Chemical Communications

www.rsc.org/chemcomm

Volume 49 | Number 18 | 4 March 2013 | Pages 17731872

ISSN 1359-7345

COMMUNICATION
Floris Helmich and E. W. Meijer
Controlled perturbation of the thermodynamic equilibrium by microfluidic
separation of porphyrin-based aggregates in a multi-component
self-assembling system

1359-7345(2013)49:18;1-5

ChemComm
View Article Online

Published on 08 November 2012. Downloaded by University of Groningen on 25/08/2015 13:15:19.

COMMUNICATION

Cite this: Chem. Commun., 2013,

49, 1796
Received 22nd September 2012,
Accepted 8th November 2012
DOI: 10.1039/c2cc36887k

View Journal | View Issue

Controlled perturbation of the thermodynamic

equilibrium by microfluidic separation of
porphyrin-based aggregates in a multi-component
self-assembling system
Floris Helmich and E. W. Meijer*

www.rsc.org/chemcomm

In a microfluidic H-cell, a multi-component self-assembled system is

brought out-of-equilibrium by changing the bimodal composition of
porphyrin stacks and pyridine-capped dimers. Driven by their dierent
diusivities, diusion-controlled separation in methylcyclohexane
reveals dierent compositions when detected in-line and o-line,
which demonstrates the kinetic behaviour of this metastable system.
The microfluidic technique also proves to be highly equipped to
determine diusion constants of the dierent assemblies.

Supramolecular polymers comprised of p-conjugated chromophores form an interesting bottom-up approach for the development of functional, nanometer-sized materials.1 Their
macroscopic properties are highly tuneable, reversible and
dynamic due to the wide versatility of self-assembled architectures. Structural control herein strongly depends on the monomer design and the thermodynamic state imposed by external
stimuli such as temperature and concentration.2 Furthermore,
the introduction of additional supramolecular interactions
causes a shift of equilibria between non-covalently linked
building blocks.3 Their highly dynamic nature makes control
over self-assembly via non-thermodynamic pathways very challenging. Reduced supramolecular dynamics already aorded
memory eects,4 interconversions of dierent aggregate types5
and size exclusion chromatography of discrete assemblies.6 The
use of multiple building blocks is an important aspect in order
to create functional materials, while such assemblies become
inherently more complex. Therefore, multi-component selfassembly requires in-depth analyses, especially when they
operate away from thermodynamic equilibrium.7
In earlier reports, microfluidic devices have been tailored to
induce stable gradients that stimulate self-assembly processes;
for instance by diusive mixing of solvents8 and pH.9 Besides
thermodynamic parameters such as temperature and monomer

Institute for Complex Molecular System, Laboratory of Macromolecular and Organic

Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven,
The Netherlands. E-mail: E.W.Meijer@tue.nl
Electronic supplementary information (ESI) available: Microfluidic procedures,
DOSY and DLS measurements. See DOI: 10.1039/c2cc36887k

1796

Chem. Commun., 2013, 49, 1796--1798

design,10 the formation of liposomes with controlled size has

also been achieved by the appropriate hydrodynamic conditions.11 In this communication, we introduce microfluidic
techniques as an approach to control the distribution of
kinetically metastable porphyrin aggregates.
Recently, we reported on the cooperative self-assembly of (S)Zn-porphyrin S-Zn in the presence of pyridine (Fig. 1).12 The
dilution-induced self-assembly of this system was fully described
in a thermodynamic model. In methylcyclohexane (MCH), the
porphyrins form helical, cofacial stacks upon the formation of
strong 4-fold intermolecular hydrogen bonds. Axial coordination
of pyridine sterically blocks one porphyrin face for hydrogen
bonding, which results in the formation of hydrogen bonded,
pyridine-capped dimers.12 The pyridine titration shows a sharp
transition from stacks to dimers and at high porphyrin concentrations a pseudo-bimodal distribution between the two is observed as
shown by an apparent isosbestic point in their UV-vis spectra.13
AFM-micrographs of the elongated stacks clearly reveal the
presence of curled fibrils, which fully disappear after the depolymerization process (Fig. 1). High association constants were
determined for both these dierent hydrogen-bonded aggregates;
hence, we conceived slow aggregate interconversion upon changing an external stimulus. And indeed upon the addition of MCH,

Fig. 1 Thermodynamic model for the S-Znpyridine system with the

corresponding UV-vis titration experiment at 2.0  10 5 M in MCH and AFMmicrographs for the stacks (left) and dimers (right).

This journal is

The Royal Society of Chemistry 2013

View Article Online

Published on 08 November 2012. Downloaded by University of Groningen on 25/08/2015 13:15:19.

Communication
dilution-induced self-assembly was observed with slow kinetics
(Bhours). This kinetic property as well as the considerable dierence in size between stacks and dimers encouraged us to investigate the possibility to control their distribution ratio by diusive
mass transport in a microfluidic H-cell,14 thereby temporarily
creating an out-of-equilibrium situation of kinetically metastable
structures. By detection at dierent timescales we probe their slow
redistribution to the thermodynamically stable state.
In our experimental setup,15 an equivolumetric flow rate is
maintained over both the inlets and outlets, by using well-calibrated
syringe pumps and flow regulators, respectively (Fig. 2A). As a result,
the contact time of the laminar interface between both flows equals
the diusion time (tdiusion), which is determined by the applied
flow rate. Additionally, in-line UV-vis detection provides quantitative
and qualitative analysis of the diused species. By combining these
features, we first use the H-cell as a tool to estimate diusion
coecients of the pure aggregates and then we continue to create
out-of-equilibrium situations by the separation of a mixture of stacks
and dimers.
In order to validate the experimental setup, we performed a
diusion experiment on the porphyrin monomer model compound S-ZnMe.16 At a concentration of 2.0  10 5 M in MCH,
S-ZnMe was eluted against pure MCH at flow rates between 0.50
and 0.05 ml s 1 (0.35 o tdiusion o 3.52 s, respectively) and the
steady-state UV-vis spectra were recorded in the extraction stream
(Fig. 2B). The absorbance at lmax = 420 nm was normalized on the
spectrum of S-ZnMe at 1.0  10 5 M and subsequently plotted
against the diusion time (Fig. 2C). The obtained diusion
profile was fitted to a 1-dimension-unsteady-state diusion
model,15 from which we estimated a diusion coecient of
3.2  10 10 m2 s 1. The latter closely resembles the value
obtained in a diusion ordered 1H-NMR (DOSY) experiment of
S-ZnMe in D14-MCH (2.2  10 10 m2 s 1).15,17
Next, we performed identical diusion studies on pure
stacks and pure dimers of S-Zn at 2.0  10 5 M, in which the
latter solution has a pyridine excess of 250. The normalized

Fig. 2 (A) Schematic representation of the H-cell, in which two 110 mm channels
with a depth of 50 mm merge and split (FM = flow meter, FR = flow regulator,
flow regulator is placed in the residual stream and in-line UV-vis measurements
are performed in the extraction stream). (B) In-line UV-vis spectra of S-ZnMe
acquired at dierent flow rates. (C) Diusion profiles for S-ZnMe, S-Zn stacks
and S-Zn : pyridine (1 : 1) dimers at lmax = 420, 390 and 427 nm, respectively,
with the corresponding fits and simulation at DDOSY = 2.2  10 10 m2 s 1
(normalized diusion = Cdi/1.0  10 5 M).

This journal is

The Royal Society of Chemistry 2013

ChemComm
diusion profiles for the stacks and dimers (constructed
from the normalized absorbance at lmax = 390 and 427 nm,
respectively)15 are highly distinctive; hardly any mass transfer is
observed for stacks, whereas a considerable amount of dimers
have diused over (Fig. 2C). Additionally, the latter diusion
profile approaches the curve of S-ZnMe; hence we estimate a
diusion coecient of 2.7  10 10 m2 s 1 for the dimers and
7.8  10 12 m2 s 1 for the stacks, both of which correspond to
their diusivities observed in dynamic light scattering (DLS)
measurements.15 Despite the fact that the porphyrins are solely
held together by supramolecular interactions, the dierent
diusion behaviour of stacks and dimers is obviously related
to their aggregate size.14,18
As a next step herein, we exploit the microfluidic setup to
separate a mixture of stacks and dimers. At a pyridine excess of
45, the molar distribution of S-Zn monomers over stacks and
dimers (stack/dimer-ratio) at thermodynamic equilibrium is
31/69, as determined from the Soret band intensity ratio of
0.54 at 390 and 427 nm, respectively (Fig. 1).13 This initial
mixture is eluted against MCH at a flow rate of 0.10 ml s 1
(tdiusion = 1.76 s) and the in-line UV-vis spectra are acquired for
both the extraction and residual stream (Fig. 3A). Compared to
the initial state, the latter spectrum shows a drop in dimer
absorbance, while the stack absorbance remains unchanged. As
a result, the stack/dimer-ratio increases to 36/64, whereas the
extraction stream reveals that predominantly dimers have
diused over; a stack/dimer-ratio of 16/84 is estimated from
the spectrum. Based on their dierent mass transport, in-line
UV-vis detection reveals that the bimodal distribution between
stacks and dimers can be moderately controlled by this microfluidic technique.
Besides porphyrin stacks and dimers, free pyridine and
(pyridine-complexed) monomers also diuse over at relatively
high rates. In addition, the diusion process in this particular
configuration leads to inhomogeneity of a two-fold diluted

Fig. 3 In-line (A) and o-line (B) UV-vis spectra of S-Zn at 2.0  10 5 M with 9.0 
10 4 M pyridine of the initial solution, residual stream and extraction stream
recorded tdet B20 s and B3 h after separation, respectively. (C) Kinetic profile of
the dilution-induced self-assembly upon 2-fold dilution of the initial solution
probed by CD (black axis, left at 390 nm) and UV-vis (red axis, right at 390 and
427 nm).

Chem. Commun., 2013, 49, 1796--1798

1797

View Article Online

Published on 08 November 2012. Downloaded by University of Groningen on 25/08/2015 13:15:19.

ChemComm
solution. With these features, the system is being pushed outof-equilibrium, leading to a complex reactiondiusion event,
which is characterized by the supramolecular dynamics for
regaining thermodynamic equilibrium. In order to obtain more
insight into this process, we compare the in-line UV-vis spectra
with the spectra obtained after sample collection, corresponding to detection times of B15 s and B3 h after separation,
respectively.15
Directly after separation, the in-line spectra seem to reveal
the sole diusion process of dimers to the other stream.
However after sample collection, highly distinctive spectra are
observed that cannot be explained by mass transport only
(Fig. 3B). Compared to the in-line spectra, both the residual
and extraction streams show an increased stack/dimer-ratio of
58/42 and 47/53, respectively. In the residual stream, the
significant enhancement of stacks is mainly due to loss of
dimers and the diusion of pyridine and pyridine-complexed
monomers that source dimerization. Despite the pyridine
enrichment in the low-concentration extraction stream, the
modest enhancement of stacks can be explained by the
dilution-induced self-assembly eect, which operates strongly
at high dilutions.12
Both short and long timescale analyses can be rationalized
on the basis of mass transport and thermodynamic equilibria.
In order to investigate how the supramolecular dynamics span
these distinctive timescales, we perform a 2-fold dilution
experiment on the initial mixture, which closely resembles
the microfluidic experiment. In this kinetic measurement, the
dilution-induced self-assembly leads to the formation of stacks,
which can exclusively be probed by CD spectroscopy while the
stack/dimer-ratio can be probed by UV-vis at 390 and 427 nm.
Directly after injecting an equivolumetric amount of MCH
to the initial mixture, the kinetic profiles clearly reveal the
redistribution over stacks and dimers. Approximately 4 hours
after dilution the new thermodynamically stable state is
reached (Fig. 3C). Likely due to the relatively slow supramolecular dynamics, thermodynamic equilibrium is achieved
while collecting the extraction and residual streams.
With reference to detection, these kinetic aspects are important to be considered when changing the thermodynamic state
e.g. by mixing-in multiple components or concentration- and
temperature jumps.19 Noteworthily, this feature has also been
recognized in the 1 : 1 complexation of Znporphyrin and
pyridine in a microfluidic mixer20 and probing fast self-assembly
kinetics of peptide ligands and quantum dots.21 On the other
hand, besides its properties to estimate size and separate
supramolecular aggregates, this microfluidic technique aords
the controlled preparation of non-thermodynamically stable
intermediates.22 Herewith, we envision the creation of kinetic
traps by freezing-in the intermediates and related experiments
are underway.
This work was supported by the Council of Chemical
Sciences of The Netherlands Organization for Scientific

1798

Chem. Commun., 2013, 49, 1796--1798

Communication
Research (CW-NWO). We thank Dr Thomas Hermans, Niels
Brankaert and Joost van Dongen for experimental contributions
to the project. Dr Takashi Hirose is acknowledged for
curve-fitting and Paul Schlebos for the DOSY experiment.

Notes and references

1 (a) A. Schenning and E. W. Meijer, Chem. Commun., 2005,
32453258; (b) E. R. Kay, D. A. Leigh and F. Zerbetto, Angew. Chem.,
Int. Ed., 2007, 46, 72191.
2 T. F. A. De Greef, M. M. J. Smulders, M. Wols, A. Schenning,
R. P. Sijbesma and E. W. Meijer, Chem. Rev., 2009, 109, 56875754.
3 (a) R. Hoogenboom, D. Fournier and U. S. Schubert, Chem. Commun.,
2008, 155162; (b) J. Hamacek, M. Borkovec and C. Piguet, Chem.Eur.
J., 2005, 11, 52175226.
4 (a) F. Helmich, C. C. Lee, A. P. H. J. Schenning and E. W. Meijer,
J. Am. Chem. Soc., 2010, 132, 1675316755; (b) L. Rosaria, A. DUrso,
A. Mammana and R. Purrello, Chirality, 2008, 20, 411419;
(c) L. J. Prins, F. De Jong, P. Timmerman and D. N. Reinhoudt,
Nature, 2000, 408, 181184.
5 R. F. Pasternack, C. Fleming, S. Herring, P. J. Collings, J. dePaula,
G. DeCastro and E. J. Gibbs, Biophys. J., 2000, 79, 550560.
6 (a) W. S. Li, K. S. Kim, D. L. Jiang, H. Tanaka, T. Kawai, J. H. Kwon,
D. Kim and T. Aida, J. Am. Chem. Soc., 2006, 128, 1052710532;
(b) M. J. Ahrens, R. F. Kelley, Z. E. X. Dance and M. R. Wasielewski,
Phys. Chem. Chem. Phys., 2007, 9, 14691478; (c) D. Furutsu,
A. Satake and Y. Kobuke, Inorg. Chem., 2005, 44, 44604462.
7 B. A. Grzybowski and C. J. Campbell, Chem. Eng. Sci., 2004, 59,
16671676.
8 A. Jahn, W. N. Vreeland, M. Gaitan and L. E. Locascio, J. Am. Chem.
Soc., 2004, 126, 26742675.
9 L. Brown, S. L. McArthur, P. C. Wright, A. Lewis and G. Battaglia,
Lab Chip, 2010, 10, 19221928.
10 J. M. Zook and W. N. Vreeland, Soft Matter, 2010, 6, 13521360.
11 A. Jahn, W. N. Vreeland, D. L. DeVoe, L. E. Locascio and M. Gaitan,
Langmuir, 2007, 23, 62896293.
12 F. Helmich, C. C. Lee, M. M. L. Nieuwenhuizen, J. C. Gielen,
P. C. M. Christianen, A. Larsen, G. Fytas, P. Leclere, A. Schenning
and E. W. Meijer, Angew. Chem., Int. Ed., 2010, 49, 39393942.
13 Due to their high association constants and cooperativity, both the
hydrogen-bonded states (stacks and pyridine-capped dimers) are
the most abundant species in solution. As a result, they barely
contribute to the UV-vis absorbance thus an isosbestic point is
observed. For the current studies, we do not consider the presence
of free porphyrin monomers and monomeric adducts.
14 J. P. Brody and P. Yager, Sens. Actuators, A, 1997, 58, 1318.
15 See ESI.
16 S-ZnMe contains a tertiary amide and due to this methyl substitution on the nitrogen, it cannot donate in intermolecular hydrogen
bonding. Therefore, this molecule does not aggregate in MCH and
serves as a discrete reference compound.
17 The dierence in diusion coecient of S-ZnMe determined by
DOSY and the microfluidic experiment is likely due to minor
contributions of convective mass transfer as a consequence of
pressure-driven flow in the latter, which therefore shows an overprediction of the diusion coecient.
18 B. H. Weigl and P. Yager, Science, 1999, 283, 346347.
19 (a) E. T. Pashuck and S. I. Stupp, J. Am. Chem. Soc., 2010, 132,
88198821; (b) A. Lohr and F. Wuerthner, Chem. Commun., 2008,
22272229; (c) F. Helmich, M. M. J. Smulders, C. C. Lee, A. P. H. J.
Schenning and E. W. Meijer, J. Am. Chem. Soc., 2011, 133,
1223812246.
20 M. Brivio, R. E. Oosterbroek, W. Verboom, A. van den Berg and
D. N. Reinhoudt, Lab Chip, 2005, 5, 11111122.
21 J. H. Wang, P. J. Jiang, Z. Y. Han, L. Qiu, C. L. Wang, B. Zheng and
J. Xia, Langmuir, 2012, 28, 79627966.
22 (a) C.-W. Wang, D. Sinton and M. G. Mott, J. Am. Chem. Soc., 2011,
133, 1885318864; (b) A. Jahn, S. M. Stavis, J. S. Hong, W. N. Vreeland,
D. L. Devoe and M. Gaitan, ACS Nano, 2010, 4, 20772087.

This journal is

The Royal Society of Chemistry 2013