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

Fig. 2.1 FT-IR Spectrum of Aspartame

Fig.2.2.1 HNMR Spectrum of Aspartame

67

Fig 2.3. F.T-IR.Spectrum of Saccharin

FIG.2.4.1HNMR Spectrum of Saccharin

68

Fig 2.5. FT-IR.Spectrum of Sucralose

FIG.2.6.1HNMR Spectrum of Sucralose

69

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