Study of the cyclodextrin and its complexation with 2,4-dinitrobenzoic acid through photophysical properties and 2D NMR spectroscopy

Article abstract of DOI:10.1016/j.molstruc.2013.11.048

The host-guest inclusion complex formation of 2,4-dinitrobenzoic acid (2,4-DNB) with nano-hydrophobic cavity of β-cyclodextrin (β-CD) in solution phase were studied by UV-visible spectrophotometer and electrochemical method (cyclic voltammetry, CV). The prototropic behaviors of 2,4-DNB with and without β-CD and the ground state acidity constant (pKa) of host-guest inclusion complex (2,4-DNB-β-CD) was studied. The binding constant of the inclusion complex at 303 K was calculated using Benesi-Hildebrand plot. The solid inclusion complex formation between β-CD and 2,4-DNB was confirmed by ¹H NMR, 2D ¹H NMR (ROESY), FT-IR, XRD and SEM analysis. A schematic representation of this inclusion process is proposed by molecular docking studies using the patch dock server.

Full text of DOI:10.1016/j.molstruc.2013.11.048

Journal of Molecular Structure 1060 (2014) 239–250
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Study of the cyclodextrin and its complexation with 2,4-dinitrobenzoic
acid through photophysical properties and 2D NMR spectroscopy
a
b
T. Stalin a, , K. Srinivasan , K. Sivakumar
⇑
a School of Chemical Sciences, Department of Industrial Chemistry, Alagappa University, Karaikudi 630 003, Tamilnadu, India
b Department of Chemistry, Sri Chandrasekharendra Saraswathi Viswa Mahavidyalaya University, Enathur, 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.
Solid complex characterized by 1H NMR, 2D 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 13 August 2013
Received in revised form 10 November 2013
Accepted 20 November 2013
Available online 28 December 2013
The host–guest inclusion complex formation of 2,4-dinitrobenzoic acid (2,4-DNB) with nano-hydropho-
bic cavity of b-cyclodextrin (b-CD) in solution phase were studied by UV–visible spectrophotometer and
electrochemical method (cyclic voltammetry, CV). The prototropic behaviors of 2,4-DNB with and with-
out b-CD and the ground state acidity constant (pKa) of host-guest inclusion complex (2,4-DNB-b-CD)
was studied. The binding constant of the inclusion complex at 303 K was calculated using Benesi–Hilde-
brand plot. The solid inclusion complex formation between b-CD and 2,4-DNB was confirmed by 1H NMR,
2D 1H NMR (ROESY), FT-IR, XRD and SEM analysis. A schematic representation of this inclusion process is
proposed by molecular docking studies using the patch dock server.
Keywords:
b-Cyclodextrin
2,4-Dinitrobenzoic acid
Patch dock server
2D 1H NMR
Ó 2013 Elsevier B.V. All rights reserved.
ROESY
1. Introduction
(van der Waals attractions, hydrogen bonding, hydrophobic attrac-
tions, etc.) between the host (CDs) and guest molecules [5,6]. The
Cyclodextrins (CDs) are cyclic oligosaccharides consisting of 6,
7, and 8 units of 1,4-linked glucose units, and are named alpha
(a), beta (b) and gamma (c)-Cyclodextrins, respectively (Scheme 1).
CDs also have several advantages in other areas, such as the food,
cosmetics industries and agro chemistry [7–9], especially owing
to their capacity to protect the guest molecules against oxidation,
light-induced reaction and loss by evaporation. Additionally, they
usually enhance the aqueous solubility of poorly soluble or even
insoluble compounds [10].
2,4-Dinitrobenzoic acid (2,4-DNB) belong to major organic pol-
lutants that have been analysed in the environment. 2,4-DNB used
as an anticorrosion protection of metals and or coated with a Cr-
laminated glass support when mixed with cross linkable buthyl-
metacrylate and then heated under defined conditions. Aqueous
emulsions of some dinitrobenzoic acids were used in order to ren-
der steel sheet and strip C-smut free after batch annealing [11].
2,4-DNB are listed as priority pollutions by the US Environmental
Protection Agency [12]. They have great potential toxicities of car-
cinogenesis, teratogenesis, and mutagenesis [13,14]. It’s released
into the air, water and soil. It may also be released into the envi-
ronment from landfill leaks and accidental spills. Consequently,
due to the harmful effects of these organic compounds, the
These macromolecules, which can be spatially represented as a
torus with wide and narrow openings corresponding to secondary
and primary hydroxyl groups respectively, can encapsulate a large
variety of compounds due to the hydrophobic character of their
internal cavity [1]. Although the depth of the cavities for the three
CDs is the same (ꢁ0.78 nm), their cavity diameters are ꢁ0.57, 0.78
and 0.95 nm respectively (Scheme 1). Due to the unique chemical
structure of CD molecules, the inner side of the cavity is hydropho-
bic and the outer side is hydrophilic. The hydrophobic nature of the
CD cavities facilitates the ability of CDs to act as host for both non-
polar and polar guests, which include small molecules as well as
polymers [1–4]. Once the inclusion compound is formed, the sta-
bility of the guest molecules increases due to the binding forces
⇑
Corresponding author. Tel.: +91 9944266475; fax: +91 4565 225202.
E-mail addresses: drstalin76@gmail.com, tstalinphd@rediffmail.com (T. Stalin).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.11.048

240
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
Scheme 1. 3D view of chemical structures of a-CD, b-CD and c-CD.
wastewaters containing them must be treated before being dis-
charged to receiving water bodies. In order to assess the fate of
2,4-DNB in wastewater and to control their mobility and reactivity
during remediation processes, the sorption behavior of these toxic
contaminants must be understood and revealed.
relaxation delay of 1 s and mixing time 300 ms under the spin lock
conditions. Powder X-ray diffraction spectra were taken by XPert
PRO PANalytical diffractometer (2Theta:0.001; Minimum step size
Omega:0.001). The surface morphology was taken by Hitachi S
3000 H SEM.
The author studied the host-guest inclusion complex mecha-
nism between 2,4-DNB and b-CD in different techniques. In this
study, we report for the spectral and electrochemical behaviors
of 2,4-DNB in different pH with b-cyclodextrin. With respect to
the formation of host-guest inclusion complex of 2,4-DNB with
b-CD in solution phase was studied by UV–Visible spectroscopy
(UV–Vis) and cyclic voltammetric technique (CV). The solid com-
plex was prepared and characterized by 1H NMR, 2D 1H NMR
(ROESY), FT-IR, XRD, and SEM techniques. The schematic represen-
tation of this inclusion process is proposed by molecular docking
studies using the patch dock server.
2.2. Molecular docking study
The most probable structure of the 2,4-DNB:b-CD inclusion
complex was determined also by molecular docking studies using
the Patch Dock server [15]. The 3D structural data on b-CD was ob-
tained from HIC-Up database (http://xray.bmc.uu.se/hicup) using
the search interface. The 3D structures of 2,4-DNB were obtained
by translating the SMILES formula of 2,4-DNB using CORINA server
(http://www.molecular-networks.com/
online_demos/cori-
na_demo).The guest molecule (2,4-DNB) was docked into the host
molecule (b-CD) cavity using PatchDock server by submitting the
3D coordinate data of 2,4-DNB and b-CD molecules. Docking was
performed with complex type configuration settings. PatchDock
server follows a geometry-based molecular docking algorithm to
find the docking transformations with good molecular shape com-
plementary. PatchDock algorithm separates the Connolly dot sur-
face representation [16] of the molecules into concave, convex
and flat patches. These divided complementary patches are
matched in order to generate candidate transformations and eval-
uated by geometric fit and atomic desolvation energy scoring [17]
function. RMSD (root mean square deviation) clustering is applied
to the docked solutions to select the non-redundant results and to
discard redundant docking structures.
2. Experimental
2.1. Instruments
The UV–Vis spectra (absorption spectral measurements) were
carried out with Shimadzu UV-2401PC double-beam spectropho-
tometer (range 1100–200 nm) with scan speed of 400 nm minꢂ1
,
the pH values in the range 1.0–12.0 were measured on Elico pH
meter LI-120; Electrochemical studies were carried out using Auto
lab electrochemical analyzer (GPES software), A conventional three
electrode cell assembly was used for the electrochemical measure-
ments. Cyclic Voltammetry measurements at a glassy carbon elec-
trode (diameter: 1 mm) were carried out at an applied potential of
ꢂ0.9 V to 0.4 V for each concentration of b-cyclodextrin at single
cycle only. Reference electrode was saturated calomel electrode
(SCE) and platinum wire as counter electrode. All experiments
were carried out at 30 1 °C. The working electrode was polished
2.3. Reagents
b-Cyclodextrin {(b-CD), were obtained from the Sd fine chemi-
cal company} and used without further purification. 2,4-Dinitro-
benzoic acid (2,4-DNB) purchased from Alfa Aesar company and
used without further purification. Triply distilled water was used
to prepare all solutions. Solutions in the pH range 2.0–12.0 were
prepared by adding the appropriate amount of NaOH and H3PO4.
Yagil basicity scale (H–) [18] for solutions above pHꢁ12 (using a
NaOH–H2O mixture) and a modified Hammett’s acidity scale (H0)
[19] for the solutions below pHꢁ2 (using a H2SO4–H2O mixture)
was employed. The solutions were prepared just before taking
measurements. The concentrations of the solutions were of the or-
der (10ꢂ4 to 10ꢂ5 mol dmꢂ3). The stock solution of 2,4-DNB and
b-CD preparation for spectral and electrochemical studies was
to a mirror with 0.05 lm alumina aqueous slurry, and rinsed with
triply distilled water before each experiment. The supporting elec-
trolyte was pHꢁ1 (0.1 M H2SO4 + 0.1 M Na2SO4) and pHꢁ7 (0.1 M
KH2PO4 + 0.1 M NaOH). FT-IR was recorded using Nicolet 380 Ther-
mo Electron Corporation Spectrophotometer using KBr pellets and
scan between 4000 and 400 cmꢂ1. The sample solutions for 1H
NMR were prepared by dissolving the dinitro compounds and their
complexes in D2O solvent to obtain the final concentration of
20 mM. Two-dimensional rotating-frame Overhauser effect spec-
troscopy (ROESY) experiments were performed using BRUKER-
NMR 400 MHz instrument operating at 300 K and the standard
Bruker program was used, DMSO-d6 was used as a solvent,

T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
241
Table 1
continuously stirred for 48 h at room temperature. The reaction
mixture was put and kept in refrigerator for 48 h. At this time,
we observed that yellow precipitate is formed. The precipitate
was filtered by G4 crucible and washed with triply distilled water.
The precipitate is taken in a Petri dish and spread over as thin layer
and then dried in oven at 50 °C for 12 h. After drying in the oven
the yellow powder was obtained. This is solid inclusion complex
of 2,4-DNB with b-CD and it further analyzed by 1H NMR, 2D 1H
NMR (ROESY), FT-IR, XRD and SEM analysis.
Various prototropic maxima (absorption spectra) and pKa values of 2,4-DNB in
without and with b-CD medium.
Species
Without b-cyclodextrin
With b-cyclodextrin
kmax (nm)
H0/pH/H_
kmax (nm)
H0/pH/H_
Monocation
Neutral
310(Sh)
243
1
310(sh)
241
1
255
208
421
2.0–15.4
15.62
252
207
421
2.0–15.4
15.62
Monoanion
290
290
219
219
3. Results and discussion
pKa
1.4
1.18
a Sh-Shoulder.
3.1. Effect of pH
The absorption of 2,4-DNB has been studied in the different pH
range and the relevant data are compiled in Table 1 and Fig. 1.
There are three prototropic species (monocation, neutral, and
monoanion) present in the prototropic behavior of this molecule.
In the pH range of 2 to H_15.4, the absorption maxima (255 nm)
resemble the spectra observed in non-aqueous solvents (e.g. in 2-
Propanol at 240 nm, and 1,4-Dioxane at 243 nm, Ethyl acetate
254 nm) and thus can be assigned to the neutral species (Fig. S1
and Table S1) in these pH ranges. Above H_15.4 the absorption
spectrum of 2,4-DNB is red shifted in comparison to the neutral
species and is assigned to monoanion formed by deprotonating
the carbonyl (ACOOH) group (290 and 421 nm). A further increase
in the base strength, which is no change in the absorption maxima
in comparison to monoanion, hence there is no possibility to form
a dianion in this molecule. When acid concentration is increased
from pHꢁ2, the absorption maxima of 2,4-DNB is red shifted in
the absorption maximum of 310 nm (shoulder peak) but another
one absorption peak observed at 243 nm. This spectrum is clearly
indicated that protonation should occur at the carbonyl group.
Here a progressive shift with an increase in the concentration of
acid and at pHꢁ1.0 a UV band appears due to the formation of
p–p
* transition is the lowest energy transition, pro-
Fig. 1. Absorption spectra of different prototropic species of 2,4-DNB at 303 K;
concentration 2 ꢃ 10ꢂ4 M (a) monocation, (b) neutral and (c) monoanion.
monocation. If
tonation of carbonyls results in a red shift of the electronic spec-
trum. As mentioned earlier,
p* is the lowest energy transition
p–
in 2,4-DNB. The red shift confirms the formation of the cation on
protonation of the carbonyl group. However, in the ground state,
a red shift is observed in neutral and monoanion, this behavior is
different from its monocation. Further the pKa value of the mono-
cation-neutral and neutral-monoanion equilibrium (pKa) values is
calculated by spectrophotometric methods (Table 1). The prototro-
pic equilibrium is shown in Scheme 2.
prepared by adopting the procedure detailed in our previous report
[20,21].
2.4. Preparation of solid inclusion complex of 2,4-DNB with b-CD
Accurately weighed 1 g of the b-CD was placed into 50 ml con-
ical flask and 30 ml triply distilled water added and then oscillated
this solution enough. After that, 0.1628 g 2,4-DNB was put into a
50 ml beaker and 20 ml ethanol added and put over electromag-
netic stirrer to stir until it was dissolved [22]. Then slowly poured
2,4-DNB solution into b-CD solution. The above mixed solution was
3.2. Effect of b-cyclodextrin
Table 2 and Fig. 2 show the absorption maxima of 2,4-DNB in
pHꢁ1.0 (monocation) and pHꢁ7 (neutral) solutions containing
Scheme 2. Prototropic equilibria of 2,4-DNB in aqueous and b-CD medium.

242
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
Table 2
Absorption maxima (nm) and log
pHꢁ1 and pHꢁ7 solutions.
e of 2,4-DNB at different concentrations of b-CD in
S. No
Concentration of b-
CD (M)
pHꢁ1
pHꢁ7
kmax
log
e
kmax
loge
(nm)
(nm)
1
2
3
4
5
6
7
0
310 sh
243
3.23 310sh
3.5
4.2
4
3.52
4.2
4
3.54
4.2
4
3.55
4.2
4
3.56
4.2
4
3.57
4.2
4
3.58
4.2
4
4.2
250
208
0.002
0.004
0.006
0.008
0.01
310sh
242
3.29 310sh
4.2
249
207
310sh
242
3.32 310sh
4.2
249
207
310sh
241
3.38 310sh
4.2
249
207
310sh
241
3.42 310sh
4.2
248
207
310sh
241
3.44 310sh
4.2
248
207
0.012
310sh
241
3.48 310sh
4.2
248
207
Binding constant
92
152
(Mꢂ1
)
D
G (kJ/mol)
ꢂ11.4
ꢂ12.7
Sh-shoulder.
different concentrations of b-CD. At pHꢁ7, 2,4-DNB exists as a neu-
tral form only, hence we also recorded spectra at pHꢁ1.0 cationic
form. The absorption spectra of these two forms (neutral,
monocation) are drastically different. In pHꢁ7 and pHꢁ1.0 no
clear isosbestic point was observed in the absorption spectra
even in the presence of higher b-CD concentration. The equilibrium
between neutral and monocation forms of 2,4-DNB in b-CD
medium.
Fig. 2. Absorption spectra of 2,4-DNB (Conc.2 ꢃ 10ꢂ4 M) in (a) pHꢁ1 and (b) pHꢁ7
solution at different b-CD (a–g Conc. 0–12 ꢃ 10ꢂ3 M).
b-CD : 2; 4-DNBHþ
ꢀ
pHꢁ1:5 b-CD þ 2; 4-DNB pꢀHꢁ7 b-CD : 2; 4-DNB ð1Þ
þHþ
In pHꢁ7 solution the absorption maxima is increased and 1:1
inclusion complex was formed, whereas the absorption spectra of
2,4-DNB in pHꢁ7 there was a blue shift (250 nm to 248 nm) in
the absorption maxima and also the absorption intensity was in-
creased with increasing the b-CD concentration. In pHꢁ7 the 2,4-
DNB located inside the b-CD cavity and the COOH group located
at above the b-CD rim, which will cause the increase of the absor-
bance. In pHꢁ1.0; 2,4-DNB exist as a cationic form with 1:1 inclu-
sion complex form, In pHꢁ1.0 the 2,4-DNB located inside the b-CD
cavity and the COOHþ2 group located in the b-CD rim. This behavior
has been attributed to the enhanced dissolution of the 2,4-DNB
molecule through the hydrophobic interaction between 2,4-DNB
and b-CD. These results indicate that 2,4-DNB molecule is en-
trapped into b-CD cavity to form an inclusion complex. In both
cases, the binding constant for the formation of 2,4-DNB: b-CD
complex has been determined by analyzing the changes in the
intensity of absorbance with the b-CD concentration. Fig. 3 plotting
1/(AꢂA0) versus 1/[b-CD] for a 1:1 host:guest inclusion complex. In
1:1 inclusion complex gives straight line at pHꢁ7, and pHꢁ1.0, The
binding constant (K) can be obtained by using the modified Bene-
si–Hildebrand equation [23] for the 1:1 complex Eq. (2) between
2,4-DNB and b-CD as shown below.
Fig. 3. Benesi-Hildebrand plot of 1/AꢂA0 Vs 1/[b-CD] for 2,4-DNB in pHꢁ1 and pH
ꢁ7 solution.
where AꢂA0 is the difference between the absorbance of 2,4-DNB in
the presence and absence of b-CD, De is the difference between the
molar absorption coefficient of 2,4-DNB and the inclusion complex,
where [G]o is [2,4-DNB]0 and [H]o is [b-CD]0 are the initial concen-
tration of 2,4-DNBand b-CD, respectively. A good correlations were
1
1
1
¼
þ
ð2Þ
A ꢂ A0
D
e
½Gꢄ0½Hꢄ0K
D
e½H0ꢄ

T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
243
plex formation between 2,4-DNB and b-CD are possible. In pHꢁ1,
the carbonyl group is protonated, moreover it available in the near
of the upper rim of the b-CD by the cationic charge structure
(Scheme 3). In pHꢁ7, the carboxyl group of 2,4-DNB is not proton-
ate or deprotonate in the b-CD solution. This is indicated in the
neutral molecule absorption maxima of 2,4-DNB is red shifted in
b-CD than protonated form. The ground state pKa values for the
neutral-monocation equilibrium are listed in Table 1. This confirms
that the environments around the COOH in b-CD are same in the
bulk aqueous medium and OH group present in the b-CD cavity.
These features indicate that the probable inclusion complex struc-
ture represented in the Scheme 3. This is further supported by
using semi empirical quantum mechanical calculations discussed
in Section 3.4. To substantiate the above discussion, the effect of
b-CD on the prototropic equilibrium, the 2,4-DNB was present as
monocation in pH 1.0, neutral in the pH range 2.0–15.4 and the
prototropic equilibrium molecular resonance structure can be
written in the Scheme 2. These results indicate that 2,4-DNB mol-
ecule is entrapped into the b-CD cavity to form a 1:1 inclusion
complex.
Scheme 3. The proposed structure of inclusion complex of 2,4-DNB with b-CD for
1:1 inclusion complex. The oxygen atoms are shown as red ball, nitrogen as blue
and carbon as gray colour balls; hydrogen atoms are white balls. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
obtained for pHꢁ7 and pHꢁ1 respectively (Fig. 3; r2 = 0.9864 and
0.978), confirm that the formation of 1:1 inclusion complexes. From
the intercept and slope values of this plot K were calculated
[pHꢁ1 = 92 Mꢂ1, pHꢁ7 = 152 Mꢂ1] at 303 K (Table 2).
3.4. Semi empirical quantum mechanical calculations
The internal diameter of the b-CD is approximately 6.5 Å and its
height is 7.8 Å (Scheme 4). Considering the shape and dimensions
of b-CD, the ground state of 2,4-DNB molecules were optimized
using AM1 method (Scheme 4). In 2,4-DNB, the vertical distances
between H19AO14 is 8.3 Å and H19AO15 is 8.0 Å. The horizontal dis-
tance between H18AO1 is 5.0 Å and H18AO3 is 5.6 Å. The vertical
distance and the horizontal distance measured from the terminal
atoms of 2,4-DNB are less than the height and vertical diameter
of b-CD. Since, the height of 2,4-DNB are lower than that of b-CD,
the insertion of 2,4-DNB in the b-CD cavity is possible as shown
in Scheme 3.
3.3. Possible inclusion complex of 2,4-DNB with b-CD cavity
The absorption spectra of 2,4-DNB have been studied in the pH
range 1 to H-15.4 in b-CD (Table 1). When compared with aqueous
medium no appreciable change is observed in b-CD in absorption
maxima of neutral and monocation. The ground state pKa values
for the neutral-monocation equilibrium are also same. Considering
the above discussions, the possible inclusion mechanism is pro-
posed as follows. Naturally, two different types of inclusion com-
Scheme 4. Schematic representation of semi empirical bond distance of (a) b-CD and (b) 2,4-DNB.

244
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
Scheme 5. Ball and stick representation of (a) b-CD, (b) 2,4-DNB, (c) 2,4-DNB:b-CD inclusion complex; the oxygen atoms are shown as red ball, nitrogen as blue and carbon as
grey balls; hydrogen atoms are white balls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. Molecular docking studies of inclusion process
Formation of the inclusion complex of 2,4-DNB with b-CD was also
confirmed by electrochemical method. In pHꢁ7 the cyclic voltam-
mograms shows an anodic peak during the forward scan (towards
positive potential) at 0.058 V and two reduction peaks during the
reverse scan (towards negative potential) at ꢂ0.198 V and
ꢂ0.855 V. These peaks are ascribed as oxidation of 2,4-DNB
(COOH; 0.058 V) and the reduction of carboxyl cation (ꢂ0.198 V)
into COOH and another one reduction peak of NO2 (ꢂ0.855 V) into
hydroxylamine.
In pHꢁ1, from the cyclic voltammograms one oxidation peak
during the forward scan (towards positive potential) at 0.329 V
and two-reduction peaks were observed during the reverse scan
(towards negative potential) at 0.283 V and ꢂ0.415 V. These peaks
were ascribed as oxidation of carboxyl cation into carboxyl group
(0.329 V) and reduction of carboxyl group into carboxyl cation
(0.283 V) and NO2 (ꢂ0.415 V) into hydroxylamine. The oxidation
and reduction mechanism of 2,4-DNB was clearly explained in
Scheme 6.
The 3D structure of b-CD, 2,4-DNB was obtained from crystallo-
graphic databases are shown in Scheme 5(a and b). The guest mol-
ecules of 2,4-DNB were docked into the cavity of b-CD using
PatchDock server. The PatchDock server program gave several pos-
sible docked models for the most probable structure based on the
energetic parameters; geometric shape complementarity score,
approximate interface area size and atomic contact energy of the
2,4-DNB:b-CD inclusion complexes. The docked 2,4-DNB:b-CD
1:1 model Scheme 5c with the highest geometric shape comple-
mentarity score 2500 for 2,4-DNB:b-CD, the approximate interface
area size of 2,4-DNB:b-CD was 266.2 Å2 and atomic contact energy
of ꢂ861 kJ/mol for the inclusion complex of 2,4-DNB:b-CD was cal-
culated. This is highly probable and energetically favorable model
and it was in good correlation with results obtained through exper-
imental methods.
The cathodic peak current (Fig. 4 and Table 3) ipc, increased with
increasing the b-CD concentration in both pH solutions (pHꢁ1.5
and pHꢁ7). The cathodic peak potential, Epc, shifted towards a po-
sitive direction in pHꢁ7 and negative direction in pHꢁ1.5 when
3.6. Electrochemical studies
The cyclic voltammograms shows an electrochemical behavior
of 2,4-DNB in pHꢁ1 and pHꢁ7 with b-CD (Fig. 4 and Table 3).

T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
245
b-CD cavity and the catalytic behavior of b-CD to the included
guest molecule (2,4-DNB).
The binding constant (K) and the stoichiometric ratio of the
inclusion complex of 2,4-DNB can be determined according to
the Benesi–Hildebrand [23] relation assuming the formation of a
1:1 host–guest complex.
1
1
1
¼
þ
ð3Þ
IHG ꢂ IG
D
I
K½2; 4-DNBꢄ0DI½b-CDꢄ0
where IG is the reduction peak current of the guest molecule of 2,4-
DNB, and IHG is the reduction peak current of the inclusion complex
of 2,4-DNB:b-CD. IHGꢂIG is the difference between the reduction
peak current of inclusion complex and 2,4-DNB.
DI is the difference
between the molar peak current coefficient of the inclusion com-
plex and 2,4-DNB. The [2,4-DNB]0 and [b-CD]0 are the initial con-
centration of 2,4-DNB and b-CD, respectively. Plot of [1/IHGꢂIG]
versus 1/[b-CD] gives a straight line for both pH solutions as shown
in Fig. 5. Good linear correlations were obtained (r = 0.950, 0.923 for
pHꢁ1 and pHꢁ7 respectively), confirm that the formation of a 1:1
inclusion complex for both pH (pHꢁ1 and pHꢁ7) solutions. From
the intercept and slope values of this plot K was evaluated, the bind-
ing constant values for 2,4-DNB:b-CD is 38 Mꢂ1and 83 Mꢂ1 in pHꢁ1
and pHꢁ7 respectively. These values indicate that 2,4-DNB mole-
cule is encapsulated in the b-CD cavity to form an inclusion
complex.
3.7. 1H NMR spectrum
The formation of the solid inclusion complex can be analyzed
from 1H NMR spectra [24]. Fig. 6; Tables 4 and 5 showed the typ-
ical 1H NMR spectra of (a) b-CD, (b) the solid inclusion complex of
2,4-DNB with b-CD (chemical shift changes with b-CD), (c) 2,4-
DNB and (d) the solid inclusion complex (chemical shift changes
with 2,4-DNB). The significant distinguish for these 1H NMR spec-
tra strongly confirmed that the solid inclusion complex formation.
The values of chemical shifts, d for different protons in b-CD with
solid inclusion complex and 2,4-DNB with solid inclusion com-
plex were listed in Tables 4 and 5. It can be seen from the 1H
NMR that in solid inclusion complex, a great upfield shift was
occurring for H3 and H5 protons, which locate in the nano hydro-
phobic cavity of b-CD (Scheme 7b). The changes of chemical shift
Fig. 4. CV for 2,4-DNB:b-CD in pHꢁ1 and pHꢁ7 buffer solution, scan rate
100 mV sꢂ1, 2,4-DNB (Conc. 2 ꢃ 10ꢂ4 M) and b-CD (a–g Conc. 0–12 ꢃ 10ꢂ3 M).
b-CD concentration was increased in both cases. The result showed
that the inclusion complex between 2,4-DNB and b-CD was formed
when 2,4-DNB was added onto b-CD aqueous solution. In addition,
the cathodic peak current was increased with increasing b-CD con-
centration; this is due to the nitro groups are encapsulated in the
(Dd) of H3 and H5 suggested that the 2,4-DNB monomer com-
pletely entered into the nano hydrophobic cavity of b-CD. The
phenyl ring of 2,4-DNB made the signals of b-CD protons (H3
and H5) upfield shift. On the contrary, the chemical shifts of H1,
Table 3
CV for 2,4-DNB:b-CD in pHꢁ2 and pHꢁ7 buffer solution, scan rate 100 mV sꢂ1, 2,4-DNB (Conc. 2 ꢃ 10ꢂ4 M) and (a–g) 0–12 ꢃ 10ꢂ3 M b-CD concentrations.
Concentration of b-CD (M)
pHꢁ1
Epa (V)
0.329
pHꢁ7
Epa (V)
0.058
Ipa (
lA)
Epc (V)
Ipc (
l
A)
Ipa (lA)
Epc (V)
Ipc (lA)
0
13.89
14.63
15.27
15.33
15.63
16.15
16.45
0.283
ꢂ14.63
ꢂ20.24
ꢂ16.21
ꢂ21.06
ꢂ17.29
ꢂ22.66
ꢂ16.4
9.461
9.346
8.89
ꢂ0.198
ꢂ0.804
ꢂ0.191
ꢂ0.804
ꢂ0.149
ꢂ0.85
ꢂ0.375
ꢂ7.49
ꢂ0.498
0.002
0.004
0.006
0.008
0.01
0.334
0.334
0.356
0.356
0.363
0.366
0.283
0.044
0.049
0.051
0.063
0.122
0.117
ꢂ0.473
ꢂ5.462
ꢂ3.685
ꢂ7.968
ꢂ3.585
ꢂ5.322
ꢂ3.629
ꢂ5.084
ꢂ3.322
ꢂ5.582
ꢂ1.624
ꢂ5.997
ꢂ0.489
0.293
ꢂ0.474
0.29
8.628
8.614
8.221
8.611
ꢂ0.152
ꢂ0.85
ꢂ0.43
0.249
ꢂ23.57
ꢂ17.5
ꢂ0.176
ꢂ0.855
ꢂ0.108
ꢂ0.855
ꢂ0.142
ꢂ0.857
ꢂ0.423
ꢂ23.69
ꢂ18.64
ꢂ23.98
ꢂ19.29
ꢂ25.26
0.254
ꢂ0.41
0.254
ꢂ0.415
0.012
Binding constant (Mꢂ1).
38
83
D
G (kJ/mol)
ꢂ9.2
ꢂ11

246
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
Scheme 6. Reaction mechanism of 2,4-DNB in pHꢁ1 and pHꢁ7 buffer solution at glassy carbon electrode.
H2, H4, and H6ab, which are on the outer surface of b-CD and the
narrow opening of b-CD as shown as in Scheme 7b, were only
slightly affected by the guest molecule. Similarly, the chemical
shifts of Ha, Hb and Hc of 2,4-DNB (Scheme 7c) are located in
the nano hydrophobic cavity of b-CD was also moved in upfield
significantly because the interaction between 2,4-DNB and b-CD.
On the other hand, as shown as in Fig. 6d, when 2,4-DNB mono-
mer entered into the nano hydrophobic cavity of b-CD, the
change of the micro-environment of 2,4-DNB protons leaded the
phenyl ring protons moved upfield shift. From the above
discussions we can conclude that of 2,4-DNB molecule was
included into nano hydrophobic cavity of b-CD.
3.7.1. 2D 1H NMR (ROESY) studies on 2,4-DNB with b-CD
The 2D 1H NMR (ROESY) is a powerful technique for investiga-
tion of inter and intra molecular interactions. The chemical shift
changes were shown by Ha, Hb and Hc protons of aromatic (hydro-
phobic part) moiety [25] of dinitrophenol may play a major role in
the inclusion process. To verify this hypothesis, 2D 1H NMR
(ROESY) spectra were recorded. The presence of NOE cross-peaks

T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
247
Table 4
1H NMR chemical shifts of b-CD in free and complexed state determined in D2O at
303 K.
Proton
b-CD d (ppm)
2,4-DNB-b-CD d (ppm)
Dd
H1
H2
H3
H4
H5
H6
4.966
3.554
3.877
3.478
3.748
3.797
4.963
3.553
3.862
3.476
3.735
3.796
ꢂ0.003
ꢂ0.001
ꢂ0.015
ꢂ0.002
ꢂ0.013
ꢂ0.001
Table 5
1H NMR chemical shifts of 2,4-DNB in free and complexed state determined in D2O at
300 K.
Proton
2,4-DNB d (ppm)
2,4-DNB-b-CD d (ppm)
Dd
Fig. 5. Benesi–Hildebrand plot of 1/IHG ꢂIG vs. 1/[b-CD] for 2,4-DNB in pHꢁ1 and
pHꢁ7 solution.
Hb
Hc
Hd
9.310
8.810
8.654
9.265
8.786
8.585
ꢂ0.045
ꢂ0.024
ꢂ0.069
the first peak was assigned to the interaction between the H3 pro-
tons of b-CD with ortho positioned protons of the 2,4-DNB and
other peak were assigned to the interaction between the H5 pro-
tons of b-CD with meta positioned protons of the 2,4-DNB. In all
cases the interaction of 2,4-DNB with only internal protons of the
b-CD were observed. In addition the H6ab protons of the b-CD were
not affected by the inclusion process. We confirmed that the 2,4-
DNB is included into the b-CD cavity via wider rim. From the above
fact the 2,4-DNB interacts with b-CD through space contact, not by
bonding.
3.8. FT-IR spectral studies
FT-IR is a very useful analytical tool to prove the existence of
both guest and host molecules in their solid inclusion complex
[26]. Fig. 8 shows the FT-IR spectra of the (a) 2,4-DNB, (b) b-cyclo-
dextrin and (c) solid complex of 2,4-DNB and b-cyclodextrin. The
FT-IR spectrum of the solid complex is almost similar to the b-
cyclodextrin molecule. It indicates the formation of the solid inclu-
sion complex, similar to an observation noticed by the same
authors [20,21]. Besides that, a broad hydroxyl band of b-cyclodex-
trin at 3371 cmꢂ1 appeared in the FT-IR spectrum of the solid
inclusion complex which is a good evidence of the formation of
the solid complex.
The wave number for the b-CD observed at 3371 cmꢂ1
,
2929 cmꢂ1, 1158 cmꢂ1, and 1030 cmꢂ1 which correspond to the
symmetric and anti-symmetric stretching of OH, CH2, CAC and
bending vibration of OAH frequencies. Meanwhile the frequencies
for 2,4-DNB were observed at 1531 cmꢂ1, 1350 cmꢂ1, 1722 cmꢂ1
,
835 cmꢂ1 and 1618 cmꢂ1 in the respective functional groups such
as NO2, COOH, CAN and vibrational stretching of AC@CA
respectively.
The solid complex formation has been verified by comparing
the spectra of b-CD and 2,4-DNB (Fig. 8a and b), because the
bands resulting from the included part of the guest molecule gen-
erally shifted or their intensities altered [26]. From Fig. 8 the
characteristic stretching bands of -NO2 are observed at
1531 cmꢂ1 and 1350 cmꢂ1 for 2,4-DNB were observed and these
bands are shifted to 1544 cmꢂ1, 1352 cmꢂ1 and the intensity of
the bands are also reduced because the nitro groups were present
(included) in the nanocavity of b-CD. The characteristic peak of
the COOH stretching band appeared at 1722 cmꢂ1 for 2,4-DNB
and it shifted to 1726 cm-1 and also the intensity of the peak
was reduced; due to the phenyl-COOH group are present in the
Fig. 6. 1H NMR spectra of (a) b-CD, (b) the solid complex (chemical shift with
respect to b-CD), (c) 2,4-DNB and (d) the solid complex (chemical shift with respect
to 2,4-DNB).
between protons of two different species in the 2D 1H NMR spec-
trum is an indication that they are in spatial contact through space
within the cavity of b-CD.
Fig. 7 shows the 2D spectrum of (a) 2,4-DNB:b-CD and (b) coun-
terpart of 2,4-DNB: b-CD systems, two groups of intermolecular
NOE cross-peaks were observed. In the complex of 2,4-DNB:b-CD,

248
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
Scheme 7. (a) The stereo-configuration of b-CD and (b) truncated-cone of b-CD and (c) 2,4-DNB.
Fig. 7. The 2D 1H NMR (ROESY) spectra of inclusion complex of (a) 2,4-DNB:b-CD, (b) Partial Counter plot of 2,4-DNB:b-CD in DMSO-d6 at 300 K.
near of the upper rim of the b-CD. The characteristics of CAN
(ANO2) stretching vibration appeared at 835 cmꢂ1 for 2,4-DNB
and it shifted to 858 cmꢂ1 for the inclusion complex also the
intensity is reduced due to the nitro groups are included into
the nanocavity of the b-CD. The characteristic peaks of AC@CA
stretching appeared at 1618 cmꢂ1 for 2,4-DNB and it shifted to
1631 cmꢂ1 and the intensities is reduced due to the hydrophobic
part of the benzene ring of 2,4-DNB were included in the nano
cavity of the b-CD.
3.9. Powder X-ray diffraction pattern
The lack of crystallinity is an added evidence for the formation
of inclusion complex [27]. Fig. 9 shows the XRD patterns for guest

T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
249
sharp high intensity peaks at different diffraction angles (2h) of
12.3, 20.3, 24.6, 25.1, 25.5, 25.9, 26.5, 28.9, 34.1, 35.1 and 37.3 sug-
gesting that the 2,4-DNB existed as crystalline nature. The b-CD
(Fig. 9b) showed a crystalline diffractogram, while a diffuse halo-
pattern was recorded for 2,4-DNB-b-CD (Fig. 9c) demonstrating
its amorphous nature. The diffraction patterns of the investigated
in the complex correspond to the correct position of those of the
pure components. The lower intensities of the diffraction peaks
indicate that the particle sizes were reduced during complex prep-
aration in solution phase of the pure crystalline components. Few
of diffraction peaks of 2,4-DNB was matched with those off b-CD
was evident. A typical diffuse pattern indicating the entirely amor-
phous nature of 2,4-DNB in complexed state.
3.10. Scanning electron microscope morphological observations
Scanning electron microscope (SEM) is well suited for visualiz-
ing the surface texture of the deposited film. The SEM analysis is
ideal for quantitatively measuring the surface roughness and for
visualizing the surface texture of the substance. First we observed
surface morphological structure of (a) 2,4-DNB shows like a broken
cement plate morphology, (b) b-CD shows as like a sheeted/bladed
morphology (Fig. 10) by SEM, and then we also observed the sur-
face morphological structure of (c) solid inclusion complex as like
a milky white rectangular sheet morphology (Fig. 10c). These pic-
tures clearly elucidated the difference of each other. Modification
of crystals can be assumed as a proof of the formation of a solid
inclusion complex with the nano hydrophobic cavity of b-CD.
Fig. 8. FT-IR Spectra of (a) 2,4-DNB, (b) b-CD and (c) solid inclusion complex of 2,4-
DNB:b-CD complex in KBr.
4. Conclusions
The following conclusion can be arrived at from the above stud-
ies; the inclusion complex of 2,4-DNB with b-CD were prepared
(aqueous and solid phases) and the structures of the complex were
investigated by UV, 1H NMR, 2D1H NMR (ROESY), FT-IR, XRD and
SEM techniques. The experimental results showed that the com-
plex was the benzene ring part of the 2,4-DNB molecules was in-
cluded into the b-CD cavity. Furthermore, the implementation of
molecular modeling confirmed that the complex of 1:1 host–guest
stoichiometry. The electrochemical study is also supporting evi-
dence for the formation of inclusion complex. In addition, the pro-
totropic reactions can be used to calculate the ground state acidity
constant (pKa) values for the 2,4-DNB molecule by spectrophoto-
metrically in the presence and absence of the b-CD.
Fig. 9. Powder X Ray diffraction spectra of (a) 2,4-DNB, (b) b-CD and (c) solid
inclusion complex of 2,4-DNB:b-CD.
Acknowledgements
(2,4-DNB), host (b-CD) and their complex systems were prepared
by co-precipitation techniques at a molar ratio of 1:1. The powder
X-ray diffraction pattern of 2,4-DNB (Fig. 9a) revealed that several
One of the authors K. Srinivasan acknowledges to UGC-BSR for
providing Research Fellowship for Science Meritorious (AU/UGC-
BSR/IC/1069/2011) student.
Fig. 10. Scanning electron microscope photographs (Pt. coated) of (a) 2,4-DNB, (b) b-CD and (c) solid inclusion complex of 2,4-DNB:b-CD.

250
T. Stalin et al. / Journal of Molecular Structure 1060 (2014) 239–250
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