Spectroscopic and electrochemical studies on the interaction of an inclusion complex of β-cyclodextrin with 2,6-dinitrophenol in aqueous and solid phases

Article abstract of DOI:10.1016/j.molstruc.2012.10.018

The inclusion complex of 2,6-dinitrophenol (2,6-DNP) with β-cyclodextrin (β-CD) in solution phase was studied by UV-visible spectrophotometer and electrochemical (cyclic voltammetry) methods. The prototropic behaviors of 2,6-DNP were studied. The binding

Full text of DOI:10.1016/j.molstruc.2012.10.018

Journal of Molecular Structure 1036 (2013) 494–504
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Spectroscopic and electrochemical studies on the interaction of an inclusion
complex of b-cyclodextrin with 2,6-dinitrophenol in aqueous and solid phases
a
b
K. Srinivasan a, T. Stalin a, , A. Shanmugapriya , K. Sivakumar
⇑
a Department of Industrial Chemistry, Alagappa University, Karaikudi 630 003, Tamilnadu, India
b Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya University, Kanchipuram 631 561, Tamilnadu, India
h i g h l i g h t s
"
"
"
"
Photophysical and electrochemical studies plays a major role in the host-guest inclusion complex process.
The binding constants and thermodynamic parameters were calculated.
Solid complex characterized by 1H NMR, FT-IR, XRD and SEM techniques.
The structure of inclusion complex proposed by molecular docking study.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 August 2012
Received in revised form 5 October 2012
Accepted 5 October 2012
Available online 15 November 2012
The inclusion complex of 2,6-dinitrophenol (2,6-DNP) with b-cyclodextrin (b-CD) in solution phase was
studied by UV–visible spectrophotometer and electrochemical (cyclic voltammetry) methods. The proto-
tropic behaviors of 2,6-DNP were studied. The binding constant of ‘b-CD:2,6-DNP’ inclusion complex was
calculated using Benesi–Hildebrand plot at 303 K. Thermodynamic parameter (DG) involved in the com-
plex formation also calculated. It indicates that the reaction is spontaneous and exergonic process. For-
mation of solid inclusion complex between b-CD and 2,6-DNP was characterized by 1H NMR, FT-IR, XRD
techniques and SEM morphological studies. The b-CD:2,6-DNP inclusion complex obtained by molecular
docking studies is in good correlation with the results obtained through experimental methods.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
2,6-Dinitrophenol
b-Cyclodextrin
Inclusion complex
Cyclic voltammetry
PatchDock server
1. Introduction
between cyclodextrin and guest molecules by several weak forces,
including Van der Waals, hydrophobic, dipole–dipole and hydro-
The supramolecular chemistry is that discipline of chemistry,
which involves all intermolecular interactions where covalent
bonds not established between the interacting species. The major-
ity of these interactions are of the host–guest type. Familiar classes
of host molecules are the crown ethers, cryptands, cyclodextrins
and spherands [1]. Among the majority of host, the cyclodextrin
seems to be the most important host to form inclusion complexes
especially with wide variety of guest molecules with suitable
polarity and dimension [2,3]. Cyclodextrins (CDs) are cyclic oligo-
saccharides consisting of 6, 7, and 8 units of 1, 4-linked glucose
gen bonding interactions [4]. Cyclodextrins are widely used in
pharmaceutical, agro and food industries.
Nitro aromatic compounds are highly toxic to humans and ani-
mals with LD50 in the range of 25–50 mg/kg. Nitrophenols belong
to major organic pollutants that have been analyzed in the envi-
ronment. 2,6-Dinitrophenol is one of the six possible dinitro phe-
nol forms. It is in the synthesis of dyes, picric acid, picramic acid,
wood preservatives, photographic developer, explosives and insec-
ticides. They have great potential toxicities of carcinogenesis, tera-
togenesis, and mutagenesis. Therefore, the U.S. Environmental
Protection Agency (EPA) [5–7] considers that as hazardous wastes
and priority toxic pollutants. It released into the air, water and soil
by landfill leaks and accidental spills. The separation mechanism
often claimed to depend on the formation of inclusion complex,
where a guest molecule enters the relatively hydrophobic interior
of the b-CD from the larger side of the opening. However, the
inclusion complex formation of 2,6-dinitrophenol was not studied.
units, and are named as alpha (a), beta (b) and gamma (c)-cyclo-
dextrins respectively (Scheme 1). The most important structural
feature of these compounds is the hydrophobic internal cavity
and hydrophilic external surfaces. The inclusion complex formed
⇑
Corresponding author. Tel.: +91 9944266475; fax: +91 4565 225202.
E-mail address: tstalinphd@rediffmail.com (T. Stalin).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.10.018

K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
495
Scheme 1. Chemical structures of a-CD, b-CD and c-CD.
2,6-Dinitrophenol is a good candidate as a model compound to
characterize inclusion formation by 1H NMR and FT-IR
spectroscopy.
fractometer and the surface morphology was taken by Hitachi S
3000 H SEM.
In this study, we report the inclusion complex formation in
solution phase by UV–visible spectroscopy and cyclic voltammetric
technique. The solid complex were prepared and characterized by
1H NMR, FT-IR, XRD, SEM techniques and molecular docking tech-
nique (using PatchDock server) in virtual state. We found that the
virtual state analysis results correlates well with the liquid and so-
lid-state analysis results.
2.2. Reagents
b-Cyclodextrin (b-CD), were obtained from Sd fine chemical
company. 2,6-Dinitrophenol (2,6-DNP) purchased from Sigma Al-
drich. Triply distilled water is using to prepare all solutions. Solu-
tions in the pH range 2.0–12.0 were prepared by adding the
appropriate amount of NaOH and H3PO4. Yagil basicity scale (H_)
[8] for solutions above pH ꢁ 12 (using a NaOHAH2O mixture) is
used. The solutions were prepared just before taking measure-
2. Materials and methods
ments. The concentrations of the guest were of the order 10ꢀ4
–
.
10ꢀ5 mol dmꢀ3. The b-CD concentration is 0–12 ꢂ 10ꢀ3 mol dmꢀ3
2.1. Instruments
The buffer solutions of pH ꢁ 3 (0.1 M H2SO4 + 0.1 M Na2SO4) and
pH ꢁ 7 (0.1 M KH2PO4 + 0.1 M NaOH) were used for electrochemi-
cal measurements.
The UV–visible spectra were carrying out with Shimadzu UV-
2401PC double-beam spectrophotometer (range 1100–200 nm)
with scan speed at 400 nm minꢀ1. The pH values were measuring
on Elico pH meter LI-120; electrochemical studies were carrying
out using Auto lab electrochemical analyzer (GPES software), the
working electrode as glassy carbon electrode, reference electrode
was saturated calomel electrode (SCE) and platinum wire as coun-
ter electrode. All experiments were carrying out at 30 1 °C. 1H
NMR spectra were taken by BRUKER-NMR 500 MHz, FT-IR was re-
corded using Nicolet 380 Thermo Electron Corporation, Powder X-
ray diffraction spectra were taken by XPert PRO PANalytical dif-
2.3. Preparation of solid inclusion complex
The solid inclusion complex is prepared between host and guest
molecules by co-precipitation method. Accurately weighed 1 g of
b-CD and 0.1628 g of 2,6-DNP then put into a 50 ml beaker add
20 ml water and 10 ml of ethanol and put over electromagnetic
stirrer to stir 48 h at room temperature [9] and followed by refrig-
eration for 48 h. The formation of yellow precipitate filtered

496
K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
Table 1
and Fig. 1. In the pH range 4.0–15.5 absorption maxima resemble
the spectra observed in non-aqueous solvents (2-propanol
435 nm, 1,4-Dioxane 435 nm) and thus can be assigned to the neu-
tral species [14] at 430 nm. The two nitro groups are present in the
ortho position with respect to the phenolic AOH group and also the
electron density spread over the phenolic AOH group and two
adjacent NO2 groups, so that the absorption occurs in the longer
wavelength range (430 nm). There is no possibility to extend the
conjugation in the benzenoid system.
With decreasing pH from 4.0, the absorption maximum of 2,6-
DNP is blue shifted in pH ꢁ 3.0. The cause of blue shift in the
absorption spectrum was corresponds to the higher hydrogen ion
concentration, it suggest that the formation of the monocation by
protonation occurs in the hydroxyl group of 2,6-DNP. The blue shift
observed only in the lower pH, because of the presence of electron
withdrawing group (nitro) in ortho position of hydroxyl group by
adjacent side, and it will cause sterric effect, so that the protana-
tion occurs in acidic solution (pH ꢁ 3) only. Further, a red shift in
the absorption spectrum at 442 nm, it shows that the monoanion
formed in the base solution (H_15.62). The formation of monoan-
ion in high concentration of alkali condition to form phenoxide
ion in 2,6-DNP. Due to the sterric hindrance of two nitro groups
in 2,6-DNP. The prototropic equilibria is shown in Scheme 2 and
the pKa value for the equilibria were calculated spectrophotomet-
rically and the pKa value is 3.67 for 2,6-DNP. This pKa value is less
than the 2,4-DNP (pKa 4.0) [9], it indicates that the 2,6-DNP mole-
cule is more acidic than the 2,4-DNP.
Various prototropic maxima (absorption spectra) and pKa values of 2,6-DNP in
without and with b-CD medium.
Species
Without b-cyclodextrin
With b-cyclodextrin
kmax (nm)
pH
3.5
kmax (nm)
pH
3.0
Monocation
348
249
206
350
250
202
Neutral
430
255 (sh)
221
4.0–15.2
15. 62
432
256 (sh)
222
3.5–15.2
15.62
Monoanion
442
442
257 sh
227
255 sh
230
pKa 2,6-DNP
3.67
3.41
sh – Shoulder.
through in its G4 crucible and again it washed with triply distilled
water for this precipitate. The precipitate spread over in a Petri
dish as a thin layer and then it dried in oven at 50 °C for 12 h. After
dried in oven the yellow powder has obtained. This is solid inclu-
sion complex of 2,6-DNP with b-CD and it further analyzed by 1H
NMR, FT-IR, XRD and SEM analysis.
2.4. Molecular docking study
The most probable structure of 2,6-DNP:b-CD inclusion com-
plex was determined also by molecular docking studies using the
PatchDock server [10]. The 3D structural data of b-CD was obtained
from the HIC-Up database (http://www.xray.bmc.uu.se/hicup)
using the search interface. The 3D structural data of 2,6-DNP was
obtained by translating its SMILES formula generated by CORINA
server (http://www.molecular-networks.com/online_demos/cori-
na_demo). Then, the 3D structure of 2,6-DNP and b-CD was energy
minimized using the ArgusLab software. The energy minimized
structure of guest (2,6-DNP) was docked into the host (b-CD) cavity
using PatchDock server by uploading the 3D coordinate data file of
(i) b-CD as receptor molecule and (ii) 2,6-DNP as ligand molecule.
The PatchDock server carries out thorough conformational search
on a geometry-based molecular docking algorithm to identify the
docking transformations with effective molecular shape comple-
mentarity. PatchDock algorithm separates the Connolly dot surface
representation [11,12] of the host and guest molecules into con-
cave, convex and flat patches. These divided complementary
patches are matched to generate candidate transformations and
evaluated by geometric fit and atomic desolvation energy scoring
[13] function. PatchDock algorithm uses root mean square devia-
tion clustering method to select the non-redundant docked models
and to omit the redundant docked models. The thorough confor-
mational search by PatchDock server yields 100 different models
of 2,6-DNP:b-CD inclusion complex. During the conformational
search process, the PatchDock server validates each conformation
and ranks all the models with a score. The highly favorable model
will have high score. The first model will be the model with high
score and it is an energetically favorable model. Hence, the first
model was selected for the analysis and compared with the model
predicted based on the experimental (UV, CV, 1H NMR, FT-IR, XRD,
SEM) investigations.
3.2. Effect of b-cyclodextrin on 2,6-DNP
Table 2 and Fig. 2 shows that the absorption maxima of 2,6-DNP
(2 ꢂ 10ꢀ4 mol dmꢀ3) in pH ꢁ 3 (monocation) and pH ꢁ 7 (neutral)
solutions containing different concentrations of b-CD. At pH ꢁ 7,
2,6-DNP exists as a neutral form. Hence, the complex behavior also
studied in pH ꢁ 3 as a cationic form. The absorption spectra of
these two forms (monocation, neutral) are drastically different.
Both pH, there is no clear isosbestic point in the absorption spectra
even in the presence of higher concentration (12 ꢂ 10ꢀ3 mol dm3)
of b-CD was used.
The absorption spectra of 2,6-DNP in pH ꢁ 7, b-CD medium is
seen to undergo a slight red shift is observed (430–432 nm) and
the absorption maxima is increased when the increase the b-CD
concentration due to the formation of 1:1 inclusion complex with
3. Results and discussion
3.1. Effect of pH on 2,6-DNP
The absorption spectra of 2,6-DNP have been investigated in the
different pH solution and the relevant data are compiled in Table 1
Fig. 1. Absorption spectra of different prototropic species of 2,6-DNP at 303 K;
concentration 2 ꢂ 10ꢀ4 M (a) monocation, (b) neutral and (c) monoanion.

K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
497
Scheme 2. Various prototropic equilibria of 2,6-DNP in aqueous and b-CD medium.
Table 2
b-CD. In pH ꢁ 7 the 2,6-DNP entrapped into inside of the b-CD cav-
ity and the OH group projected in the upper part (rim) of the b-CD
molecule, which will cause the enhancement of absorbance value
in the absorption spectra.
Absorption maxima (nm) and loge of 2,6-DNP at different concentrations of b-CD in
pH ꢁ 7 and pH ꢁ 3 solutions.
S. no.
1
Concentration of b-CD (M)
pH ꢁ 3
pH ꢁ 7
When pH ꢁ 3, the absorption spectra has observed at 348 nm
and slightly shifted to a longer wavelength at 350 nm in higher
b-CD concentration. 2,6-DNP and b-CD concentration were held
constant for both pH (pH ꢁ 7 and pH ꢁ 3) solutions. Even though,
the pH ꢁ 7 (neutral) and pH ꢁ 3 (cation) was changed from the
influence of pH on the absorbance values of the complexation pro-
cess. The absorbance value of this inclusion complex varied at both
pH solutions. In spite of absorbance value of 2,6-DNP-b-CD com-
plex was lower in pH ꢁ 3 than in neutral (pH ꢁ 7). Because in the
protonation takes place on OH group of the 2,6-DNP and the pro-
tonated 2,6-DNP was included into the nanohydrophobic cavity
of b-CD to formed an inclusion complex. Hence, pH ꢁ 3 compared
to pH ꢁ 7 and their absorption intensities has decreased. This
behavior has been attributed to the enhanced dissolution of the
2,6-DNP molecule through the hydrophobic interaction between
2,6-DNP and b-CD. These results implies that 2,6-DNP molecule
is entrapped into the b-CD cavity to form inclusion complex in
ph ꢁ 3 solution also.
kmax (nm)
log
e
kmax (nm)
loge
Without b-CD
0.002
348
249
206
3.67
3.96
4.01
430
255
221
3.57
3.57
4.00
2
3
4
5
6
7
348
250
207
3.67
3.96
4.02
432
256
222
3.72
3.77
4.18
0.004
349
250
207
3.67
3.96
4.02
432
256
222
3.77
3.78
4.19
0.006
349
250
206
3.67
3.96
4.02
432
256
222
3.78
3.78
4.20
0.008
349
250
206
3.68
3.97
4.03
432
256
222
3.79
3.79
4.20
0.010
350
250
207
3.68
3.97
4.03
432
256
222
3.81
3.80
4.21
The formation of inclusion complex between 2,6-DNP and b-CD
the equilibrium can be written as:
0.012
350
250
207
3.68
3.97
4.03
432
256
222
3.82
3.83
4.22
b-CD þ 2; 6-DNP ¢ b-CD : 2; 6-DNP
ð1Þ
Binding constant (Mꢀ1
G (kJ molꢀ1
)
29
ꢀ8.5
52
ꢀ10
D
)
The formation of 1:1 host: guest inclusion complex, the binding
constant (K) can be obtained by using the modified Benesi–Hilde-
brand equation [15] for the 1:1 complex {Eq. (2)} between 2,6-DNP
and b-CD as:
determined by analyzing the changes in the absorption intensity
of the absorption maxima with respect of b-CD concentration. From
the intercept and slope values of this plot K was calculated
[pH ꢁ 3 = 29 Mꢀ1 and pH ꢁ 7 = 52 Mꢀ1] at 303 K (Table 2).
The determination of the thermodynamic parameters in this
inclusion process in the free energy change can be calculate from
the binding constant ‘K’ by the following equation:
1
1
1
¼
þ
ð2Þ
A ꢀ A0
D
e
K½2; 6-DNPꢃ0D ½b-CDꢃ0
e
where A – A0 is the difference between the absorbance of 2,6-DNP in
the presence and absence of b-CD, is the difference between the
De
molar absorption coefficient of 2,6-DNP and the inclusion complex
[2,6-DNP]0 and [b-CD]0 are the initial concentration of 2,6-DNP and
b-CD, respectively. Fig. 3 depicts a plot of 1/A ꢀ A0 as a function of 1/
[b-CD] for 2,6-DNP. A good linear correlation has obtained, confirm-
ing the formation of 1:1 inclusion complex. In both cases, the bind-
ing constant for the formation of 2,6-DNP:b-CD complex has been
D
G ¼ ꢀRT ln K
ð3Þ
As per the inclusion complex for the thermodynamic parameter,
D
G is negative which suggests that the inclusion process pro-
ceeded spontaneously at 303 K and the value given in Table 2.

498
K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
Fig. 2. Absorption spectra of 2,6-DNP (Conc. 2 ꢂ 10ꢀ4 M) in (a) pH ꢁ 7 and (b) pH ꢁ 3 solutions at different b-CD (a–g Conc. 0–12 ꢂ 10ꢀ3 M).
of the b-CD rim. For the formation of 1:1 host: guest complex
clearly exposed in Scheme 3. Such as 1:1 inclusion complex struc-
ture is gains further stabilization energy by releasing of water from
hydrophobic cavity.
In 1:1 complex, the binding constants for 2,6-DNP in pH ꢁ 7 are
considerably greater than cationic form (pH ꢁ 3). The binding con-
stant are very sensitive to change of pH values, which further sup-
ported the selective inclusion associated with the species form of
2,6-DNP. Of the two forms (neutral, cation), the protonated form
(2,6-DNPH+) should be included more efficiently into the b-CD cav-
ity than neutral form of 2,6-DNP, but unusually neutral species in-
cluded more efficiently, because the protonated species (in pH ꢁ 3)
can have the sterric hindrance than the neutral species (Scheme 3).
The pH dependent changes in the absorption spectra of the 2,6-
DNP molecule in b-CD solution have also been recorded (Table 1).
The effect of b-CD on the prototropic equilibrium, the 2,6-DNP
formed a monocation at pH ꢁ 3.0, monoanion at H_15.62 and neu-
tral form in between the pH ꢁ 3.5–15.0 (Scheme 2). The absorption
spectra of 2,6-DNP in ground state shows the above pH as, neutral
to monocation are blue shifted and neutral to monoanion are red
shifted in b-CD medium is similar to its in aqueous medium
Fig. 3. Benesi–Hildebrand plot of 1/A ꢀ A0 vs. 1/[b-CD] for 2,6-DNP in pH ꢁ 3 and
pH ꢁ 7 solutions.
3.3. Possible inclusion complex of 2,6-DNP in b-CD cavity
In 1:1 inclusion complex, the aromatic moiety encapsulated in
b-CD cavity and the hydroxyl group projected in the upper part
Scheme 3. The proposed structure of inclusion complex of 2,6-DNP with b-CD for 1:1 inclusion complex. The oxygen atoms are shown as red ball, nitrogen as blue and carbon
as golden yellow balls; hydrogen atoms are not shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
499
Scheme 4. Ball and stick representation of (a) b-CD (b) 2,6-DNP (c) 2,6-DNP:b-CD inclusion complex; the oxygen atoms are shown as red ball, nitrogen as blue and carbon as
gray balls; hydrogen atoms are white balls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. CV for 2,6-DNP:b-CD in (a) pH ꢁ 7 and (b) pH ꢁ 3 buffer solution, scan rate 100 mV sꢀ1, 2,6-DNP (Conc. 2 ꢂ 10ꢀ4 M) and b-CD (a–g Conc. 0–12 ꢂ 10ꢀ3 M).

500
K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
Scheme 5. Reaction mechanism of 2,6-DNP in pH ꢁ 7 and pH ꢁ 3 buffer solutions at glassy carbon electrode.
(Scheme 2). The formation of monocation (2,6-DNPH+) in b-CD
medium was in higher hydrogen ion concentration (pH ꢁ 3) when
compared to without b-CD medium (pH ꢁ 3.5). Due to the 2,6-DNP
was included into the nanohydrophobic cavity of b-CD and the H+
ion was very difficult to protonate at pH ꢁ 3.5 in the hydroxyl
group of 2,6-DNP. So that the inclusion complex (2,6-DNP:b-CD)
become more acidic (pKa = 3.41) than 2,6-DNP (pKa = 3.67). Its
indicating 2,6-DNP molecule encapsulated in b-CD cavity.
ergy ꢀ182.88 kJ/mol) was the highly probable and energetically
favorable model.
3.5. Electrochemical studies
The cyclic voltammograms shows an electrochemical behavior
of 2,6-DNP in pH ꢁ 7 and pH ꢁ 3 with b-CD (Fig. 4). The formation
of inclusion complex of 2,6-DNP and b-CD was also confirmed by
electrochemical method.
3.4. Molecular docking study of inclusion process
In pH ꢁ 7 the cyclic voltammograms shows an anodic peak dur-
ing the forward scan (towards positive potential) at 0.101 V and
two reduction peaks during the reverse scan (towards negative po-
tential) at ꢀ0.260 V and ꢀ0.705 V. These peaks are ascribed as oxi-
dation of phenolic OH (0.101 V) and the reduction of phenoxide ion
(ꢀ0.260 V) into phenolic OH and another one reduction peak of
NO2 (ꢀ0.705) into hydroxylamine.
The 3D structure of b-CD and 2,6-DNP shown in Scheme 4a and
b. The guest molecule (2,6-DNP) was docked into the cavity of b-CD
using PatchDock server. The PatchDock server program gave sev-
eral possible docked models for the most probable structure based
on the energetic parameters; geometric shape complementarity
score, approximate interface area size and atomic contact energy
[13] of the 2,6-DNP:b-CD inclusion complex. The docked 2,6-
DNP:b-CD model (Scheme 4c) with the highest geometric shape
complementarity score 2354 (computed based on the approximate
interface area size of the complex 237.8 Å2 and atomic contact en-
In pH ꢁ 3, from the cyclic voltammograms two-reduction peaks
was observed during the reverse scan (towards negative potential)
at ꢀ0.336 V and ꢀ0.534 V. These peaks are ascribed as reduction of
phenolic OH (ꢀ0.336 V) to monocationic form of 2,6-DNP and
reduction of NO2 (ꢀ0.534 V) into hydroxylamine. The oxidation

K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
501
Table 3
where IG is the reduction peak current of guest molecule of 2,6-DNP,
and IHG is the reduction peak current of inclusion complex of 2,6-
DNP:b-CD. IHG ꢀ IG is the difference between the reduction peak
CV for 2,6-DNP:b-CD in pH ꢁ 7 and pH ꢁ 3 buffer solution, scan rate 100 mV sꢀ1, 2,6-
DNP (Conc. 2 ꢂ 10ꢀ4 M) and (a–g) 0–12 ꢂ 10ꢀ3 M b-CD concentrations.
b-CD
concentration
(M)
pH ꢁ 3
Epc (V)
pH ꢁ 7
current of inclusion complex and 2,6-DNP.
DI is the difference be-
tween the molar peak current coefficient of the inclusion complex
and 2,6-DNP. The [2,6-DNP]0 and [b-CD]0 are the initial concentra-
tion of 2,6-DNP and b-CD, respectively.
Ipc
(lA)
Epa
Ipa
Epc (V)
Ipc (lA)
(V)
(lA)
Plotting of [1/IHG ꢀ IG] verses 1/[b-CD] gives a straight line for
both pH solution as shown in Fig. 5. Good linear correlations were
obtained (r = 0.9881, 0.9841 for pH ꢁ 3 and pH ꢁ 7 respectively),
confirm that the formation of a 1:1 inclusion complex for both
pH (pH ꢁ 3 and pH ꢁ 7) solutions. From the intercept and slope
values of this plot K was evaluated, the binding constant values
for 2,6-DNP:b-CD is 16 Mꢀ1and 36 Mꢀ1 in pH ꢁ 3 and pH ꢁ 7
respectively. These values indicate that 2,6-DNP molecule is encap-
sulated in the b-CD cavity to form an inclusion complex.
Without b-CD
0.002
ꢀ0.336 ꢀ2.557
ꢀ0.534 ꢀ8.629
ꢀ0.334 ꢀ3.467
ꢀ0.531 ꢀ8.641
ꢀ0.331 ꢀ4.053
ꢀ0.529 ꢀ8.917
ꢀ0.329 ꢀ4.072
ꢀ0.529 ꢀ9.289
ꢀ0.319 ꢀ4.162
ꢀ0.519 ꢀ9.434
ꢀ0.309 ꢀ4.233
ꢀ0.512 ꢀ9.443
0.101 1.832 ꢀ0.260 ꢀ1.063
ꢀ0.705 ꢀ7.981
0.103 1.827 ꢀ0.263 ꢀ1.795
ꢀ0.700 ꢀ8.306
0.103 1.810 ꢀ0.263 ꢀ1.940
ꢀ0.697 ꢀ8.836
0.103 1.774 ꢀ0.265 ꢀ2.164
ꢀ0.692 ꢀ10.100
0.103 1.673 ꢀ0.268 ꢀ2.420
ꢀ0.688 ꢀ10.170
0.106 1.436 ꢀ0.268 ꢀ2.583
ꢀ0.688 ꢀ11.030
0.004
0.006
0.008
3.6. 1H NMR spectrum
0.010
The prepared solid inclusion complex can be analyzed by 1H
NMR spectra [16]. Fig. 6 shows the typical 1H NMR spectra of (a)
b-CD, (b) the solid inclusion complex of 2,6-DNP with b-CD (chem-
ical shift changes with b-CD), (c) 2,6-DNP and (d) the solid inclu-
sion complex (chemical shift changes with 2,6-DNP). The values
of chemical shifts, d for different protons in b-CD with solid inclu-
sion complex and 2,6-DNP with solid inclusion complex were
listed in Tables 4 and 5. The changes in chemical shift (d) of H3
and H5 protons suggested that the 2,6-DNP guest is encapsulated
into the nanohydrophobic cavity of b-CD. The phenyl ring of 2,6-
DNP shift the signals of b-CD protons (H3 and H5) upfield, due to
the anisotropic effect of the aromatic ring. On the contrary, the
chemical shifts of H1, H2, H4, which are exposed to the solvent
and H6ab, which are on the narrow opening of b-CD, as shown in
Scheme 6b, are slightly affected by the guest molecule. On the
other hand, the chemical shifts of Hb and Hc of 2,6-DNP
(Scheme 6c) which are inserted in the nanocavity of b-CD are also
shifted upfield due to the interaction between 2,6-DNP and b-CD.
(On the other hand, as shown in Fig. 6d, when 2,6-DNP monomer
entered into the nanohydrophobic cavity of b-CD, the change of
the microenvironment of 2,6-DNP protons leads to the phenyl ring
protons was moved into upfield shift. The significant distinguish
for these 1H NMR spectra strongly confirmed that the solid com-
plex was formed in the inclusion process.
0.012
ꢀ0.287 ꢀ5.884
ꢀ0.482 ꢀ11.760
16
0.106 1.005 ꢀ0.273 ꢀ2.693
ꢀ0.683 ꢀ12.000
Binding constant
36
(Mꢀ1
)
D
G (kJ molꢀ1
)
ꢀ7
ꢀ9
Fig. 5. Benesi–Hildebrand plot of 1/IHG ꢀ IG vs.1/[b-CD] for 2,6-DNP in pH ꢁ 7 and
pH ꢁ 3 solutions.
3.7. FT-IR spectral studies
and reduction mechanism of 2,6-DNP was clearly explained in
Scheme 5.
The solid complex formation confirmed by FT-IR spectroscopy
(Fig. 7), because the bands resulting from the included part of
the guest molecule generally shifted or their intensities altered
[9]. In 2,6-DNP the two peaks are observed at 1537 cmꢀ1 and
1346 cmꢀ1 due to the characteristics stretching of aromatic nitro
group. The characteristics peak of ACAN stretching vibration ap-
peared at 855 cmꢀ1 for aromatic nitro group. The AC@CA stretch-
ing vibration appeared at 1622 cmꢀ1 and 1589 cmꢀ1 for aromatic
nuclei. The @CAO stretching vibration appears at 1154 cmꢀ1. In
the spectrum for complex the characteristics stretching peaks of
aromatic nitro group intensities are decreased and shifted to
1524 cmꢀ1 and 1337 cmꢀ1, this is due to the nitro groups were in-
cluded into the b-CD cavity. The characteristic ACAN stretching
vibration was shifted to 856 cmꢀ1 and it intensity also decreased,
this is confirmed that the nitro group was included into the b-CD
cavity. The characteristic stretching vibration of AC@CA intensities
are reduced and shifted to 1633 cmꢀ1 and 1591 cmꢀ1 this is due to
the benzene ring encapsulated into the b-CD cavity. The character-
istic @CAO stretching vibration slightly shifted to 1156 cmꢀ1 this
indicates the OH group is projected above the b-CD rim. From
The cathodic peak current (Fig. 4 and Table 3) ipc, increased with
increasing the b-CD concentration in both pH solutions (pH ꢁ 3
and pH ꢁ 7). The cathodic peak potential, Epc, shifted in positive
direction when b-CD concentration increased in both case. The re-
sult showed that the inclusion complex between 2,6-DNP and b-CD
was formed when 2,6-DNP was added into b-CD aqueous solution.
In addition, the cathodic peak current was increased with increas-
ing b-CD concentration; this is due to the nitro groups are encap-
sulated in the b-CD cavity and the catalytic behavior of b-CD to
the included guest molecule (2,6-DNP). This would lead the catho-
dic peak potential shift in positive direction and the anodic peak
potential shift in positive direction.
The binding constant (K) and stoichiometric ratios of the inclu-
sion complex of 2,6-DNP can be determined according to the Bene-
si–Hildebrand [15] relation assuming the formation of a 1:1 host–
guest complex.
1
1
1
¼
þ
ð4Þ
IHG ꢀ IG
D
I
K½2; 6-DNPꢃ0DI½b-CDꢃ0

502
K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
Fig. 6. 1H NMR spectra of (a) b-CD, (b) the solid complex (chemical shift of b-CD from 3 to 5 ppm); (c) 2,6-DNP, and (d) the solid complex (chemical shift of 2,6-DNP from 6.5
to 8.5 ppm).
the above task, the nitro group and benzene ring is complete
sions it can be concluded that of 2,6-DNP molecule was included
encapsulated into the b-CD cavity. As can be seen above discus-
into b-CD cavity.

K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
503
Table 4
1H NMR chemical shifts of b-CD in free and complexed state determined in D2O at
303 K.
Proton b-CD d
2,4-DNP-b-CD d
(ppm) [xx]
D
d
2,6-DNP-b-CD
d (ppm)
Dd
(ppm)
H1
H2
H3
H4
H5
H6
4.966
3.554
3.877
3.535
3.748
3.797
4.960
3.548
3.860
3.528
3.720
3.795
ꢀ0.006 4.964
ꢀ0.006 3.550
ꢀ0.017 3.850
ꢀ0.007 3.531
ꢀ0.028 3.738
ꢀ0.002 3.796
ꢀ0.002
ꢀ0.004
ꢀ0.027
ꢀ0.004
ꢀ0.010
ꢀ0.001
Table 5
1H NMR chemical shifts of 2,6-DNP in free and complexed state determined in D2O at
300 K.
Proton
2,6-DNP d (ppm)
2,6-DNP-b-CD d (ppm)
Dd
Hb
Hc
8.324
7.053
8.300
6.999
ꢀ0.024
ꢀ0.054
Fig. 8. Powder X Ray diffraction spectra of (a) 2,6-DNP, (b) b-CD and (c) solid
inclusion complex of 2,6-DNP:b-CD.
3.8. Powder X-ray diffraction spectrum
The formation of the solid complex is can be confirmed by X-ray
diffractometer [17,18]. Due the crystalline nature of the guest mol-
ecule is shift to semicrystaline structure in solid inclusion complex.
Fig. 8 is the powder X-ray diffraction pattern of (a) 2,6-DNP, (b) b-
CD and (c) solid inclusion complex. The X-ray diffraction pattern of
the (c) solid inclusion complex was evidently different from that of
(a) 2,6-DNP and (b) b-CD monomer itself, because the (a) 2,6-DNP
lost its crystalline structure and it formed as semicrystaline struc-
ture. The difference is observed between these pattern is indicated
that the solid inclusion complex was formed between b-CD and
2,6-DNP.
3.9. Scanning electron microscope morphological observations
Scanning Electron Microscope (SEM) are well suited for visual-
ize the surface texture of materials. The SEM analysis is ideal for
Fig. 7. FT-IR Spectra of (a) 2,6-DNP, (b) b-CD and (c) solid inclusion complex of 2,6-
DNP:b-CD complex in KBr.
Scheme 6. (a) The stereo-configuration, (b) truncated-cone of b-CD and (c) 2,6-DNP.

504
K. Srinivasan et al. / Journal of Molecular Structure 1036 (2013) 494–504
quantitatively measuring the surface roughness and for visualizing
the surface texture of the compounds. First, the surface morpho-
logical structure of (a) 2,6-DNP, (b) b-CD (Fig. 9) observed by
SEM, and then we observed the surface morphological structure
of (c) solid complex. These pictures clearly elucidated the differ-
ence of each other. Modification of crystals can be proved as a
one of the evident for the formation of a solid inclusion complex.
4. Conclusions
2,6-DNP forms a inclusion complex with b-CD in aqueous solu-
tion. The formation of the inclusion complex got a clear results was
obtained from UV spectroscopy and cyclic voltammetry measure-
ment. The inclusion complex was formed as a 1:1 stoichiometry
of 2,6-DNP to the b-CD, while NMR, FT-IR, XRD and SEM analysis
showed that the ANO2 group attached aromatic ring of the 2,6-
DNP molecule completely encapsulated in the b-CD cavity. The
protonated AOH2 group projected in the outside of the b-CD cavity.
The binding constant (K) value was calculated using Benesi–Hilde-
brand equation. The energetically favorable complex obtained by
molecular docking studies is in good correlation with the inclusion
model predicted through experimental investigations.
Acknowledgements
The corresponding author Dr. T. Stalin acknowledge to Alagappa
University, Karaikudi, the sanction of AURF research Project fund
(A13/AURF-IC/1264/2010) for this research work and thankful to
Dr. C. Sanjeeviraja, School of Physics, Alagappa University, Karaik-
udi for utilizing the XRD analysis and also SAIF IIT Madras for uti-
lizing the 1H NMR spectra analysis.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2012.10.
018.
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Fig. 9. Scanning electron microscope photographs (Pt. coated) of (a) 2,6-DNP (b) b-
CD and (c) solid complex of 2,6-DNP:b-CD.