NMR evidence of slow monomer-micelle exchange in a calixarene-based surfactant

Article abstract of DOI:10.1021/jp910984g

The self-aggregation of amphiphilic p-sulfonatocalix[4]arene was studied by fluorescence and NMR techniques. Pyrene fluorescence spectra were used to determine the critical micelle concentration and the value obtained was confirmed using Diffusion-Ordered NMR Spectroscopy (DOSY). It was found from the ¹H NMR spectra that exchange between the monomers in the bulk and in the micelles is slow on the NMR time scale. This finding was confirmed by 2D EXSY experiments and the rate constants of the process at different temperatures were extracted using the latter technique.

Full text of DOI:10.1021/jp910984g

4816
J. Phys. Chem. B 2010, 114, 4816–4820
NMR Evidence of Slow Monomer-Micelle Exchange in a Calixarene-Based Surfactant
Nuno Basilio,† Luis Garc´ıa-R´ıo,*,† and Manuel Mart´ın-Pastor‡
Departamento de Qu´ımica F´ısica, Facultad de Qu´ımica, Laboratorio Integral de Dina´mica e Estructura de
Biomole´culas Jose´ R. Carracido, Unidade de Resonancia Magne´tica, RIAIDT, Edif. CACTUS, UniVersidad de
Santiago, 15782 Santiago, Spain
ReceiVed: NoVember 19, 2009; ReVised Manuscript ReceiVed: March 4, 2010
The self-aggregation of amphiphilic p-sulfonatocalix[4]arene was studied by fluorescence and NMR techniques.
Pyrene fluorescence spectra were used to determine the critical micelle concentration and the value obtained
was confirmed using Diffusion-Ordered NMR Spectroscopy (DOSY). It was found from the 1H NMR spectra
that exchange between the monomers in the bulk and in the micelles is slow on the NMR time scale. This
finding was confirmed by 2D EXSY experiments and the rate constants of the process at different temperatures
were extracted using the latter technique.
Introduction
that assembled into completely uniform and structurally precise
micelles, which are spontaneously formed by exactly seven
monomers in aqueous solution and are nondeformable upon
drying. Of special interest is the case of stimuli-responsive
assemblies. It was shown that vesicles formed by calixarene
derivatives transform into micelles on dropping the pH from 7
to 5 with subsequent release of encapsulated guests.15 Houmadi
et al. presented amphiphilic calix[6]arene-based systems with
an aggregation architecture that responded both to pH and metal
ions.16
When dissolved in water, surfactant molecules tend to form
aggregates above their critical micelle concentration (cmc).
Micelles are the most common and the most thoroughly studied
aggregates observed in surfactant solutions.1 Micellar aggregates
are short-lived dynamic species. Rapid exchange occurs between
surfactant molecules in the micelles and those in the surrounding
bulk solution. For single chain surfactants, the exchange is
relatively fast with characteristic times of the order of 1 ns to
1 ms, and rapid techniques such as ultrasonic absorption,2,3
pressure-jump,4 temperature-jump,5 and stopped-flow dilution
techniques are required for their measurement.6 Since the
exchange rate is too fast on the NMR time scale, the NMR
spectra of surfactant molecules show averaged signals for free
and self-assembled monomers.5 However, it has been found that
for some cationic gemini7,8 and fluorocarbon-hydrocarbon
hybrid9 surfactants the exchange is slow on the NMR time scale
and it is possible to observe separate signals for monomers in
the bulk solution and in the micelles. This situation allows the
rate constants for the process to be determined by NMR
techniques.
Recently, it has been reported that water-soluble nucleotide-
calix[4]arene self-assembles into micelles and that the exchange
between dimers and micelles is slow on the NMR time scale.17
To the best of our knowledge, this example and the water-
soluble guanidinium calixarene derivatives18 are the only
examples of this phenomenon in amphiphilic calixarene systems
to have been reported.
1
In this work, we present the results of combined H NMR,
diffusion NMR, and fluorescence studies on the micellization
of p-sulfonatocalix[4]arene bearing hexyl chains at the lower
rim (SC4TH).
Because of their facile modification,10 calixarenes are par-
ticularly attractive for the construction of “surfactants with a
host-guest recognition site”.11 Shinkai et al. showed that
p-sulfonatocalixarenes bearing appropriate alkyl groups at the
lower rim form micelles in aqueous solution.11 The same
researchers also demonstrated that the conformation of the
calixarene is a crucial parameter in modulating the aggregation
behavior12 with the cone shaped conformation being ideal for
the formation of globular micelles. After Shinkai’s pioneering
work, several examples of amphiphilic calixarenes appeared in
the bibliography. For instance, Strobel et al.13 showed that the
nature of the ionic headgroup is a key parameter in the
aggregation behavior of calixarene amphiphiles; carboxylated
calixarenes form vesicles whereas calixarenes with trimethy-
lammonium head groups form micelles. Hirsch’s group14 used
calix[4]arene as a building block to construct an amphiphile
Experimental Section
All commercial reagents were of the highest purity available
and were used as received. p-Sulfonatocalix[4]arene (SC4) was
prepared by ipso-sulfonation of p-tert-butylcalixarene in H2SO4
at 80 °C. SC4TH was synthesized from SC4 following literature
procedures.19 The product was characterized by HNMR and
1
13CNMR spectroscopy and MALDI-TOF mass spectrometry.
1
SC4TH, H NMR (400 MHz, D2O, ppm) δ ) 0.96 (t, 12H),
1.42 (br, 24H), 2.02 (q, 8H), 3.43 (d, 4H), 3.93 (t, 8H), 4.44 (d,
4H), 7.35 (s, 8H); 13C NMR (400 MHz, D2O, ppm) 158.0, 137.3,
134.5, 126.0, 75.6, 32.0, 30.30, 30.1, 25.9, 22.7, 13.8; MALDI-
TOF-MS m/z (M + 4H - 3Na+) 1103.4; calcd 1103.4.
The fluorescence spectra of pyrene were measured on a Cary
Eclipse instrument with an excitation wavelength of 334 nm.
The pyrene concentration was kept constant at 0.4 µM. 1H NMR
and Diffusion-Ordered NMR Spectroscopy (DOSY) spectra
were recorded at 25 °C on a Varian Inova 400 spectrometer.
The DOSY spectra were acquired with the standard stimulated
echo pulse sequence using LED and bipolar gradient pulses.20
Square-shaped pulsed gradients of 2 ms duration were applied
* To whom correspondence should be addressed. E-mail: luis.garcia@
usc.es.
† Departamento de Qu´ımica F´ısica, Facultad de Qu´ımica.
‡ Laboratorio Integral de Dina´mica e Estructura de Biomole´culas Jose´
R. Carracido, Unidade de Resonancia Magne´tica, RIAIDT, Edif. CACTUS.
10.1021/jp910984g  2010 American Chemical Society
Published on Web 03/19/2010

Monomer-Micelle Exchange in a Calixarene-Based Surfactant
J. Phys. Chem. B, Vol. 114, No. 14, 2010 4817
Figure 2. Variation of observed diffusion coefficient, Dobs, for SC4TH
with concentration in D2O at 25 °C.
Figure 1. Plot of the I1/I3 ratio in pyrene fluorescence vs concentration
of SC4TH; [pyrene] ) 0.4 × 10-3 mM, excitation at 334 nm.
thus indicating the presence of aggregates in solution. The
obtained value of 0.32 mM is lower than that obtained by
fluorescence and this discrepancy can be attributed to the
differences expected between D2O and H2O. It is worth noting
that the diffusion coefficients level off at about 10 mM and
therefore micellar growth can be ruled out. The diffusion
coefficients can be used to determine the hydrodynamic radius
of the species in solution using the Stokes-Einstein equation.
When micelles are present in solution the diffusion coefficient
of monomers and aggregates is given by the following expression
with a power level linearly incremented from 2.1 to 64.3 G
cm-1 in 20 steps. To obtain reliable results for the diffusion
coefficient, the diffusion time ∆ of the experiment was optimized
for each sample to a value between 60 and 200 ms. The raw
data were processed using the MestreC program (Mestrelab
Research Inc.). Two-dimensional EXSY experiments were
carried out on a Varian Inova 750 spectrometer at 25 °C with
mixing times 0 (reference) and 200 ms. Rate constants were
calculated from the peak intensities using the EXSYCALC
program (Mestrelab Research Inc.).
cmc
[SC4TH]
([SC4TH] - cmc)
Dobs
)
Dm +
DM
(1)
[SC4TH]
where Dm and DM refer to the diffusion coefficients of the
monomer and the micelle, respectively. The hydrodynamic
radius of the monomer and/or micelle was calculated with the
Stokes-Einstein equation from the diffusion coefficient at
concentrations below and above the cmc. The hydrodynamic
radii obtained below and above the cmc were 9.6 (monomer)
and 42.7 Å (aggregates), respectively, and the latter radius is
consistent with the formation of micellar assemblies. The radius
of 42.7 Å seems too much for a spherical micelle composed of
C6 chains and suggest that the micelles have another geometry,
rather than spherical. For an oblate ellipsoidal aggregate we
obtain a length for the major semiaxis, a ) 46 Å, being the
length minor semiaxis, b, equal to the length of the surfactant
molecule.
In previous studies, the 1H NMR mutiplicities of the
methylene protons, Hendo and Hexo, of calix[4]arenes have been
used as an indication of the calixarene conformation.22 In
the present study, the signals for Hendo and Hexo of SC4TH
appear as doublets at concentrations above and below the
cmc, indicating that SC4TH is fixed in the cone conformation
both in the monomeric and micellized states.
Results and Discussion
First, steady state fluorescence spectra of pyrene were used
to determine the cmc of SC4TH. The relative changes in the
intensities of the first and the third vibronic bands in the pyrene
fluorescence spectrum as a function of SC4TH concentration
were used, since this ratio is very sensitive to the polarity of
the microenvironment.21 A plot of the results obtained from the
fluorescence study of pyrene in the presence of SC4TH is shown
in Figure 1. The breakpoint at 0.54 mM was identified as the
cmc.
In an effort to corroborate the cmc and to elucidate the various
types of aggregates that SC4TH can form, a DOSY study was
carried out at different calixarene concentrations. The results
are represented in Figure 2. The cmc was taken as the point at
which the diffusion coefficient abruptly drops to lower values,
1
Shinkai et al.23 found that the H NMR chemical shifts of
an amphiphilic p-sulfonatocalix4 arene bearing butyl groups
at the lower rim at concentrations below the cmc remain
constant, but above the cmc the Hendo and OCH2 proton
signals are shifted to higher field on increasing the calixarene
concentration. The authors suggested that the butyl groups
form the micellar core and the calixarene rings form stacks
at the micellar surface.
1
A region of the H NMR spectra of SC4TH at a series of
concentrations is shown in Figure 3. Below the cmc (0.23
mM), the spectrum essentially shows a single set of sharp

4818 J. Phys. Chem. B, Vol. 114, No. 14, 2010
Basilio et al.
Figure 3. 1H NMR spectra of SC4TH at different concentrations in D2O at 25 °C. The doublet at 4.60 ppm, triplet at 4.1 ppm, and doublet at 3.45
ppm correspond to the protons Hendo, OCH2, and Hexo, respectively.
signals and, as mentioned above, these show the multiplicity
expected for this compound in the cone conformation. When
the concentration is above the cmc, a new set of broad signals
is also observed in the spectrum and the intensity of these
signals increases linearly with the concentration. Eventually,
at 2.5 mM only the set of broad signals is visible.
A combination of NMR experiments permitted the assign-
ment of the set of broad signals of SC4TH. The signals for
protons Hendo and OCH2 (see Figure 3) were shifted most
toward higher field with respect to the analogous signal in
the other set. For the other protons, the broadening was also
evident but the signals were essentially unshifted with respect
to those of the other set (e.g., Hexo in Figure 3).
The results of 2D EXchange Spectroscopy (EXSY), in
addition to DOSY experiments, confirmed that the set of
broad signals in the spectra corresponded to monomer units
of SC4TH that are attached to/incorporated into the micelle
and undergo a slow chemical exchange equilibrium (on the
chemical shift time scale) with the set of sharp signals of
the free monomer in bulk solution. The EXSY spectra shown
in Figure 4 were acquired with a short mixing time (40 ms)
to avoid NOE effects. These spectra clearly show the presence
of cross peaks between the two sets of signals for Hendo (or
OCH2), thus confirming the existence of slow chemical
exchange between them. For two site chemical exchange,
the peak amplitude in 2D-EXSY spectra is related to the
exchange rate constant, k′, the relaxation rate, and the mixing
time, tm, by expression 2,24 where A and R are (3) and (4),
respectively.
A ) e(-Rt )
(2)
(3)
m
I11/M1 I12/M2
I21/M1 I22/M2
A )
|
|
-R1 - k'1
k'-1
R )
(4)
|
|
k'1
-R2 - k'-1
In the amplitude matrix A, I11, I12, I22, and I21 are the two-
dimensional peak amplitudes measured for sites 1 and 2 in an
experiment with a certain mixing time, and M1 and M2 are the
equilibrium magnetization values obtained from the analogous
2D-EXSY experiment acquired with a mixing time of zero.
The exchange matrix R contains the kinetic parameters to
be determined, namely the exchange rate of the direct and
reverse exchange reaction equilibrium (k′1 and k′-1) and the
proton longitudinal relaxation rates of the two sites (R1 and R2).
These parameters can be obtained directly by first diagonalizing
A and then calculating the eigenvector matrix X and its inverse
X-1 so that XD X-1 ) A where D is the diagonal eigenvalue

Monomer-Micelle Exchange in a Calixarene-Based Surfactant
J. Phys. Chem. B, Vol. 114, No. 14, 2010 4819
Figure 4. Two-dimensional EXSY spectrum of SC4TH (1 mM in D2O) acquired with a mixing time of 200 ms at 25 °C. The full view spectrum
is on the left. The spectrum on the right is an expansion showing the region for protons Hendo, OCH2, and Hexo
.
TABLE 1: Monomer-Micelle Exchange Rate Constants for
matrix. The solution to this equation is given 5 where ln D )
diag (ln λi), with λi the ith eigenvalue of A. Thus R can be
directly calculated from A
SC4TH (1 mM) in D2O at Different Temperatures
T/K
k′1/s-1
k-1/s-1
k′/s-1
298.15
308.15
313.15
318.15
2.51
3.55
6.85
7.85
7.42
9.90
10.42
11.85
9.93
13.45
17.27
19.69
ln A
tm
X(ln D)X-1
R ) -
) -
(5)
tm
calix[4]arenes17 and are up to 9 orders of magnitude slower than
those for conventional surfactants.
The two magnetization exchange rate constants, k′1 and k′-1
,
were obtained from 2D-EXSY experiments with a mixing time
of 200 ms and these values correspond to the exchange from
monomer to micelle and from micelle to monomer, respectively.
The global exchange rate constant (k′) can be calculated from
the sum of k′1 and k′-1 (an approximation due to the system
being in equilibrium24). In this paper, the monomer exchange
between aggregate and solution can be represented by reaction
6. Since the magnetization exchange between monomers in the
bulk and the micelle is a first order reaction, the relationship
between the magnetization rate constants (k′) and the reaction
rate constants (k) can be found in eqs 7 and 8
On considering the equilibrium for the monomer exchange
(eq 6), one can write k1[S][Sn-1] ) k-1[Sn]. As micelles with
aggregation numbers n - 1 and n have approximately the same
probability and the concentration of surfactant monomers in
equilibrium with the micelles equals the cmc, then k1cmc )
k-1. On the basis of this assumption, we can calculate a value
for the forward bimolecular rate constant, k1, k1 ) 7.8 × 103
dm3 mol-1 s-1 at 25.0 °C. For conventional surfactants, it is
considered that the micelle behaves as a liquid-like drop25 and
the monomer association reaction does not involve slow
structural reorganization or bond formation, meaning that k1 is
close to the diffusion-limited rate. Our results show that for
SC4TH the forward rate constant is several orders of magnitude
slower than the diffusion rate. The explanation for this
unconventional result presumably lies in the fact that the sole
barrier felt by the monomers entering the micelle arises from
long-range electrostatic repulsions due to the micellar charge.25
As SC4TH is a preorganized surfactant with four negative
charges in the upper-rim, these effects could be much more
important in this system and this could increase the activation
barrier and consequently slow down the rate constant for the
monomer association.
S + Sn-1 yk1z Sn
(6)
k-1
k'1 ) k1[S]
k'-1 ) k-1
(7)
(8)
Two-dimensional-EXSY experiments were repeated at a
series of temperatures and the exchange rates determined are
given in Table 1. The calculated values for the global exchange
rate constants (k′) are of the same order of magnitude as those
obtained for gemini surfactants8 and amphiphilic nucleotide-
As expected, the exchange rate constants increase with
temperature. A graphical representation of ln k against the
inverse of temperature (1/T) is represented in Figure 5. As can

4820 J. Phys. Chem. B, Vol. 114, No. 14, 2010
Basilio et al.
lower than those of conventional surfactants and comparable
to those of other amphiphilic calixarenes and gemini surfactants.
Acknowledgment. This work was supported by the Minis-
terio de Ciencia y Tecnolog´ıa (Project CTQ2008-04420/BQU)
and Xunta de Galicia (PGIDIT07-PXIB209041PR). N.B. ac-
knowledges FCT for a Ph.D. Grant (SFRH/BD/29218/2006).
References and Notes
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nucleotide-calix[4]arene dimer-micelle exchange rates given in
ref 17. The representation of ln k against 1/T shows linear
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Conclusions
In this study, we examined the aggregation properties of
p-sulfonatocalix[4]arene tetrahexyl ether by means of NMR
techniques and using pyrene as a fluorescence probe. The cmc
was determined by fluorescence and DOSY techniques. The
slight discrepancy between the two values obtained was
attributed to known differences between H2O and D2O. In
addition, diffusion experiments allowed us to confirm that this
surfactant forms micelles in solution with a hydrodynamic radius
of 4.27 nm. We also found that in contrast to conventional
surfactants the exchange rate between free monomers in solution
and those in the micelle is slow on the NMR time scale. The
rate constants were determined using 2D EXSY experiments
and these constants were found to be several orders of magnitude
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JP910984G