Optics and Photonics Journal, 2011, 1, 59-64
doi:10.4236/opj.2011.12009 Published Online June 2011 (http://www.SciRP.org/journal/opj/)
Studies on the Inclusion Behavior of Amphiphilic
p-Sulfonatocalix[4]arene with Ascorbic Acid by
Spectrofluorometric Titrations
Yunyou Zhou, Xueping Ding, Xiaoli Fang, Tao Li, Dongbao Tang, Qin Lu
Key Laboratory of Chemo-Biosensing, College of Chemistry and Materials Science, Anhui Normal University, Wuhu,
China
Received April 5, 2011; revised May 6, 2011; accepted May 16, 2011
Abstract
The aqueous solution of tetrabutyl ether derivatives of p-sulfonatocalix[4]arene (SC4Bu) and ascorbic acid
(AA) complex has been studied based on fluorescence and 1H NMR spectroscopic results. It was found that
the fluorescence intensity of SC4Bu quenched regularly upon the addition of AA. A 1:1 stoichiometry for the
complexation was established and was verified by Job’s plot. The temperature-dependent inclusion constants
were calculated, form which H and S values were calculated. Meanwhile the proposed interaction
mechanism of the inclusion complex was discussed based on 1 H NMR results. The various factors (ionic
strength, and surfactants) effecting the inclusion process were examined in detail.
Keywords: P-Sulfonatocalix[4]arene, Ascorbic Acid, Fluorescence Spectrometry, Inclusion Interaction
1. Introduction
Calixarenes and their derivatives have attracted consid-
erable attention in host-guest chemistry due to their ex-
cellent recognition ability [1,2]. The water-soluble
p-sulphonated calyx[n]arenes (SCnA, n = 4, 6, 8) deriva-
tives display wider applications in supramolecular chem-
istry science because they allow the study of host-guest
interactions in a solvent where most biological processes
take place [3-9]. P-sulphonated calix[n]arenes (SCnA, n
= 4, 6, 8) have been reported to interact with specific
drugs or their intermediates [10-12]. However, SC4A
and SC6A have very weak fluorescence and absorption
signals in an aqueous solution [10]. They are difficult to
be used for the investigation of inclusion behavior of
non-fluorescent or weakly fluorescent guest molecules
[13]. Therefore, it is of great importance to synthesize
fluorescent water-soluble calixarene derivatives for
studying the inclusion behavior of these non-fluorescent
molecules.
Ascorbic acid (AA, Scheme 1), commonly known as
Vitamin C, is one of the most important water-soluble
vitamins in foods and drinks [14,15]. It is also widely
used as a food additive and antioxidant [16], which can
prevent scurvy and reduce the incidence and mortality
from cardiovascular disease and cancer [17].
Herein, we have synthesized the tetrabutyl ether de-
rivative of p-sulfonatocalix[4]arene (SC4Bu, Scheme 1)
with strong fluorescence in aqueous solution and also
investigated the inclusion behavior of SC4Bu with AA in
aqueous solution using fluorescence spectrometry. The
results indicate that SC4Bu and AA form a complex (1:1
mole ratio), with a binding constant of 1.78 × 103 L·M-1.
The interaction mechanism of the inclusion process was
discussed. This work may extend the application of
SC4Bu in the biochemistry area.
Scheme 1 chemical structure of ascorbic acid (1) and
SC4Bu (2)

Y. Y. ZHOU ET AL.
60
2. Experimental
2.1. Apparatus
Fluorescence spectra and relative fluorescence intensities
were performed on a model F-4600 fluorescence spectro-
photometer (Hitachi, Japan) using a 1 cm × 1 cm quartz
cell. The slit widths for excitation and emission were both
set to 5.0 nm. The fluorescence lifetimes were measured
on a FLS920 combined steady-state lifetime fluorescence
spectrometer (Edinburgh Instrument). All measurements
were carried out at desired temperature adjusted by a
thermostatic cell holder. 1H NMR was performed on an
Avance Bruker-300 MHz spectrometer. IR was obtained
using a PE983 infrared spectrophotometer (Perkin Elmer).
2.2. Chemicals
SC4Bu was synthesized according to the literature [18,
19] and identified by IR and 1H NMR. AA was pur-
chased from Shanghai Chemical Reagent Co. All other
reagents were of analytical reagent grade and used with-
out any further purification. Doubly distilled water was
used throughout.
2.3. Procedure
A 1.0 mL of 1.00 × 10–4 mol·L–1 SC4Bu, and an appro-
priate amount of 1.00 × 10–3 mol·L–1 AA were trans-
ferred into a 10 mL volumetric flask. The mixture was
diluted to the final volume with water and mixed thor-
oughly. Fluorescence spectra were measured at 25.0˚C.
3. Results and Discussion
3.1. Characteristics of Fluorescence Spectra
Figure 1 displays the fluorescence spectra of 1.00 × 10−5
mol· L–1 SC4Bu in aqueous solution. It is clear that
SC4Bu shows strong fluorescence in aqueous solution
with excitation and emission wavelengths at 230 and 315
nm, respectively. When an appropriate amount of 1.00 ×
10–3 mol·L–1 AA was added to SC4Bu, the fluorescence
quenching of SC4Bu was observed, as shown in Figure
2, indicating the formation of the inclusion complexes.
The formation of inclusion complex was also revealed
by the fluorescence decay curves obtained at different
concentrations of AA. The fluorescence lifetimes of the
SC4Bu molecule in aqueous and in AA were determined
from the decay curves and the results are shown in Table
1. The fluorescence decay of SC4Bu without AA ob-
tained from monitoring the emission at 312 nm is a sin-
gle-exponential decay with lifetime value of 0.72 ns (τ1).
Figure 1. Fluorescence excitation (a) and emission (b) spec-
tra of SC4Bu (1.00 × 10−5 mol·L−1) in solution.
Figure 2. Fluorescence spectra of SC4Bu with different
concentrations of AA. The concentrations from (1) to (15)
of AA were: (1) 0.00, (2) 0.20, (3) 0.40, (4) 0.60, (5) 0.80, (6)
1.00, (7) 1.20, (8) 1.40, (9) 1.60, (10) 1.80, (11) 2.00, (12) 2.50,
(13) 3.00, (14) 3.50 and (15) 4.00 × 10−4 mol·L−1. SC4Bu =
1.00 × 10–5 mol·L−1, 25.0◦C.
Table 1. Time-resolved fluorescence spectral data of SC4Bu
in the absence and presence of different concentrations of
AA (λex = 250nm, λem = 310 nm; [SC4Bu] = 1.0 × 10−5 M)
Concentration of
AA (M)
Lifetime
(ns)
Relative ampli-
tudes (%) χ2 Standard devia-
tion (ns)
0 0.72 100 1.018 0.02
0.2 × 10−5 0.78
2.52
84.12
15.88 1.062 0.02 0.16
0.4 × 10−5 0.77
2.62
82.53
17.47 1.002 0.02 0.17
0.8 × 10−5 0.76
2.50
81.86
18.14 1.021 0.02 0.14
Upon the addition of AA, single exponential decay
changes to biexponential decay with lifetime τ1 and τ2, re-
spectively, suggesting the presence of two species, SC4Bu
and its complex. It is also evident from Table 1 that the
short-lived species (τ1) is close to the measured lifetime of

Y. Y. ZHOU ET AL.
61
SC4Bu in pure water, and that of the long-lived species τ2
are correspond to the inclusion complex. The amplitude of
the complex form also increases due to an increase in
complex formation and the free species decreasing.
3.2. Stoichiometry and Inclusion Constant
The formation of 1:1 SC4Bu-AA complex was con-
firmed by the continuous variation method (Job’s plot)
using fluorescence spectrometry. Here, the solutions of
SC4Bu and AA were mixed in different mole ratio keep-
ing the total of the SC4Bu and AA concentration con-
stant. The maximum relative fluorescence intensity was
observed when [SC4Bu]/([SC4Bu] + [AA]) = 0.5 as
shown in Figure 3, this is in agreement with the double
reciprocal plot and confirmed the formation of a 1:1 in-
clusion complex in the system.
Assuming that SC4Bu and AA forms a 1:1 ratio com-
plex, according to our earlier work [12, 20] the equation
of host–guest inclusion complex can be expressed as:


010 10
11 1
F
FFFKGFF

 
(1)
Here F0 is the intensity of fluorescence of SC4Bu with-
out AA, F is the observed fluorescence intensity at differ-
ent AA concentration tested, F1 is the intensity with the
highest concentration of AA and K is the binding constant.
The double reciprocal plots of


0
1
F
F versus 1/[AA]
are depicted in Figure 4. The plot displays a linear relation
with a relative correlation constant of 0.9993, indicating
the formation of a 1:1 stoichiometry between SC4Bu and
AA. The association constant (K) obtained from the ratio
of the intercept to the slope is 1.78 × 103 L· M–1.
3.3. Thermodynamic Parameter Value
The binding constants at various temperatures were inves-
tigated by measuring the fluorescence spectra of SC4Bu in
the presence of AA, and the results were summarized in
Table 2. By plotting lnK against 1/T (the Van’t Hoff
method) [19] (see Figure 5), the enthalpy (
H
) and en-
tropy (S) were calculated to be 25.25 kJ·mol–1, 146.75 J
mol–1K–1, respectively. These results suggested that the
entropic driving force favored the formation of SC4Bu–AA
complex. The relevant free energy change for this system is
G = –18.48 kJ·mol–1 at 298 K, indicating that this inclu-
sion process is an energetically favored process.
3.4. 1H NMR Studies
Chemical shift variations of specific host or guest nu-
cleus could provide evidence for the formation of inclu-
sion complexes in solution, since significant changes in
Figure 3. Job’s plot of SC4Bu–AA system, [SC4Bu] + [AA]
= 1.00 × 10−5 mol·L−1, 25.0◦C.
Figure 4. A plot of 1/(F–F0) versus 1/[SC4Bu] for SC4Bu–
AA complex.
Table 2. Inclusion constants of complexes at different tem-
peratures.
Temperature (K)288 298 208 318
Inclusion constant
（×103） 1.26 ± 0.141.78 ± 0.09 2.32 ± 0.11 3.38 ± 0.05
R 0.99960.9993 0.9997 0.9969
Figure 5. The plot of ln K versus 1/T for the SC4Bu–AA
complex.

Y. Y. ZHOU ET AL.
62
Table 3. Chemical shifts

and


of protons of AA in free guest and inclusion complex.
H
1
H
2
H
3
H
4
H
a
H
b
H
c

(AA) 3.7185 4.0281 4.9374

(SC4Bu) 7.6359 4.1250/3.4247 3.9762 1.4676

(SC4Bu-AA) 7.5850 4.0354/3.3976 3.9607 1.4657 3.7463 4.0354 4.9212

 –0.0509 –0.0896/–0.0271 –0.0155 –0.0019 +0.0278 +0.0073 –0.0162
microenvironment are known to occur between the free
and bound states [21]. The interaction between SC4Bu and
AA were studied by 1H NMR spectra. The significant dis-
tinction between the 1H NMR spectra of SC4Bu and the
inclusion complex of AA with SC4Bu in D2O confirms
that the inclusion complex was formed and the value of
chemical shifts for different protons in SC4Bu, AA and
SC4Bu-AA inclusion complex as shown in Table 3.
The 1H NMR shifts are in agreement with those re-
ported in the literatures [21-22], which mentioned that
downfield shifts should be observed for guest protons
and upfield shifts for host protons upon hydrophobic
interactions between both partners. The complexation
causes an upfield shift of SC4Bu protons: a great upfield
was recorded for H1and H2. Since both of them are lo-
cated inside the cavity. The upfield shift observed for H2
and H1 confirms the inclusion inside the cavity. Further
confirmation was obtained by observing the changes in
the chemical shifts of AA.
The H-chemical shifts in both the free and the com-
plexed state are reported in Table 3. We can see that Hc
proton was shifted upfield, while Ha and Hb were de-
shielded upon complexation. In fact, the downfield shifts
of the guest protons have been attributed to a variation of
local polarity when these protons are inside the cavity
[21]. The shielding of Hc proton has been attributed to it
is close to the cavity of SC4Bu. As expected, the prob-
able binding mode in the SC4Bu inclusion complexes
involves the insertion of less part of the guest into the
cavity and exposure of more groups to the bulk solvent
outside.
3.5. Discussion of Interaction Mechanism
Calixarenes and their derivatives have been known to be
able to form non-covalent inclusion complexes with
various guest molecules through many interactions, such
as the electrostatic interaction, cation–π interactions,
hydrogen bonding, van der Waals and hydrophobic in-
teractions [23]. In this context, considering the size/shape
fitting, multiple recognition and 1H NMR results, pattern
inclusion manner between SC4Bu and AA was proposed
as show in Figure 6. AA partially goes into the cavity of
SC4Bu with the help of hydrogen bonding, which
formed by the hydroxyl groups of AA bonding with sul-
phonyl groups of SC4Bu, respectively.
The main driving force for the inclusion of AA by
SC4Bu was investigated. The electrostatic effect was
studied by varying the ionic strength of NaCl and the
results were shown in Figure 7. As can be see when the
NaCl concentration is 0.01 mol·L-1, the fluorescence in-
tensity of the SC4Bu–AA system had no significant
changes compared with the absence of NaCl in the sys-
tem. Thus the main driving force for inclusion of AA by
SC4Bu should not be electrostatic interaction. The hy-
drogen bonding and the hydrophobic interaction should
play the main driving force for this inclusion process.
This is different from what the literature suggests, in
which electrostatic interaction was thought to play im-
portant part in the inclusion process [12].
Figure 6. The proposed inclusion pattern of the SC4Bu-AA.
complex.
Figure 7. Influence ionic strength of NaCl on the fluores-
cence intensity of SC4Bu–AA system; [SC4Bu] = 1.00 × 10−5
mol·L−1, CNaCl = 0.01 mol·L−1.

Y. Y. ZHOU ET AL.
63
Figure 8. Influence of surfactants concentration on the
fluorescence intensity of SC4Bu–AA system; (1)
SC4Bu+TritonX-100; (2) SC4Bu+AA+TritonX-100; (3)
SC4Bu+CTAB; (4) SC4Bu+AA+ CTAB; (5) SC4Bu+SDS;
(6) SC4Bu+AA +SDS; [SC4Bu] = 5.00 × 10–6 mol·L−1, [AA]
= 6.00 × 10–5 mol·L–1.
3.6. Influence of Surfactants
The influence of surfactants on this inclusion process was
also investigated. Three types of surfactants: the cationic
surfactant cetytrimethyammonium bromide (CTAB), the
anionic surfactant sodium dodecyl sulfate (SDS) and the
non-ionic surfactant octylophenylpolyoxyethylene ether
(Triton X-100) were chosen to study their effect on
SC4Bu and AA complex. Among those, SDS showed no
influence on the fluorescence intensity of SC4Bu and the
SC4Bu-AA complex as shown in Figure 8, while Triton
X-100 and CTAB enhanced the fluorescence intensities
of SC4Bu and the corresponding SC4Bu-AA complex.
Triton-100 itself has been reported to be able to enhance
the fluorescence intensity of certain types of sulfuric
fluorophores [24], Similarly, it may also lead to the in-
crease of the fluorescence intensity of SC4Bu and the
SC4Bu-AA complex. In our previous work, we have
reported that SCnA can form a 1:1 complex with CTAB,
which lead to the enhancement of the fluorescence inten-
sity of SCnA [24]. Similarly, CTAB may lead to the in-
crease of the fluorescence intensity of SC4Bu through
the inclusion process. Upon addition of AA, the quench
of the fluorescence intensity of the SC4Bu-CTAB com-
plex was observed, indicating the partial replacement of
CTAB with AA [25].
4. Conclusions
In this work, a water-soluble p-sulfonatocalix[4]arene
derivative (SC4Bu) with strong fluorescence was synthe-
sized and inclusion process between SC4Bu and AA in
aqueous solution was also investigated by fluorescence
spectrometry. AA and SC4Bu were found to form a 1:1
host-guest inclusion complex. The possible interaction me-
chanism was discussed. Similar to cyclodextrins, SC4Bu
and its inclusion complexes show potential for biological
and medical applications.
5. Acknowledgements
The authors thank the financial supports of the National
Natural Science Foundation of P. R. China (20875002).
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