Spectral characteristics of sulfa drugs: Effect of solvents, pH and β-cyclodextrin

Article abstract of DOI:10.1007/s10953-010-9559-0

The absorption and fluorescence spectra of sulfamethoxazole (SMO), sulfisoxazole (SFO), sulfathiazole (STO) and sulfanilamide (SAM) in different solvents, pH and β-cyclodextrin (β-CD) have been analyzed. The inclusion complexes of the above sulfa drugs wi

Full text of DOI:10.1007/s10953-010-9559-0

J Solution Chem (2010) 39: 1061–1086
DOI 10.1007/s10953-010-9559-0
Spectral Characteristics of Sulfa Drugs:
Effect of Solvents, pH and β-Cyclodextrin
A. Antony Muthu Prabhu · G. Venkatesh ·
N. Rajendiran
Received: 12 December 2009 / Accepted: 19 February 2010 / Published online: 14 July 2010
© Springer Science+Business Media, LLC 2010
Abstract The absorption and fluorescence spectra of sulfamethoxazole (SMO), sulfisox-
azole (SFO), sulfathiazole (STO) and sulfanilamide (SAM) in different solvents, pH and
β-cyclodextrin (β-CD) have been analyzed. The inclusion complexes of the above sulfa
drugs with β-CD were investigated by UV-visible spectroscopy, fluorometry, DFT, SEM,
FT-IR and 1H NMR. The solvent study indicates that the position of the substituent (oxazole
or thiazole group) in the SAM molecule (R–SO2–NH-group) is not the key factor to change
the absorption and emission behavior of these sulpha drug molecules. In aqueous solution,
a single fluorescence band (340 nm) was observed whereas in solutions of β-CD dual emis-
sion (430 nm) was noticed in sulpha drug compounds. Formation of the inclusion complex
in SMO, SFO and STO should result dual emission which is due to a Twisted Intramolecu-
lar Charge Transfer band (TICT). The β-CD study indicates that (i) sulpha drugs form 1:1
inclusion complexes with β-CD and (ii) the red shift and the presence of TICT in the β-CD
medium confirms heterocyclic ring encapsulated in the β-CD cavity with the aniline ring
present on the out side of the β-CD cavity.
Keywords Sulfa drugs · Solvent effects · β-Cyclodextrin · TICT · Inclusion complex
1 Introduction
In recent years, molecules of β-cyclodextrin (β-CD) have been shown to be interesting
micro vessels for several molecules and the resulting supramolecular species serve as ex-
cellent miniature models for enzyme substrate complexes [1–3]. The reduced polarity and
the restricted space provided by the β-CD cavity markedly influence a number of photo
physical properties of the drug molecules. In addition, especially in pharmaceutical indus-
tries, the inclusion process of drug molecules in β-CDs leads to important modifications of
pharmaceutical properties of drug molecules. For example, the pharmaceutical interest in β-
CDs extends to enhanced solubility, chemical stability and bioavailability of poorly soluble
A.A. Muthu Prabhu · G. Venkatesh · N. Rajendiran (
)
ꢀ
Department of Chemistry, Annamalai University, Annamalai nagar 608 002, Tamilnadu, India
e-mail: drrajendiran@rediffmail.com

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drugs, to reduced toxicity and to control of the rate of release. The size of the β-CD and the
drug molecule plays an important role to determine the complex type or stoichiometry.
Further, it is known that cyclodextrins (CD) have the property of forming inclusion com-
plexes with guest molecules that have suitable characteristics of polarity and dimension
[1–3]. The inclusion complex formation in the CD systems is favored by substitution of
the high-enthalpy water molecules located inside the CD cavity, with an appropriate guest
molecule of low polarity. An overview of the non-chromatographic analytical uses of CDs
has been presented by Szejtli [3]. As the complexation process implies an interaction pro-
ducing a protection of the included species, the CDs have been used in the pharmaceutical
industry to encapsulate drugs that are photosensitive. There are a few reports dealing with
the analytical determination of sulphonamides by using CD and they employ, in general,
a capillary electrophoresis technique. In the case of SMO, Okamoto et al. [4] used dimethyl-
β-cyclodextrin as a modifier of the mobile phase in micellar electro-kinetic chromatography
and β-CD is used in capillary electrophoresis. Muñoz de la Peña et al. [5] made fluorescence
measurements in pharmaceutical preparations of the inclusion complex of SMO in β-CD.
Among the many and different families of organic inorganic chemicals being currently
investigated because of their applications, sulfonamides and their N-derivatives are one of
the outstanding groups. Sulfonamides were the first effective chemotherapeutic agents em-
ployed systematically for the prevention and cure of bacterial infections in humans. After
the introduction of penicillin and other antibiotics, the popularity of sulfonamides decreased.
However, they are still considered useful in certain therapeutic fields, especially in the case
of ophthalmic infections as well as infections in the urinary and gastrointestinal tract. Be-
sides, sulfa drugs are still today among the drugs of first election (together with ampicillin
and gentamycin) as chemotherapeutic agents in bacterial infections by Escherichia coli in
humans. The sulfanilamides exert their antibacterial action by the competitive inhibition of
the enzyme dihydropterase synthetase towards the substrate p-aminobenzoate.
Sulphonamides belong to the group of antibacterial drugs which are used for human and
animal therapy, to cure infectious diseases of digestive and respiratory systems, infections
of the skin (in the form of ointments) and for prevention or therapy of coccidiosis of small
domestic animals [6]. Quality control of sulphonamide formulations and their quick system-
atic monitoring in body fluids are important analytical tasks. A number of articles have been
published concerning the determination of sulphonamides by different analytical methods.
Sulphamethoxazole (SMO) is widely used as an antibacterial, mainly in combina-
tion with trimethroprim. This is a well-recognized preparation, as the combination of a
sulphonamide with an inhibitor of the dihydropholate reductase increases the bacteriosta-
tic effect of the sulphonamide, by blocking the metabolic pathways of the microorgan-
isms at two different points. Different methods have been described for determining the
sulphonamides, but, in general, they are based on separation methods using different detec-
tion types. Its determination in urine has been made by micellar liquid chromatography and
by supported liquid membrane with high pressure liquid chromatography-electrospray mass
spectrometry detection [7].
The fluorescence characteristics of different sulphonamides have been studied [8] and
proposed for the determination of several of these compounds. Thus, sulphanilamide can
be determined by reaction with homophthaldehyde. The analysis of sulphadrugs [9, 10] has
been performed in foods and pharmaceuticals using the fluorescamine reaction. The reaction
of 9-cloroacridine with sulphonamides produces a fluorescence quenching which allows the
determination of sulphonamides [11]. Sulpha drugs were determined in milk and pharma-
ceutical preparations by photochemically-induced fluorometry [12]. Fluorescence has also

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been used as HPLC detection for determining sulpha drugs in milk and eggs [13] and re-
cently, fluorescence after pre-column derivatization with fluorescamine has been applied as
a detection technique [13].
In this work, we not only studied spectral properties of sulpha drug–β-CD complexes
by UV-visible and fluorescence, but also prepared their solid inclusion complexes by the
co-precipitation method and determined their formation by FT-IR, 1H NMR, SEM and
DFT methods. The formation constant of the inclusion complex were obtained according
to UV-visible and fluorescence data using the modified Benesi-Hildebrand equation. The
solid studies on the inclusion complex of β-CD with guest molecules have been performed
to obtain direct evidence for the formation of the inclusion complexes. The obtained results
indicate that the solid structure of these complexes is designable by appropriately selecting
of the type, length and functional substituent group in the guest.
2 Materials and Methods
2.1 Instruments
Absorption spectral measurements were carried out with a Hitachi Model U-2010 UV-
visible spectrophotometer and fluorescence measurements were made using a Shimadzu
RF 5301 spectrofluorimeter. In both instruments for measurement of the readings we used a
quartz cell and the width of this cell is 1 cm and the height is 4.5 cm. We used the excitation
wavelength 270 nm for all the sulpha drugs. The pH values in the range 2.0–12.0 were mea-
sured on Elico pH meter model LI-120. FT-IR spectra were obtained with Avatar-330 FTIR
spectroscopy using KBr pelleting. The range of spectra was from 500 to 4000 cm−1. Micro-
scopic morphological structure measurements were performed with a JEOL JSM 5610 LV
scanning electron microscope (SEM).
2.2 Reagents and Materials
SMO, SFO, STO, SAM, β-CD and all the spectrograde solvents were obtained from the
Sigma-Aldrich chemical company and used as such. The purity of the compound was
checked by similar fluorescence spectra when excited with different wavelengths. Triply
distilled water was used for the preparation of aqueous solutions. Solutions in the pH range
2.0–12.0 were prepared by adding appropriate amounts of NaOH and H3PO4. A modified
Hammett’s acidity scale (H0) [14] for the solutions below pH ∼2 (using a H2SO4–H2O mix-
ture) and Yagil’s basicity scale (H) [15] for solutions above pH∼12 (using a NaOH–H2O
mixture) were employed. The solutions were prepared just before taking measurements. The
concentration of the sulfa drug solutions was of the order 2 × 10−4 to 2 × 10−5 Mol·L−1
.
The concentration of the β-CD solution was varied from 1 × 10−3 to 1 × 10−2 Mol·L−1
2.3 Preparation of Solid Complex of Sulfa Drugs with β-CD
.
Accurately weighed β-CD (1.2 g) was taken in a 50 mL conical flask and dissolved in 30 mL
distilled water. Then, sulpha drugs were taken in a 50 mL beaker and 20 mL distilled water
was added and stirred on an electromagnetic stirrer until it was dissolved. The β-CD solution
was slowly poured into the sulpha drugs solution. This mixed solution was continuously
stirred for 48 hrs at 50 ◦C. The reaction mixture was refrigerated for 48 hrs. White crystal
were precipitated, which were filtered with a G4 sand filter funnel and washed with distilled
water. After drying in an oven at 60◦C for 12 hrs a white powder product was obtained,
which was the inclusion complex of the sulpha drug with β-CD.

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3 Results and Discussion
3.1 Effect of Solvents
Table 1 depicts the absorption maxima, log10 ε and fluorescence maxima of sulfisoxa-
zole (SFO, N1-(3,4-dimethyl-5-isoxazolyl) sulfanilamide), sulfamethoxazole (SMO, N1-
(5-methyl-3- isoxazolyl)sulfanilamide), sulfathiazole (STO, N1-(2-thiazolyl) sulfanilamide)
and sulfanilamide (SAM, 4-aminobenzene sulfonamide) in the solvents of different po-
larity and hydrogen bond forming tendency as well as at different proton concentrations
(Scheme 1). Because of the very low solubility of sulfa drugs in cyclohexane, their max-
ima were obtained using 1% dioxane solutions of cyclohexane. These maxima will be very
near to the maxima obtained from pure cyclohexane as the polarity of dioxane is close to
cyclohexane. Furthermore the trend observed in the maxima of sulfa drugs in cyclohexane
is similar to the trend in other solvents. Data in Table 1 clearly indicate that the absorption
spectra of the above molecules are red shifted from cyclohexane to methanol, but compared
to methanol a blue shift is observed in water. The red shift increases according to the follow-
ing sequence: SAM < SMO = STO = SFO. The above sequence indicates the position of
the substituent (oxazole or thiazole group) in the SAM molecule (R–SO2–NH)-group is not
the key factor to change the absorption behavior of these molecules. When compared to ani-
line [16] (cyclohexane, λabs ∼ 283, 235 nm, λflu ∼ 320 nm, methanol, λabs ∼ 278, 230 nm,
λflu ∼ 335 nm), the absorption spectra of the sulpha drug molecules are blue shifted but the
fluorescence spectra are red shifted in all solvents. In polar solvents, only a small spectral
shift was observed in the absorption and emission maxima of the above sulpha drugs. This is
because a tautomeric structure is present in sulpha drug molecules [17] (Scheme 2). For this
reason, sulpha drug molecules are insoluble in the non-polar cyclohexane solvent. Though
there is no discernible effect (not more than a few nm) of the solvent polarity on the absorp-
tion maxima, the spectral shifts observed in the absorption spectra of the above molecules
in solvents are consistent with the characteristic behavior of the amino group [16–20].
The fluorescence spectra of the above molecules are displayed in Fig. 1. In all solvents,
the above compounds have one broad structureless fluorescence maxima. The fluorescence
spectra of the sulpha drug molecules are red shifted as the solvent polarity and proton
donor/acceptor capacity increases. However, the emission maxima are almost the same in
polar solvents; this is because the oxazole and thiazole groups are isoelectronic and their
electronic spectra are expected to be similar. Hence, the spectral shifts of these molecules
indicates that the emission maxima at 340 nm originate from the phenyl ring [20, 21]. Ear-
lier, spectral studies of 2-arylbenzoxazoles in solvents [21] indicated that the aryl group acts
as the main chromophore and its electronic transitions are perturbed by the oxazole moiety.
The present experimental results in Table 1 also confirmed earlier results [19–21].
The electronic spectra of aryl amines have been studied extensively [16–20] and the red
shift produced in the absorption spectra of the parent hydrocarbon is due to the resonance
interaction of the lone pair of the amino nitrogen with the π-cloud of the parent hydro-
carbon. This interaction increases if an electron withdrawing group is attached in the para
position. Thus, the nature of the electron withdrawing group in the para position decides
the percentage charge transfer character in the π–π∗ transition as well as the value of the
band maximum. The sulpha drug molecules also have a –SO2– group as an electron with-
drawing group in para position. The amino group conjugated with the electron withdrawing
group (Scheme 2) can also inhibit more interaction between the solvents and these mole-
cules. Further it is well known that the amino proton becomes a strong acid and the tertiary
nitrogen and SO2 groups become strong bases in S1 their state. This is reflected in sulpha
drug molecules, hence no large red shift is observed in protic solvents.

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H1-H8=12.481
H3-H5=5.071
N1-N2=6.923
N1-S2=6.201
H2-H8=11.854
H1-S2=11.345
N1-N3=9.252
H9-S1=6.332
Scheme 1 CAChe-DFT structure—SFO, SMO, STO and SAM
Scheme 2 Various resonance structures of SMO
In this study, the Stokes shifts of SFO, SMO, STO and SAM determined in different
solvents of varying polarity have been correlated with the Biolet-Kawaski (BK) [22] and
ET (30) [23] parameters. Table 1 gives list of solvents and their corresponding polarity para-

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Fig. 1 Emission spectra of SFO
and SAM in selected solvents at
303 K: 1—cyclohexane, 2—ethyl
acetate, 3—acetonitrile,
4—2-propanol, 5—methanol,
6—water
meters and the Stokes shifts. Figure 2 in aprotic solvents indicates that nonspecific interac-
tions are the key factors in shifting the fluorescence maxima to the red for these molecules.
As it is evident from the slops of the plots, these interactions are large in the case of SAM and
may be attributed to its increase of dipole moment of excitation. In the excited state, rigid
molecules with limited internal degrees of freedom change the structure of the solvent cage
by their dipole moment change. This process induces a large Stokes shifts in SAM in polar
solvents. A good correlation of the Stokes shift with the ET (30) scale in Fig. 2 indicates the
fact that the dielectronic solute-solvent interactions are responsible for the solvatochromic
shifts in these molecules.
3.2 Effect of Hydrogen ion Concentration
The absorption and fluorescence spectra of the SFO, SMO, STO and SAM sulpha drug sam-
ples were studied in various acid-base concentrations in the H0/pH/H range of −10 to 16.0
−
at room temperature (303 K). The spectral properties for their different prototropic species
are listed in Table 2 and the spectra are shown in Figs. 3 and 4. The behavior of these mole-
cules in the above range of acid-base regions are similar. With a decrease in pH from 7, the
absorption and emission spectra of SAM are largely blue shifted (220 nm) at H0 −0.80 with
very weak absorption shoulder at 269, 266 nm, which indicates that a monocation is formed.
No further change is noticed even at H0 −10. When the pH is increased from 10, due to the

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Fig. 2 Plot of Stokes shifts
−1
(cm ) of SFO, SMO, STO and
SAM versus E (30) and BK
T
solvent parameters:
1—cyclohexane, 2—diethyl
ether, 3—1,4-dioxane,
4—ethylacetate,
5—dichloromethane,
6—acetonitrile, 7— t-butyl
alcohol, 8—2-butanol,
9—2-propanol, 10—ethanol,
11—methanol, 12—water
formation of a monoanion, the emission intensity of SAM is quenched at the same wave-
length. In very high basicity (H 16) a red shifted emission maximum is obtained indicating
−
that a dianion is formed in the SAM molecule. None of the molecules show any change of
absorption characteristics in the region pH ∼7 to 12, indicating that only neutral species are
present in this pH range. Above pH ∼11, the fluorescence intensity decreases at the same
emission wavelength suggesting that monoanions are formed. In general, the monoanions
of many aromatic amino compounds are reported to be a non-fluorescent [24, 25]. A regular
red shift is noticed in both absorption and fluorescence spectra in these pH ranges from 7.0
to 4.0, indicating the formation of a monocation. The monocation can be formed by protona-
tion either at the ring nitrogen or in the –NH2 group. The experimental results (Table 3) and
those of Dogra et al. [24, 25] have clearly established that the first protonation takes place at
the tertiary nitrogen atom. A similar behavior (i.e. red shift in absorption spectrum) has been
observed during the first protonation of 2-(4ꢀ-aminophenyl) benzoxazoles [24, 25]. Further,
when the hydrogen ion concentration is increased below pH ∼3, the absorption spectrum is
largely blue shifted which indicates that protonation takes place in the –NH2 group. It is a
well known fact that protonation of an amino group gives blue shifted absorption maxima
[27–30]. Furthermore, the absorption maxima of SMO, STO and SFO molecules resemble
those of SAM, also indicating that the protonation takes place in the NH2 group.
The dication absorption maxima of these molecules are largely blue shifted (220 nm)
with very week shoulders that appear at 272 nm and 266 nm, which resemble those of the
SAM monocation. The above absorption and fluorescence spectra of these molecules at H0
−0.80, which resemble the monocation spectrum of SAM, can be assigned to the dication
formed by protonation of the amino group. It is observed that the fluorescence intensity

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Fig. 3 Absorption spectra of
different prototropic species of
SFO and SAM at 303 K:
concentration
−5
−1
4 × 10 Mol·L . 1—dication,
2—monocation, 3—neutral
of the monocation starts decreasing at pH ∼3 and a red shifted fluorescence band starts
appearing only at H0 −0.26. The decreased fluorescence intensity at the same emission
wavelength could be due to either proton induced fluorescence quenching or formation of a
non-fluorescence monocation.
3.3 Acidity Constants
The pKa values for different prototropic reactions determined by using absorption data are
listed in Table 3. Since the amino group in a sulpha drug molecule is resonance stabilized,
the electron density at the amino group will be decreased (Scheme 2). Thus, a regular red
shifted absorption maximum is observed in neutral to monocation species. The very low pKa
value for the dication-monocation equilibrium is due to the presence of a nearby positive
charge on the amino group which reduces the electron density in the oxazole nitrogen atom.
The pKa∗ values for different prototropic equilibria have been determined by fluorimetric
titrations and the values clearly indicate that the amino group becomes a stronger acid upon
excitation.
3.4 Interaction of Sulpha Drugs with β-CD
The absorption and emission spectra of SMO, SFO, STO and SAM were recorded in solu-
tion of β-CD at different concentrations (pH ∼ 7) and are shown in Table 4, Figs. 5 to 8.
The absorption maxima of the above sulpha drug molecules are red shifted with a gradual
increase in the molecular coefficient. The increase in the absorbance is due to the encap-

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Fig. 4 Emission spectra of
different prototropic species of
SFO and SAM at 303 K:
concentration
−5
−1
4 × 10 Mol·L . 1—dication,
2—monocation, 3—neutral,
4—dianion
sulation of these molecules into the β-CD cavity and it is attributed to the detergent action
of β-CD [31–34]. After preparing these drug-β-CD solutions, we also recorded the absorp-
tion spectra after 12 hrs. But no significant absorbance change is observed in these drug
solutions, revealing that these drugs are encapsulated in the β-CD cavity and the inclusion
complexes do not decompose upon storage. The above behavior may be attributed to the en-
hanced dissolution of the guest molecule through hydrophobic interaction between the guest
and the non-polar cavity of the β-CD. These results indicate that sulpha drugs are entrapped
in the β-CD to form inclusion complexes.
No clear isosbestic point is observed in the absorption spectra, but the changes that are
observed in the absorbance are very small. In general, the existence of an isosbestic point in
the absorption spectra is indicative of the formation of a well defined 1:1 complex [35–42].
These possibilities are proposed for this deviation: (i) more than one guest molecule can be
accommodated within a single β-CD cavity, (ii) due to the space restriction of β-CD cavity,
more than one type of complex with each having 1:1 stoichiometry may be formed, and
(iii) the changes detected in the absorption spectra when β-CD is added to a sulpha drug
solution containing methanol (1%) can also give rise to interaction between these compo-
nents. Since, in these experiments the concentration of methanol is practically constant with
respect to β-CD concentration, it may affect the isosbestic point. Further, in sulpha drug
inclusion complexes, the detergent action of β-CD is so strong that no isosbestic point was
observed in the absorption spectra. In the following discussions we propose two distinct
complexes, one is the aniline part encapsulated and the other is the heterocyclic (i.e. five
member ring) partly encapsulated into the β-CD cavity.

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Fig. 5 Absorption spectra of
SFO and SMO in different β-CD
−1
concentrations (Mol·L ): 1—0,
2—0.001, 3—0.002, 4—0.004,
5—0.006, 6—0.008, 7—0.01.
Insert figure: absorbance versus
β-CD concentration
The binding constant for inclusion complex formation has been determined by the
changes in the absorbance and fluorescence intensities with the β-CD concentrations. The
binding constant (K) values were determined by using the Benesi-Hildebrand relation [43]
and indicate that 1:1 complexes are formed between β-CD and these compounds. In all
cases, k values 1:1 complexes formed between β-CD and these molecules were determined
using the following equation:
1
1
1
=
+
(1)
I − I0
Iꢀ − I0 K(Iꢀ − I0)[β − CD]0
where [β-CD]0 represents the initial concentration of β-CD, I0 and I are the absorbance
and fluorescence intensities in the absence and presence of β-CD, respectively, and Iꢀ is the
limiting intensity of absorbance and fluorescence. The ‘K’ values were obtained from the
slope and intercept of the plots (Figs. 9 and 10). The Benesi-Hildebrand plot shows excel-
lent linear regression supporting the formation of the 1:1 inclusion complex. The binding
constant values are very small compared with other guest molecule/β-CD complexes such
as dialkyl aminobenzonitrile and its derivatives [40–42]. This is probably because (i) sulpha
drug molecules are partially included into the cavity, (ii) the oxazole/thiazole group may be
included in the cavity, and (iii) these molecules are not tightly encapsulated in the β-CD
cavity.
The change in standard Gibbs energy of this complex can be calculated from the:
ꢀG = −RT lnK
(2)

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Fig. 6 Absorption spectra of
STO and SAM in different β-CD
−1
concentrations (Mol·L ): 1—0,
2—0.001, 3—0.002, 4—0.004,
5—0.006, 6— 0.008, 7—0.01.
Insert figure: absorbance versus
β-CD concentration
As can be seen from Table 4, the ꢀG values are negative which suggests that the inclusion
process proceeded spontaneously at 303 K. The negative values at the experimental temper-
ature indicate that the inclusion process is an exothermic and enthalpy controlled process.
The hydrophobic interaction between the internal wall of β-CD and guest molecules is an
important factor for the stability of inclusion complexes. In sulpha drugs, it may be consid-
ered that the difference in the magnitude of the hydrophobic interaction is related to that of
the contact area of the guest molecule with the internal wall of β-CD.
Several driving forces have been postulated for the inclusion complexation of CD with
guest compounds: (1) Van der Waals forces; (2) hydrophobic interactions; (3) hydrogen
bonding; (4) release of distortional energy of CD by binding of the guest; and (5) extrusion
of ‘high energy water’ from the cavity of CD upon inclusion complex formation. Based
on the thermodynamic parameter (ꢀG) calculated for the inclusion of drugs, we conclude
that the hydrogen bonding interaction, van der Waals interaction, and breaking of the water
cluster around this polar drug compound mainly dominate the driving force for inclusion
complex formation.
It is well known that the strength of interaction is also dependent on the size of the
CD cavity and size of the substituent in the complex. This means that the interaction is
more sensitive to the size of substituents and the CD in the complexation. The CDs are
truncated, right-cylindrical, cone-shaped molecules, 7.8 Å in height with a central cavity.
The diameters of the narrower and wider rim of the cavity for β-CD are 6.5 Å and 5.8 Å,
respectively. It is well known that the van der Waals force including the dipole-induced
dipole interactions are proportional to the distance between the drug and the wall of the CD

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Fig. 7 Emission spectra of SFO
and SMO in different β-CD
−1
concentrations (Mol·L ): 1—0,
2—0.001, 3—0.002, 4—0.004,
5—0.006, 6—0.008, 7—0.01.
Insert figure: fluorescence
intensity versus β-CD
concentration
cavity and to the polarizabilities of the two components. It is thus a short range interaction;
therefore, the drug may be embedded deeper in β-CD. The phenyl moiety may achieve
a maximum contact area with the internal surface of the cavity of the β-CD. Hence, the
interaction of the phenyl ring with β-CD would play an important role.
In general, the inclusion of CDs with guest compounds is also affected by hydrophobic
and electronic interactions. Since CDs have a permanent dipole, the primary hydroxyl end is
positive and the secondary hydroxyl end is negative in the glucose units of CDs. The stability
of binding by hydrophobic interaction is partly the result of the van der Waals force but is
mainly due to the effects of entropy produced on the water molecules. In aqueous solution,
a hydrophobic guest compound is restricted by the water shell formed by the hydrogen
bonding network. It has a strong tendency to break down the water cluster and penetrate the
non-polar cavity of CD. This process is exothermic due to an entropic gain. The association
constants for the inclusion of β-CD with guest compounds were observed to be proportional
to the substituent hydrophobic constant of the guest.
Figures 7 and 8 show the fluorescence spectra of the sulfa drugs SMO, SFO, STO and
SAM in aqueous solution as a function of the β-CD concentration. Since no clear isosbestic
point was observed in the absorption spectrum of a neutral molecule, the excitation wave-
length is selected in such a manner that the absorbance changes are very small. In aqueous
solution, a single fluorescence maximum was observed in the above sulpha drug molecules.
Upon addition of β-CD, the emission band at 342 nm was gradually increased with an ap-

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Fig. 8 Emission spectra of STO
and SAM in different β-CD
−1
concentrations (Mol·L ): 1—0,
2—0.001, 3—0.002, 4—0.004,
5—0.006, 6—0.008, 7—0.01.
Insert figure: fluorescence
intensity versus β-CD
concentration
pearance of dual emission at 430 nm in SMO/SFO/STO molecules. In contrast in SAM, the
emission intensity at 340 nm was gradually decreased without any dual emission.
The β-CD dependent emission spectra of sulpha drugs show that the shorter wavelength
(SW) band is more sensitive in β-CD solutions, whereas the longer wavelength (LW) band
shows a small enhancement with a regular bathochromic shift (Fig. 6). The LW intensity
in β-CD medium is lower than the SW intensity. With addition of β-CD both LW and SW
intensities are increased; however, the rate of enhancement of the SW emission is greater
than that for the LW band. Further, with an increasing β-CD concentration, a regular red
shift is observed in the LW band (420–430 nm) whereas no significant change is observed
in the SW band (340 nm). This can be seen more clearly from observing Fig. 6, where the
SW intensity in 0.010 Mol·L−1 β-CD solution is increased to 5 times that of the aqueous
medium and the LW emission is enhanced by approximately 0.5 times. The enhancement
of both bands of sulpha drug molecules in β-CD solutions may be explained as follows
[44, 45]. The enhancement of the SW band in β-CD may be due to lowering of the sol-
vent polarity at higher β-CD concentration [44, 45]. Inside the β-CD cavity sulpha drug
molecule feels a much less polar environment and the main non-radiative path of the SW
band (through intramolecular charge transfer (ICT) or twisted intramolecular charge trans-
fer (TICT)) is restricted, which also causes an enhancement of the SW band. Further, the
geometrical restriction of the cavity would restrict the free rotation of the heterocyclic ring

J Solution Chem (2010) 39: 1061–1086
1079
Fig. 9 Absorption spectra of the
Benesi-Hildebrand plots for the
complexation of SFO, SMO,
STO and SAM with β-CD. Plot
of 1/ꢀA versus 1/[β-CD]
Fig. 10 Emission spectra of the
Benesi-Hildebrand plots for the
complexation of SFO, SMO,
STO and SAM with β-CD. Plot
of 1/I − I versus 1/[β-CD]
0
or methyl group in the β-CD cavity and thus hinder the formation of an ICT state causing
an enhancement of the normal SW band.
From the above findings it is clear that in β-CD solutions, the surrounding polarity of
SO2NH-group does not change very much as there is no hypsochromic shift of the most
polar (ICT) state [46], but the relative intensity (IICT/ILE) increases with the increase of β-
CD concentration. This may be possible if the orientation of sulpha drugs is such that part
of the aromatic ring goes inside the β-CD cavity (Scheme 3). This is because, if the amino
group is present in the interior part of the β-CD cavity, a hypsochromic shift or ICT emission
should not be present in the β-CD medium. We found that our earlier results, with aqueous
β-CD solutions, hydrophobicity is the driving force for encapsulation of the molecule inside
the cavity and naturally the hydrophobic part likes to go inside the deep core of the nonpolar
cavity and the polar group will be projected in the hydrophilic part of the β-CD cavity
[40–42].
The above results indicate that part of a SMO, SFO and STO molecule is entrapped in
nonpolar β-CD cavity whereas SAM is completely included in the β-CD cavity to form an
inclusion complex. Formation of the inclusion complex in SMO, SFO and STO with β-CD
should result in dual emission. Since the size of SAM is smaller than the β-CD cavity size,
this molecule may be completely included in the cavity; therefore, the emission intensity is
decreased. However, the sizes of SMO, SFO and STO molecules are larger than the β-CD
cavity size, and these molecules are partially included in the β-CD cavity (Schemes 1 and 3).
Further, it is noteworthy that the LW emission intensity gradually increased along with the
red shift upon increasing the β-CD concentrations. Thus, it can be suggested that the LW
emission band is related to the formation of a β-CD inclusion complex. Such a spectral shift

1080
J Solution Chem (2010) 39: 1061–1086
Scheme 3 The proposed inclusion complexes of SFO, SMO, STO and SAM
may correspond to an energy stabilization of the emitting state and is characteristic for the
fluorescence of an ICT or TICT state. The LW emission from above sulfa drugs originate
from the TICT state with twisting occurring at the amide S–N bond between the aniline
moiety (electron donor) and the SO2 moiety (electron acceptor). Further, Modiano et al.
[47] reported whenever two aromatic rings are separated by groups like SO2, CH2, CO, NH
etc. they form a TICT state. Thus it can be speculated that the enhanced 430 nm emission in
the above sulfa drugs should originate from the TICT state. The TICT emission is observed
in β-CD suggesting that the inclusion process plays a major role in the TICT emission.
Supporting this implication, the excitation spectra for the 440 nm emission is similar to that
of the 340 nm emission.
3.5 Prototropic Reactions in β-CD Medium
To know the effect of β-CD on the prototropic equilibrium between neutral, monocation
and dication, pH dependent changes in the absorption and emission spectra of the sulpha

J Solution Chem (2010) 39: 1061–1086
1081
drug molecules in aqueous solution containing β-CD have been recorded and are shown
in Table 2. The absorption and emission maxima of these molecules have been studied in
6 × 10−3 Mol·L−1 β-CD solutions in the pH range from 0.1 to 11. On comparison with
aqueous medium, in β-CD solutions the absorption and emission maxima of these neutral
drug molecules are red shifted (Table 2). The ground and excited state pKa (pKa∗) values of
these molecules in β-CD medium are slightly lower than those in aqueous medium (Table 3).
However, a red shift is observed in the β-CD solutions compared to the aqueous solutions,
suggesting the sulpha drug molecules are encapsulated in the β-CD.
3.6 Possible Inclusion Complex
Considering the above discussions, the possible inclusion mechanism is proposed as follows.
Naturally, two different types of inclusion complex formation between SMO, SFO and STO
molecules with β-CD are possible: (i) the aniline group is captured in the β-CD cavity and
(ii) the oxazole group is captured in the β-CD cavity. Let us consider the type I arrangement;
if the aniline part is entrapped into the β-CD cavity, like SAM, the fluorescence intensities
should decrease at higher β-CD concentrations. In this case, SMO, SFO and STO molecules
should not show dual emission in β-CD medium. Further, if the amino group is entrapped
into the β-CD cavity, the dication maxima [40–42] (i.e. protonation of amino group) should
be blue shifted in β-CD compared to aqueous medium. But the results in Table 3 shows no
significant spectral shifts either in the β-CD and aqueous medium; i.e., the dication maxima
follow the same trend both in aqueous and β-CD medium.
For type II encapsulation, the oxazole group may be included in the β-CD cavity. If this
type of complex is formed, the neutral maxima of the sulpha drugs (protonation of tertiary
nitrogen atom) should be red shifted in β-CD compared with aqueous medium. The red
shift observed in pH∼7 β-CD solution indicates that the tertiary nitrogen atom interacts
with β-CD hydroxyl groups, because it is well known that CDs are good hydrogen donors
[31–34]. Furthermore, the results observed for sulpha drugs are similar to amino benzoic
acids [27–29] and 2,6-diaminopyridine [48]. The tendencies of these shifts in λabs and λflu
of these molecules are attributable to the inclusion into the β-CD cavity, and are explained
in Scheme 3. This is because of the formation of inclusion complex, i.e. in the inclusion
complexes, pKa (pKa∗) values are known to change depending upon relative the affinity of
the guest and host [40–42].
Further, in Type II complexes, the β-CD cavity will impose a restriction on the free ro-
tation of the CH3 group or oxazole and thiazole groups in its excited state. For this type, the
TICT emission should increase in β-CD solutions. The question may arise why TICT emis-
sion is not observed in SAM. This is because even this complex formation does not affect
the free rotation of –SO2–NH2 group because the size of SAM is smaller than the β-CD
cavity size. Further, another question may arise why SMO, SFO and STO do not exhibit the
TICT emission in polar and nonpolar solvents because of a weak dipole–dipole interaction
between R–SO2–NH-group with solvent and fast back-charge transfer. These features sup-
port the idea that the TICT state in β-CD is stabilized through complex formation between
SMO/SFO/STO with β-CD. This confirms that the environments around the anilino group
in β-CD medium are the same as in the bulk aqueous medium. These features indicate that
in Scheme 3, type-II complex is favored more than type-I.
This is further supported by semi-empirical quantum mechanical calculations: the ground
state geometry of the sulpha drugs were optimized by using the DFT method (CaChe 7.5
program). The internal diameter of β-CD is approximately 6.5 Å and its height is 7.8 Å. The
vertical distance between the sulpha drug molecules are higher than the β-CD cavity size

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J Solution Chem (2010) 39: 1061–1086
and the horizontal distance is smaller than the β-CD cavity size (Scheme 1). Considering
the shape and dimensions of β-CD and sulpha drugs, the only way sulpha drugs can enter
into the β-CD is lengthwise. Under this situation, it is impossible for the sulpha drugs to be
encapsulated completely in one β-CD molecule. Taking into account the dimensions of the
sulpha drugs and β-CD, the complex can be located as shown in Scheme 3.
4 Solid Inclusion Complex Studies
4.1 Microscopic Morphological Observation
First we recorded the powdered form of the above sulfa drugs and β-CD by scanning elec-
tron microscope and then we also observed the powdered form of the inclusion complex
(Fig. 11). These pictures clearly elucidate the difference of all sulfa drugs and their inclu-
sion complex. As seen from the SEM figures, (i) β-CD is present in a platted form, and
(ii) sulfa drugs are present in different forms from their inclusion complexes. Modification
of these structures can be assumed as a proof of the formation of a new inclusion complex.
The different structure of pure sulfa drugs and their inclusion complex again support the
presence of a solid inclusion complex.
Fig. 11 Scanning electron microscope photographs (Pt coated) of (A, B) β-CD, (C) SFO, (D) SMO,
(E) STO, (F) SAM, (G) SFO–β-CD complex, (H) SMO–β-CD complex, (I) STO–β-CD complex,
(J) SAM–β-CD complex

J Solution Chem (2010) 39: 1061–1086
4.2 FTIR Spectra of Sulfa Drugs
1083
(i) SAM: The FTIR spectra of SMO, SFO, STO and SAM and the solid inclusion com-
plex were also studied. The sharp amino stretching at 3477 cm−1 and amide NH stretch-
ing at 3373 cm−1 shifts to 3473 cm−1, while the NH2 deformation at 1629 cm−1 shifts to
1639 cm−1 in the inclusion complex. In addition, the C–N stretching at 1312 cm−1 moves
to 1335 cm−1 and CH bending at 899–622 cm−1 moves to 858–706 cm−1. The CH stretch
at 3065 cm−1, however, is absent in the complex, as well the C=C stretching at 3098 cm−1
is not present in the inclusion complex. The aromatic ring stretching at 1593 cm−1 does
not appeare in the inclusion complex. The S=O stretch 1094 cm−1 moves in the inclusion
complex to 1080 cm−1 and the SO2NH2 symmetric stretch 1148 cm−1 and anti-symmetric
stretch 1312 cm−1 are moved in the inclusion complex to 1156 cm−1 and 1335 cm−1, re-
spectively. The overtone frequencies around 2600 to 2000 cm−1 are lost in the inclusion
complex. The above finding indicates that a SAM molecule is included in the β-CD cavity
and form an inclusion complex.
(ii) SMO: The amino sharp stretch at 3378 cm−1 and the amido sharp stretch at
3467 cm−1 changed in the inclusion complex to a broad peak that appears at 3378 cm−1. The
SO2 anti-symmetric stretch at 1366 cm−1 is shifted in the inclusion complex to 1368 cm−1
and the symmetric stretch 1144 cm−1 is moved to 1157 cm−1. Aromatic CH stretch at
3144 cm−1 does not appear in the inclusion complex. The CH bending peak at 885 to
684 cm−1 moves in the inclusion complex to 860–707 cm−1. The aromatic C=C stretch
at 3239 cm−1 is not present in the inclusion complex. The aromatic ring stretching at
1621 cm−1 moves in the inclusion complex to 1638 cm−1. The CH stretching in C–CH3
group at 2930 cm−1 is also not present in this complex. The CH3 anti-symmetric stretch at
1472 cm−1 moves in the inclusion complex to 1413 cm−1. The CN stretch at 1266 cm−1
is moved in the inclusion complex to 1244 cm−1. The SO2 stretch at 1026 cm−1 moves
to 1029 cm−1. Further the absorption intensity ratio between the drugs and their inclusion
complexes are variable. The above results confirm that this molecule is encapsulated in the
CD cavity.
(iii) SFO: The sharp NH stretch at 3381 cm−1 and amido stretch at 3485 cm−1 become
broad in the inclusion complex. The CH stretch at 3095 cm−1 is lost and the bending at 876–
688 cm−1 moves in the inclusion complex to 862–707 cm−1. The C=C stretch at 3003 cm−1
does not appear in the inclusion complex. The C=C bending at 1596 cm−1 shifts in the inclu-
sion complex to 1596 cm−1. The methyl stretching at 1345 cm−1, 1438 cm−1 is not shifted
but the intensities are greatly reduced in this complex. The SO2 stretching at 1345 cm−1 ap-
pears at the same peak. The C–NH2 stretch at 1301 cm−1 is moved in the inclusion complex
to 1345 cm−1, 1330 cm−1. The aromatic ring stretching at 1323 cm−1 is moved in the inclu-
sion complex to 1330 cm−1. Further, the aromatic overtone frequencies at 2000–1800 cm−1
are lost in the inclusion complex. The above results confirm this molecule is encapsulated
in the CD cavity.
4.3 1H NMR Spectral Studies
Proton nuclear magnetic resonance (1H NMR) spectroscopy has proved to be a powerful tool
1
in the study of inclusion complexes [49, 50]. H NMR spectroscopy provides an effective
means of assessing the dynamic interaction site of β-CD with that of the guest molecules.
The basis of information gained from NMR spectroscopy is located in this shifts, loss of
resolution and broadening of signals observed for the host and guest protons [49, 50].
The resonance assignments of the protons of β-CD are well established [49, 50] and
consist of six types of protons. The chemical shift of β-CD protons reported by different

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J Solution Chem (2010) 39: 1061–1086
authors [49, 50] are very close to those reported in this work. The H-3 and H-5 protons are
located in the interior of the β-CD cavity and it is, therefore, likely that the interaction of
the host with the β-CD inside the cavity will affect the chemical shifts of the H-3 and H-5
protons. A minor shift is observed for the resonance of H-1, H-2 and H-4 located on the
exterior of β-CD.
The addition of sulpha drugs into the β-CD results in a downfield chemical shift for
the sulpha drug protons in DMSO are given below (Scheme 3): SAM (inclusion complex)
values in ppm: NH2 ∼ 5.76 (5.78), SO2NH2 ∼ 6.86 (6.92), H/6H ∼ 7.45 (7.50), H/5H
∼ 6.59 (6.64); SMO (inclusion complex) values in ppm: NH2 ∼ 6.10 (6.12), SO2NH ∼
10.95 (11.02), 2H/6H ∼ 7.485 (7.498), 3H/5H ∼ 6.598 (6.602), CH3 ∼ 2.290 (2.297), 2Hꢀ ∼
6.107 (6.111); SFO (inclusion complex) values in ppm: NH2 ∼ 6.15 (6.17), SO2NH ∼ 11.25
2
3
ꢀ
ꢀ
2
3
(11.30), H/6H ∼ 7.480 (7.484), H/5H ∼ 6.604 (6.607), 3 CH3 ∼ 2.295 (2.299), 4 CH3 ∼
2.303 (2.307); STO (inclusion complex) values in ppm: NH2 ∼ 5.90 (5.92), SO2NH ∼ 12.41
(12.47), 2H/6H ∼ 7.456 (7.462), 3H/5H ∼ 6.579 (6.583), 2Hꢀ ∼ 7.196 (7.201), 3Hꢀ ∼ 6.746
(6.750). As can be seen from the above values, the chemical shifts data for the inclusion
complex are different from the free compound. A small downfield shift on sulpha drugs was
observed, suggesting that the studied molecules are encapsulated into the β-CD cavities.
5 Conclusion
From the above studies we find out the following points: Solvent studies reveal that the po-
sition of the substituent (oxazole or thiazole group) in the SAM molecule (R–SO2–NH–)
group is not the key factor to change the absorption and emission behavior of these mole-
cules. In sulpha drugs, a single fluorescence band was observed in aqueous solution and dual
emission appears in β-CD solution, indicating that the heterocyclic ring is encapsulated into
the β-CD cavity. Formation of the inclusion complex in SMO, SFO and STO should result
in dual emission, which is due to the presence of TICT. β-CD studies indicate (i) sulpha
drugs form 1:1 inclusion complexes with β-CD, and (ii) the red shift in the β-CD medium
confirms heterocyclic ring encapsulated in the β-CD cavity and the aniline ring is present
on the outside of the β-CD cavity.
Acknowledgements This work was supported by the Department of Science and Technology, New Delhi
(Fast Track Proposal Young Scientist Scheme No. SR/FTP/CS-14/2005) and University Grants Commission,
New Delhi (Project No. F-31-98/2005 (SR). One of the authors A. Antony Muthu Prabhu is thankful to CSIR,
New Delhi for the award of Senior Research Fellowship (SRF).
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