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CHAPTER II
EXPERIMENTAL METHODS
In this chapter, a detailed account of various materials used, general experimental
procedures for the preparation of some of the starting materials, preparation of
cyclodextrin complexes, characterization of the starting materials and products,
spectroscopic methods and analytical techniques such as TLC, UV-visible, FT-IR, and 1H-
and 13C-NMR used in this study are described.
2.1 Materials
-, !- and "-CDs used at various stages of this work were purchased from
American Maize Products, Indiana and Aldrich. The other chemicals and their sources are
listed below
Chemicals Suppliers Chemicals Suppliers
- Cyclodextrin Aldrich m-Nitrobenzaldehyde Merck
!- Cyclodextrin Aldrich p-Bromobenzaldehyde Merck
"- Cyclodextrin Aldrich o-Chlorobenzaldehyde Merck
Aspartame Himedia p-Bromobenzaldehyde Merck
Saccharin Himedia Potassium chloride Merck
Sucralose Himedia Potassium iodide Merck
Benzaldehyde Merck Sodium hydroxide Merck
p-Hydroxy benzaldehyde Merck Sodium Hydrogen phosphate Merck
p-Methoxybenzaldehyde Merck Acetone Merck
m-Methoxybenzaldehyde Merck Zinc Acetate Merck
p-Chlorobenzaldehyde Merck Acetonitrile Merck
p-Nitrobenzaldehyde Merck n- Hexane Merck
mChlorobenzaldehyde Merck Ethanol Merck
p-Methylbenzaldehyde Merck Diethyl ether Merck
52
Liquid samples and common solvents (Merck, AR and HPLC grade) like
chloroform, diethyl ether, carbon tetrachloride, acetone, acetonitrile and methanol etc.,
were purified according to standard procedures1,2 and solid samples were purified by
recrystallization before use. The purity of the compounds was confirmed by M.Pt / B.Pt
and also by spectral methods. Doubly distilled water was used throughout this study.
2.2 Preparation of CD complexes
To a saturated solution of CD in water, equimolar amount of substrate dissolved in
minimum amount of methanol was added and stirred for 24 hrs at room temperature. The
resultant white crystalline precipitate was filtered, washed with diethyl ether to remove
any uncomplexed substrate and dried in an air oven for 4 hrs at 60oC. The dried white
crystalline powder was used for further studies. Higher order complexes (2:1 or 1:2 host:
guest) were also prepared using the above procedure with excess amount of CD or
substrate.
2.3.Characterization of the CD-substrate complexes
The physicochemical properties of the guest molecules vary upon the formation of
the inclusion complexes with CDs. The orientation of the substrates inside the CD cavity
is governed by the size, substituents, mode of inclusion of the substrate and also by the
interaction between the substrate and CD. Hence, FT-IR, electronic absorption, and NMR
technique were used to study the incorporation of the guest into the CD cavities.
2.3.1Host-Guest ratio
Host-guest ratio was estimated by adopting the following procedure
A known amount of the solid CD inclusion complex was dissolved in minimum
amount of distilled water and the guest molecule was extracted with warm chloroform. 2
53
2.3.2 Determination of binding constants
Stock solution of the substrate (1 x 10-2 or 1 x 10-3 M) was prepared by weighing a
known amount of the substrate and dissolving it in minimum amount of pure methanol
(HPLC grade). For water-soluble compounds, the substrate was dissolved in doubly
distilled water and then sonicated. 0.1 mL of this was added to a known volume of
appropriate CD (1 x 10-2 M) stocksolution in water and then diluted to 10 mL. The
solution was stirred for 24 hrs unless noted otherwise. Absorption and emission spectra
were recorded to calculate the equilibrium constants for the complexes. Benesi-
Hildebrand5 equation (eqn. 2.1) was used to calculate the equilibrium constants for the 1:1
inclusion, complexes formed between the substrate and the CD from UV and emission
studies.
...(2.1)
In some cases, the binding constants were calculated using non-linear curve fitting
by using the following equations 2.2 and 2.3 for 1:1 and 1:2 complexation of guest with
CD respectively.
...(2.2)
...(2.3)
#$%&%'()*+(
Absorbance value of the substrate in presence and absence of cyclodextrin; [S0]
and [CD] are concentrations of substrate and ,-,./0%12&345(&%56%,237%.-8(9SCD :40(9S are
;9K
1
;9
][substrate[CD]
;*+
rate][CD][subst
f
##
$
[CD]K1
][CD][SK]9[9;*+
1
o1SSCD o
#
%$
2211
o2
21SS(CD)o1SSCD
[CD]KK[CD]K1
][S[CD]K]K9[9][CD][SK]9[9;<+ o2o
##
%#%$
54
molar extinction coefficients of the complexes and substrates. K1 and K2 are binding
constant values of the 1:1 host-guest complexes.
2.3.3 Calculation of equilibrium constants
The fluorescence enhancement F/F0 measured as a function of host concentration
can be used to obtain the association constant for the host-guest inclusion process. In the
case of 1:1, host: guest inclusion, a single equilibrium is involved, with association
constant K.
Where, S = Substrate,
In this case, the dependence of F=/F0 on added host concentration, [CD] 0, is given
by the following equation 2.4.
K[CD]1
K[CD]1)(F/F1
F
F
o
oo
o #
%#$ ...(2.4)
Where F=/F0 is the maximum enhancement, when all guests are complexed within a host.
If only 1:1 complexes are formed, then the double-reciprocal plot of
1/( F& /F0-1) versus l/[CD]0 will be linear; a non-linear double reciprocal plot indicates
the presence of higher-order inclusion complexes. Considering the 1:2 complexation of
substrate with two molecules of cyclodextrin, the binding constant was calculated by the
following stepwise mechanism.
CD + CD : S CD2 : SK
[CD][S]
S]:[CDK $
55
CD + S CD : SK
These two equilibria are described by the equilibrium constants K1 and K2
[CD][S]
S]:[CDK1 $
S]:[CD[CD]
S]:[CDK 2
2 $
with the overall equilibrium constant for 2:1 complexation equal to the product K1 and K2.
The dependence of F/F0 on [CD] 0 for this complexation mechanism is given by eqn. 2.5.
2o21o1
2o21o2o1o1
o [CD]KK[CD]K1
[CD]KK/FF[CD]]K/FF[1
F
F
##
##$ ...(2.5)
Equilibrium constants in the excited states were calculated using the method of
Tahara et al.6
2.3.4. Instrumentation and other methods of analysis
The FT-IR spectra for the complexes were recorded in a JASCO FT-IR 410
spectrometer using pressed KBr pellets.
1H- NMR spectra were recorded in CDCl3 (300 MHz) and DMSO-d6 (75 MHz) in
a Bruker NMR instrument using TMS as an internal standard. UV absorption spectra were
recorded using a UV/VIS double beam spectrophotometer 2201. Optical densities were
monitored at appropriate wavelengths ranging from 190-900 nm in aqueous medium at
room temperature.
56
Instrument Make and model
NMR Bruker 300 MHz instruments with TMS as internal standard
FT-IR JASCO FT/IR-410
UV-Vis Double beam spectrophotometer 2201
Powder XRD
Bruker AXS-D8 advanced step-scanning diffractometer (Cu K -
radiation, ' = 1.54178 Å, step/step time = 0.045o/0.5 sec, power
= 40 kV/3mA, Rane 2( = 2-80o)
SEM and EDX HITACHI S-3400N
Spectrofluorimetry JASCO 550
TEM JOEL-JEM-1200EXMI, Japan Electro Optic Laboratory
Corporation, Japan.
2.4. Aspartame
2.4.1 Binding behavior of aspartame with cyclodextrin: structure and
characterization of cyclodextrin complexes of aspartame
The aspartame sample purchased from Himedia was doubly recrystallised from
methanol and characterized by 1H NMR. Sodium monohydrogen phosphate and di
hydrogen phosphate (Merck) were used for preparation of phosphate buffers. KCl and HCl
used for buffer solutions of lower pH were also Merck samples.
FT-IR (KBr) cm-1 : 3316,2949. 1737, 1663,1589,699.07
(Fig.2.1)
1H NMR : 3.51 (t, 2H), 3.7 7(s, 3H), 3.80
(s, 1H), 4.99 (d)
(Fig.2.2)
57
2.4.2 Determination of dissociation constant of aspartame-CD complexes
The following scheme represents the complex formation equilibria8
where, HA denotes the free acid or neutral (-COOH form) and A¯ denotes the
deprotonated or anionic (-COO¯ form)
Ka = Acid dissociation constant of the acid
K = Acid dissociation constant of the inclusion complex of the neutral
guest, CD-HA.
Ka is acid dissociation constant, pKa = -log Ka and K = dissociation constant of the
34,.>53/4(,/?6.%1(/@(4%>2&:.(5>A52&:2%(#32$( (-'(!(- :40("(- CDs. KA¯ and KHA being the
binding constant values of the substrate in anionic (pH = 12.5) and in neutral forms (pH =
2.4) respectively. pK = -log K and it relates to the acid strength of the inclusion complex
according to the following equation
HA
Aa
K
KKK
%
$
2.4.3. Preparation of stock solutions for spectrophotometric studies
Stock solution of Aspartame (1x10-3 M) was prepared by dissolving a known
amount of the compound in water and methanol then by sonication. The respective
cyclodextrin solutions were added from the CD (1x10-3M) stock solution freshly prepared
in water and the volume was made upto 10 mL by using respective buffers of appropriate
HA + CD A¯ + H+ + CDKa
CD - HA CD - A¯ + H+K
KHA KA¯
58
pH values and then stirred for 24 hrs. Absorption spectra were recorded for the substrate in
varying CD concentration and also in absence of CD. Emission spectra were also recorded
at room temperature. Excitation wavelength was fixed as the absorption maximum of the
substrate. The scan speed was 200 nm/min and emission slit widths were set at 10 nm for
the emission spectra.
2.4.4. Aspartame !–"-#$%!&–-CD Solid Complexes.
Solid inclusion complexes of Aspartame with !-CD were prepared in 1:1 molar
ratios using a kneading method. -, !-, "- Cyclodextrins (CDs )and Aspartame were
weighed in the ratios of 1:1 ( CDs:Asp). The CDs were dissolved in minimum quantity of
water with constant stirring and this solution was added to a solution of Aspartame. The
powdered forms of CDs and Aspartame were blended in dry conditions first and then a
small volume of water in which both CD and Aspartame partly dissolve, was added,
stirred magnetically for 24 hours. The inclusion complex was formed as slurry which was
then dried to give a composition containing the inclusion complex. In this included form,
Aspartame is stabilized against hydrolysis. If such a composition was subjected to
conditions which ordinarily cause hydrolysis of Aspartame, the degradation was found to
be reduced. The obtained solid mass was further dried under vacuum to a constant weight
at room temperature and pulverized, sieved through mesh no. 100 and stored in a
desiccators and characterized by spectral techniques such as FT-IR, 1HNMR and XRD
studies.
2.4.5. FT-IR spectra
Solid CD complexes of aspartame were prepared of 1:1 ratio H-G ratio also and
were analyzed by pressed KBr pellet technique. As aspartame contains several
59
chromophores giving IR active vibrations, FT-IR spectra were found highly useful in
characterization of the CD complexes of aspartame.
2.4.6. X-ray diffraction studies
Solid CD complexes of aspartame were prepared of 1:1 ratio H-G ratio also and
were analyzed by X-ray diffraction technique. The X-ray diffraction technique has been
used to identify the inclusion complexation in the solid state using monochromatized Cu-
ka( =1.5056) on x-ray diffractometer Bruker AXS-D8 advanced step-scanning
diffractometer (Cu K -radiation, ' = 1.54178 Å, step/step time = 0.045o/0.5 sec, power =
40 kV/3mA, Rane 2( = 2-80o) available at MS university Tirunelveli. The X-ray
diffraction technique were found highly useful in characterization of the CD complexes of
aspartame.
2.4.7. 1HNMR Spectra
Solid CD complexes of aspartame were prepared of 1:1 ratio H-G ratio also
and are analyzed using DMSO-d6 (75 MHz) in a Bruker NMR instrument using TMS as
an internal standard.
2.5.Saccharin- Cyclodextrin Complexes-Synthesis and Characterization
2.5.1.Saccharin and Cyclodextrin
-, !-, "- Cyclodextrins were purchased from Aldrich and Saccharin was
purchased from Himedia. Sodium monohydrogen phosphate and di hydrogen phosphate
(Merck) were used for preparation of phosphate buffers. KCl and HCl used for buffer
solutions of lower pH were also Merck samples.
60
Characterisation of Saccharin was done by NMR and FT-IR methods.
1H NMR : 8.8 (s, 1H), 8.1-8.83 (m, 3H, Ar-H)
(B ppm, DMSO-d6, 300 MHz):
(Fig.2.3)
FT-IR (KBr) cm-1 : 3399.89, 1719, 1592.91, 1357, 1296.89, 1177 .33
(Fig 2.4)
2.5.2 Preparation of stock solutions for UV and Fluoresence emission studies
Stock solution of saccharin (1x10-3 M) was prepared by dissolving a known
amount of the compound in water and then by sonication. The respective cyclodextrin
solutions were added from the CD (1x10-3M) stock solution freshly prepared in water and
the volume was made up to 10 mL by using respective buffers of appropriate pH values
and then stirred for six hrs. Absorption spectra were recorded for the substrate in varying
CD concentration and also in absence of CD. Emission spectra were also recorded at room
temperature. Excitation wavelength was fixed as the absorption maximum of the substrate.
The scan speed was 200 nm/min and emission slit widths were set at 10 nm for the
emission spectra.
2.5.3. Saccharin !–"-#$%!&–-CD Solid Complexes.
Solid inclusion complexes of Saccharin #32$(!-CD were prepared in 1:1 molar ratios
using a kneading method. -, !-, "- Cyclodextrins (CDs )and Aspartame were weighed in
the ratios of 1:1 ( CDs:Sac).The CDs were dissolved in minimum quantity of water with
constant stirring and this solution was added to a solution of Saccharin. The powdered
forms of CDs and Saccharin were blended in dry conditions first and then a small
volume of water in which both CD and Saccharin partly dissolve, was added, stirred
61
magnetically for 24 hours. The inclusion complex was formed as slurry which was then
dried to give a composition containing the inclusion complex. In this included form,
saccharin is stabilized. If such a composition is subjected to conditions which ordinarily
cause hydrolysis of Sac, the degradation is found to be reduced. The obtained solid mass
was further dried under vacuum to a constant weight at room temperature and pulverized,
sieved through mesh no. 100 and stored in a desiccators and characterized by spectral
techniques such as FT-IR, 1HNMR and XRD studies.
2.5.4. FT-IR spectra
Solid CD complexes of Saccharin were prepared of 1:1 ratio H-G ratio also and
were analyzed by pressed KBr pellet technique. FT-IR spectra were found highly useful in
characterization of the CD complexes of aspartame.
2.4.5. X-ray diffraction studies
Solid CD complexes of Saccharin were prepared of 1:1 ratio H-G ratio also and were
analyzed by X-ray diffraction technique. The X-ray diffraction technique has been used to
identify the inclusion complexation in the solid state using monochromatized Cu-ka(
=1.5056) on x-ray diffractometer Bruker AXS-D8 advanced step-scanning diffractometer
(Cu K -radiation, ' = 1.54178 Å, step/step time = 0.045o/0.5 sec, power = 40 kV/3mA,
Rane 2( = 2-80o). The X-ray diffraction technique were found highly useful in
characterization of the CD complexes of saccharin.
2.5.6.1HNMR Spectra
Solid CD complexes of saccharin were prepared of 1:1 ratio H-G ratio also
and are analyzed using DMSO-d6 (75 MHz) in a Bruker NMR instrument using TMS as
an internal standard.
62
2.5.7 Determination of 1:1 binding constants
Binding constant of 1:1 H-G complexes (K11) of saccharin at pH values 1.5 and 4
are calculated by using Benesi-Hildebrand equation .
2.6. Cyclodextrin Inclusion Complexes of Sucralose and their Spectral
characterization
-, !-, "- Cyclodextrins and Sucralose (SL) were obtained from Alchemy
Laboratory, Mumbai. Double distilled water was used throughout the study and all other
chemicals were used of analytical grade and used without further purification.
Characterisation of Sucralose (SL) was done by NMR and FT-IR methods.
1H NMR : 3.4 (s, 1H), 3.8.3,3.68 (m, 3H, Ar-H)
(B ppm, DMSO-d6, 300 MHz):
(Fig.2.5.)
FT-IR (KBr) cm-1 : 3010, 1727, 1547, 1357, 1277, 1187
(Fig 2.6)
2.6.1 Preparation of stock solutions for UV –vis and Fluoresence emission studies
Stock solution of sucralose(1x10-3 M) was prepared by dissolving a known amount
of the compound in water and then by sonication. The respective cyclodextrin solutions
were added from the CD (1x10-3M) stock solution freshly prepared in water and the
volume was made upto 10 mL and then stirred for twelve hrs. Absorption spectra were
recorded for the substrate in varying CD concentration and also in absence of CD. Optical
densities were measured at appropriate wavelengths ranging from 200-500 nm at room
temperature. Emission spectra were also recorded at room temperature. Excitation
63
wavelength was fixed as the absorption maximum of the substrate. The scan speed was
200 nm/min and emission slit widths were set at 10 nm for the emission spectra.
2.6.2. Preparation of molecular inclusion complexes
Solid inclusion complexes of sucralose #32$( !-CD were prepared in 1:1 molar
ratios using a kneading method. -, !-, "- Cyclodextrins (CDs )and Sucralose (SL )were
weighed in the ratios of 1:1 ( CDs: SL) .The CDs were dissolved in minimum quantity of
water with constant stirring and this solution was added to a solution of SL. The powdered
forms of CDs and SL were blended in dry conditions first and then a small volume of
water in which both CD and SL partly dissolve, was added, stirred magnetically for 24
hours. The inclusion complex was formed as slurry which was then dried to give a
composition containing the inclusion complex. In this included form, sucralose is
stabilized against decomposition. If such a composition is subjected to conditions which
ordinarily cause decomposition of SL, the degradation is found to be reduced. The
obtained solid mass was further dried under vacuum to a constant weight at room
temperature and pulverized, sieved through mesh no. 100 and stored in a desiccators and
characterized by FT-IR, 1HNMR and XRD studies.
2.6.3. FT-IR spectra of Sucralose
Solid CD complexes of Sucralose were prepared of 1:1 ratio H-G ratio also and
were analyzed by pressed KBr pellet technique. FT-IR spectra were found highly useful in
characterization of the CD complexes of sucralose.
2.6.4. X-ray diffraction studies of Sucralose
Solid CD complexes of Sucralose were prepared of 1:1 ratio H-G ratio also and
were analyzed by X-ray diffraction technique. The X-ray diffraction technique has been
used to identify the inclusion complexation in the solid state using monochromatized Cu-
64
ka( =1.5056) on x-ray diffractometer Bruker AXS-D8 advanced step-scanning
diffractometer (Cu K -radiation, ' = 1.54178 Å, step/step time = 0.045o/0.5 sec, power =
40 kV/3mA, Rane 2( = 2-80o). The X-ray diffraction technique were found highly useful
in characterization of the CD complexes of sucralose.
2.6.5. 1HNMR Spectra of Sucralose
Solid CD complexes of Sucralose were prepared of 1:1 ratio H-G ratio also
and are analyzed using DMSO-d6 (75 MHz) in a Bruker NMR instrument using TMS as
an internal standard.
2.6.6 Calculation of binding constants
Benesi-Hildebrand equation (B.H equation) was used to calculate the equilibrium
constants for the 1:1 inclusion complexes formed between the substrate and the CD from
UV and emission studies.
;9K
1
;9
][substrate[CD]
;*+
rate][CD][subst
f
##
$
Binding constants were calculated from Benesi-C3.0%A&:40(6./25(/@(DCE(DFE(G()*+(
Vs [H] + [G].
2.7. Experimental Section
2.7.1. Typical Procedure for the Preparation of benzochromene
To a magnetically stirred solution of benzaldehyde (0.10 g, 1.0 mmol) and
malononitrile (0.07 g, 1.0 mmol) in a screw capped vial, which contained CH3CN (5 mL),
ZnO nanoparticles (50mg) was added, and it was stirred for 30 min; then, 2-
hydroxynaphthalene- 1,4-dione (0.17 g, 1.0 mmol) was added to the reaction mixture at
room temperature. The reaction mixture was stirred for 20 hrs. After completion of the
65
reaction (monitored by TLC method), the precipitated product was separated from reaction
mixture by filtration and was washed with 5 mL of n-hexane. The desired product was
obtained as an orange powder.
2.7.2. FT-IR spectra of benzochromene
Benzochromenes were synthesized and were analyzed by pressed KBr pellet
technique. FT-IR spectra were found highly useful in characterization of
benzochromenes.
2.7. 2.1HNMR Spectra of benzochromene
Benzochromenes were synthesized and were analyzed by pressed KBr pellet
technique. 1HNMR spectra were found highly useful in characterization of
benzochromenes.
.
66
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