanced Grant
highly acidic zeolite H-ZSM-5 crystals. (321140). M.B.J.R. and J.H. thank the “Fonds voor
The maximum recorded rate of fluorescent reaction product Wetenschappelijk Onderzoek”(Grant G0197.11), the KU
formation is in the order 10 events per μm3 s−1. If all Al atoms Leuven Research Fund (OT/12/059), Belspo (IAP-VII/05),
are considered catalytically equally active, this number and the Flemish government (long term structural funding,
approximately translates into the turnover frequencies in the Methusalem funding CASAS2 METH/15/04). M.B.J.R.
order of 10−8 s−1 (red line in Figure 8a). The low turnover acknowledges the ERC for financial support (Starting Grant
numbers of 10−12 s−1 (blue line Figure 8a) indicate the LIGHT 307523). Dr. Machteld Mertens (ExxonMobil,
detection rate of <0.01 events μm−3 s−1. Based on the Machelen, Belgium) is thanked for providing the parent
presented results, it is clear that a subset of the acid sites are ZSM-5 crystals.
probed with our approach and this number is determined by
the frequency and density of detected fluorescent events
(Figure 8b). The approach does not determine the total
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13596 DOI: 10.1021/jacs.6b06083

J. Am. Chem. Soc. 2016, 138, 13586−13596