Spectro Photometric Studies of Charge Transfer Complexes

THESISSubmitted in Partial fulfilment of the

requirement for the award of degree of

Doctor of Philosophyin

C h e m i s t r y

Submitted By

Z u l k a r n a i n

D e p a r t m e n t o f C h e m i s t r y

Faculty Of ScienceAligarh Muslim University

Aligarh -202002

2017

Department of Chemistry F a c u l t y o f S c i e n c e

A l i g a r h M u s l i m U n i v e r s i t y aligarh-202002 (India)

C e r t i f i c a t eThis is certify that the work embodied in this thesis entitled “ Spectro Photometric Studies of Charge Transfer Complexes .” is original contribution of ‘ Mr.Zulkarnain ’ carried out under the guidance and supervision of undersigned and is suitable for the award of the degree of Doctor of philosophy in Chemistry of Aligarh Muslim University, Aligarh.The matter embodied in this thesis has not been submitted in part or full to any other university or institute for the award of any degree.

Prof. Afaq Ahmad(Signature)

Department of Chemistry

Aligarh Muslim University, Aligarh−202002 (India)

Dated : 29th December 2017

ACKNOWLEDGMENTAll thanks and praises to almighty Allah for giving me the strength and patience to work through all these years. Nothing is possible without his blessings including this work. The work presented in this thesis would not have been possible without my close association with many people who were always there when I needed them the most.

I am a mediocre person but I have always been privileged to have teachers of highest vision and compassion. Number of teachers were involved in my academic career who provide me intellectual and moral attention to me as a teacher.Although learning is an endless process but formally it is now reaching its destination through Prof. Afaq Ahmad. Words are indeed poor slaves to pen down about Prof. Afaq Ahmad, a true technocrat, great motivator, always available literally 24×7 except for few hours in the night. The academic freedom He provide is his way of encouragement that has always raised my enthusiasm and confidence. Out of gratitude I can only say “Thank you, Afaq Sir”

I am highly grateful to Prof. Mohammad Shakir, Chairman, Department of Chemistry, Aligarh Muslim University, Aligarh, for his constant support and for providing research facilities to carry out experimental work in the department.

I would like to thank University Sophisticated Instrument Facility (USIF), Instrumentation centre for providing research facilities. I would also like to thank SAIF, Punjab University Chandigarh for providing ¹H NMR and ESI-Mass spectra.

I am indebted to several faculty members at our department for their assistance and cooperation during the course of my research tenure, especially Prof. Sartaj Tabassum, Prof. M. Muneer, Prof. Rafiuddin, Prof. Riyazuddin, Prof. Abdul Rauf, Prof. Shamsuzzaman, Dr. Md. Shahid Nayeem, Prof. Nafisur Rahman, and Prof. (Mrs.) Farrukh Arjmand, Dr. Aminul Islam, Dr Ishaat Mohamaad Khan, Dr. Mohammad Zain Khan, Dr. Mohd. Shahid Dr. Md. Palashuddin Sk and Noorul Hasan Bhai for their immense help and encouragement.

I am virtually speechless to pay words of gratitude towards my parents namely Mrs. Mukhtari begum (mother) and Mr. Majid Ali Khan (father), what should I write, I have come to a conclusion that enough words have not been coined yet to express true feeling of children towards parents and vice-versa. Out of this whole episode if any one took to brunt it was my family; my brothers Mr. Mohammad Haroon Khan, Mr. Wajid Ali Khan and Mr. Abdullah Aamir Khan and sisters Mrs. Fatima Khan, Miss. Salma Khan and Miss. Nargis Khan

In AMU I have been blessed with a friendly and cheerful group of fellow students namely Dr. Neelam Singh, Mr. Lal Miyan, Mr. Ziya Afroz, Miss. Faria Khatoon Naqvi, Mrs. Sumbul Qamar, Khhkashan Alum and Sonam Shakya. That have opportunity to work them. They provided a friendly and cooperative atmosphere at work and also useful feedback and insightful comments on my work.

Beyond academics, beyond family and relatives, and the moments one is pissed off there are friends (har friend zaroori hota hai). How fortunate I am I possess friends like Mr. Arsalan Parvez, Dr Jahngeer Chouhan, Dr. Ziaul Haque Dr. Rami Khan, Dr. Urfi Ishrat, Mr. Azfar Shaida, Dr. Zoya Zaheer, Mrs. Subhi Hasan,

Mr. Mohd. Shoeb, Dr. Shariqe Imaz. Mr. Mohammad Umair, Mr. Mohammad Sarver, Mr. Syed Saud Ali and Mr. Mohammad Shoeb for their constant help in carrying out experimental works and finalizing the report.

Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the thesis.

It will be incomplete if I will not mention my ideals namely Sir Syed Ahmad Khan, Mohandas Karamchand Gandhi (rashtrapita) and Maulana Sayyid Abul Kalam Ghulam Muhiyuddin Ahmed Azad.

I gratefully acknowledge to award of fellowship by U.G.C. Government of India New Delhi for the financial support during my research period.

Above all I am thankful to Allah, the ultimate source of knowledge; with his blessings in completing this thesis.

Zulkarnain

The charge transfer interaction between (i) p-nitroaniline (PNA) and chloranilic acid (CAA), (ii) 2-aminopyrimidine (AP) with chloranilic acid (CAA) (iii) 2-amino-4-methylthiazole (AMT) and chloranilic acid (CAA) was studied spectrophotometrically in methanol and acetonitrile at different temperatures within the range 298-328 K. This experimental work explores the nature of charge-transfer interactions that play a significant role in chemistry and biology.

The CT complexes act as intermediate in wide variety of reactions involving electron rich and electron deficient molecules. An organic molecular complexes consist of constituent held together by weak forces of the donor-acceptor interaction or through hydrogen bond. These molecular interaction with formation of intensely colored charge transfer complexes accompanying with absorption of radiation in UV-Visible region. The spectral analysis of the CT complex is indicating that N and NH2 groups are playing a major role in determining the orientation in the reaction mechanism. Structure of synthesized charge transfer (CT) complex was investigated by different technique such as X-ray crystallography, FTIR, 1HNMR, ESI−MS, Elemental Analyses, UV-visible spectroscopy, XRD and TGA-DTA, which indicates the presence of N+-H---O- bond between donor and acceptor moieties. Spectrophotometric studies of CT complexes were carried out in methanol at different temperatures to estimate thermodynamic parameters such as formation constant (KCT), molar absorptivity ( εCT), free energy change (ΔG), enthalpy change (ΔH), resonance energy (RN), oscillator strength (f), transition dipole moment (μEN) and interaction energy (ECT) were also calculated.

The effect of temperatures on all the parameters was studied in solvent. 1:1 stoichiometric of CT-complex was ascertained by Benesi-Hildebrand plots giving straight line, which are good agreement with other analysis. Synthesized CT complex was screened for its antimicrobial activity such as antibacterial activity against two gram-positive bacteria, Staphylococcus aureus and bacillus subtilis and two gram negative bacteria Escherichia coli and pseudomonas aeruginosa, and antifungal activity against fungi Fusarium oxysporum, and Aspergillus flavus. . The fluorescence study shows that Ct-DNA interacted with CT-complex and quenched its intrinsic fluorescence in a static quenching process. The results of CD and UV–Vis spectroscopy showed that the binding of CT-complex to Ct-DNA induced conformational changes in Ct-DNA. Furthermore, the drug induces detectable changes in its viscosity. Stern–Volmer equation was used to determine the binding ability of the CT complex with in vitro calf thymus DNA.

ABSTRACT

C O N T E N T S

CHAPTER−1 Page No

GENERAL INTRODUCTION 2-42

1.1 Introduction 2-4

1.2 Historical Background of Charge Transfer Complexes 4-6

1.3 Mulliken’s Theory 6-9

1.4 Dewar’s Theory 10-13

1.5 Nature of Charge Transfer among Different Molecules 13-17

1.6 Proton Interaction between Acceptor and Donor Molecule. 17-21

1.7 Spectrophotometric Studied of Charge Transfer Complexes. 22-24

1.8 Determination of Formation Constant and Molar Extinction Coefficient.

24-27

1.9 Study of Charge Transfer Complexes using Infrared Spectroscopy.

28-30

1.10 Application of Charge Transfer Complexes 30-32

References 33-42

CHAPTER−2 43-70

Synthesis of Charge Transfer Complex of Chloranilic Acid as Acceptor with

p-nitroaniline as Donor: Crystallographic, UV-visible Spectrophotometric and

Antimicrobial Studies

2.1 Introduction 44-45

2.2 Experimental Study 45

2.2.1 Material and Methods 45

2.2.2 Synthesis of (CT) Complex 46

2.2.3 Synthesis of Single Crystal 46

2.2.4 Preparation of standard stock solution 46

2.2.5 Biological Evaluation 47-48

2.2.5.1 Antibacterial activity 47

2.2.5.2 Antifungal activity 47-48

2.3 Results and Discussion 48-65

2.3.1 Observation of Electronic Spectra 48-51

2.3.2. Determination of transition energy 51

2.3.3 Ionization Potential of the Donor 51

2.3.4 Determination of Resonance Energy (RN) and Free Energy (ΔG°)

52-53

2.3.5 Determination of Oscillator strength (ƒ) and Transition Dipole

Moment (μEN) 53-54

2.3.6. Determination of entropy change (ΔS) and enthalpy change (ΔH)

54-55

2.3.7 Comparative study of FTIR spectra of CT complex and reactants

55-57

2.3.8 1H-NMR spectrum 57-59

2.3.9 Powder X-ray diffraction studies of the CT complex. 59-61

2.3.10 Single Crystal Studies. 60-62

2.3.11. Thermal Stability 62-64

2.4 Pharmacology 64-66

2.4.1 Antibacterial activities. 64-65

2.4.2 Antifungal activity. 66

2.5 References 67-70

CHAPTER−3 71-119

Synthesis, Single-Crystal, DNA Interaction, Spectrophotometric and

Spectroscopic Characterization of the Hydrogen-Bonded Charge Transfer

Complex of 2-Aminopyrimidine with π-Acceptor Chloranilic Acid at different

Temperature in Acetonitrile

3.1 Introduction 72-73

3.2 Experimental Study 73

3.2.1 Material and Methods 73-74

3.2.2 Synthesis of Solid (CT) Complex 74

3.2.3 Single Crystal Growth 74

3.2.4 Preparation of Standard Stock Solutions 74-75

3.2.5 Instrumental Measurements 75

3.2.5.1 Electronic Spectra 75

3.2.5.2 FTIR Spectra 75

3.2.5.3 TG/DTA Analysis 75

3.2.5.4 1H NMR Spectra. 75

3.2.5.5 X−ray Single Crystal Measurement 76

3.2.5.6 Mass Spectra 76

3.2.5.7 DNA Binding Studies 76

3.2.5.7.1. Sample Preparation for DNA Binding Studies 76

3.2.5.7.2 UV−Visible Spectroscopy. 77

3.2.5.7.3 Fluorescence Studies 77

3.2.5.7.4. Circular Dichroism (CD) Measurments 78

3.2.5.7.5 Relative viscosity measurements 78

3. 3 Results and discussion 79

3.3.1 Observation of CT Electronic Spectra 79-83

3.3.2 Determination of Ionization Potentials of the Donor (ID) 83

3.3.3 Determination of Resonance Energy (RN) 83-84

3.3.4 Determination of Oscillator Strength (f ) and Transition Dipole

Moment (µEN) 84

3.3.5 Determination of Thermodynamics Parameters of the CT Complex

85-87

3.3.6 FTIR Spectral Study 87-89

3.3.7 1H NMR Spectra 90-91

3.3.8 ESI−Mass Spectra 91-94

3.3.9 X˗ray Crystallographic Study of the (AP: CLA) CT Complex 95-102

3.3.10 TG/DTA Analysis 102-104

3.3.11 DNA Binding Studies 105

3.3.11.1 Absorption Spectral Titrations 105

3.3.11.2 Steady State Fluorescence 105-110

3.3.11.3 Binding Parameter of Charge Transfer Complex with Ct-DNA

110

3.3.11.4 Ethidium Bromide Displacement Assay 111

3.3.11.5 Circular Dichroism (CD) studies 112

3.3.11.6 Relative Viscosity studies 112-113

3.4 References 114-119

CHAPTER−IV 120-155

Synthesis, Spectral, UV–visible, DNA interaction and Thermodynamic Studies

of the Hydrogen-Bonded Charge-Transfer Complex between 2-Amino-4-

methylthiazole and Chloranilic Acid at different Temperatures

4.1. Introduction 121-112

4.2. Experimental Studies. 122-125

4.2.1 Chemical and Reagents 122

4.2.2 Synthesis of Solid CT Complex 122-123

4.2.3 Preparation of Standard Stock Solutions 123

4.2.4 Instrumental Measurements 123

4.2.4.1 Electronic Spectra 123

4.2.4.2 FTIR Spectra 123

4.2.4.3 TG/DTA Analysis 124

2.5.4 1H NMR Spectra 124

4. 2.5.6 Mass Spectra 124

4. 2.4.7 DNA Binding Studies 124

4. 2.4.7.1 UV–Visible Spectroscopy 124-125

4. 2.4.7.2 Fluorescence Studies 125

4. 3 Results and discussion 125-150

4.3.1 Observation of CT Spectra 125-130

4.3.3 Determination of Resonance Energy (RN) 130-131

4. 3.2 Determination of Ionization Potentials of the Donor (ID) 131

4.3.4 Determination of Oscillator Strength (f) and Transition Dipole

Moment (µEN) 131-132

4.3.5 Determination of Thermodynamic Parameters of CT-Complex.

132-135

4.3.7 FTIR Spectral Study 134-136

4.3.8 1H NMR Spectra 136-138

4.3.9 ESI-Mass Spectra 138-140

4.3.11 TG/DTA Analysis 141-144

4.3.10 DNA binding studies 144-147

4.3.10.1 Steady state fluorescence 148-150

4.3.10.3 Binding parameter of charge transfer complex with Ct-DNA

149

4.3.10.4 Ethidium bromide displacement assay 149-150

4.4. References 151-155

Conclusion. 156-158

Appendix 159

Vita 160

List of Abbreviations,

UV

D

A

CT

HOMO

LUMO

MLCT

LMCT

NMR

DNA

RNA

EDA

Kf

ε

CAA

XRD

FTIR

TG/DTA

PNA

Ultra Violet

Donor

Acceptor

Charge Transfer

Highest Occupied Molecular Orbital

Lowest Occupied Molecular Orbital

Metal to Ligand Charge Transfer

Ligand to Metal Charge Transfer

Nuclear Magnetic Resonance

Deoxyribonucleic acid

Ribonucleic acid

Electron Donor Acceptor

Formation Constant

Molar Absorptivity

Chloranilic acid

Powder X-Ray diffraction

Fourier Transform Infrared

Thermogravimetric analysis/ Differential

thermal analysis

p-nitroaniline

2-Aminopyrimidine

AP

Ct

ESI−MS

TMS

EB

CD

ID

RN

AMT

AP

Calf thymus

Electrospray Ionization Mass Spectrometry

Tetramethylsilane

Ethidium Bromide

Circular Dichroism

Ionization Potentials

Resonance Energy

2-Amino-4-methylthiazole

2-Aminopyrimidine

1

Chapter -1

Introduction

2

1.1 INTRODUCTION

It was the year of 1940 when Benesi and Hildebrand [1] observed two absorption band in the

region of 500 nm and 297 nm (UV- Visible) region that could neither be attributed to the

benzene monomer nor to the iodine molecule. He observed that change in color of the

solution when iodine complexes with benzene, initially it was considered that the color

change of the iodine molecule was due to change in molecular weight [2]. Bachmann explains

the color of I2-molecule in different solvent depend upon the polarity of the solvent [3]. Such

absorption of radiation is due to transfer of charge through electron i.e. charge transfer

transition (CT-transition) [4]. The source molecule from which charge is transferred termed

as a donor (benzene) (D) and receiving molecule as an acceptor (iodine molecule) (A) [5].

Various experimental studies had been done to explore I2 – benzene interaction[6-14].After

certain interval of time Mulliken proposed resonance model of charge transfer based on

valence bond approximation to describe the interaction between donor and acceptor moieties

to form a relatively stable intermediate charge transfer complex (CT-Complex)[15], Dewar

(1961) considered the charge transfer complex as a hybrid resonating between polar (charged

hybrid I) structure and non-polar (uncharged hybrid II) structure [16].

A B A+ B-

(I) (II)

He proposed molecular orbital (MO) model to explain charge transfer complexes [17]. Where

the transfer of charge takes place between the highest occupied molecular orbital (HOMO)

of the donor and the lowest unoccupied molecular orbital. (LUMO) of the acceptor. Murrel

considered donor-acceptor interaction is too strong, presented a very sophisticated MO

3

perturbation treatment of charge transfer interactions to calculate the energy as the total of

the contributions from different types of interactions (Van der Waals, electrostatic etc.) [18,

19]. Flurry studied these complexes as a new molecule whose wave function is a linear

combination of the HOMO and LUMO from the donor and the acceptor respectively [20].

Moseley considered these complexes as a “supermolecule “made up by the donor and

acceptor molecules [20].Foster and his coworkers advanced the extensive development of

charge transfer interaction to form CT-Complex, considered the formation of excimer [22,

23]. Molecular interaction between electron donor and acceptor molecules play a vital role

in the diverse field of science such as protein folding, drugs design, pharmaceutical, material

science, biosensor [24-34]. This interaction governs physiosorption in Vander-wall system

and control self-assembly and self-organization process as in supramolecular systems [35,

36]. The present work focused on two closely related 1:1 CT-Complexes between acceptor

with different donor molecules, namely Chloranilic acid as an acceptor and donor moieties

namely 2-aminopyrimidine, p-nitroaniline and 2-amino-4-methylthiazole. Chemical

reorganization in acceptor component of the complex is expected to produce fascinating

consequences on the crystal structure stabilized in the semicrystalline state which is caused

by weak interaction. Hydrogen bonding play an interesting role in the formation of relatively

stable complex [37]. Other molecular interaction also provide extra stability to CT-Complex

through electrostatic interaction (dipole-dipole interaction and dipole-induced dipole

interaction) and through the back donation of electron density between the molecules. [38,

39]. The interaction between electronegative and electropositive species are characterized by

spectrophotometric technique. The excitation of these transitions occurs mainly in UV-

Visible region in the electromagnetic spectrum. On the recording, the spectra of CT-Complex

new absorption maxima are observed that are not attributed to any one of the two

components. It is assumed that formation of CT-Complex has been accomplished between

4

acceptor and donor molecule. Qualitatively change in color after mixing of two solutions

between acceptor and donor molecule should be possible.

1.2 HISTORICAL BACKGROUND OF CHARGE TRANSFER COMPLEXES

A number of reviews have appeared related to the formation of stable molecular complexes

from acceptor and donor moieties. The numerous efforts had been done to explain nature of

interaction among the CT-Complex. The earliest example of CT-Complex is crystalline

picrate resulted from the association of aromatic hydrocarbon and picric acid, which was

synthesized by Fritzsche [40]. After synthesis of such complex large number of CT-

complexes were isolated with different acceptor and donor molecules, such as hydrocarbon

and aromatic hydrocarbon (donor) and nitroaromatics, phenols, amines, quinines,

nitrobenzene etc. (acceptor). Other substituted hydrocarbons and aromatic hydrocarbons has

attracted also a great interest to charge transfer scientists, examples of complexes are (i)

substituted hydrocarbon and Trinitrobenzene donor and acceptor respectively (ii) aromatic

amines and nitroaromatics are also a considerable examples which can involve in the

formation of CT-Complex [41-43]. The stability of CT-Complex were explained by Buehler

and his co-workers considering that it increases with electronegativity of acceptor molecule

(2, 4-dinitrobenzene) [44]. Many scientific activities had been done to explain bonding

between acceptor and donor molecule. The crystal structure of anthracene-trinitrobenzene

and fluorine-trinitrobenzene found to be lying in the parallel plane [45]. Current bonding

theories did not meet this assumption and give an alternative explanation. Bennett and his

coworkers considered that electron donor and acceptor were components in an ordinary

complex [46-48]. Moore and his coworker proposed covalent type linking between

nitroaromatic (acceptor) and hydrocarbon (donor) component [49]. Hammiick and Gibson

5

proposed polarization mechanism to the emergence of CT-complex between aromatic amines

and nitro compound. The complex formed between hydrocarbon-nitroaromatic results from

dipole-induced dipole interaction, which stabilizes the complex [50, 51]. But all these

interactions does not provide any clarification to color of CT-Complex on mixing of acceptor

and donor moieties. Weitz and Weiss gave an idea of interaction among the acceptor and

donor molecule. Donor molecule having low ionization potential and acceptor with high

electron affinity formed stable dipolar ion A+D- type complex [52-54]. Stabilization of

complex was explain in term of complete transfer of electron from donor to acceptor

molecule. Weiss model successfully explained color of CT-Complex by considering lower

transition energy of complex as compare to neutral acceptor and donor molecule respectively,

from which rate of formation constant, intermolecular distance and dipole moment could also

be calculated. This theory actually predicted the nature of charge transfer phenomenon.

Mulliken put forward his interpretation of color in charge transfer by considering iodine

molecule dissolve in various organic solvents [55]. Lachmann classified iodine solution into

two category brown and violet [56]. Iodine with saturated solvents show violet color while

with unsaturated solvent give brown solution. Brown solution of iodine were considered to

be in equilibrium with adduct solution. It is considered that Iodine was chemically bound

with pure solvent and that solvent acted as an electron donor [57]. Fairbrother explain that

brown solution of Iodine had non-zero dipole moment which was an additional evidence for

solvent interaction with Iodine molecule [58]. Brakhmanne proposed resonance model

between “no-bond and a bonded” structure [59]. Benesi and Hildebrand explore his vision to

explain color spectrum of hydrocarbon-iodine solution in visible region. He also found

absorption peak in ultraviolet region that could neither be assigned any of two components.

This was attributed to formation of new CT-Complex between acceptor and donor moieties.

6

Later Mulliken formulated a quantum mechanical model to characterize formation of CT-

Complex.

1.3 M

The characteristic properties of formed CT-complex were explained by many scientists

before Mulliken, but Mulliken provided the more acceptable concept of charge transfer based

on quantum mechanical treatment. He also calculated spectral intensities, provide selection

rule for spectral transition, and explained hyperconjugation in organic molecules [60-63].

Mulliken (1925) considered diatomic molecule to show two type of multiplicity in band

spectra. In the year of 1927 Mulliken and Friedrich Hund described electron through

mathematical function delocalizes over an entire molecule. In 1934, he explore his concept

and calculated average ionization potential and electron affinity of various organic

molecules. In 1952, he explained the interaction between acceptor (Lewis acid) and donor

(Lewis base).

In the year of 1960, he explained spectra of various diatomic or monoatomic molecules such

as dihydrogen, dinitrogen, and helium. Mulliken (1950) calculated various physical

parameter by considering his charge transfer theory [64]. The general valence bond

description of CT complexes was developed by Mulliken. The 1:1 complex is considered to

be formed by the weak interaction of an electron donor (D) and an electron acceptor (A). The

complex Molecule D+—A− is formed by the transfer of electrons from a donor to the acceptor

molecule in a singlet state with wave function ψ1 = ψ (D+—A−).

Similarly, singlet wave function of unassociated molecules is given as ψ0 = ψ (D, A), which

includes no intermolecular terms. From the above definitions, the true wave functions of the

ground and excited states of the CT complex can be represented as.

7

ΨN (DA) = a ψ0 + b ψ1 (1)

ΨE (DA) = a* ψ0 − b* ψ1 (2)

Where D refers to the donor and A the acceptor. The ΨN and ΨE are the wave functions of

the ground and excited state in which the electron transfer from D to the A molecule. Ψ0 and

ψ1 are regarded as basic functions with which the system is described. The relation a*˃˃b*

is the necessary for the orthogonality of wave functions ΨE and ΨN. This implies that the

ground state is primarily of "no-bond" character, while the excited state has predominantly

dative characteristics. The eigenvalues associated with the above equations are obtained by

solving the secular determinant of the form was obtained.

Where E0 = ∫ ψ0 H ψ0 dr

E1 = ∫ ψ1 H ψ1 dr

H01 = ∫ ψ0 H ψ1 dr

H10 = ∫ ψ1 H ψ0 dr

S01 = ∫ ψ0 ψ1 dr

S10 = ∫ψ1ψ0 dr

The solution of the above secular determinant gives a quadratic equation in W with two roots.

One of these roots corresponds to the ground-state energy

WN = E0 − (H01− S01E0)2

(E1−E0) (4)

8

Whereas the second root yields the excited-state energy

WE = E1 +(H01− S01E1)2

(E1−E0) (5)

The approximate results of this procedure can be used to obtain a ratio of the coefficients,

b/a, by the application of second-order perturbation theory [65].

𝑏

𝑎 = −

(H01− S01E0)

(E1−E0) (6)

𝑏∗

𝑎∗ = −(H01− S01E1)

(E1−E0) (7)

The importance of this equation has been discussed in detail by Mulliken [66]. If ψ0 and ψ1

are not of the same symmetry then the integrals H01 and S01 both vanish, making b/a = 0,

meaning that b = 0, or in other words, there is no interaction. The above equations suggests

that the complex is stabilized for small energy separations (El – E0) and for large values of

(H01 – S01E0). This large value increases with increasing the value of S01 which was

formulated by Mulliken and Person.

S01 = √2SDA (1 + SDA2)1/2 (8)

Where SDA = ∫ ψD ψA dr, which is the overlap integral between the highest occupied level in

the donor and the lowest empty orbital of the acceptor. The higher value of SDA stabilized the

maximum stability of the ground state of the complex. This was not a complete requirement

for the stability of the complex; however, since steric hindrance, strong localized interactions

or hydrogen bonding may cause SAD to be other than a maximum. The absorption band

known as the CT band arises from a transition ΨN→ΨE. This transition has been obtained

from the energy difference of equations (4) and (5) which is given as:

hνCT = WE − WN = (E1 − E0) + [(H01− S01E0)2+ (H01− S01E1)2]

(E1−E0) (9)

9

The energies E1 and E0 can be obtained from ground state energy E0 = E∞ − G0 and excited

state energy E1 = E∞ + ID – EA – G1.

E1 – E0 = (E∞ + ID –EA – G1) − (E∞ − G0) (10)

E1 − E0 = ID − EA − G1 + G0 (11)

Where E∞ is the energy at infinite separation of A and D, ID is the vertical ionization potential

of the donor, EA is the vertical electron affinity of the acceptor, G0 is the sum of all

electrostatic energies of A and D in the ground state and G1 is the electrostatic energy of D+

and A− .

The energy of the CT complex can be obtained by substituting the value of E1 – E0 in equation

(9) we get

hνCT = ID − EA − G1 + G0 + [(H01− S01E0)2+ (H01− S01E1)2]

ID−EA−G1+G0 (12)

Which is the desired form expressing the functional dependencies upon ID and EA. Mulliken

introduced an explanation as to the possible nature of the terms G0 and G1 values. He

suggested that G0 was a non-bonding stabilization term which largely consisted of

contributions from London dispersion forces.

The G1 was a term which had to include major contributions from coulombic and exchange

forces due to the nature of the excited state.

The equation (11) can be simplified to become.

hνCT = ID – EA + W (13)

Where W is approximately constant for a series of complexes with a given acceptor (donor).

Mullikan’s theory is consequently seen to explain some of the observed properties of the CT

absorption band.

10

1.4 D

The interaction of charge transfer complex between donor and acceptor can also be explained

in term of molecular orbital treatment in which CT-Complex formed due to interaction

between highest occupied molecular orbital (HOMO) of donor component to lowest

unoccupied

Fig. 1.1 An energy-level diagram explaining the formation of a charge-transfer complex and occurrence of an

intermolecular charge-transfer transition in terms of interaction between molecular orbitals of the two

components. D, an electron donor; A, an electron acceptor.

11

Molecular orbital (LUMO) of acceptor component [67-69]. Since these interactions are very

weak, which can be considered simply by perturbation theory [16]. There is no change in

total amount of energy on interaction between occupied molecular orbital of donor and

acceptor moieties respectively. On the other hand interaction of occupied molecular orbital

of donor molecule to the unoccupied orbital of acceptor (a*) molecule leads to transition of

negative charge from donor to acceptor molecule and stabilizes the system.

Simultaneously transfer of charge in opposite directions also possible from filled orbital of

acceptor to unoccupied orbital of donor (d*). The extent of interaction is inversely

proportional to the difference in energy between participating molecular orbital and it is

directly proportional to square of overlap integral between two molecular orbital. Fig. 1.1

illustrated schematically represent highest occupied orbital of donor (D) ϕ+1(D) and lowest

unoccupied orbital of acceptor molecule represented as ϕ-1(A). By the interaction between two

orbitals, ϕ+1(D) lowered their energy by X amount and produced perturbed orbital ϕp+1(D).

Simultaneously ϕ-1(A) increase their energy by the amount of X. gave rise to perturbed orbital

ϕp-1(A). The perturbed orbital can be represented as.

ϕp+1(D) = N (ϕ+1(D) + C ϕ-1(A)) (14)

ϕp-1(A) = N (ϕ-1(A) - C ϕ+1(D)) (15)

The coefficient “C” is much smaller then unity. And hence perturbation theory is

approximately expressed as,

∁ ≑ −𝐻 [𝜀−1(𝐴) − 𝜀+1(𝐷)]⁄ (16)

Where H represent perturbation operator between ϕ+1(D) and ϕ-1(A), and ε+1(D) ε-1(A) represents

energies of ϕ+1(D) and ϕ-1(A respectively the energy X is given by

12

𝑋 ≑ −𝐻2 ⁄ [𝜀−1(𝐴) − 𝜀+1(𝐷)] (17)

Here H is approximately proportional to overlap integral between interacting molecular

orbitals. The promotion of an electron from HOMO of donor molecule (ϕ+1(D)) to LUMO of

acceptor molecule (ϕ-1(A) ) accompanied partial charge transfer from Donor to Acceptor

molecule known as intermolecular charge transfer transition. This is due to a linear

combination of molecular orbital of donor and acceptor.

∆𝐸 = 𝜀−1(𝐴) − 𝜀+1(𝐷) + 2𝑋 = 𝐼𝐷 − 𝐴𝐴 + 2𝑋 (18)

Where ID and AA are ionization potential of donor and electron affinity of acceptor molecule

respectively. The ionization energy of donor molecule is equal to a highest occupied

molecular orbital of donor i.e. ε+1(D) and electron affinity of acceptor molecule is equal to

LUMO of acceptor i.e. ε-1(A), for the validation of above energies of charge transfer transition

predicted from molecular orbital theory. Instead of this transition, other multiple transitions

are also possible which is arise due to the transfer of an electron from HOMO of the acceptor

(ϕ+1(A)) to LUMO of donor molecule (ϕ-1(D)). A number of cases where more than one CT-

Band were observed in the spectrum of charge transfer complexes due to the formation of

new charge transfer complex the absorption band of an individual component is shifted from

their original position. Intermolecular transition in donor as well as in acceptor from (ϕ+1(D))

to (ϕ-1(D)) and (ϕ+1(A)) to (ϕ-1(A)) respectively which are locally excited also contribute to

additional intensity of the CT-transition.

Dewar explained number of cases in which such type of transition are possible. Few of them

are as follows halogen- aromatic compound, trans-stilbene -1,3,5-trinitrobenzene , N,N-

dimethylaniline-p-toluidine, chloroanil-hexamethylbenzene etc. He considered the CT-

Complex of Iodine-Benzene in which CT-band shifted to longer wavelength (lower

wavenumber) and the CT-band of chloroanil-hexamethylbenzene also shifted towards longer

13

wavelength (red shift) and increased intensity in both cases by a factor of 1.2 and 1.7

respectively under the pressure of 20,000-50,000 atm [70].

1.5 NATURE OF CHARGE TRANSFER AMONG DIFFERENT MOLECULES

Enormous inorganic and organic molecule undergo charge transfer transition by photo-redox

processes [71-73]. Inorganic coordination complexes show color via three processes (i) d-d

transition (ii) metal to ligand charge transfer (MLCT) (iii) ligand to metal charge transfer

(LMCT). Metal complexes show color spectrum in the ultraviolet-visible region. This color

is imparted due to d-d transition among the metal complexes in which promotion of electron

occurred from t2g-level to eg –level. These transitions appear as weakly intense on spectrum

because they are Laporte forbidden [74]. The color of transition metal complexes in solution

is affected by various parameter such as nature of metal ion, oxidation state of metal, nature

of the ligand and number of electrons present in d-orbital. In some cases transition metal

complexes imparted intense color in solution but they had no d-electron, This is due to the

distribution of electron metal and ligand molecules give rise to charge transfer bands [75].

Charge transfer phenomena gives intense absorption peak in UV-Visible region. Such

transition is Loporte allowed. The molar extinction value of such transition is around 50,000

Lmol-1cm-1 or greater than this value. Where as in d-d transition in octahedral complexes this

value is very small which nearly equal to 20 Lmol-1cm-1 or less than this, indicating weak

transition. A charge transfer transition involves internal oxidation-reduction processes [76,

77], if metal have empty or partially filled d-orbital and a ligand having filled σ, σ*, π, π*

and non-bonding. If ligand molecular orbital are fully filled by electrons, transfer of charge

occurred from filled ligand molecular orbital to empty or partially filled d-orbital of metal.

Such transition is known as ligand to metal change transfer (LMCT). Fig. 1.2 LMCT

14

transition produces intense band. Here d-d transition is also possible but it is very weak

transition. In LMCT metal undergoes reduction [78]. Metal to ligand charge transfer (MLCT)

also give rise to intense band if metal present in their lowest oxidation state and ligand having

empty low lying π* orbital (e.g. CO, CN- , NO+ etc.). Such ligand are known as π-accepting

ligand. These transition are common in metal carbonyl, metal nitrosyl and some coordination

compounds. Electron present in molecular orbital of metal get excited upon absorption of

radiation to empty orbital of the ligand.

The interaction between donor and acceptor species are explained on the basis of HOMO-

LUMO interaction. High-lying HOMO energy level of donor molecule interact with low-

lying LUMO energy level of acceptor molecule and form an intermediate copolymer which

is relatively stable. Formed intermediate have small band gap. Number of theories and

comprehensive surveys had been done previously [79-81].

Fig. 1.2 Ligand to Metal Charge Transfer (LMCT) involving an Octahedral d6 Complex.

15

Fig. 1.3 Metal to Ligand Charge Transfer involving an octahedral d5 –Complex.

But still it needs a great attention and explanation to charge transfer interaction. Most

accepted explanation in term of directional overlapping of highest occupied molecular orbital

of donor with lowest unoccupied molecular orbital creating bonding molecular orbital with

low energy and antibonding molecular orbital with high energy, respectively (Fig 1.4).

Fig. 1.4 Molecular energy levels.

16

Interaction between donor and acceptor molecules explained in term of electrostatic

attraction, stabilizes resultant CT-Complex. These coulombic interactions depend on

ionization potential of donor molecule and electron affinity of acceptor molecule.

Other than electrostatic forces Van der-Waall forces also play a vital role in deciding the

stability of CT-Complex. Various sigma and pi-donor have been studied with different

acceptor. Sigma donor are generally nitrogen, oxygen, phosphorous or halogen containing

molecule. These may be amines, ether, phosphines etc. These donor molecules may be

neutral or may be negatively charged (eg. Halogenide, cynide, and hydroxyl anions). π donor

are generally neutral having localized or delocalized multiple bond, electron donating

substituents enhance donating tendency of donor. Electron acceptor must contain vacant

atomic orbital having ability to expand their coordination polyhedra through coordinate or

dative bond or the molecule which have empty π* orbital also interact with filled orbital of

donor. These antibonding molecular orbitals associated with localized/ delocalized multiple

bond, electron withdrawing substituents (such as NO2, CN-, SO3, etc.) increase accepting

tendency of acceptor molecule. HOMO-LUMO energy gap describe stability of CT-Complex

formed HOMO-LUMO gap are small enough so that wavelength of U.V-Visible light can

excite electron bonding molecular orbital to antibonding molecular orbital. This can be

explained by UV-VIS spectrophotometer [82].

The HOMO-LUMO gap of various molecules have been studied through quantum-chemical

methods have been increasingly applied to predict the band gap of conjugated systems [83].

The HOMO and LUMO energy levels of the donor and acceptor components for various

complexes are very important parameter to determine whether effective charge transfer will

happen between donor and acceptor. The π- π* transition arises due to transfer of electron

17

from electron rich (donor) molecule to electron deficient (acceptor) molecule, observe in

aromatic and π-conjugated system [84, 85]. Example of donor moieties are generally

polycyclic aromatic hydrocarbon possess one of the heteroatom (N, O, S etc.) and phenol

derivatives [86, 87] such as 2-hydroxy-1, 4-napthaquinone, 1, 2-napthaquinone-4-sulphonic

acid, 2, 4-dimethyl-1, 10-phenanthroline [88]. All above donor molecule formed CT-

Complex with acceptor molecule such as Chloranilic acid and picric acid in different polar

solvent (CH3OH, CHCl3, CH3CN, CH2Cl2 etc.). The stoichiometry of donor acceptor is

always in the ratio of 1:1, which should not be changing during complexation. The acceptor

molecule are generally quinine, nitro/ carboxylic/ keto derivative of aromatic compound.

1.6 PROTON INTERACTION BETWEEN ACCEPTOR AND DONOR MOLECULE.

Hydrogen bonding plays crucial role in chemistry as well as in biology [89], which is

considered a relatively weak interaction [91]. Proton donor are those molecule which contain

ionisable proton. The hydrogen atom attached to more electronegative atom/groups is

assumed to be ionisable proton, like hydroxyl and amino groups. Acceptor molecules are

those which contain lone pair of electrons so that they can attract proton of donor.

Electronegative atom or electron withdrawing groups increase attracting tendency of

acceptor molecule [92]. These interaction is believed to be electrostatic in nature [93].

Hydrogen atom attached to high electronegative atom, attract covalent bonded electron pair

towards nucleus and away from hydrogen and create a partial positive charge at hydrogen

which is attracted by electron acceptor molecule leading to dipole- dipole interaction. These

interaction are weaker than covalent interaction but stronger than Van der-Waall interaction.

18

Due to these interactions boiling point of liquid is comparatively high. 8-Hydroxyquinoline

complexes with citric acid in different polar solvent such as ethanol, methanol, chloroform

etc. are good examples [94-95]. The proton transfer from citric acid to 8-Hydroxyquinoline

was confirmed by H1 –NMR [94 & 96]. The role of hydrogen bonding in biological system

is ubiquitous. It is important in organization of complementary chains of base pair in DNA

and RNA [97-98]. Kinetic study of proton transfer in various molecules in aqueous solution

were discussed by Eigen et al. in several publications [99-101]. By considering the salicylic

derivative molecule in which phenolic hydrogen involved in internal hydrogen bonding with

carboxylic group [102-104]. Strong base like OH- can attack only with rate constant well

below those of diffusion controlled process [105]. This analysis indicated that two step

mechanism of proton transfer firstly H-bond become loose followed by attack of base takes

place [106]. Magnitude of deviation from diffusion controlled rate is considered to be a

measure of the strength of intramolecular H-bond. Later, Eyring and co-workers described

alternate mechanism in which proton directly attached at bridging proton; breaking of H-

bond and proton transfer to the base accrued in concerted manner [107-109].

Recent research concluded that both the mechanism contribute to the overall reaction. The

influence of intramolecular H-bonding on proton transfer reaction can be understood by a

temperature-jump study of acid-base reaction [110]. The temperature-jump relaxation

technique has been used to study the kinetic of proton transfer between salicylic acid and

their derivatives (3, 5-dintrosalicylic acid, thiosalicylic acid etc.). The kinetic study of proton

transfer observed within the PH-range of 6.0 to 9.0. Investigation results showed that proton

transfer may take place directly or via hydrolytic/protolytic path way which depend on

concentration [111].

19

The value of rate constant for proton transfer from salicylate anion to the proton acceptors

NH, cacodylate and OH are relatively low than the diffusion controlled value, even this type

of reaction are thermodynamically favoured [112]. The low value of rate constant indicates

that proton engage in intramolecular H-bonding in salicylic acid. The extent of internal H-

bonding in thiosalicylic acid is much smaller than that of 3, 5-dinitrosalicylic acid [113].

Fig. 1.5 Intramolecular Hydrogen Bonding

Since electronegativity value of hydrogen is too smaller (2.2) and other alkali metal (Na= 0.9

and K= 0.8) are not polarizable, they are involved in purely electrostatic interaction and

possess very little covalent nature [114]. However hydrogen generally is polarized by high

electronegative atom such as fluorine, oxygen, and nitrogen and possess a dipole leading to

dipole-dipole interaction. These interaction are relatively stable which has significant bond

strength ranging from 9 KJ/mol to about 30 KJ/mol depending upon nature of

electronegativity of acceptor and donor atom. The value of bond enthalpies of certain H-bond

in vapour phase are given bellow [115-116].

20

Table 1.1

Quantum mechanical calculation done by D.R. Hartree in association with V. Fock and J.C.

Slater (Hatree-Fock (HF) Methods) revealed that water existed as a dimer in which O-H

bond length is slightly shifted. But Hatree-Fock (HF) Methods does not make good

prediction about bond strength [117-122]. PM3 Quantum-Mechanical Method shows charge

transfer from donor to acceptor molecule. This method predict that ammonia is a good

hydrogen bond acceptor and a poor hydrogen donor when interacting with neutral molecules.

Electronegativity differences between F, N, and O predict that donor strength follows the

order F > O > N and acceptor strength follows the order N > O > F. It is observed that

electronegativity differences, predicting the F–H‧‧‧N bond to be the strongest and the N–

H‧‧‧F bond the weakest. Pseudospin-electron model explain proton-electron charge transfer

in the complex through hydrogen bond. A series of experimental data and result of quantum

chemical calculations are in support of formation of proton-electron existence [123-129].

Type of H-bond

F–H‧‧‧F

O–H‧‧‧N

O–H‧‧‧O

N–H‧‧‧N

N–H‧‧‧O

HO–H‧‧‧OH3 +

Energy of bond in KJmol-1

161.50

29.00

21.00

13.00

8.00

18.00

21

It is evident that electrostatic interaction play decisive role in formation of charge transfer

complexes in solution as well as in solid state. Formation of organic charge-transfer complex

between tetrathiafulvlene (TTF) as a donor and 9H-fluorenone derivative as an acceptor: for

example 4,5,7-trinitro-9H-fluorene-9-one-carboxylic (C2TNF) complexes with

tetrathiafulvlene through classical H-bonding involving carboxylic group, these H-bonds

arrange acceptor as centrosymmetric dimer in the crystal[130]. This hydrogen bonding

confirmed by Infrared and Raman spectroscopy [131-132].

The design of non-covalent supramolecular assemblies strongly explained by hydrogen bond,

suggested that construction of large aggregates by combining smaller molecules [133]. These

rule state that six-membered intramolecular H-bond are generally unperturbed by presence

of other functional groups leading to formation of complex, indeed, quasi-ring formed

through intramolecular H-bond [134-135]. Imidazole-4, 5-dicarboxylic acid derivative (such

as Imidazole-4, 5-dicarboxamide (145DCs)) extensively studied which formed strong

intramolecular H-bonding in solution [136]. This interaction is confirmed by 1H-NMR,

chemical shift of amide proton observed at 11.5 and 8.5 ppm [137]. Imidazole-4, 5-

dicarboxylic acid itself shows crystallization properties with amino acid [138]. In similar

way, the field of crystal engineering had been developed [139].

22

1.7 SPECTROPHOTOMETRIC STUDIED OF CHARGE TRANSFER COMPLEXES.

A sensitive spectrophotometric method was employed to study the intermolecular charge

transition in charge transfer complexes [140-141]. Charge transfer complexes have unique

absorption bands in the ultraviolet-visible region. The molecular interactions between

electron donors and acceptors are generally associated with the formation of intensity colored

charge transfer complexes, which absorb radiation in the visible region [142]. An electronic

spectrum should consist one or more sharp peak, corresponding to promotion of electron

from one energy level to another energy level [144]. On the other hand organic complex have

band spectrum instead of sharp peak. This is because during excitation vibration as well as

rotation motion of the molecule are also be possible.

There are four major transition possible in UV-Visible region.

(i) σ→ σ* Highest E

(ii) n→ σ* 2nd Highest E

(iii) n→ π* Lowest E

(iv) π→ π* Also fairly low

The possibility of σ→σ* transition is generally observed in homo/hetero diatomic

molecules, saturated hydrocarbon, and other single bonded compounds such as dihydrogen,

methane etc. H2 absorb radiation having energy equal to HOMO-LUMO energy gap. ΔE

for this electronic transition is 1079 KJ/mol, corresponding to light with a wavelength of

111 nm. π→π* transition are generally possible in those compound which possess double

bond in their structural formula [143]. It has smaller HOMO-LUMO energy gap than that

23

of σ→ σ* transition and therefore they absorb radiation with smaller energy (ΔE of 464.4

KJ/mol) with longer wavelength radiation.

Fig. 1.5 Possible Electronic Transitions of π, σ, and n electrons in UV-Vis region

Hence UV-VIS spectroscopy becomes very functional tool for organic and biological

chemists for the study of molecules wich possess conjugated π system. π→π* transitions

generally shifted to lower energy or longer wavelength in polar solvents termed as

bathochromic shift (red-shift). The molecule having extended double bond absorbs radiation

in visible region rather than the UV region of the electromagnetic spectrum. Because the

HOMO-LUMO energy gap becomes so smaller in such molecules. The energy of

intermolecular charge transfer in solution is given by the equation [145].

𝐸𝐶𝑇 = N0h𝜈𝐶𝑇𝑚𝑎𝑥 = N0 h𝐶 𝜆𝐶𝑇𝑚𝑎𝑥⁄ (19)

24

Where 𝐸𝐶𝑇 transition energy for CT-Complex, N0 is Avogadro’s number, h is plank constant,

𝜈𝐶𝑇𝑚𝑎𝑥 is maximum frequency at which CT-transition is observed, C is speed of light in

vacuum and 𝜆𝐶𝑇𝑚𝑎𝑥 wavelength corresponding to maximum absorption [146],.

1.8 Determination of Formation Constant and Molar Extinction Coefficient

Several critical discussions regarding the formation of CT-Complexes are available. The

thermodynamic and spectroscopic properties of the formed CT-Complexes depend on donor

and acceptor concentration. The Benesi-Hildebrand (BH) graphical method (In 1949), for

the evaluation of formation constants and molar extinction coefficient for molecular

complexes by electronic spectroscopy was the first and peradventure the most popular

method. The BH equation does describe accurately the average properties of 1: 1 complexes,

in the absence of solvent competition. Benesi and Hildebrand applied this method to

calculate formation constant and molar extinction coefficient of 1:1 electron donor acceptor

(EDA) complex formed between aromatic hydrocarbon and iodine molecule using inert

solvent such carbontetrachloride and heptane[147]. The electronic spectra of donor (D),

acceptor (A) and the resulting CT-Complex (DA) measured in different solvents using UV-

Visible spectrophotometer in the range of 200-700nm. In deriving this equation, one should

assume that the activity coefficients for the species D, A and DA are all unity. The following

equation has been used for the determination of formation constant (Kƒ) and

molar absorptivity (ε) of DA association complex.

D + A [D‐ ‐ ‐ A]CT

25

Kf = [DA]CT

[D] [A] (20)

Where 𝐾𝑓 is formation constant, [𝐷𝐴]𝐶𝑇 [D] and [A] the concentration of CT-complex, donor

and acceptor components in mole-litre-1 at equilibrium position respectively.

Since the absorbance of CT-complex is explained by the Beer’s law.

Absorbance (A) = log 𝐼0

𝐼 = ε b [DA] (21)

Here ε is molar extinction coefficient of CT-complex, b is the path length which is equal to

1 centimetre, the value of absorbance increase with concentration of donor. The relation

depicted in Fig. 1.6.

Fig.1.6. the concentration of the complex as a function of donor concentration for a fixed concentration of

acceptor [A] 0. Region I: [AD] is approximately a linear function of donor [D]. Region III: saturation has been

reached, and [AD] is constant and equal to [A] 0.

26

The Benesi­Hildebrand analysis of Kf involves the measurement of the [D­­A]CT

absorbance

[A] as a function of varied [A] when [A]>>[D]. A plot of x = 1/[A]o vs y = [D]0/A gives an

in-tercept = 1/ε and slope = (1/Kfε) as defined by the Benesi­Hildebrand Equation.

[𝐷]0

𝐴=

1

𝐾𝐶𝑇 ∈𝐶𝑇.

1

[A]o+

1

∈𝐶𝑇 (22)

OR

[𝐴]0

𝐴=

1

𝐾𝐶𝑇 ∈𝐶𝑇.

1

[D]o+

1

∈𝐶𝑇

Where [D]o = total concentration of the donor (fixed)

A = CT absorption of DA complex at wavelength λ

[A]o = Total concentration of the acceptor (varied)

𝐾𝐶𝑇 = Equilibrium constant for DA complex formation

∈𝐶𝑇 = Molar absorptivity of DA complex at maximum wavelength

(λMAX)

Equation (20) is modified to equation (22) called modified Benesi­Hildebrand equation.

Kf = [DA]

[D][A]

As

[D]o = total D (uncomplexed and complexed) = [D] + [DA]

[A]o = total A (uncomplexed and complexed) = [A] + [DA]

Substituting these values in to the equation (22) we get

27

Kf = [DA]

{[D]0− [DA]}{[A]0−[DA]} (23)

If [A]o >> [D]o, then {[A]o − [DA]} ≈ [A]o,

So that

Kf = [DA]

{[D]0− [DA]}[A]0 (24)

Rearranging gives,

[DA] = Kf[A]0[D]0

1+Kf[A]0 (25)

CT absorbance by DA according to Beer's law is

Abs = ɛ l [DA] = ɛ l(Kf[A]0[D]0)

(1+Kf[A]0) (26)

Where, l = sample path length in cm (typical 1 cm). The value of Absorbance [A]

must increase as the concentration of [D] increase, which has been shown in Fig. 1.7

Fig. 1.7 Benesi–Hildebrand plots of charge transfer complex of PNA with CAA [A]o / A vs 1 / D in methanol.

28

1.9 STUDY OF CHARGE TRANSFER COMPLEXES USING INFRARED

SPECTROSCOPY

Infrared (IR) spectroscopy extensively used for the confirmation of structural changes when

reacting molecules are converted into product/adduct [148-149]. Absorption of radiation

possible in the range of 50-12500 cm-1 in electromagnetic spectrum. 400-4000 cm-1

frequency region is very important for organic molecule because large number of functional

groups absorb radiation in this region, 400-1250cm-1 region consider as fingerprint region

and 1250-4000cm-1 consider as a functional group region. Different functional group absorb

different radiation (i.e. no two compounds can have similar infrared spectra except

enantiomers). Infrared spectroscopy is a technique based on the vibrations of the atoms of a

molecule. The absorption of IR-radiation causes an excitation of molecule from lower to

higher vibrational level. Molecules which possess change in permanent dipole moment are

IR-active molecule. And the molecule which have zero dipole moment are IR-inactive. This

is the selection rule for IR-spectroscopy. The absorption energy in infrared-region depend

upon masses of linked atoms, strength of bonds and their geometric interrelationships. The

applications are overwhelmingly in the field of organic chemistry. Organic molecules

produce profuse spectra because of the large number of atoms involved. One of the changes

experienced by both donor and acceptor molecules in a CT-complex is a change in bond

lengths, which produces changes in the vibration spectra; thus infrared spectra can indicate

the formation of such complexes. Charge transfer arises basically in the excited state and

stabilizes to the ground state by electrostatic interaction between donor and acceptor

moieties. Hydrogen bonding also play diverse role in structural changes when reacting

molecule interact to each other.

29

On comparing IR-spectra of CT-Complex with donor and acceptor molecules, certain

changes are observed i.e. frequencies of donor and acceptor are slightly shifted. If interaction

between reacting molecules is weak then replicate IR-spectra of CT-Complex in both portion

are obtained. Initial peak of individual component is disturbed on complex formation, bond

order is also changed.

In general, the bands corresponding to the acceptor molecule are shifted to lower wave

number region in comparison to the donor molecule shifted to higher wave number region.

This decrease in energy of a particular bond indicate that charge transfer complex formation.

Example of such interaction are Chloranilic acid (CAA) as a acceptor complexes with

different donor molecule containing amino group (NH2), such as p-nitroaniline (PNA), 2-

aminopyrimidine, 2-amino-4-methylthiazole, 2-amino-4-methoxy-6-methyl pyrimidine etc.

the symmetric and asymmetric stretching vibration of amino group (NH2) appear at (3326,

3205 cm-1), (3399, 1465 cm-1) and (3216, 3075 cm-1) in IR-spectrum of donors, suggested

that Hydrogen of amino group does not involve in H-bonding with OH group of CAA[150-

151]. Whereas proton of CAA interacted by amino group and formed NH3+ which appear at

2900-2500 cm-1 , described in the next chapter. The σ-donor- σ-acceptor observed large

change in energy and intensity IR-spectrum than that of π-π interaction of CT-complexes.

New absorption band appear at lower energy region due to formation of complex through the

reacting molecules. The compound which are IR-inactive such as Cl2, Br2, and I2 may become

IR-active on complexation. Cl-Cl vibration band appear at 541 cm-1 in Raman spectrum while

in IR-spectrum it is observed at 513 cm-1 in benzene. IR spectroscopy also helpful to

determine association constant by applying Benesi-Hildebrand equation

[𝐴]0

𝐴 =

1

𝜀 + (

1

[𝐷]0) (

1

𝑘𝜀) (27)

30

The validity of this equation is only for those systems which is isolated at maximum peak

that should not be overlapping by other peaks. IR-Spectroscopy is very versatile technique

in distinguishing charge transfer phenomenon and hydrogen bonding.

1.10 APPLICATION OF CHARGE TRANSFER COMPLEXES.

Charge transfer interaction play a promising role in development of biosensor [152]. Golam

Faruque Khan (1996) developed a TTF: TCNQ based organic sensor by delineating a tree

shaped CTC crystal structure on a conductive polymer film such as poly(Sulphur nitride)

[(SN)x], tetracyano and tetraoxalato-platinates and Poly-paraphenylene on the surface of

the crystal glucose oxidase is adsorbed and it cross-linking with glutaraldehyde [153].

Graphene oxide (GO) holds great potential for optical biosensors, great attention has been

paid to the development of GO-based sensors for DNA, protein, ATP, duplex DNA

unwinding, glucose and metal ions. The near-IR fluorescence of GO as a label-free PCT-

based biosensor for dopamine (DA). The multiple noncovalent interactions between GO and

DA resulted in effective self-assembly of DA on the surface of graphene oxide [154]. The

DNA biosensor is able to detect target DNA in the subnanomolar range within 1 h. Charge

transfer interaction play an important role designing new drugs [155]. Charge transfer

complexes are also actively precipitated in various chemical reaction such as addition

substitution and condensation reaction [156, 157]. The complexes acquiring great attention

for non-linear optical material and electrical conductivities. Now a day’s organic material are

gaining great advantages over inorganic materials due to their low cost, high flexibility, large

area capability and easy processing [158], organic solar cell are the third generation solar

cell, they are good absorbent of light, large-area, inexpensive organic solar cells on flexible

31

substrates[159-161]. Organic photovoltaics (OPV) is a rapidly increasing new solar cell

technology [162]. Conductive organic polymers are of great interest for construction of

organic photovoltaic cell, on which absorption of light is possible followed by conversion

into electricity, poly (3,4-ethylendioxythiophene) (PEDOT) show a remarkable high

stability, high electrical conductivity and low optical band gaps[163, 164]. Several EDOT

based donor-acceptor conjugated polymer with small band gap have been reported [165]. In

solar cell separation of charge takes place under the influence of external electric field. The

donor–acceptor interaction play diverse role in physiological and pharmacological science

[166]. Plenty of CT-complex had been synthesized which are physiologically and

pharmacologically active to cure various disease [167]. Imidazole and its derivatives are

extensively used in this field. Imidazole and epiperazine derivatives observed to have

anticancer activity, the most active compound in this series. When cis-platin is considered as

reference drug [168]. Transfer of electrons between protein molecules play an essential role

in biological energy production. Ethyl carbamate (EC) is a multi-step genotoxic carcinogen

of wide spread occurrence in fermented food and alcoholic beverages. Charge transfer

complexes of ethyl carbamate with various acceptors were synthesized and screened for their

antifungal and antibacterial activity [169]. Charge transfer complex of EC-Quinol exhibited

strong antimicrobial activities against various bacterial and fungal strains compared with

standard drugs. Intermolecular charge transfer complex can exhibit various absorption band,

implying a possibility of use of these molecules as a pigment and dyes. The study of such

interaction between drug and DNA is one of the most important aspect in biological

investigation aimed at discovering and developing a new type of antiproliferative agent [170],

since DNA is the molecular target in the design of anticancer compounds [171-173]. Charge

transfer complex of o-phenylenediamine with 3, 5-dinitrosaycylic acid exhibited

antimicrobial activity and interacted with DNA which may be used in developing a variety

32

of neoplastic drugs [174]. Recent significant attempts had been made to the synthesis and

studies of supramolecular donor–acceptor systems in relation to their possible use as

components of molecular electronic devices for solar energy conversion and sensor and

catalytic applications [175, 176]. Supramolecular CT complexes between bis (18-crown-6)

stilbene and dipyridylethylene derivatives act as fluorescence sensors for alkaline-earth metal

ions [177]. In view of these, the studies selected for the studies is justified and significant as

the CT-complex formed are biologically active molecules.

33

REFERENCES.

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Chapter -2

Synthesis of Charge Transfer Complex of Chloranilic Acid

as Acceptor with p-nitroaniline as Donor: Crystallographic,

UV-visible Spectrophotometric and Antimicrobial Studies

44

2. 1 INTRODUCTION

Charge transfer (CT) complexes have been studied exclusively due to their wide applications

in various fields such as biosensor [1], organic semiconductor [2], optoelectronics, optical

communication [3-4], photo catalyst [5], dendrimer, [6]. Some of the CT-Complexes also

show electrical properties [7-10] and their applications in the field of electronics and in

organic solar cell [11-12] CT-Complexation mechanism has currently achieved a great

importance in biological systems in the field of DNA binding, antibacterial, antimicrobial,

antifungal, antitumor, anti-inflammatory, and drug receptor binding mechanism [13].

Charge transfer mechanism originated from weak molecular interaction between donor and

acceptor moieties [14-17]. This weak interaction was first reported by Matsunaga and his

coworkers [18] and proposed proton transfer interaction. Further Pauling proposed that

charge transfer phenomenon is possible due to hydrogen bonding [19], Atkins showed

dipole-dipole interaction among donor and acceptor molecules [20]. Chloranilic acid is

widely used in the analysis of various drugs showing π-accepting tendency in charge transfer

complexation reactions [21-23]. Nitrogen containing compounds are special type of σ-donor

or π-donor [24]. The molecular interaction between pharmaceutical compounds and electron

accepting reagents are generally associated with the formation of intensely colored CT-

complexes, which usually absorb radiation in the visible region. Considerable attention has

recently been devoted to the formation of stable CT complexes at various temperatures.

Effect of solvent polarity on spectroscopic and thermodynamic properties of CT-Complexes

of various acceptor and donor has been widely investigated [25-27].

45

The aim of these present study is to synthesize new CT complex of donor and acceptor and

to describe the stability of CT-Complex at various temperatures using UV–visible

spectroscopy and Benesi-Hildebrand equation. The properties of CT complexes have been

investigated in methanol at various temperatures in terms of various important

thermodynamic parameters such as formation constant (KCT), molar absorptivity (εCT), free

energy change (ΔG), enthalpy change (ΔH), resonance energy (RN), oscillator strength (f),

transition dipole moment (μEN) and interaction energy (ECT). The molecular structure, charge

transfer interactions and thermal stability of CT complex have been investigated by various

technique such as 1H-NMR, FTIR, UV–Vis spectra, powder-XRD, X-ray crystallography

and TG/DTA thermal analyses. One of the aims of this paper is also to investigate the

antimicrobial activity of synthesized CT Complex.

2.2 EXPERIMENTAL DETAILS

2.2.1 Materials and methods

All the required chemicals used were chemically pure and AR grade. Solvents (methanol)

were purified and dried according to the standard procedures. The FTIR spectrum was

recorded as KBr pellet method in the 400–4000 cm-1 region with a Perkin Elmer FT-IR 8000

Spectrophotometer. Electronic spectrum was recorded in methanol solution with a

Systronics Double Beam UV–vis spectrophotometer lamda-850 in the range 200–800 nm.

1H-NMR spectra were recorded on a Bruker AV III 500 MHz instrument using TMS as an

internal reference and DMSO as solvent. The thermal analyses (TG and DTA) were carried

out under nitrogen atmosphere with a heating rate of 200C/min by NETZCHSTA 409C

Analyzers.

46

2.2.2 Synthesis of CT complex

Solid CT complex was synthesized by using slow evaporation of solvent from the solution.

The p-nitroaniline and Chloranilic acid were dissolved in methanol and mixed together. The

solution was stirred well using a magnetic stirrer to get homogenous mixture and filtered

using Whatmann filter paper. The clear filtrate obtained was kept aside unperturbed in a

dust-free environment. A good transparent needle shaped crystals were collected in a growth

period of 20–25 days. The purity of the synthesized crystals was further improved by

repeated recrystallization process by using methanol as solvent.

2.2.3 Single crystal

Single crystal X-ray data of CT-complex was collected at 293K on a Bruker SMART APEX

CCD diffractometer using graphite monochromated Mo Kα radiation (λ= 0.71073 Å). The

linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion

corrections were referred from the International Tables for X-ray Crystallography [28]. The

data integration and reduction were worked out with SAINT [29] software. Empirical

absorption correction was applied to the collected reflections with SADABS [30] and the

space group was determined using XPREP [31]. The structure was solved by the direct

methods using SHELXTL-97 [32] and refined on F2 by full-matrix least-squares using the

SHELXTL-97 program package [33]. All non-hydrogen atoms were refined anisotropically.

The H-atoms attached to carbon atoms were positioned geometrically and treated as riding

atoms using SHELXL default parameters.

2.2.4 Preparation of Standard Stock Solutions

A standard stock solution of p-nitroaniline 10 − 2 M (donor) was prepared by dissolving

0.13812 g of PNA in a 100 ml volumetric flask using methanol. Solutions of different

47

concentrations of donor (PNA) (1 × 10 − 4 M, 1.5 × 10 − 4 M, 2.0 × 10 − 4 M, 2.5 × 10 − 4 M,

3.0 × 10 − 4 M, 1 × 10 − 3 M, 1.5 × 10 − 3 M) were prepared in individual volumetric flask by

serial dilution method from standard stock solution of donor 10 − 2 M solution with the same

solvent. Standard methanolic solution of 10 − 2 M Chloranilic acid (CAA) (acceptor) was

prepared by dissolving 0.10449 g of CAA in 100 ml volumetric flask using methanol.

Solution of 1 × 10 − 4 M CAA was prepared in 100 ml volumetric flask by diluting 10 − 2 M

solution with the same solvent.

2.2.5 Biological evaluation

2.2.5.1 Antibacterial activity

The antibacterial activity of newly synthesized CT complex was treated in vitro using agar

well plate diffusion method. Bacterial inocula were obtained by growing a single colony

overnight in Mueller–Hinton broth and adjusting the turbidity to 0.5 McFarland standards.

100 μl of bacterial test pathogens (Klebsiella pneumoniae, Escherichia coli, Bacillus cereus,

and Staphylococcus aureus) were spread on Mueller–Hinton agar (MHA) plates, and

different concentrations (2, 4, 6, and 8 µg/mL) of CT complex were added into wells (of 5

mm size). These plates were incubated at 370C for 24 hours and the zones of inhibition were

measured. The results are depicted in Fig. 2.11 and Table 2.6

2.2.5.2 Antifungal activity

The new CT complex was also screened for its antifungal assay at for antifungal assay at

different concentration of CT-complex (5, 10, 15, and 20 µg/mL) were poured into growth

media before plating, and incubated at room temperature. After 48 h of incubation, the agar

plugs of uniform diameter (8 mm) containing respective fungi (Fusarium oxysporum, and

Aspergillus flavus) were inoculated simultaneously at the center of each petri dish containing

synthesized CT-complex, followed by incubation at 28±2oC for 8 days. Growth inhibition

48

was measured after incubation. All experiments were concurrently performed in triplicates.

The results are depicted in Fig. 2.12and Table 2.7.

2.3 RESULT AND DISCUSSION

2.3.1 Observation of Electronic spectra

The electronic absorption spectrum of donor p-nitroaniline (PNA) solution, acceptor

Chloranilic acid (CAA) solution and CT complex solution of methanol were recorded in

UV-visible region at different temperature ranging from 298K to 328K as shown in Fig. 2.1.

The concentration of the donor in the reaction mixture was kept greater then acceptor ([D]

>>> [A]) [34-35], and changed over a wide range of concentration from 1. 3 ×10−4 M to 4

× 10−4 M while concentration of acceptor in each of the reaction mixture was kept fixed at

4 × 10−4 M at various temperature. It was observed that the two new absorption maxima

bands at 310.04 nm and 377.06 nm were observed in visible region which is not due the

absorption of any of the reactants and considered to be due to formation of new CT complex.

The absorption maxima of CT complex is slightly shifted towards longer wavelength (i.e.

red shift) and low energy absorption observed in solution containing both donor and acceptor

have been described by Mullikan [36] as a charge transfer transition involving the excitation

of an electron from the donor to the empty orbital of the acceptor in visible region was

evidence that of n → π ∗ type transition. Charge transfer phenomenon was further explained

by Foster [37]. Molar absorptivity, formation constant, enthalpy change and free energy

change of CT complex in methanol at various temperatures were evaluated by using Benesi-

Hildebrand equation [38].

49

[𝐴]0

𝐴=

1

𝐾𝐶𝑇 ∈𝐶𝑇.

1

[D]o+

1

∈𝐶𝑇

Where [A]0 and [D]0 are the initial concentration of acceptor and donor respectively and KCT

and ∈𝐶𝑇 are formation constant and molar absorptivity of CT-complex respectively. The

straight line was obtained when [𝐴]0

𝐴⁄ was plotted against 1

[𝐷]0 which supports the 1:1

formation of CT-complex as shown in Fig. 2.2 and correlation coefficients (r) for BH plots

at various temperature have been listed in Table 2.2. The slope and intercept of the plot are

equal to 1

𝐾𝐶𝑇 ∈𝐶𝑇 and

1

∈𝐶𝑇 respectively.

It is observed that molar absorptivity increases with increasing temperature that indicates the

stability of CT complex decreases with rise in temperature and negative value of ΔG suggest

spontaneity of complexation process. Obtained values of formation constants of CT complex

at various temperatures suggested that formed CT complex is strong enough and not

dissociated easily at elevated temperature from 298K to 308K. The formation constant and

molar absorptivity of CT complex were estimated by from the intercepts and slopes at

various temperature of reaction mixture. The values of formation constant, molar

absorptivity and their thermodynamic parameters are summarized in Table 2.1.

50

250 300 350 400 450 500 550 600

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Abs

oban

ce (a

.u.)

Wavelength (nm)

% (CAA)

% (PNA)

% (CT)

Fig. 2.1 Electronic absorption spectra of (A) PNA, donor (1 × 10−4 M), (B) CAA, acceptor (1 × 10−4 M) and

(C) donor–acceptor CT complex (1 × 10−4 M + 1 × 10−4 M) in methanol at room temperature.

2000 4000 6000 8000 10000

2.5x10-5

3.0x10-5

3.5x10-5

4.0x10-5

4.5x10-5

5.0x10-5

[A] 0/A

bsor

banc

e

1/[D]

% (298K)

% (308K)

% (318K)

% (328K)

Fig. 2.2 Benesi–Hildebrand plots of charge transfer complex of PNA with CAA [A]o / A vs 1 / D in methanol

at different temperature.

51

Table 2.1

Absorption data for spectrophotometric determination of stoichiometry and formation

Constant (KCT), molar extinction coefficient (ƐCT) and thermodynamic parameter of the

[(PNA) + (CAA) −] complex at different temperature.

Temperature(K) KCT (mol-1) ΔG0(joule/mole) ΔH0(joule/mole) ΔS0(joule/mole)

Molar

absorptivity

298

11674.98

-23202.97

-10.96234

40.81476

40358.296

308 9366.589 -23417.47 44147.180

318 8505.032 -23922.67 44610.419

328 7708.3257 -24406.73 47118.985

2.3.2. Determination of transition energy

The energy of CT complex (ECT) was calculated using the following relation [39].

ECT=1243.667/λCT nm

Where λCT is the wavelength of CT complex at which maximum absorbance is obtained.

2.3.3 Ionization potentials of the donor

Ionization potential of donor in the charge transfer complex was calculated by Aloisi and

Piganatro equation [40].

ID (eV) = 5.76 +1.53 x 10−4 νCT

Where ID is the ionization potential of the donor molecule. The νCT is wave numbers in cm−1

of the complex. The values of ionization potentials thus resolved are given in Table 2.2.

52

Table 2.2

Wavelength (λCT), ionization potential (ID), energy of interaction (ECT), resonance energy

(RN), oscillator strength (f), dipole moment (μEN), free energy (ΔG) and correlation

coefficient (r)of the [(PNA)+(CAA)-] CT complex in methanol.

Temperature

(K)

Wavelength

(nm)

Ionization

potential ID

(eV)

Energy of

interaction

ECT (eV)

Resonance

energy RN

Oscillator

strength f

×10-3

Dipole

moment

μEN (D)

Correlation

coefficient

(r)

298

308

318

328

310.04 10.69 4.0113

0.74256

0.76572

0.76838

0.78210

2.576917

40.7887105

42.6604006

42.8836456

44.0726759

0.99843

0.98578

0.99808

0.99716

2.3.4 Determination of resonance energy (RN

Resonance energy (RN) was determined by using following relation, which was derived by

Brieglab and Czekalla [41].

CT= 7.7 x 104/ [hνCT/ RN −3.5]

Where εCT is the molar extinction coefficient of the complex at the maximum absorption of

CT complex, νCT is the frequency of the CT peak and RN is the resonance energy of the

complex

53

The standard free energy of the CT complex (ΔG0) was calculated [42] from the formation

constant using the following equation.

ΔG0 = −2.303 RT log KCT

Where ΔG0 is the free energy of the CT- complex (KJmol−1), R is the gas constant (8.314 J

mol−1 K−1), T is the temperature in Kelvin and KCT is the formation constant of donor–

acceptor complex at different temperature. The values are represented in Table 1, which

indicate that ΔG0 decreases with increases temperature.

EN)

The oscillator strength (f), a dimensionless quantity, is used to express the transition

probability of the CT- band [43]. We can extract that the oscillator strength (f) from the CT

absorption spectra is estimated using the formula.

𝑓 = 4.32 x 10−9∫ εCTdv1/2

Where ∫εCTdv is the area under the curve of the extinction coefficient of the absorption band

in question vs. frequency to a first approximation

𝑓 = 4.32 x 10−9 εCTdv1/2

Where εCT is the maximum extinction coefficient of the band and Δv1/2 is the half-width, i.e.,

the width of the band at half the maximum extinction. The observed oscillator strengths of

the CT are summarized in Table 2.2. The extinction coefficient is related to the transition

dipole moment. The transition dipole moment of charge transfer complex is calculated by

using following equation, which was given by Tsubumora and Lang [44].

54

μEN = 0:0958 [εCTΔν1/2/Δν] 1/2

Δν where Δν ≈ ν at εCT in wave number unit and μEN is defined as −e ∫ ψex Σiriψgdτ. Δν1/2 is

half-width in wave number unit and μEN for the complex of PNA with CAA is given in

Table 2.2.

2.3.6. Determination of entr

Enthalpy change (ΔH) and entropy change (ΔS) were calculated using the following Van’t

Hoff’s equations at different temperatures.

log 𝐾𝐶𝑇 = 𝛥𝑆

2.203𝑅 −

ΔH

2.303RT

Where R is an ideal gas constant (8.314 J K-1 mol-1) and T is the absolute temperature (in

K). Plotting log KCT against 1000/T [45] gives straight lines as shown in Fig. 2.3 with slope

and intercept equal to -ΔH/2.303R and +ΔS/2.303R were calculated. These thermodynamic

parameters are reported in Table 1. The negative values of ΔH confirm the exothermic nature

of compounds and the decomposition of chemical bonds. The positive values of ΔS indicate

a less orderly structure. Results indicate that there is no remarkable effect of temperature on

enthalpy change (ΔH) and entropy change (ΔS) whereas formation constant and free energy

change decreases and molar extinction constant increases with the increase in temperature

as shown in Table 2.1.

55

Fig. 2.3 Relation between ln KCT and 1000/T K for CT complex of p-nitroaniline (PNA) with Chloranilic

acid (CAA).

2.3.7 Comparative study of FTIR spectra of CT complex and reactants

FTIR spectra of p-nitro aniline (PNA), Chloranilic acid (CAA), and CT complex were

recorded with the help of Perkin Elmer FT-IR Spectrometer (Spectrum Two) using the KBr

pellets. FTIR spectra of the organic compounds have been obtained in the region 4000-650

cm-1. FTIR spectra of PNA, CAA and resulting CT complex were depicted in Fig. 2.4 and

their frequencies are assigned in Table 2.3. In general, the bands corresponding to the

acceptor molecule are shifted to lower wave number region in comparison to the donor

molecule. In the FTIR spectra of CT complex, the band appeared at 3420 cm-1 is due to the

3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35 3.408.9

9.0

9.1

9.2

9.3

9.4

log

KC

T

(1/T)

56

aromatic ν (O-H) stretching vibration which was observed at 3235 cm-1 in the FTIR spectra

of free CAA molecule. The band appeared at 3320 cm-1 in the spectrum of CT complex is

due to ν (O─···H─N+) stretching vibration reveals the proton transfer mechanism as shown

in scheme 2.1.

Appearance of H-bonded ion pair O─···H─N+ confirms the existence of new CT complex of

PNA and CAA. This band indicates that hydrogen bond is relatively weak in comparison to

the reactant molecule. C-H aromatic band in the resulting CT spectrum has been observed

at 3070 cm-1. This was appeared at 3217 cm-1 in the spectrum of PNA due to the transfer of

electron from HOMO to the LUMO of the acceptor.

The band for ν (C=O) at 1715 cm-1 is shifted to 1664 cm-1 in the spectrum of CT complex.

The absence of some peak for PNA and CAA in the spectrum of in the FTIR spectrum of

CT complex is due to the charge transfer from donor to acceptor molecule. The stretching

vibration of NO2 group observed at 1481 cm-1 in PNA spectra is remarkably appeared at

1438 cm-1 in the spectrum of CT complex. The appearance of bands at different wave

numbers of CT complex as compared to the donor and acceptor molecules alone is

supporting for the formation of a new CT complex.

57

Fig. 2.4 FTIR spectra of (A) Acceptor; (B) Donor; (C) CT charge transfer complex.

2.3.8 1H NMR spectrum

1H NMR spectrum of the CT complex was measured in DMSO-d6 using Bruker Avance II

400 NMR spectrometer. The 1H NMR spectrum of CT complex has been promising tools for

detecting proton shifts. In 1H NMR spectrum of CT complex, signals of the protons of

acceptor are shifted towards up field, while those of donor are shifted towards down field as

shown in Fig. 2.5.

This has been attributed to increase shielding of the proton of acceptor and decrease of those

of the donor, which reveals transfer of electron from donor acceptor. The 1H NMR spectrum

of the CT complex formed from the interactions between 4-nitroaniline as electron donor

with π acceptor Chloranilic acid, it is evident that nitrogen atom of donor forms

intermolecular hydrogen bonding with the proton of OH group of Chloranilic acid [46-51].

58

All the protons of 4-nitroaniline and Chloranilic acid indicate their signals at the appropriate

positions in the spectrum of the H-bonded CT-complex. The aromatic protons of the 4-

nitroaniline appeared at δ 7.91 and 6.93 ppm in CT complex whereas the three proton of -

NH3+ observed at δ 4.61 ppm because of the intermolecular hydrogen bonding with phenolic

protons of Chloranilic acid increases the shielding effect by the lone of pair electrons of the

nitrogen atom of 4-nitroaniline, while the non-hydrogen bonded phenolic protons appears at

δ 9.59 ppm due to the anisotropic effect of the benzene ring [52-56] as shown in proposed

scheme 2.1 which has been attributed for the formation of the CT complex.

Fig. 2.5 (A)

59

Fig. 2.5 (B)

Fig. 2.5 (A) and (B) 1H NMR spectrum of.1:1 CT complex in DMSO.

2.3.9 Powder X-ray diffraction studies of the CT complex

The characteristic powder X-ray spectra has been observed on a P-Analytical XPRT-PRO

(Nederland) diffractometer with anode material Cu, Kα1, Kα2 and Kβ radiations of

wavelength λ = 1.540 Å, 1.544 Å and 1.392 Å respectively in the range of angles from 5o–

70o at generator settings 35 m A, 40 kV. Kα1 has a slightly shorter wavelength and twice the

intensity as Kα2. The specific wavelengths are characteristic of the target material. The

PXRD pattern of the synthesized CT complex is shown in Fig 2.6.

The strongest Bragg’s peak was observed at diffraction angles 2θ of 27.01o for the CAA-

PNA complex confirms that resulting CT complex is crystalline in nature. The amorphous

materials do not give sharp peaks rather the pattern has noise signals. The data of the PXRD

pattern is depicted in Table- with good agreement for the formation of newly CT complex.

Proton transfer mechanism between the hydroxyl group of π-acceptor (CAA) and donor

60

10 20 30 40 50 60 70 80

Inten

sty (a

.u.)

20

CAA

CT

PNA

10 20 30 40 50 60 70 80

intens

ity(a.

u)

20

CAA

(PNA) also plays a very important role in the self-assembly and crystallization processes in

the molecules.

Fig. 2.6 Powder XRD Pattern of Semi Crystalline CT Complex of PNA with CAA.

2.3.10 Single Crystal Studies

To obtain the unit cell parameters and to confirm the crystallinity of grown crystals, both

single crystal and powder crystal X-ray diffraction studies were carried out. From the single

crystal X-ray diffraction data, it is confirmed that the crystal belongs to monoclinic with

space group of P21/n. The cell parameters were calculated by data reduction and the structure

was solved. The unit cell parameters were measured at 293 K (Table 2.4) where the cell

volume is 984.51 Å3 with a molecular weight of 485.23g. The crystal data, experimental

conditions and structural refinement parameters of crystal are presented in Table 2.4. The

amino substituted p-nitroaniline have potential to make stable CT-complex with Chloranilic

acid through extensive hydrogen bonding network. The selected hydrogen bonding

interactions exist as C6-H6---O2 = 2.512 Å, C8-H8---O2 = 2.352 Å and C10-H10---O5 =

61

2.292 Å. These hydrogen bonding interactions are intermolecular i.e., between donor and

acceptor moieties resulting in a consolidated supramolecular framework (Fig. 2.7, 2.8 &

2.9). In addition to the hydrogen bonding interaction, C-H---π interaction is also present as

C10-H10---C8 = 2.791 Å between the aromatic rings of the donors which are properly

stacked. These non-covalent interactions (H-bonding and C-H---π interactions) result in the

robust network of the CT complex. Interatomic distances and angles are listed in Table 2.5.

Fig. 2.7 Representation of molecular structure of CT complex (a) pictured in ortep view (50% thermal

ellipsoid probability level) and (b) Ball and Stick model.

Fig. 2.8 Diagrammatic representation of weak extensive hydrogen bonding network between the donor and

acceptor moieties of CT-complex

62

Fig. 2.9 2D view of CT-complex.

2.3.11 Thermal Stability

The thermal stability of CT complex as well as acceptor (Chloranilic acid) and donor (P-

Nitroaniline) was illustrated by thermogravimetric and differential thermal analysis (TGA

and DTA). The study is performed under nitrogen flow of heating rate of 200C/min with in

temperature range 25–7500C. The TGA and DTA curves of CT complex, donor and acceptor

are depicted in Fig. 2.10 (A), (B) and (C). Thermal data exhibited that the most of the CT

complex is decomposed in one step with in temperature range 200–232 0C (Fig. 2.10 (C) and

about 71.25 % of the compound is lost at around 2320C. The compound shows phase

transition at around 252.980C.

63

This can also be revealed by the existence of corresponding endothermic peak (DH = 1005.6

J/g). Another endothermic peak observed at around 1980C this endothermic peak shows that

compound is almost completely decomposed at 3520C. It is clearly observed in thermogram

(Figs. A and B) of CAA and PNA that 88.12% of CAA is lost at around 2510C and 88.31%

of PNA is lost at around 2480C. By comparing the thermogram of CAA, PNA and the CT

complex have been made which suggested that the formation of stable CT-complex of

acceptor and donor.

Fig. 2.10 (A)

Fig. 2.10 (B)

64

Fig. 2.10 (C)

Fig. 2.10 (A) Thermogram of the acceptor (A), donor (B) and the formed CT complex (C).

2.4 Pharmacology

2.4.1 Antibacterial activities

The antibacterial activities of synthesized CT-complex have been studied against four

pathogenic microorganisms, B cereus, S aureus, as Gram-positive and K pneumoniae, E

coli, as Gram-negative bacteria of clinical significance. The newly CT-complex of various

concentrations (2, 4, 6, and 8 µg/mL) has exerted significant inhibitory activity against the

growth of bacteria strains as shown in Fig. 2.11. The zones of inhibition are given in Table

2.6 were observed even at very low concentration 2 µg/mL in case of Gram-negative bacteria

where as in case of Gram-positive bacteria the zone of inhibition was observed around 4

µg/mL.

The highest zone of inhibition was observed for K pneumonia and then for B cereus. On the

other hand, E coli and S aureus exhibited comparatively lower inhibition; however, it showed

65

the sharpest rise in inhibitory effect as a function of added CT-complex amount. This was

attributed that the antibacterial potential may be due to the intrinsic physiochemical

properties of the CT-complex (Loo et al). Additionally, the inhibition due to the CT-

complex likely arises from a combined action of the disruption of the cell membrane and

resultant CTC penetration into the cell, and surface reactions that yield ROS, which leads to

oxidative stress and cell damage.

Given the high affinity of the NP for phosphorus and nitrogen, the entry of CTC into the cell

adversely impacts the DNA by inhibiting its replication as well as binding with proteins that

impede cellular metabolism. Taken together, the non-specific mode of action of CTC against

pathogenic bacteria and the current method of synthesis of CTC provides a sustainable and

clean route for the development of antimicrobial agents.

Fig. 2.11. The growth inhibitions against the growth of bacteria at various concentrations are shown.

Control (C)

5 µg/mL

10 µg/mL

15 µg/mL

20 µg/mL

66

2.4.2 Antifungal activity

In case of fungi, the growth inhibition was observed for both fungi Fusarium oxysporum,

and Aspergillus flavus when treating it with different concentration (5, 10, 15, and 20 µg/mL)

of CTC as shown in Fig. 2.12 and listed in Table 2.7.

The visible growth inhibition was observed at low concentration 5 µg/mL. There was a

sharp decrease in inhibitory effect as a function of added CTC amount. CTC exhibited potent

antifungal effects on fungi tested, probably through destruction of membrane integrity;

therefore, it was concluded that CTC has considerable antifungal activity, deserving further

investigation for clinical applications.

Fig. 2.12 The growth inhibitions against the growth of fungi at various concentrations are shown.

Control (C)

5 µg/mL

10 µg/mL

15 µg/mL

20 µg/mL

67

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[52] Shamsuzzaman, K A. A. A. Baqi, M. Asif, A. Ali, H. Khanam, A. Mashrai, A. Ahmad, A.

U Khan, Eur. Chem. Bull.,4 (2015) 154.

[53] Shamsuzzaman, M. Asif, A. Ali, A. Mashrai, H. Khanam, A. Sherwani, M. Owais, Eur.

Chem. Bull. 3 (2014) 1075.

[54] M. S. Refat, H. A. Saad, A. M. A. Adam, Russian J. General Chem. 84 (2014) 1417.

[55] A. S. AL-Attas, M. M. Habeeb, D. S. AL-Raimi, J. Mol. Struct. 928 (2009) 158.

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71

Chapter-3

Synthesis, Single-Crystal, DNA Interaction,

Spectrophotometric and Spectroscopic Characterization of

the Hydrogen-Bonded Charge Transfer Complex of 2-

Aminopyrimidine with π-Acceptor Chloranilic Acid at

different Temperature in Acetonitrile

72

3. 1 INTRODUCTION

Theory of charge transfer complex first introduced by Mulliken [1], considered the complex

as a hybrid resonating between the non˗polar and polar structure as a result of the transfer of

one electron from a donor to an acceptor molecule [2]. Most of the charge transfer complexes

have been formed under the hydrogen bonding proton transfer beside electron transfer,

which gives extra stability for the formation of CT complexes [3−6].

The formation of the CT−complexes between donor [AP] and π˗electron acceptor [CAA] has

been widely investigated by spectrophotometric and spectroscopic techniques in acetonitrile

at different temperature. [7−9]. Charge transfer complex has been studied to investigate the

geometries, binding constants, dipole moments, and spectral properties [10−14]. The CAA

is a strong π˗acceptor and readily formed radical anion by accept the pair of electrons from

AP (scheme-1). The review of literature in the last decade focused on the spectral studies of

the CT−complexes [3−6]. The charge transfer complex are formed during the reaction

between strong π˗acceptor (CAA) and donor (AP), containing different sites of the donation,

exhibited in the form of intense colour that absorb radiation in the visible region. These

interactions have been attributed to the transfer of charge from donor to acceptor [15−17].

The interaction of DNA with various types of drugs, proteins and enzymes is the most

important aspect in the biological field [18, 19]. DNA structure has many mode of

interactions in which various drugs interact with it, which can be analysed with the help of

UV−Visible and fluorescence spectrophotometric [20]. In vitro Ct−DNA interaction with

CT complex is usually accompanied by shift in the wavelength upon absorbtion [21]. The

investigation of CTC−Ct-DNA interaction is importance for understanding the molecular

mechanism of CTC action. Charge transfer complexation has been played an important role

73

in biochemical and bio electrochemical energy transfer processes [22]. The redox properties

of CT complexes have been investigated to promote DNA−binding [23]. The Ct−DNA

interaction with CT complex also provide under study of nature and binding pattern. The

important role of CT complexes play in many biological systems such as drug acceptor

binding mechanism, DNA−binding, Antioxidant, Antibacterial, Antifungal, antibacterial,

antifungal, and insecticides [6, 21, 24−28]. Furthermore, the research applications of charge

transfer complexes are in fields like photo catalysts [29], dendrimers [30], surface chemistry

[31], non-linear optical materials and electrical conductivity [32], solar energy storage [33],

organic semiconductors [34] as well as in studying redox processes. Pyrimidine and its

derivatives have important numerous biodynamic properties and biological activities such

as bactericides, fungicides, vermicides, insecticides, and medicines [25, 35−38]. Pyrimidines

are also of immerse use as corrosion inhibitors and chelating agents [39]. The experimental

investigation tends to elucidate mainly the study of the molecular interaction between donor

(AP) and acceptor (CLA) and interpreting the nature of this interactions using various

spectroscopic techniques such as FTIR, 1H NMR, TGA−DTA, UV−Visible studies,

ESI−MS and single X-ray crystallography. We have also studied to investigate about the

binding property of CTC with calf thymus (Ct)-DNA using circular dichroism spectroscopy

and dynamic viscosity measurements.

3.2 EXPERIMENTAL STUDY.

3.2.1 Materials and method.

All chemical used were of analytical grade. The donor AP, acceptor CAA and Calf thymus

DNA (Ct−DNA) were purchased from Sigma–Aldrich Chemical Company (USA) with a

stated purity of greater than 98% and these were used without any further purification.

Spectroscopic grade solvents were supplied by Merck Chemical Company and were used

74

without any purification. The charge transfer complex has been synthesized using

acetonitrile as a solvent.

3.2.2 Synthesis of Solid CT Complex

The synthesized (AP : CAA) CT complex was obtained by mixing a saturated solution of AP

(0.19020 g, 2 m. mol) and CAA (0.41796 g, 2 m. mol) in acetonitrile. A blue colour solution

was formed upon the mixing. The saturated solution of AP and CAA in the same solvent was

stirred continuously for about 50 minutes at room temperature. The solutions were allowed

to evaporate slowly at room temperature. The solid CT complex was filtered and washed

several times with little amounts of the solvent as blue crystals and dried under vacuum over

anhydrous calcium chloride. The results of elemental analysis of CT complex with

theoretical values are: C10H9Cl2N3O5 (M.W = 322.10 g, M.P. = 270 oC), C = 37.26 % (37.52

%), H = 2.81 % (2.94 %), N = 13.04 % (13.12 %), O = 24.84 % (24.88 %), Cl = 22.01 %

(23.09 %).

3.2.3 Single Crystal Growth

A saturated solution of the synthesized solid [(AP)+ (CAA)−] CT was prepared by dissolving

in methanol. The Whatmann: 41 grade filter paper has been used to remove the suspended

impurities from the solution. The clean filtrate was kept unperturbed in a dust free chamber

without any disturbance for one week. Transparent, dark blue, needle shaped crystals were

harvested on the last day of the week.

3.2.4 Preparation of Standard Stock Solutions

Standard stock solutions of AP (10−2 M) and CAA (10−2 M) were prepared by dissolving

0.0475 g (AP) and 0.10449 g (CAA) each in separate volumetric flasks of 50 ml using

75

acetonitrile as a solvent. Solutions of the concentration (1 x 10−4 M) of the donor and

acceptor were prepared each in 25 ml volumetric flask by diluting 10−2 M solution. The

various concentrated solutions were prepared by mixing the fixed concentration of the

acceptor (1 x 10−4), different concentrations of donor (1 x 10−4 M, 1.5 x 10−4 M, 2, 0 x 10−4

M, 2.5 x 10−4 M, 3.0 x 10−4 M and 3.5 x 10−4 M each in 25 ml individual volumetric flask.

The other solutions were also prepared using same procedure in the same solvent.

3.2.5 Instrumental Measurements

3.2.5.1 Electronic Spectra

Spectrophotometric measurement were made using a Perkin Elmer Lambda−850 nm UV–

visible spectrophotometer in the region 200–650 nm at different temperature ranging from

25 oC to 50 oC quartz cell with 1 cm path length.

3.2.5.2 FTIR Spectra

The FTIR measurement of donor, acceptor and CT complex were recorded employing Perkin

Elmer FTIR Spectrometer (Spectrum Two) using the KBr pellets, evacuated to avoid water

and CO2 absorption.

3.2.5.3 TG/DTA Analysis

Thermal analysis (TG/DTA) of donor, acceptor and their resulting CT complex were

recorded employing the instrument EXSTAR TG/DTA 6300 model with heating rate of 20

0C/ min in nitrogen atmosphere.

3.2.5.4 1H NMR Spectra

The 1H NMR spectra was carried out in DMSO using the BRUKER AVANCE II 400 MHz

spectrometer with TMS as an internal reference.

76

The crystallographic data for the 1:1 CT complex of AP with CAA was recorded on a Bruker

(SHELXL−2014/7 ) APEX−II CCD diffractometer, with graphite-monochromatic Mo−Kα

(λ = 0.71073 Å) at 298 K.

3.2.5.6 Mass Spectra

The electron spray ionization (ESI)–mass spectra of CT complex was obtained using

WATERS−Q−TOF MICROMASS (ESI−MS) mass spectrometer.

3.2.5.7 DNA Binding Studies

The binding affinity and orientation pattern between Ct-DNA and CT complex was

investigated by the UV-Visible, fluorescence, circular dichroism (CD) spectroscopy and

relative viscosity measurement.

3.2.5.7.1. Sample Preparation for DNA Binding Studies

The stock solution of Ct-DNA was prepared by dissolving DNA in 10 mM Tris-HCl buffer

(pH 7.2) at room temperature with gentle stirring to ensure the formation of a homogeneous

solution. Purity of the DNA stock solution check by taking absorbance ratio of A260/A280.

If absorbance ratio was found to be in the range of 1.8–1.9, indicates that the no

contamination of protein in Ct DNA solution [40] . The concentration of Ct-DNA was

determined from its absorption intensity at 260 nm with a molar extinction coefficient of

6600 M−1 cm−1 [41]. The stock solution of CT complex is prepared by dissolved in 50%

DMSO solution and for interaction studies further diluted in working buffer (10 mM Tris-

HCl of pH 7.2) in such a way maximum 6% of DMSO present in each titrating sample.

77

3.2.5.7.2 UV−Visible Spectroscopy

Interaction analysis of CT complex with Ct-DNA was done by using UV-1800 Shimadzu

spectrophotometer equipped with Peltier temperature programmer-1 (PTP-1). The

absorption spectra of CT complex complexed with DNA were measured in the wavelength

range from 250–300 nm. The absorption titration experiments were carried out by keeping

the fixed concentration of Ct-DNA (50 μM) in 10 mM Tris-HCl buffer of pH 7.2 with

varying concentration of CT complex (0-50 μM). CT complex solutions of the same

concentrations without Ct-DNA were used as the blank and final spectrum of each

concentration were reported after correcting with respective CT complex-Ct-DNA

complexed spectrum.

3.2.5.7.3 Fluorescence Studies

All fluorescence experiments were carried out by fluorometric titration using a Shimadzu

spectroflurometer˗5301 (Japan) equipped with constant temperature holder attached with

Neslab RTE−110 water bath with an accuracy of ±0.1 °C. The fluorescence titration was

performed against fixed concentration of complex (30 µM) with varying concentration of

Ct−DNA (0−30 µM). The emission spectra complex was recorded in the range of (350−450

nm) by exciting at 325 nm with slit width for both the excitation and emission are 5 nm and

10 nm respectively. In EB (ethidium bromide) displacement assay, a solution containing EB

(5 μM) and Ct−DNA (20 μM) was titrated with increasing concentration of CT complex.

EB−Ct−DNA complex was excited at 478 nm and emission spectra were recorded from 500

to 700 nm. In another experiment, 5 μM of Hoechst 33258, a well−known groove binder

was added to 20 μM of Ct−DNA. Ct−DNA−Hoechst 33258 complex was excited at 363 nm

and emission spectra were recorded from 350 to 600 nm.

78

3.2.5.7.4. Circular Dichroism (CD) Measurments

The structural changes (secondary) induced in Ct-DNA by CT Complex was investigated by

using a JASCO-J 813 spectropolarimeter. The spectropolarimeter was equipped with a

Peltier-type temperature controller, maintained 25oC temperature during experiment and a

quartz cell with a path length of 0.1 cm. Two scans were accumulated at a scan speed of 100

nm min-1 and spectra were recorded in the range of 220-320 nm. The CD spectra of Ct-

DNA in the absence and presence of varying concentration of CT complex were recorded

and the respective blanks were subtracted from each spectrum.

3.2.5.7.5 Relative viscosity measurements

Viscosity measurements were carried out for further investigation of binding modes of CT

complex with Ct-DNA. The change in the viscosity of Ct-DNA due to interaction of CT

complex was measure by using Ubbelohde viscometer, kept in water bath at 25oC. In this

process we were mix the varying concentration of CT complex to fixed concentration of Ct-

DNA (50 μM) and average flow rate was measured by using a digital stopwatch. The data

obtained were presented as (η/η0)1/3 versus [CT complex] / [Ct-DNA]. Where, η0 and η are

represents the viscosity of Ct-DNA solution in absence and presence of CT complex

respectively. The values of viscosity were calculated using the relation η = (t-t0)/t0, where t

is the time of flow of samples containing Ct-DNA and t0 is the time of flow of buffer alone.

Each sample was investigated in triplicate.

79

3. 3 RESULTS AND DISCUSSION

3.3.1 Observation of CT Electronic Spectra

Spectrophotometric spectra were observed from the fixed concentration (100 µL) of the

acceptor with various concentrations of donor (1 x 10−4 M, 1.5 x 10−4 M, 2,0 x 10−4 M, 2.5

x 10−4 M, 3.0 x 10−4 M and 3.5 x 10−4 M) from the concentrated solutions (1 x 10−4 M) of

the donor and acceptor [41]. The CT spectrum was also recorded by taking the equal

concentrated solution (1 x 10−4 M + 1 x 10−4 M) of the acceptor and donor in 3 ml cuvette

without any further diluting. The new CT absorption peaks were observed in the ultraviolet

region, which are different from the absorption peaks of the reactant molecules. The

electronic absorption spectra of the donor, acceptor and the formed CT complexes were

shown in Fig 1. The maximum absorbance peaks of the CT complex were appearing at 225

nm in acetonitrile. The broad peak observed at 516 nm in the visible region, which is

corresponding to the blue colour of the complex. The indication of the formation of CT

complex was observed during the changing colour of the reaction mixture between donor

and acceptor molecules. The maximum absorbance peak of the CT complex (λCT) was

depicted in Table 1. The absorption intensity has been changed to the higher side

continuously by increasing the concentration of the donor with the fixed concentration of the

acceptor which is given in Table 1. The CT absorption spectra were analysed by fitting to

the Gaussian function y = y0 + [A/(w√(π/2))] exp[−2(x − xc)2/w2] where x and y denote

wavelength and absorbance, respectively. The Gaussian analysis results for the all systems

under study, are reported in Table 2. The absorption bands at a maximum wavelength

attributed to the formation of a CAA radical anion resulting from complete transfer of charge

from AP to CLA (Scheme˗1). The radical anion nature of C AA has been confirmed by

80

electron spin resonance spectral measurements. The hydrogen bonded structure of the CT

complex is shown in scheme˗2.

200 250 300 350 400 450 500 550 600 650

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

(C)

(B)

(A)

Ab

so

rban

ce

Wavelength (nm)

Donor (AP)

Acceptor (CLA)

CT complex

Fig.3.1 UV–Visible spectra of (A) AP, donor (1 × 10−4 M); (B) CAA, acceptor (1 × 10−4 M) and (C)

donor˗acceptor, CT complex (1 × 10−4 M + 1 × 10−4 M) in acetonitrile at 25 oC temperature.

2 4 6 8 10

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

At 25 oC in acetonitrile

At 30 oC in acetonitrile

At 35 oC in acetonitrile

At 40 oC in acetonitrile

At 45 oC in acetonitrile

At 50 oC in acetonitrile

[A] o

/A

1/[D]o

Fig. 3.2 Benesi-Hildebrand plots of [A]o/A against 1/[D]o for AP−CAA complex in acetonitrile at different

temperature in the range of 25−50 oC.

81

Table 3.1 Absorption data for spectrophotometric determination of stoichiometry, absorption maxima (λCT),

formation constant (KCT) and molar absorptivity (ɛCT) of the [(AP)+(CAA)−] complex in methanol at

different temperature.

Concentration of

donor

Concentration

of acceptor

Absorbance

at CT (nm)

Formation constant

(KCT) (104) mol-1

Molar absorptivity (CT)

(103) l cm-1 mol-1

At 25 oC At 225 nm

1.8785 26.289

1.0 x10−4 1.0 x10−4 0.21193

1.5 x 10−4 1.0 x10−4 0.37764

2.0 x 10−4 1.0 x10−4 0.48195

2.5 x 10−4 1.0 x10−4 0.59760

3.0 x 10−4 1.0 x10−4 0.93122

3.5 x 10−4 1.0 x10−4 1.35938

At 30 oC At 225 nm

1.7926

25.693

1.0 x10−4 1.0 x10−4 0.19313

1.5 x 10−4 1.0 x10−4 0.32078

2.0 x 10−4 1.0 x10−4 0.41462

2.5 x 10−4 1.0 x10−4 0.50589

3.0 x 10−4 1.0 x10−4 0.56204

3.5 x 10−4 1.0 x10−4 0.62814

At 35 oC At 225 nm

1.7048

22.065

1.0 x10−4 1.0 x10−4 0.17245

1.5 x 10−4 1.0 x10−4 0.26743

2.0 x 10−4 1.0 x10−4 0.31255

2.5 x 10−4 1.0 x10−4 0.36147

3.0 x 10−4 1.0 x10−4 0.40489

3.5 x 10−4 1.0 x10−4 0.42110

At 40 oC At 225 nm

1.6560 17.540

1.0 x10−4 1.0 x10−4 0.16307

1.5 x 10−4 1.0 x10−4 0.27233

2.0 x 10−4 1.0 x10−4 0.33861

2.5 x 10−4 1.0 x10−4 0.37973

3.0 x 10−4 1.0 x10−4 0.42602

3.5 x 10−4 1.0 x10−4 0.46601

At 45 oC At 225 nm

1.6362 17.214

1.0 x10−4 1.0 x10−4 0.15322

1.5 x 10−4 1.0 x10−4 0.23077

2.0 x 10−4 1.0 x10−4 0.28958

2.5 x 10−4 1.0 x10−4 0.32572

3.0 x 10−4 1.0 x10−4 0.35442

3.5 x 10−4 1.0 x10−4 0.38190

At 50 oC At 222 nm 1.6158

16.417

1.0 x10−4 1.0 x10−4 0.14020

1.5 x 10−4 1.0 x10−4 0.20753

2.0 x 10−4 1.0 x10−4 0.25683

2.5 x 10−4 1.0 x10−4 0.29105

3.0 x 10−4 1.0 x10−4 0.31434

3.5 x 10−4 1.0 x10−4 0.32971

82

Table 3.2 Gaussian curve analysis for the [(AP)+(CAA)−] spectrum in acetonitrile at different temperatures.

Solvent Area of the curve (A) Width of the curv (w) Centre of the curve (xc) yo

Acetonitrile

At 25 oC 33.426 ± 1.466 23.426 ± 1.178 221.821 ± 0.554 0.068 ± 0.007

At 30 oC 33.368 ± 1.465 23.543 ± 1.193 221.887 ± 0.561 0.068 ± 0.007

At 35 oC 33.297 ± 1.467 23.694 ± 1.206 221.785 ± 0.566 0.067 ± 0.007

At 40 oC 32.665 ± 1.448 23.445 ± 1.198 221.905 ± 0.564 0.067 ± 0.007

At 45 oC 32.427 ± 1.439 23.455 ± 1.199 221.902 ± 0.565 0.067 ± 0.007

At 50 oC 32.332 ± 1.435 23.578 ± 1.207 221.831 ± 0.567 0.066 ± 0.007

The formation constant (KCT) molar extinction coefficient (εCT) and other physical

parameters of the CT complex were calculated at different temperature using Benesi–

Hildebrand equation [42] equation is reported in Table 1 and 3. This equation is applicable

only when either concentration of donor or acceptor should be larger than the other.

Formation constant (KCT) calculated of D···A CT absorbance (A) as a function of variability

[D]o >> [A]o [43, 44]. A plot of X = 1/[D]o vs Y = [A]o/A gives a Y−intercept = 1/ɛCT and

slope =1/ɛCT KCT as defined by the Benesi–Hildebrand equation:

[A]o

A =

1

KCTɛCT∙

1

[D]o +

1

ɛCT

where [A]o and [D]o are the initial total concentration of the acceptor (fixed) and donor

(varied), A is the CT absorption of the DA complex at wavelength (λCT) against the solvent

as reference, ɛCT is the extinction coefficient and KCT is the formation constant of the CT

complex. The straight line plots were obtained at different temperatures by plotting the

values [A]o/A versus 1/[D]o which supports the formation of the 1:1 charge transfer complex

as shown in Fig. 2. KCT and ɛCT of the CT Complex were calculated from the slopes and

intercepts of all linear plots at different temperatures.

83

These parameters of the CT complex are decreasing with increasing temperature because the

donor and the acceptor molecule gain freedom [45, 46] which are reported in Table 1. The

G. Briegleb and Z. Angew [47] equation has been used for the calculation of energy (ECT)

of the π→π* interaction between the donor (AP) and acceptor (CAA) molecules as given

below.

ECT = 1243.667

λCTnm

Where λCT is the wavelength of the CT band. The energy of the interaction between donor

and acceptor depend upon the maximum absorbance of the CT complex. The calculated

value of ECT is given in Table 3.

3.3.2 Determination of Ionization Potentials of the Donor (ID).

Aloisi and Pignataro drive an empirical equation for the calculation of ionization potential

of the donor in the CT complex given below [48]:

ID = 5.76 + 1.53 × 10−4 νCT

Where νCT is the wavenumber in cm−1 of the CT complex determined in acetonitrile. The

values of ionization potentials are summarized in Table 3. Ionization potential is useful

quantity for measurement of the electron donating power of a donor molecule.

3.3.3 Determination of Resonance Energy (RN).

The resonance energy of the CT complex was calculated using relation theoretically derived

by Brieglab and Czekalla [49].

ɛCT = 7.7 × 104

[hνCT

|RN|− 3.5]

84

where εCT is the molar extinction coefficient of the complex at the maximum absorption of

the CT complex (λCT), νCT is the frequency of the CT peak. Resonance energy is the

favourable factor to determine the stability of the complex. The values of the resonance

energy under study at different temperature are listed in Table 3.

3.3.4 Determination of Oscillator Strength (f) and Transition Dipole Moment (µEN).

The oscillator strength (ƒ) is a dimensionless quantity which is used for the expression of

transition probability of the CT bands. The oscillator strength (ƒ) was obtained from the

approximate formula.

ƒ = 4.32 × 10−9 ɛCT Δν1/2

Where Δν1/2 is the half˗band width of the absorbance and εCT is the extinction coefficient at

maximum absorption of the CT band. The relative high values of (ƒ) for charge transfer

complexes indicate a strong interaction between donor and acceptor molecules. Oscillator

strength of the complex decreases with increasing temperature. The calculated values of

oscillator strengths of the CT complex are listed in Table 3.

Transition dipole moment (µEN) for the CT complex, which is related to the extinction

coefficient has been calculated using the relation:

µEN = 0.0958√ɛCTΔν1/2

Δν

Where Δν ≈ ν at ɛCT and µEN is defined as –e ∫ ψex ∑i riψi dτ for the complex. Δν1/2 is half -

width in wave number unit. The values of μEN for the CT complex at various temperatures

are given in Table 3. Transition dipole moment has been used for the allowed transitions

between the HOMO of the donor and LUMO of the acceptor moieties.

85

3.3.5 Determination of Thermodynamics Parameters of the CT Complex.

The free energy change of the CT complex (ΔG) related to the formation constant at various

temperatures was evaluated using the following equation.

ΔG = −2.303RT logKCT

Where ΔG is the free energy change of the CT complex at the standard condition (kJ mol−1),

R is the gas constant (8.314 J mol−1 K−1), T is the absolute temperature measured in Kelvin

(273 + °C) and KCT is the formation constant of the CT complex at a definite temperature.

The progressive negative values of the free energy change (ΔG) indicate the strong

interaction between donor and acceptor molecules [46, 50, 51]. The thermodynamics

parameters (ΔH) and (ΔS) were calculated using the slope and intercept of the linear plot

based on logKCT versus 1/T using Van’t Hoff’s equation [52, 53].

logKCT = −ΔH

2.303RT +

ΔS

2.303R

Where ΔH is the enthalpy change (kJ mol−1) of the CT complex, R is gas constant (8.314 J

mol−1 K−1), T is the absolute temperature in Kelvin (273 + °C), and KCT is the formation

constant of the CT complex at different temperature. The linear plot is shown in Fig 3. The

values of thermodynamic parameters such as ΔG, ΔH and ΔS of the complex at different

temperature were reported in Table 4. The high values of the formation constants (KCT), free

energy changes and entropy explain the spontaneity of the complexation. The negative

values of enthalpy and free energy change are the significance to the spontaneous formation

of the CT complex and its stability. The ΔH < 0 and ΔS > 0 values indicated that the force

acting between the reacting molecules was electrostatic interactions [50].

86

2.0 2.5 3.0 3.5 4.0

0.20

0.21

0.22

0.23

0.24

0.25

0.26

0.27

0.28

R2 = 0.9859

logKCT

= 0.13766 + 0.03396. X

log

KC

T

1/Tx10-2 (K)

Fig. 3. 3 Vant's Hoff plot of AP−CAA complex logKCT vs 1/T in acetonitrile at various temperature.

Table 3.3 Physical parameters of the [(AP)+(CAA)−] CT complex in acetonitrile at different temperatures.

Solvent Wavelength

λCT (nm)

Ionization

Potential

ID (ev)

Energy of

interaction

ECT (ev)

Resonance

energy

RN × 10−3

Oscillator

strength

ƒ × 10−6

Dipole

moment

μEN (D)

Correlation

coefficient

r

Acetonitrile 222 12.56 5.563

At 25 oC 1.887 1.330 2.153 0.99874

At 30 oC 1.884 1.307 2.123 0.99868

At 35 oC 1.584 1.129 1.961 0.99694

At 40 oC 1.259 0.888 1.758 0.99866

At 45 oC 1.236 0.872 1.741 0.99822

At 50 oC 1.200 0.836 1.695 0.99810

87

Table 3.4 Thermodynamic parameters for the [(AP)+(CAA)−] CT complex in acetonitrile at different

temperatures.

Solvent Free energy ΔG

(kJ mol-1)

Enthalpy ΔH x 10−3

(kJ mol-1)

Entropy ΔS x 10−3

(kJ K-1 mol-1)

Acetonitrile

At 25 oC −1.5623 −0.6502 2.636

At 30 oC −1.4706

At 35 oC −1.3662

At 40 oC −1.3128

At 45 oC −1.3020

At 50 oC −1.2887

3.3.6 FTIR Spectral Study

Infrared spectra of donor (AP), acceptor (CAA) and [(AP):(CAA)] CT complex were shown

in Fig. 3.4 The band assignments of the donor, acceptor and the resulting CT complex are

given in (ESI, Table 5S). In the spectrum of the CT complex the observation of the main

infrared bands of the AP and CAA with small shifts in frequency values from those of the

free molecules supports the formation of the CT−Complex. The two sharp asymmetric and

symmetric stretching vibrations of the amino group exhibited at 3431 and 3305 cm−1 in the

spectrum of the CT complex, whereas, these were observed at 3351 and 3244 cm−1 in the

spectrum of the free donor molecule. The shift of the amino group stretching frequencies

confirms the charge transfer from AP towards CAA. The appearance of a broad band at 2932

cm−1 attributing to NH+ including in the hydrogen bond bridge (N+—H∙∙∙∙∙O−) between AP

and C AA molecules [54−56]. Consequently, one suggests the involvement of both ring

nitrogen and amino group in H−bonding with chloranilic acid. This type of hydrogen

bonding was good agreement for the existence of the new CT complex. The strong band of

88

C−H (aromatic) of CT complex is shifted to 3022 cm−1 in the spectrum of the CT complex

compared with 3169 cm−1 for free donor molecule.

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

120

140

160

180

(C)

(B)

(A)

Tra

nsm

itta

nce (

%)

Wavenumber (cm-1

)

CT complex

Acceptor

Donor

Fig.3.4 FTIR spectra of (A) AP; (B) CAA; and (C) 1:1 CT complex of AP with CAA in the range of 4000−400

cm-1. The strong band at 3236 cm−1 has been assigned to the OH group in the spectrum of the acceptor molecule

which was appearing at 3431 cm−1 in the spectrum of CT complex. In the plane and out of the plane bending

vibration motion of –NH2 group were appeared at 1649 and 997 cm−1 respectively, which were observed at

lower frequency region at 1632 and 980 cm−1 in the moiety of the CT complex due to the decreased electron

density of the amino group by transfer of electrons from the nitrogen atom of AP to CAA through

intermolecular hydrogen bonding. The carbonyl stretching, vibration ν(C=O) is shifted to 1687 cm−1 for the

complex compared with 1664 cm−1 for CAA alone. From the (ESI, Table 5S) it was observed that small shift

of the vibrational frequencies of ν(C=N), ν(C−C), ν(C−N), ν(C−O) and ν(C−Cl) of the complex to 1423, 1377,

1349, 1219 and (870,781) cm−1 compared with 1357, 1224, 1177, 1369 and (854, 753) cm−1 for the free reactant

molecules. These small shifts of the frequencies are strongly favored for the formation of new CT complexes.

89

Table 3.5

Tentative assignments and infrared frequencies of donor (AP), acceptor (CAA) and [(AP) : (CAA)]

CT complex in cm−1.

Compound Frequency Assignments

AP

3351 (as), 3244 (sy) ν(−NH2)

3169 (s) ν(C–H); (aromatic)

1649 (w) δ(−NH2); in plane bending

1578 (ν), 1560 (m), 1478(ν) ν(C=C) (ring)

1357 (s) ν(C=N) (ring)

1224 (ν) ν(C−C) (ring)

1177 (s) ν(C−N) (ring)

997 (w) δ(−NH2); out of plane bending

CLA

3236 (s, br) ν(OH)

1664 (ms) ν(C=O)

1632 (vs) ν(C=C) (ring)

1539 (s) ν(C−C) (ring)

1369 (w) ν(C−O)

854 (s), 753(m) ν(C−Cl)

(AP:CLA) CT complex

3431 (as), 3305 (sy) ν(NH2)

3105 (s) ν(OH) hydrogen bonded

3022(s) ν(C−H); (aromatic)

2932 (br) ν(N+−H∙∙∙∙∙∙O−) bonding

1687 (s) ν(C=O)

1632 (w) δ(−NH2); in plane bending

1607 (vs) ν(C=C); (aromatic ring)

1423 (w) ν(C=N); aromatic ring

1377(w) ν(C−C)

1349 (s) ν(C−N)

1219 (w) ν(C−O)

980 (s) δ(−NH2); out of plane bending

870 (s), 781(m) ν (C−Cl)

s, strong; w, weak; br, broad; v, stretching; vs, very strong; m, medium; sh, shoulder; as, asymmetric;

sy,symmetric; def, deforming

90

3.3.7 1H NMR Spectra

1H NMR spectra of the CT complex in DMSO displays distinct signals with appropriate

multiplies and depicted in Fig. 3.5 (A)–(B). 1H NMR analysis of donor and acceptor in order

to make the comparison with CT more convenient and obviously. The chemical shift values

of the CT complex have been changed in comparison to the donor and acceptor alone, which

is a good agreement for the formation of new CT complexes. The doublet of doublets was

recorded at δ = 8.51 ppm for two equivalent protons H4 and H6 in the AP moiety of the CT

complex due to shielding effect produce by amino group. The singlet of triplet resonance

signal at δ = 6.85 ppm is attributed to H5 proton in the AP moiety of the CT spectrum. The

resonance signal at δ = 8.15 ppm is presumably attributed to the protons of the amino group.

The appearance of a new broad singlet resonance signal near δ = 5.25 ppm and is attributed

to the proton on −NH3+ group due to migration of phenolic proton of CAA moiety to AP

moiety (scheme 3.2) [57−59]. Hence, one concludes the existence of a proton transfer beside

charge transfer during complexation. The 1H NMR spectrum of the complex reveals 1:1

composition.

Scheme -3.1 Formation of AP and CAA radical ions with proton transfer in acetonitrile at room temperature.

91

Scheme -3.2 The molecular structure of charge transfer transition of hydrogen bonded [(AP)+(CAA)−] CT

complex.

The stability of the synthesized [(AP):(CAA)] CT complex was estimated using electron

spray ionization-mass spectrum. More precisely mass spectrometry determines the mass and

elemental composition of the complex. The ESI˗mass spectra of the formed CT complex is

depicted in Fig. 6 (A) −(C). Both the –OH and –NH2 substituent groups, which lower the

ionization potential, increase the relative abundance of the CT complex. The molecular ion

peaks at m/z = 208.98, 207.97 and 206.98 were assigned to [CTC−AP]+, [CTC−AP−H]+ and

[CTC−AP−2H]+ in the ESI−mass spectrum of the CT complex, respectively..

Table 3.6

The main characteristic molecular ion peaks corresponding to their fragmentations of the CT

complex.

Fragmentation Peak assignment (m/z values)

[CTC − 3H]+ 301.10

[CTC − CLA + H]+ 96.09

[CTC − CLA + 2H]+ 97.09

[CTC − CLA + 3H]+ 98.99

[CTC − AP − Cl]+ 172.97

92

[CTC − AP −2H]+ 206.98

[CTC − AP − H]+ 207.97

[CTC − AP]+ 208.98

[CTC − AP + H]+ 209.97

[CTC − AP + 2H]+ 210.98

The mass spectra of CT complex shows intense peaks at at m/z = 96.09, 97.09 and 98.99

correspond to [CTC−CAA+H]+, [CTC−CLA+2H]+ and [CTC−CAA+3H]+ moieties

respectively. The mass spectrum exhibited a molecular ion peak at m/z = 310.10

corresponding to the [CTC−3H]+ fragment, which revealed the existence of 1:1

[(AP)+(CAA)−] complex. The main characteristic molecular ion peaks were reported in 6

(ESI, Table 6S). This stoichiometry ratio of the formed complex can also be confirmed by

various remaining fragment peaks at m/z = 210.98, 172.97, 149.96, 123.12, 115.07 and

100.99. These most abundance peaks are in favoured for the formation of new CT

complexes.

Fig. 3.5 (A)

93

Fig. 3.5 (B)

Fig. 3.5 (A) and (B) 1H NMR spectrum of 1:1 [(AP)+(CAA)−] CT complex in DMSO.

Fig. 3.6 (A)

94

Fig. 3.6 (B)

Fig. 3.6 (C)

Fig. 3.6 (A) − (C) electron spray ionization-mass spectra (ESI−MS) of [(AP)+ (CAA)−] CT complex.

95

3.3.9 X˗ray Crystallographic Study of the (AP: CAA) CT Complex

The crystallographic data and refinement results for [(AP)(CAA)] complex are listed in (ESI

Table 7S). An ortep view of crystal structure with atoms numbering and the hydrogen

bonding network of the CT complex are shown in Figs. 3.7 and 3.8. It is clear from the

crystal structure of the CT complex that a proton from the −OH group of CAA has been

transferred to the –NH2 group of AP, which has been ascertained that hydrogen bonded

network stabilized between donor and acceptor moieties. This type of hydrogen bonding is

present between the hydrogen of O3 and O2 of CAA molecule and N1, N2 and N3 of AP

molecule moieties (Table 3.8). The adduct like structure has been formed through the

intermolecular hydrogen bonding [59] (scheme 2) and Triclinic packing diagram of the

hydrogen bonded CT complex is shown in (ESI, Fig. 9S). The helix like crystal structure of

the complex ascertained the 1:1 stoichiometry. It was also observed that bond distance and

bond angles in the moiety of the CT complex become relatively shorter compared to the

optimized structures of CAA and AP [50, 51] due to the extensive hydrogen bonding, which

reveals the stability of the of the HBCT complex. One can also observe from Table 3.9, the

shortening of the C (7)−Cl (2) and C (10)−Cl (1) bond lengths by 0.0121 and 0.0031 (Å)

after complex formation compared with the C−Cl bond length (1.73 Å) of CAA alone due

to increasing the electron density on the acceptor moiety supporting the participation of AP

in CT process with CLA [55]. The shortening of C(6)−O(3) bond length by 0.0572 (Å)

during the complexation compared with original C(6)−O(3) bond length (1.36 Å) of free

CAA due to proton transfer through intermolecular hydrogen bonding O(3)−H(3A)···N(2)

[55]. Selected bond lengths, bond angles and the hydrogen bonding data are reported in

Tables 3.8 and 3.9. The isotropic, equivalent isotropic and anisotropic displacement

parameters are summarized in Tables 3.10, 3.11 and 3.12.

96

Fig. 3. 7 An ortep view of crystal structure of CT complex at 296 K with 40 % probability level, showing

the atom numbering scheme.

Fig. 3.8 The hydrogen bonding network between the donor (AP) and acceptor (CAA) moieties of CT complex

97

Table 3.8

Crystal data and structure refinement for CT complex.

Empirical formula C10 H9 Cl2 N3 O5

Formula weight 322.10

Temperature 296 (2) K

Wavelength 0.71073 Å

Crystal system Triclinic

Space group P˗1

Unit cell dimensions

a = 6.7633(3) Å, α = 106.125(2)°

b = 9.4151(4) Å, β= 105.826(3)°

c = 11.0205(5) Å, γ = 102.004(2)°

Volume 617.27(5) Å3

Z 2

Density (calculated) 1.733 Mg/m3

Absorption coefficient 0.550 mm-1

F (000) 328

Crystal size 0.350 x 0.250 x 0.200 mm3

Theta range for data collection 2.366 to 36.262°.

Index ranges −10<=h<=10, −15<=k<=13, −18<=l<=16

Reflections collected 19978

Independent reflections 5167 [R (int) = 0.0281]

Completeness to theta = 25.242° 100.0 %

Absorption correction Semi-empirical from equivalents

Max. and min. transmission 0.7471 and 0.7107

Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 5167 / 8 / 205

Goodness-of-fit on F2 1.018

Final R indices [I>2sigma(I)] R1 = 0.0407, w R2 = 0.1007

R indices (all data) R1 = 0.0744, w R2 = 0.1214

Extinction coefficient n/a

Largest diff. peak and hole 0.369 and −0.615 e.Å-3

98

Table 3.9

Bond lengths (Å) and bond angles (o) in crystal of CT complex

Bond Bond length (Å) Bond Bond angle(o)

C(1)-N(3) 1.312(2) N(3)-C(1)-N(2) 118.83(13)

C(1)-N(2) 1.3520(18) N(3)-C(1)-N(1) 120.94(13)

C(1)-N(1) 1.3534(17) N(2)-C(1)-N(1) 120.21(14)

C(2)-N(1) 1.343(2) N(1)-C(2)-C(3) 119.77(14)

C(2)-C(3) 1.355(2) N(1)-C(2)-H(2) 120.1

C(2)-H(2) 0.9300 C(3)-C(2)-H(2) 120.1

C(3)-C(4) 1.384(2) C(2)-C(3)-C(4) 116.79(16)

C(3)-H(3) 0.9300 C(2)-C(3)-H(3) 121.6

C(4)-N(2) 1.318(2) C(4)-C(3)-H(3) 121.6

C(4)-H(4) 0.9300 N(2)-C(4)-C(3) 123.99(14)

C(5)-O(4) 1.2499(15) N(2)-C(4)-H(4) 118.0

C(5)-C(10) 1.3906(19) C(3)-C(4)-H(4) 118.0

C(5)-C(6) 1.5187(19) O(4)-C(5)-C(10) 126.45(13)

C(6)-O(3) 1.3028(16) O(4)-C(5)-C(6) 115.13(12)

C(6)-C(7) 1.3599(18) C(10)-C(5)-C(6) 118.42(11)

C(7)-C(8) 1.4298(18) O(3)-C(6)-C(7) 122.21(13)

C(7)-Cl(2) 1.7179(13) O(3)-C(6)-C(5) 116.33(11)

C(8)-O(1) 1.2272(15) C(7)-C(6)-C(5) 121.46(12)

C(8)-C(9) 1.5390(18) C(6)-C(7)-C(8) 120.36(12)

C(9)-O(2) 1.2397(16) C(6)-C(7)-Cl(2) 121.35(10)

C(9)-C(10) 1.3997(17) C(8)-C(7)-Cl(2) 118.29(10)

99

C(10)-Cl(1) 1.7231(13) O(1)-C(8)-C(7) 123.60(13)

N(1)-H(1) 0.886(15) O(1)-C(8)-C(9) 117.25(12)

N(3)-H(3B) 0.842(13) C(7)-C(8)-C(9) 119.14(11)

N(3)-H(3C) 0.889(14) O(2)-C(9)-C(10) 125.88(13)

O(3)-H(3A) 0.921(18) O(2)-C(9)-C(8) 116.24(11)

O(5)-H(5A) 0.870(16) C(10)-C(9)-C(8) 117.88(11)

O(5)-H(5B) 0.900(15) C(5)-C(10)-C(9) 122.63(12)

C(5)-C(10)-Cl(1) 118.80(10)

C(9)-C(10)-Cl(1) 118.55(10)

C(2)-N(1)-C(1) 121.45(13)

C(2)-N(1)-H(1) 119.7(13)

C(1)-N(1)-H(1) 118.8(13)

C(4)-N(2)-C(1) 117.75(13)

C(1)-N(3)-H(3B) 123.3(12)

C(1)-N(3)-H(3C) 117.2(13)

H(3B)-N(3)-H(3C) 119.3(17)

C(6)-O(3)-H(3A) 107(3)

H(5A)-O(5)-H(5B) 105.2(18)

100

Table 3.10

Hydrogen bonds (Å) in crystal structure of CT complex.

D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

N(3)-H(3B)...O(5)#1 0.842(13) 1.984(15) 2.799(2) 162.5(17)

N(3)-H(3C)...O(4)#2 0.889(14) 2.098(15) 2.9329(18) 156.0(19)

N(1)-H(1)...O(1)#1 0.886(15) 1.885(15) 2.7649(16) 172(2)

O(5)-H(5B)...O(2) 0.900(15) 1.987(17) 2.8581(16) 162(2)

O(5)-H(5B)...O(1) 0.900(15) 2.30(2) 2.8514(16) 119.4(18)

O(5)-

H(5A)...Cl(1)#3 0.870(16) 2.93(2) 3.4876(12) 123.7(19)

O(5)-

H(5A)...O(2)#3 0.870(16) 1.971(18) 2.8078(16) 161(2)

O(3)-H(3A)...N(2)#2 0.921(18) 1.98(3) 2.7633(16) 142(3)

O(3)-H(3A)...O(4) 0.921(18) 2.08(4) 2.6276(17) 117(3)

Symmetry transformations used to generate equivalent atoms:

#1 ˗x+1,˗y,˗z #2 ˗x+2,˗y+1,˗z+1 #3 ˗x+2,˗y+1,˗z

Table 3.11

Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 10 3) for the

CT complex.

Hydrogen atom x y z U (eq)

H(2) 2247 584 −3034 44

H(3) 4767 3021 −2099 48

H(4) 6364 4079 222 44

H(1) 1420(30) −601(19) −1640(20) 48(6)

H(3A) 13950(60) 6240(20) 7430(30) 143(15)

H(3B) 1870(30) −605(17) 558(17) 35(5)

H(3C) 3510(30) 740(20) 1735(15) 51(6)

H(5A) 9730(40) 3090(30) −1027(19) 73(8)

H(5B) 10370(40) 3360(20) 363(16) 65(7)

101

Table 3.12

Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for the CT

complex. U (eq) is defined as one third of the trace of the orthogonalized Uij tensor.

Atom x y z U (eq)

C(1) 3300(2) 1003(2) 70(1) 28(1)

C(2) 2908(3) 1058(2) -2102(2) 36(1)

C(3) 4390(3) 2486(2) -1560(2) 40(1)

C(4) 5319(3) 3111(2) -167(2) 37(1)

C(5) 13025(2) 6739(2) 5454(1) 26(1)

C(6) 12867(2) 5181(2) 5632(1) 26(1)

C(7) 12035(2) 3827(2) 4555(1) 25(1)

C(8) 11252(2) 3837(2) 3215(1) 25(1)

C(9) 11475(2) 5403(2) 3004(1) 27(1)

C(10) 12322(2) 6758(1) 4151(1) 26(1)

N(1) 2396(2) 330(1) -1290(1) 30(1)

N(2) 4810(2) 2415(1) 642(1) 33(1)

N(3) 2778(3) 285(2) 854(2) 39(1)

O(1) 10388(2) 2654(1) 2205(1) 41(1)

O(2) 10871(2) 5341(1) 1817(1) 43(1)

O(3) 13566(2) 5220(1) 6869(1) 39(1)

O(4) 13795(2) 7893(1) 6525(1) 39(1)

O(5) 9953(2) 2612(1) -453(1) 40(1)

Cl(1) 12556(1) 8522(1) 3935(1) 40(1)

Cl(2) 11869(1) 2068(1) 4760(1) 37(1)

Table 3.13

Anisotropic displacement parameters (Å2x 103) for the CT complex. The anisotropic displacement

factor exponent takes the form: -2p2 [h2 a*2U11 + ... + 2 h k a* b* U12]

Atom U11 U22 U33 U23 U13 U12

C(1) 28(1) 24(1) 29(1) 6(1) 6(1) 11(1)

C(2) 43(1) 33(1) 28(1) 7(1) 8(1) 12(1)

C(3) 46(1) 34(1) 40(1) 14(1) 16(1) 9(1)

C(4) 33(1) 25(1) 42(1) 6(1) 8(1) 4(1)

102

C(5) 27(1) 25(1) 22(1) 4(1) 7(1) 6(1)

C(6) 26(1) 28(1) 21(1) 7(1) 7(1) 7(1)

C(7) 28(1) 22(1) 26(1) 11(1) 9(1) 8(1)

C(8) 28(1) 22(1) 23(1) 6(1) 9(1) 4(1)

C(9) 32(1) 22(1) 22(1) 7(1) 7(1) 4(1)

C(10) 30(1) 18(1) 25(1) 5(1) 7(1) 4(1)

N(1) 32(1) 20(1) 28(1) 2(1) 4(1) 5(1)

N(2) 33(1) 28(1) 27(1) 3(1) 3(1) 7(1)

N(3) 45(1) 34(1) 37(1) 14(1) 13(1) 10(1)

O(1) 63(1) 22(1) 26(1) 5(1) 11(1) 1(1)

O(2) 70(1) 26(1) 23(1) 9(1) 7(1) 7(1)

O(3) 57(1) 32(1) 22(1) 10(1) 6(1) 11(1)

O(4) 52(1) 28(1) 24(1) -1(1) 5(1) 8(1)

O(5) 59(1) 34(1) 29(1) 13(1) 13(1) 19(1)

Cl(1) 54(1) 21(1) 38(1) 10(1) 13(1) 7(1)

Cl(2) 47(1) 28(1) 40(1) 18(1) 13(1) 13(1)

3.3.10 TG/DTA Analysis

The composition and thermal stability of the CT complex in order to compare with donor

and acceptor were evaluate using TG/DTA analysis. TG/DTA curves of the donor, acceptor

and their resulting CT complex were depicted in (ESI, Fig. A−C 10S). The data of thermal

analysis has been reported in Table 7. The TGA thermogram data of the CT complex shows

step wise degradation. In the first step most of weight of the compound lost 99.53 % at 99.9

0C and in the last step remaining 2.05% weight lost at 899.3 0C.In the DTA thermogram

spectrum of the CT complex, the exothermic peaks were observed at ∆H = −4.400 uV/gm,

∆H = −10.507 uV/ gm, ∆H = −10.809 uV/gm and ∆H = −9.336 uV/mg corresponding to the

temperature at 218.5 0C, 634.4 0C, 644.4 0C and 658.2 0C, respectively. The exothermic peak

appeared at ∆H = −9.336 uV/mg in the spectrum of the CT complex shows that the crystal

structure of the compound completely change at around 658.2 0C. From the DTA

thermogram spectrums of the donor (AP) and acceptor (CAA) that endothermic peaks were

103

observed at ∆H = +1.1274 uV/mg, +0.0588 uV/mg and +1.045 uV/mg at 129 0C, 181 0C and

285 0C temperatures , respectively, which corresponds to their melting points. From the DTA

thermogram, it is clearly observed that exothermic peaks were not detected in the spectrum

of neither donor and nor acceptor, which were observed in the spectrum of the CT complex.

Fig. 3.10 (A)

Fig. 3.10 (B)

104

Fig. 3.10 (B)

Fig. 3.10 A-C (ESI-10): TG/DTA spectrum for the (A) donor (AP), (B) acceptor (CAA) and (C)

[(AP)+(CAA)−] CT complex.

This is strongly favoured for the formation of the CT complex. From the TG curve of the

donor it is inferred that the decomposition of the compound takes place in two stages. In the

first stage, 98.5% weight eliminates corresponding to the temperature in the range of

100−183 oC and in the second stage decomposition noticed between the temperature in the

range of 183−500 °C incurs a weight loss of 2.0 % of the donor into gaseous. From the

comparative study of the donor, acceptor and CT complex, it was found that the weight of

the complex lost with change of temperature at different rate compared to the donor and

acceptor molecules, which is strongly supported to the formation of newly CT complex.

105

3.3.11 DNA Binding Studies

DNA is a significant biological target for number of drugs and the alterations of the DNA

structure often are linked with cytotoxic activity due to its chief role in cellular replication

[20, 62]. Metal complexes can interact to DNA through either covalent bonding where the

labile ligand of the metal ion can be replaced by a nitrogen base of DNA such as guanine N7

and/or through noncovalent interactions i.e., intercalative, electrostatic or groove binding of

complexes to DNA helix along major or minor grooves [63−65].

3.3.11.1 Absorption Spectral Titrations

Electronic absorption spectroscopy is one of the most suitable tools to scrutinize the

interaction of small molecules with protein and DNA [66, 67]. The interaction between the

complex and DNA is expected to perturb the ligand cantered transitions of complex. In the

UV region, the Ct−DNA exhibit intense absorption bands at 260 nm attributed to π→π* intra

ligand transition. Upon addition of charge transfer complex, Ct−DNA exhibited decrease in

molar absorptivity without any significant shift in band position (Fig. 11), which suggest

that the charge transfer complex might be interacts with Ct−DNA via intercalative mode.

In general, hyperchromism demonstrated that the binding of any small molecules to

Ct−DNA could be ascribed to electrostatic interaction with the exterior phosphates of DNA

duplex while the degree of observed hypochromism is commonly related with the potency

of intercalative interaction [64, 65].

3.3.11.2 Steady State Fluorescence

To further clarify the interaction of charge transfer complex with Ct−DNA, steady state

fluorescence experiment was carried out. The fluorescence intensity of a compound usually

reduces by a number of molecular interactions (excited-state reactions, molecular

106

rearrangements and energy transfer) [68], resulting into fluorescence quenching. Stern–

Volmer constant (Ksv) is used to estimate the fluorescence quenching efficiency.

Fig 3.11 Absorption spectra of Ct−DNA (50 µM) in Tris-HCl buffer of PH 7.2 on addition of increasing

concentration of CT complex (0−50 µM).

Fig 3.12 Effect of increasing concentration (0−30 µM) of Ct−DNA on the fluorescence spectra of CT

complex at room temperature.

107

Fig 3.13 (A) Stern-volmer plot (B) Modified Stern-Volmer plot steady state fluorescence quenching of

Ct−DNA with increasing concentration of CT complex.

Fig 3.14 (A) Fluorescence quenching spectra of fluorogenic dyes Ct−DNA complex in presence of increasing

concentration of CT complex (0−40 µM). (B) Stern-Volmer plot of EB−Ct DNA complex with CT complex.

108

Fig 3.15 Displacement analysis. Flourescence quenching of Ct−DNA−hoechst complex with increasing

concentration (0−60 µM) of CT˗ complex.

Fig.3.16 CD Spectra of Ct-DNA (50 µM) in 10 mM Tris-HCl buffer of pH 7.2 with varying concentration of

CT complex. Each spectrum is obtained at 25oC.

109

Fig.3.17. Effect of CT complex on the viscosity of Ct-DNA. Concentration of Ct-DNA kept constant and

varying the concentration of CT complex at 25oC.

A decrease in relative fluorescence intensities (RFI) was observed, and the data were

analyzed according to the Stern˗Volmer equation [69].

𝐹0

𝐹= 1 + 𝐾𝑠𝑣[𝑄] = 1 + 𝐾𝑞𝜏0[𝑄]

where Fo and F are the fluorescence intensities in the absence and presence of Ct−DNA

(quencher), KSV is the Stern˗Volmer constant, [Q] is the molar concentration of quencher,

and kq and τo are the bimolecular quenching rate constant and the lifetime of the protein

fluorescence in the absence of quencher, respectively. Fig. 12 shows the characteristic

changes in fluorescence emission spectra during the titration of complex (30 µM) with

Ct−DNA. The results illustrate that on consequent addition of Ct−DNA the fluorescence

intensity of charge transfer complex is decreased gradually at 363 nm without any noticeable

shift in the emission wavelength, indicating direct interaction between charge transfer

complex and Ct−DNA. To quantify the results of fluorescence quenching, Ksv was calculated

110

from the slope of the Fig. 13(A) and it was found to be 3.29 x 103 M−1, which is consistent

with earlier studies [40, 62]. A linear Stern–Volmer plot as obtained in indicates that only

one kind of quenching process occurs, either static or dynamic quenching [64]. Bimolecular

rate constant (Kq), which gives information pertaining to the quenching process, was

calculated by the following equation

𝐾𝑞 =𝐾𝑠𝑣

𝜏0

The value of Kq 3.29 x 1011 M−1 Sec-1 is found to be higher than the limiting diffusion rate

constant (2.0 × 1010 M−1 Sec−1) [64]. Therefore, interaction of charge transfer complex with

Ct−DNA might have occurred by complex formation rather than by dynamic collision.

So far, it is clear that from the previous steady state fluorescence study charge transfer

complex form static complex with Ct−DNA. We determined the binding constant (Kb) and

the number of binding sites (n) by using the following modified Stern˗Volmer equation:

𝐿𝑜𝑔(𝐹0 − 𝐹)

𝐹= 𝐿𝑜𝑔 𝐾𝑏 + 𝑛𝐿𝑜𝑔[𝑄]

This equation show a linear temperature dependence of log [(Fo − F)/F] versus log [Q] plots,

the slopes and intercepts of which were equal to n and log Kb values, respectively. In our

case value of Kb found to be 6.75 x 105 M-1 at 298 K and there was only one binding site.

111

3.3.11.4 Ethidium Bromide Displacement Assay

In order to further explore the interaction mode of charge transfer complex with Ct−DNA, a

competitive binding experiment using EB as a probe was carried out. EB

(3,8˗diamino˗5˗ethyl˗6˗phenylphenanthrium bromide) is a conjugate planar molecule with

very weak fluorescence intensity due to fluorescence quenching of the free EB by solvent

molecules but it is greatly enhanced when EB is specifically intercalated into the adjacent

base pairs of double stranded DNA [70]. The increased fluorescence can be quenched upon

the addition of the second molecule which could replace the bound EB or break the

secondary structure of the DNA. On addition of charge transfer complex to Ct−DNA pre-

treated with EB ([DNA]/[EB] = 1) a significant reduction in the emission intensity at 595

nm was observed, indicating that the replacement of the EB fluorophore by the complex

which results in a decrease of the binding constant of ethidium bromide to DNA (Fig. 14 A).

The extent of quenching of the emission intensity gives a measure of the binding propensity

of the interacting molecule to Ct−DNA. The Stern–Volmer quenching constant value, Ksv,

obtained as a slope of F0/F vs Q ([complex]/[DNA]) was evaluated for complex was found

to be 8.90 x 103 M−1 (Fig. 14 B ). The high Ksv value of the charge transfer complex

indicated their significant binding with Ct−DNA. The mode of interaction is further

confirmed by another experiment in which Hoechst 33258 dye was taken as a marker for

groove binding [71].Hoechst Ct−DNA complex titrated with increasing concentration of

charge transfer complex (0−60μM) no quenching of the fluorescence of hoschet˗Ct˗DNA

complex was observed (Fig. 15 ). Hence, it can be verified that charge transfer complex

exhibit intercalative binding mode with the Ct−DNA.

112

3.3.11.5 Circular Dichroism (CD) studies

CD spectroscopy is the one of the sensitive technique by which we can detect any changes

in the conformation of DNA/protein upon interaction of small molecules or ligand

[42,72,73]. In general CD spectrum of B-DNA consists of two major peaks at 243 nm

(negative) and 275 nm (positive) which correspond to right hand helicity and base pair

stacking respectively [74]. It is also well documented, there are negligible or no changes in

the both the peaks when ligand binds to non-intercalative mode to DNA, whereas

intercalative ligand change the intensity of both the bands. When we incubate the different

concentration of CT complex with Ct-DNA and spectra were recorded. It was observed that

(Fig. 16), when the concentration of CT complex increases both the peak intensity of Ct-

DNA get changed. The planer ligand molecules such as pyridine, thiadiazole rings could

partially insert into the DNA base pair and form partial stacking of the complexes with the

base pairs [75]. It is evident from figure CT complex having both π-acceptor molecule

Chloranilic acid and planer molecule in the form of 2-aminopyridine which interfere with

the both the helicity and base pair stacking of Ct-DNA. So, from that observation our CT

complex might be intercalates into the Ct-DNA.

3.3.11.6 Relative Viscosity studies

To further investigate the mode of interaction of CT complex with Ct-DNA, relative

viscosity analysis was done. It is a very sensitive and reliable technique to confirm any

change in the length of DNA upon addition of small molecule or ligands [76]. In general a

classical intercalator molecule increase the overall viscosity of DNA, which intercalate

inside the nitrogenous bases and separate the nitrogenous bases and overall increase the

length of DNA helix whereas groove binder and electrostatic interacting molecule could bent

and kink to the DNA helix, thereby reducing the overall length of the DNA helix and

113

consequently its relative viscosity [77, 78]. It is evident from the Fig.17 , when we increases

the ratio of CT complex to Ct-DNA its relative viscosity increases. So from that observation

we can conclude that nature of interaction of CT complex with Ct-DNA is intercalative.

114

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Chapter -4

Synthesis, Spectral, UV–Visible, DNA interaction and

Thermodynamic Studies of the Hydrogen-Bonded Charge-

Transfer Complex between 2-Amino-4-methylthiazole and

Chloranilic Acid at different Temperatures

121

4.1 INTRODUCTION

In the last few decades, numerous studies were devoted to the synthesis and characterization

of new complex molecular assemblies containing electron donor and π˗acceptor organic

molecules have been reported either in liquid or in solid states [1-9]. Enormous amount of

work has been reported on the CT-complex of organic acceptor (CAA) with various organic

donors [10-14]; in many areas of research. The CT complexes act as intermediate in wide

variety of reactions involving electron rich and electron deficient molecules. We repute in

this paper CAA and AMT have been selected because they are chemically highly reactive

compounds. The CAA is a strong π-acceptor and readily forming anion radical during the

complexation between CAA and AMT molecules and formed free radical ion pairs (scheme-

1) [10, 15]. Charge transfer complex are formed through intermolecular hydrogen bonding

(N+---H—O−), i.e., the acidic proton of the acceptor moiety will be transferred to the donor

moiety, which is generally routed for the formation of ion pair adducts (scheme-2) [14−27].

Hydrogen bond plays an extremely significant role on determining the structure stability of

many chemical complexes and biological macro molecules. The charge transfer interaction

between organic donor and acceptor has also involved as electron transfers from the HOMO

of the donor to the LUMO of the acceptor through resonance in many photophysical

processes and photochemical reactions [6, 28]. The magnitude of the charge transferred

depends on the donor, acceptor and solvent properties. The molecular association between

donor and acceptor molecules is influenced not only by the functional group containing

nitrogen, oxygen or Sulphur atoms present at their positions but also by the nature of the

solvents [8, 29]. The strong association will be more in less polar solvent since less polar

solvent has high dielectric constant. A lot of research has been carried out for the synthesis

122

of different analogs of donor and acceptor moieties to improve the chemical and

pharmacological potentials of these nuclei [30]. The study of the molecular interaction

between donor and acceptor is due to wide applications [30-37]. These organic molecules

exist in different isomeric forms, having enormous synthetic and biological importance such

as antibacterial, antifungal, antioxidant, anticancer, DNA-binding/cleavage and ion transfer

through lipophilic membranes [30, 38-47]. Charge transfer complexes also play an important

role in the field of magnetic, electrical conductivity and optical properties [48, 49]. The

potential of the synthesis CT complex has been obtained in terms of the DNA binding

affinity by UV-Visible and fluorescence spectroscopy [50-52]. The aim of my study directed

to find the stability, stoichiometry, structure and modes of the interaction of Ct-DNA with

CT complex. Various types of techniques have been used for the experimental results.

4.2 EXPERIMENTAL STUDIES.

4.2.1 Chemical and reagents

The AMT (98 %) and CAA (99 %) were of analytical grade and used without further

purification. These chemicals were purchased from Sigma Aldrich (USA chemical

company). The spectroscopic grade acetonitrile (Merck) has been used throughout the

investigation.

4.2.2 Synthesis of Solid CT Complex.

The 1:1 solid CT complex was synthesized after mixing equimolar amounts of AMT

(0.22834 g, 2 mmol) and C AA (0.41796 g, 2 mmol) in acetonitrile. During mixing, the

saturated solution has undergone a change. After 2 hours, stirring of a saturated solution, the

solid CT complex was filtered and washed several times with little amounts of the

acetonitrile. The solid CT complex dried under vacuum over anhydrous calcium chloride.

The formed CT complex was characterized by elemental analysis (theoretical values are

123

shown in brackets): (theoretical values are shown in brackets): CT complex C10H8N2Cl2O4S

(M.W = 323.15 g, M.P. = 270 0C), C = 37.17 % (37.22 %), H = 2.49 % (2.53 %), N = 8.67%

(8.56 %), O = 19.99 % (18.81 %), Cl = 131.65 % (131.73 %), S = 9.92 % (9.98 %).

4.2.3 Preparation of Standard Stock Solutions.

Standard stock solutions of donor (AP) 10−2 M and acceptor (CAA) 10−2 M were prepared

by dissolving 0.082115 g of (AP) and 0.10449 g of (CAA) each in separate 50 ml volumetric

flasks in acetonitrile. Solution of the fixed concentration (1.0 x 10−4 M) of the donor was

prepared in 25 ml volumetric flask by diluting 10−2 solution and the solution of different

concentration of acceptor (1.0 × 10−4 M, 1.5 × 10−4 M, 2.0 × 10−4 M, 2.5 × 10−4 M, 3.0 ×

10−4 M) was prepared in a 25 ml individual volumetric flask by diluting 10−2 M solution with

polar solvent. By applying the same procedure the other solution of different concentrations

were also prepared in the same solvent.

4.2.4 Instrumentation Measurements.

4.2.4.1 Electronic Spectra

The electronic spectra of the donor, acceptor and CT complex at constant concentration of

the donor were recorded on Perkin Elmer Lambda-850 nm UV–visible spectrophotometer

in the region 200–700 nm at the different temperature in the range of 25 0C to 45 0C with a

1 cm quartz cell path length.

4.2.4.2 FTIR Spectra

The Perkin Elmer FT-IR Spectrometer (Spectrum Two) with KBr pellets was used to

evaluate the FTIR spectra of donor, acceptor and CT complex.

124

4.2.4.3 TG/DTA Analysis

The thermogravimetric (TG) and differential thermal analysis (DTA) of the donor, acceptor

and their resulting CT complex were carried out employing EXSTAR TG/DTA 6300 modal

with heating rate of 20 0C/min in nitrogen atmosphere.

4.2.4.4 1H NMR Spectra

The 1H NMR spectra for the CT complex was recorded in DMSO using the BRUKER

AVANCE II 400 MHz spectrometer with TMS as an internal reference.

4. 2.4.6 Mass Spectra

WATERS-Q-TOF MICROMASS (ESI-MS) mass spectrometer was used to obtain the

composition of novel 1:1 CT complex.

4. 2.4.7 DNA Binding Studies

The binding affinity and orientation pattern between Ct−DNA and CT complex was

investigated by the UV−Visible and fluorescence spectroscopy.

4. 2.4.7.1 UV–Visible Spectroscopy

The UV−Visible absorption spectra of the Ct−DNA on increasing concentrations of CT

complex were recorded on UV−1800 Shimadzu spectrophotometer. The ratio of the

absorbance of Ct−DNA at 260 and 280 nm in 10 mM Tris-HCl buffer (pH 7.2) was 1.85,

indicating the DNA was sufficiently free of protein [51]. The concentration of Ct-DNA was

determined from its absorption intensity at 260 nm with a molar extinction coefficient of

6600 M−1 cm−1 [52]. The DNA binding experiments were carried out by titration of

increasing concentrations of complex (0-40 μM) to a fixed concentration of Ct-DNA (40

μM) in the range of 230-320 nm. The blanks were used for each tube containing same amount

of complex present in sample without Ct-DNA. Thus, after baseline correction using Ct-

125

DNA solution as a blank, any measured absorbance is due to the presence of the CT complex

complexed with Ct-DNA.

4. 2.4.7.2 Fluorescence Studies

All fluorescence experiments were carried out by fluorometric titration using a Shimadzu

spectroflurometer˗5301 (Japan) equipped with constant temperature holder attached with

Neslab RTE−110 water bath with an accuracy of ±0.1°C. The fluorescence titration was

performed against fixed concentration of complex (40 µM) with varying concentration of

Ct-DNA (0-40 µM). The emission spectra complex was recorded in the range of (335-450

nm) by exciting at 325 nm and slit width for both the excitation and emission are 5 nm and

10 nm respectively. In EB (ethidium bromide) displacement assay, a solution containing EB

(5 μM) and Ct-DNA (40 μM) was titrated with increasing concentration of CT complex. EB-

Ct-DNA complex was excited at 478 nm and emission spectra were recorded from 500 to

700 nm. In another experiment, 5 μM of Hoechst 33258, a well-known groove binder was

added to 20 μM of Ct−DNA. Ct-DNA-Hoechst 33258 complex was excited at 363 nm and

emission spectra were recorded from 350 to 600 nm.

4. 3 RESULTS AND DISCUSSION

4.3.1 Observation of CT spectra.

The UV–Visible absorption spectra of the AMT, CAA and their CT complex were recorded

on increasing concentrations of donor and fixed concentration (1.0 x 10−4 M) of acceptor.

The absorption spectra of the CT complex have been recorded using equal concentrated

solution of donor and acceptor without any further diluting. The samples were incubated at

various temperatures in the range of 25−45 0C. The absorbance measurements of (Ct−DNA)

− CTC interaction were performed by increasing concentration of the CT complex and the

spectra were recorded in the range of 230–320 nm (Fig.4. 9). The characteristic absorption

126

spectra of the acceptor, donor and their resulting CT complex along with their maximum

absorbance peaks were shown in Fig. 4. 1. These absorption peaks are associated with the

strong change in colour observed upon mixing of the reactants. The maximum absorbance

peak of the CT complex was observed at 212 nm in acetonitrile (Table-4.1). A new broad

peak appeared at maximum wave length 531 nm in the visible region, corresponding to the

color of the CT complex (Fig. 4.1). The observed electronic spectra have been investigated

is due to a transformation of the initially formed outer complex into an inner complex

followed by a fast reaction of the resulting inner complex (scheme-4.1). The results strongly

confirmed the formation of the proposed CT complex. The Gaussian function y = y0 + [A/

(w√ (π/2))] exp [−2(x − xc) 2/w2] has been used to analyses the CT absorption spectra, where

x and y denote wavelength and absorbance, respectively. The experimental results of the

Gaussian analysis are reported in Table-4.2. Benesi–Hildebrand [55] equation has been used

to determine the various physical parameters at different temperature, which are depicted in

Table-4.3. This equation is valid only when either concentration of donor or acceptor should

be larger than the other.

200 300 400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

(C)

(B)

(A)

Ab

so

rban

ce

Wavelength (nm)

Donor (AMT)

Acceptor (CLA)

CT complex

Fig. 4. 1 UV–Visible spectra of (A) AMT, donor (1 × 10−4 M); (B) CAA, acceptor (1 × 10−4 M) and (C)

[(AMT)+(CAA)−], CT complex (1 × 10−4 M + 1 × 10−4 M) in acetonitrile at 25 oC temperature.

127

200 250 300 350 400

0.0

0.2

0.4

0.6

0.8

1.0

Ab

so

rban

ce

Wavelength (nm)

CT at 25 oC in acetonitrile

CT at 30 oC in acetonitrile

CT at 35 oC inacetonitrile

CT at 40 oC in acetonitrile

CT at 45 oC in acetonitrile

Fig. 4. 2 UV-Visible spectra of [(AMT)+(CAA)−] CT complex (1 × 10−4 M + 1 × 10−4 M) in

acetonitrile at different temperature in the range of 25−45 0C.

2 4 6 8 10

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50 At 25

oC in methanol

At 30 oC in methanol

At 35 oC in methanol

At 40 oC in methanol

At 45 oC in methanol

[D] o

/A

1/[A]o

Fig.4.3 Benesi-Hildebrand plots [(AMT)+(CAA)−] CT complex [D]o/A vs 1/[A]o in acetonitrile.

128

The Benesi-Hildebrand analysis of formation (KCT) involves the measurement of the D--A

CT absorbance (A) as a function of varied [A]o when [A]o>> [D]o.

[D] + [A] ⇌ [DA]CT → [D+•+ A−•]

A plot of x = 1/[A]o vs y = [D]o/A gives a y-intercept = 1/εCT and slope = (1/KCTεCT)

defined as:

[D]o

A =

1

KCTɛCT∙

1

[A]o +

1

ɛCT

where [D]o is the total of donor (fixed), A is the CT absorption of DA complex at wavelength

λCT, [A]o is the total concentration of acceptor (varied) KCT is the equilibrium constant for

DA complex formation ε = molar absorptivity of the DA complex at λCT. From Fig. 4. 2, it

was observed that the absorption intensity of the complex decreasing with increasing

temperature. The physical parameters, KCT and ɛCT of the CT Complex were calculated from

the slopes and intercepts of the all linear plots at different temperature (Table-4.1). The all

straight line plots are shown in Fig. 4. 3. The energy (ECT) for the formed CT complex can

be calculated using G. Briegleb and Z. Angew [56] equation:

ECT = 1243.667

λCTnm

Where λCT is the wavelength of the CT band of the complex. The values of the ECT are given

in Table-4.3.

129

Table 4. 1

Absorption data for spectrophotometric determination of stoichiometry, absorption maxima (λCT),

formation constant (KCT) and molar absorptivity (ɛCT) of the [(AMT)+(CAA)−] complex in acetonitrile

at different temperature.

Concentration of

acceptor (M)

Concentration

of donor(M)

Absorbance

at CT (nm)

Formation constant (KCT)

l mol-1

Molar absorptivity

(CT)

l cm-1 mol-1

At 25 oC At 212 nm

2.6783 x 104 19.186 x 104

1.0 x 10−4 1.0 x 10−4 0.28687

1.5 x 10−4 1.0 x 10−4 0.40882

2.0 x 10−4 1.0 x 10−4 0.46243

2.5 x 10−4 1.0 x 10−4 0.49644

3.0 x 10−4 1.0 x 10−4 0.51538

At 30 oC At 212 nm

2.2080 x 104

18.225 x 104

1.0 x 10−4 1.0 x 10−4 0.24935

1.5 x 10−4 1.0 x 10−4 0.42241

2.0 x 10−4 1.0 x 10−4 0.58450

2.5 x 10−4 1.0 x 10−4 0.70442

3.0 x 10−4 1.0 x 10−4 0.82715

At 35 oC At 212 nm 2.0368 x 104

16.597 x 104

1.0 x 10−4 1.0 x 10−4 0.23610

1.5 x 10−4 1.0 x 10−4 0.40001

2.0 x 10−4 1.0 x 10−4 0.54380

2.5 x 10−4 1.0 x 10−4 0.68673

3.0 x 10−4 1.0 x 10−4 0.74962

At 40 oC At 212 nm

1.6365 x 104 15.272 x 104

1.0 x 10−4 1.0 x 10−4 0.21878

1.5 x 10−4 1.0 x 10−4 0.38275

2.0 x 10−4 1.0 x 10−4 0.50459

2.5 x 10−4 1.0 x 10−4 0.66565

3.0 x 10−4 1.0 x 10−4 0.71763

At 45 oC At 212 nm 1.6068 x 104

14.582 x 104

1.0 x 10−4 1.0 x 10−4 0.20444

1.5 x 10−4 1.0 x 10−4 0.36293

2.0 x 10−4 1.0 x 10−4 0.48416

2.5 x 10−4 1.0 x 10−4 0.58643

3.0 x 10−4 1.0 x 10−4 0.68652

130

Table 4.2

Gaussian curve analysis for the [(AMT)+(CAA)−] spectrum in acetonitrile at different temperatures.

Solvent Area of the curve (A) Width of the curv (w) Centre of the curve (xc) yo

Acetonitrile

At 25 oC 120.179 ± 5.891 151.726 ± 6.998 237.351 ± 3.884 0.0327 ± 0.005

At 30 oC 119.264 ± 5.738 150.425 ± 6.870 238.469 ± 3.765 0.0323 ± 0.005

At 35 oC 116.693 ± 5.428 148.693 ± 6.636 239.782 ± 3.582 0.0327 ± 0.005

At 40 oC 111.035 ± 4.895 145.657 ± 6.282 242.608 ± 3.286 0.0323 ± 0.005

At 45 oC 104.663 ± 4.344 141.872 ± 5.905 246.137 ± 2.971 0.0319 ± 0.005

Table 4. 3

Physical parameters of the [(AMT)+(CAA)−] CT complex in acetonitrile at different temperature.

Solvent Wavelength

λCT (nm)

Ionization

Potential

ID (ev)

Energy of

interaction

ECT (ev)

Resonance

energy

RN × 10−4

Oscillator

strength ƒ

× 10−6

Dipole

moment

μEN (D) x

10-2

Correlation

coefficient

(r)

Acetonitrile 317 10.586 3.923

At 25 oC 9.7774 6.269 85.778 0.99694

At 30 oC 9.2881 5.922 83.962 0.99801

At 35 oC 8.4590 5.331 80.589 0.99788

At 40 oC 7.7842 4.805 78.274 0.99767

At 45 oC 7.4327 4.468 77.334 0.99983

4. 3.2 Determination of Resonance Energy (RN)

The values of the resonance energy were calculated with the help of the following equation

[57].

ɛCT = 7.7 × 104

[hνCT

|RN|− 3.5]

131

Where εCT is the molar extinction coefficient of the complex at the maximum absorption of

the CT complex and νCT is the frequency of the CT absorption peak. The calculated values

of the formed CT complex at various temperatures are summarized in Table-3.

4.3.3 Determination of Ionization Potentials of the Donor (ID)

The ionization potential of the donor in the CT complex were calculated using the following

equation, which derived by Aloisi and Pignataro [58].

ID = 5.76 + 1.53 × 10−4 νCT

The νCT is wave numbers in cm−1 of the complex were resolved in acetonitrile. Ionization

potential value of the donor stabilizes the stability of the formed CT complex and values are

reported in Table-4.3.

4.3.4 Determination of Oscillator Strength (f) and Transition Dipole Moment (µEN)

The oscillator strength of the CT band, which is related to the transition probability, has been

calculated using the following relation given below [59].

ƒ = 4.32 × 10−9 ɛCT Δν1/2

Where Δν1/2 is the half-band width of the absorbance and εCT is the extinction coefficient at

maximum absorption of the CT band. Oscillator strength of the complex decreases with

increasing temperature. The calculated values of oscillator strengths of the CT complex at

different temperatures are depicted in Table-4.3.

The extinction coefficient is related to the transition dipole given as:

µEN = 0.0958√ɛCTΔν1/2

Δν

132

Where Δν ≈ ν at ɛCT and µEN is defined as –e ∫ ψex ∑i riψi dτ for the complex. Δν1/2 is half -

width in wave number unit. The calculated values of μEN for the CT complex at various

temperatures are given in Table-4.3.

4.3.5 Determination of Thermodynamics Parameters of the CT Complex

Evaluation of the formation constant (KCT) for the CT complex at different temperatures

allows us to determine the values of the Gibbs free energy change (ΔG) using the following

relation [60]:

ΔG = −2.303RT logKCT

Where ΔG is the free energy change of the CT complex (kJ mol−1), R is the gas constant

(8.314 J mol−1 K−1), T is the absolute temperature measured in Kelvin (273 + 0C) and KCT is

the formation constant of the CT complex at a definite temperature. It can be seen that the

negative value of ∆G revealed the donor-acceptor interaction process is spontaneous. The

values of the ∆G are reported in Table-4.4.

The plot of log KCT versus 1/T (Fig. 4. 4) allows calculating the values of enthalpy (∆H) and

entropy (∆S) at different temperature using van’t Hoff equation:

logKCT = −ΔH

2.303RT +

ΔS

2.303R

Where ΔH is the enthalpy change (kJ mol−1) of the CT complex, R is gas constant (8.314 J

mol−1 K−1), T is the absolute temperature in Kelvin (273 + °C), and KCT is the formation

constant of the CT complex at different temperature.

133

2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

R2 = 0.98122

logKCT = -0.05892 + 0.12239. X

log

KC

T

1/Tx10-2 (K)

Fig. 4. 4 Vant's Hoff plot of [(AMT)+(CAA)−] complex logKCT vs 1/T in acetonitrile at various temperature.

Table 4.4 Thermodynamic parameters of [(AMT)+(CAA)−] CT complex at different temperatures.

Solvent Free energy ΔG

(kJ mol-1)

Enthalpy ΔH x 10−3

(kJ mol-1)

Entropy ΔS x 10−3

(kJ K-1 mol-1)

Acetonitrile

At 25 oC −2.4413 −2.3434 −1.1282

At 30 oC −1.9957

At 35 oC −1.8219

At 40 oC −1.2820

At 45 oC −1.2540

Ross and Subramanian [61] reported that (1) when ∆H > 0 and ∆S > 0, hydrophobic forces;

(2) ∆H < 0 and ∆S < 0, van der Waals interaction and hydrogen bonds and (3) when ∆H < 0

and ∆S > 0, electrostatic interactions [62-63]. Therefore, van der Waals interactions or

hydrogen bonds are the main forces between the donor and acceptor in the investigated CT

complex. The values of thermodynamic parameters such as ΔH and ΔS of the complex at

different temperature were reported in Table-4.4.

134

4.3.6 FTIR Spectral Study

The infrared spectra of the donor, acceptor and their resulting complex were shown in Fig.

4.5. The IR absorption bands of the reactant molecules along with CT complex were also

summarized in the Table-4.6. The infrared spectrum of the formed CT complex could be

interpreted based on the expected electronic structure change upon complexation. It was

observed that most of the frequencies of donor decrease while most of the frequencies of

acceptor increase with changes in their intensities in the FTIR spectrum of CT complex. This

shift indicates that the charge transfer from donor to the acceptor during complex formation.

It was further supported to the formation of the CT complex by decreasing in the infrared

stretching frequency of the OH group of the acceptor molecule due to the intermolecular

hydrogen bond has been formed between donor and acceptor moieties. Consequently the OH

broad band appeared at 3351 cm−1 in the spectrum of CT complex, whereas in free CLA this

was observed at 3236 cm−1.

4000 3500 3000 2500 2000 1500 1000 500

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

(C)

(B)

(A)

Tra

nsm

itta

nce (

%)

Wavenumber (cm-1

)

Donor

Acceptor

CT complex

Fig. 4. 5 FTIR spectra of (A) donor (AMT); (B) acceptor (CAA); and (C) 1:1 [(AMT)+(CAA)−] CT complex

in the range of 4000−500 cm−1.

135

Table 4.4

Characteristic infrared frequencies and band assignments for donor (AMT), acceptor (CAA)

and [(AMT) + (CAA)−] CT complex in cm−1.

Compound Frequency Assignments

AMT

3432 (as), 3303 (sy) ν(−NH2)

3118 (s) ν(C–H); (aromatic)

2982 (ms), 2919 (mw), 2919 (w) ν(C−H) in –CH3 group

1619 (w) δ(−NH2); in plane bending

1519 (νs), ν(C=C) (ring)

1441(m) ν(C=N) (ring)

1379 (w) ν(C−N) (ring)

1323 (s) ν(C−S) (ring)

970 (w) δ(−NH2); out of plane bending

CLA

3236 (s, br) ν(OH)

1631 (ms) ν(C=C)

1538 (vs) ν(C=O) (ring)

1369 (s) ν(C−C) (ring)

1264 (w) ν(C−O)

753 (m) ν(C−Cl)

(AMT:CLA) CT complex

3351 (s ,br) ν(OH)

3225 (as), 3179 (sy) ν(NH2)

3092(s) ν(C−H); (aromatic)

2931 (br) ν(C−H) in –CH3 group

2848 (ms) ν(N+−H∙∙∙∙∙∙O−) bonding

1639 (s) ν(C=C); (aromatic ring)

1624 (m) ν(C=N)

1574 (s) ν(C=O)

1411 (w) ν(C−C); (aromatic ring)

1383 (m) ν(C−N)

1347 (w) ν(C−O)

838 (s) δ(−NH2); out of plane bending

772 (m) ν (C−Cl)

s, strong; w, weak; v, stretching; vs, very strong; m, medium; br, broad; sh, shoulder; as, asymmetric;

sy,symmetric

136

The asymmetric and symmetric stretching frequencies of the amino group are shifted to 3225

and 3179 cm−1 for the complex compared with 3432 and 3303 cm−1 for donor alone. Upon

complexation, a new band in the spectrum of the CT complex exhibited at 2848 cm−1, which

conforming the involvement of the both amino group and ring nitrogen of the AMT in

hydrogen bonding (N+−H∙∙∙∙O−) with the OH group of the CAA (scheme-2). The stretching

frequency of ν(C=C)(aromatic ring), ν(C=O) and ν(C−Cl) appears at 1639, 1574 and 772

cm−1 increases in the spectrum of the CT complex compared with free CAA, which were

appearing at 1631, 1538 and 753 cm−1 respectively. These shifts have been attributed due to

transfer of electron density from AMT to CAA moiety during the complex formation, which

is strongly supported to the formation of newly 1:1 CT complex.

4.3.7 1H NMR spectra

1H NMR spectra of 1:1 CT complex has been recorded in DMSO shown in Fig. 6. A sharp

signal at δ = 5.356 ppm was shifted downfield assigned to the H5 proton of the AMT in the

spectrum of the CT complex. An appearance of the new broad signal at δ = 8.622 ppm was

also shifted downfield attributed to the –NH+ species due to the change in the electronic

structure upon complexation through the proton transfer mechanism (scheme-2).

Fig.4.6 (A)

137

Fig. 4.6 (B)

Fig. 4.6 (A) and (B) 1H NMR spectrum of.1:1 [(AMT)+(CAA)−] CT complex in DMSO.

The singlet signal was recorded at δ = 2.1606 ppm assigned to the protons of methyl group

of AMT moiety. The 1H NMR analysis of the resulting CT complex ascertained that the

existence of proton transfer from –OH group of CAA to the ring nitrogen of AMT besides

charge transfer during the complex formation, which gives the extra stability to the formation

of the complexes.

Scheme -4.1 Mechanism of the CT reaction with proton transfer in acetonitrile at room temperature.

138

Scheme -4. 2 Molecular structure of the charge transfer transition of the hydrogrn bonded CT complex.

4.3.8 ESI-mass spectra

The electron spray ionization (ESI)-mass spectral analysis was ascertained the 1:1

stoichiometry of the formed CT complex. The substituent groups like –NH2 and –OH, which

lower the ionization potential increases the relative abundance of the characteristic

fragmentations of the CT complex. The molecular fragment [CTC−AMT]+ was ascertained

by the presence of a peak at m/z = 208.97. The high relative abundance peaks were appearing

at m/z = 210.98 and m/z = 206.97 corresponding to the molecular ion fragments

[CTC−AMT+2H]+ and [CTC−AMT−2H]+, respectively. These fragments proposed the

molecular structure of the resulting compound. In addition to these peaks, the molecular ion

peak also appeared at m/z = 115.06 assignable to the fragment [CTC-CAA+H]+, which

confirm the formation of [(AMT):(CAA)] charge-transfer complex. The ESI-mass spectrum

of the complex shown in Fig. 4. 7 and data for the peak assignment along with their

fragmentations has been summarized in Table-4.5. The other remaining molecular ion

fragmentation peaks at different m/z values, which are shown in the mass spectrum of the

complex were also revealed 1:1 stoichiometry of the CT complex.

139

Fig. 4.7 (A)

Fig. 4.7 (B)

140

Fig. 4.7 (C)

Fig. 4.7 (A) − (C) electron spray ionization-mass spectra (ESI-MS) of [(AMT)+(CAA)−] CT complex.

Table 4.5

The main characteristic molecular ion peaks corresponding to the fragmentations of the CT

complex.

Fragmentation Peak assignment (m/z values)

[CTC−CAA+H]+ 115.06

[CTC−AMT−Cl]+ 173.97

[CTC−CAA−CH3]+ 98.99

[CTC−AMT]+ 208.97

[CTC−AMT−2H]+ 206.97

[CTC –AMT+2H]+ 210.98

141

4.3.9 TG/DTA Analysis

The decomposition and thermal stability of the synthesis CT complex compared with donor

and acceptor molecules were confirmed using thermogravimetric (TG)/differential thermal

(DTA) analysis. It was observed that both the donor and acceptor decompose in the different

steps as shown in Fig. 4.8 (A) and (B), respectively. The data for the weight loss, degradation

enthalpy and temperatures of degradation were recorded in Table-4.7. From the thermal data

of the donor, acceptor and CT complex, it was concluded that CT complex degrades at a

higher temperature than that of the donor and acceptor, which gives the highest thermal

stability to the newly formed CT complex.

Fig. 4.8 (A)

142

Fig. 4.8 (B)

Fig. 4.8 (C)

Fig. 4.8 TG/DTA spectrum for the (A) donor (AMT), (B) acceptor (CAA) and (C) [(AMT)+(CAA)−] CT

complex.

143

The step wise degradation of the CT complex depicted in Fig. 4.8 (C). The formation of the

CT complex can also be expected due to change the value of the degradation enthalpy of the

CT complex compared with the donor and acceptor molecules. From the DTA thermogram

of the CT complex, it was observed that one exothermic peak (∆H = −3.552 uV/mg) was

appeared at 214.2 oC and another exothermic peak (∆H = −13.059 uV/mg) was observed at

632.1 0C each, which corresponds to the crystallization point of the compounds. In the DTA

thermogrames of the donor and acceptor the endothermic peaks (∆H = +1.235 uV/mg, ∆H

= +1.045 uV/mg) were observed at 182.2 0C and 285 0C, respectively, which corresponds to

the melting point of the donor and acceptor compounds. In the TG thermogram of the CT

complex, it was found that CT complex decompose in many steps at the different

temperature in the range of 28.65−899.2 0C with weight loses about in the range of

100.00−1.04% compared to the reactant molecules. This suggests the stability of the formed

CT complex.

Table 4.7

Weight loss, enthalpy (H), and degradation temperature (T) for AMT, CAA and [(AMT)+(CAA)−]

CT complex.

Step

AMT CLA CT Complex

Weight

loss (%)

H

(uV/gm)

T

(oC)

Weight

loss (%)

H

(uV/gm)

T

(oC)

Weight

loss (%)

H

(uV/gm)

T

(oC)

I 100 +1.235 28.18 100 +1.045 20.84 100 −3.552 28.65

II 95.25 99.76 99.75 199.9 99.81 −13.059 100.0

III 9.4 228.0 2.7 294.0 71.2 176.7

IV 8.459 300.1 − 0.899 499.8 63.26 300.0

V 7.539 400.1 −1.597 898.9 50.39 399.9

144

4.3.10 DNA binding studies

DNA is a significant biological target for number of drugs and the alterations of the DNA

structure often are linked with cytotoxic activity due to its chief role in cellular replication

[64, 65]. We have used aqueous media for the interaction of CT complex with DNA because

DNA present inside the cell is mostly hydrophilic in nature. Metal complexes can interact to

DNA through either covalent bonding where the labile ligand of the metal ion can be

replaced by a nitrogen base of DNA such as guanine N7 and/or through non-covalent

interactions i.e., intercalative, electrostatic or groove binding of complexes to DNA helix

along major or minor grooves [66-68].

4.3.10.1 Absorption spectral titrations

Absorption spectroscopy is one of the most extensively used techniques to decipher the

interaction of small molecules with protein and DNA [69, 70]. The interaction between the

complex and DNA is expected to perturb the ligand centred transitions of complex. In the

UV region, the Ct−DNA exhibit intense absorption bands at 260 nm attributed to π→π*

intraligand transition.

VI 6.415 500.4 33.68 500.3

VII 5.02 600.2 10.83 599.9

VIII 4.46 700.0 − 0.3 662.8

IX 4.32 800.0 −1.04 899.2

145

Fig. 4.9 Absorption spectra of Ct−DNA (40 µM) in Tris-HCl buffer of PH 7.2 on addition of increasing

concentration of CT complex (0−40 µM).

Fig. 4.10 Effect of increasing concentration (0−40 µM) of Ct−DNA on the fluorescence spectra of CT

complex at room temperature.

146

Fig. 4.11 (A) Stern-volmer plot (B) Modified Stern-Volmer plot steady state fluorescence quenching of

Ct−DNA with increasing concentration of CT complex.

Fig. 4.12 (A) Fluorescence quenching spectra of fluorogenic dyes Ct−DNA complex in presence of

increasing concentration of CT complex (0−30 µM). (B) Stern-Volmer plot of EB−Ct DNA complex with

CT complex.

147

Fig. 4.13 Displacement analysis. Fluorescence quenching of Ct−DNA−hoechst complex with increasing

concentration (0−30 µM) of CT˗ complex.

Upon addition of charge transfer complex, Ct-DNA exhibited decrease in absorption profile

without any spectral shift (either red or blue) (Fig. 4. 9), which suggest that there is

intercalation mode of interaction between charge transfer complex to Ct-DNA. In general,

hyperchromism demonstrated that the binding of any small molecules to Ct-DNA could be

ascribed to electrostatic interaction with the exterior phosphates of DNA duplex while the

degree of observed hypochromism is commonly related with the potency of intercalative

interaction [67, 68].

148

4.3.10.2 Steady state fluorescence

To further explore the interaction of charge transfer complex with Ct−DNA, steady state

fluorescence experiment was carried out. Stern–Volmer constant (Ksv) is used to estimate

the fluorescence quenching efficiency. A decrease in relative fluorescence intensities (RFI)

was observed, and the data were analyzed according to the Stern˗Volmer equation [71].

𝐹0

𝐹= 1 + 𝐾𝑠𝑣[𝑄] = 1 + 𝐾𝑞𝜏0[𝑄]

Where Fo and F are the fluorescence intensities in the absence and presence of Ct−DNA

(quencher), KSV is the Stern˗Volmer constant, [Q] is the molar concentration of quencher,

and Kq and τo are the bimolecular quenching rate constant and the lifetime of the protein

fluorescence in the absence of quencher, respectively.It was observed that when we

increasing the concentration of Ct-DNA to fixed concentration of CT complex, there is

quenching of fluorescence intensity of CT complex. It was also observed that there is no

noticeable shift in fluorescence intensity of CT complex with gradual increase in Ct-DNA

(Fig. 4.10). To quantify the extent of fluorescence quenching, Ksv was calculated from the

slope of the curve between Fo/F and Ct-DNA concentration. Fig. 4. 11 (A). The value of Ksv

was found to be 4.62 x 103 M−1, which is consistent with earlier studies [30, 52]. There are

two types of quenching either dynamics and static, in the case of dynamics quenching there

are molecular collision between ligand and acceptor molecules and increasing with

increasing temperature, whereas in the case of static quenching there is formation of complex

between ligand and acceptor molecule and decreases with increasing temperature [67].

Bimolecular rate constant (Kq), which gives information pertaining to the quenching

process, was calculated by the following equation

𝐾𝑞 =𝐾𝑠𝑣

𝜏0

149

The value of Kq 4.62 x 1011 M−1 Sec-1 was found to be higher than the limiting diffusion rate

constant (2.0 × 1010 M−1 Sec−1) [67]. Therefore, it was confers that the interaction of charge

transfer complex with Ct-DNA through formation of complex between these two

components that is static mode of quenching.

4.3.10.3 Binding parameter of charge transfer complex with Ct-DNA

It was observed from the previous steady state fluorescence study charge transfer complex

form static complex with Ct-DNA. We determined the binding constant (Kb) and the number

of binding sites (n) by using the following modified Stern˗Volmer equation:

𝐿𝑜𝑔(𝐹0 − 𝐹)

𝐹= 𝐿𝑜𝑔 𝐾𝑏 + 𝑛𝐿𝑜𝑔[𝑄]

This equation show a linear temperature dependence of log [(Fo − F)/F] versus log [Q] plots,

the slopes and intercepts of which were equal to n and log Kb values, respectively (Fig. 4.11

B). In our case value of Kb found to be 7.24 x 103 M-1 at 298 K and there was only one

binding site.

4.3.10.4 Ethidium bromide displacement assay

To study the mode of drug-DNA interaction we used several fluorescent dyes whose binding

modes were well established [72]. Any small molecule that competitively replaces a bound

dye from DNA helix is expected to bind with DNA in the similar fashion as that of the bound

dye [73]. Thus on addition of a small molecule, any change in the fluorescence intensity of

the dye–DNA complex can give us valuable information regarding their mode of interaction.

EB (3,8˗diamino˗5˗ethyl˗6˗phenylphenanthrium bromide) is a conjugate planar molecule

with very weak fluorescence intensity due to fluorescence quenching of the free EB by

solvent molecules but it is greatly enhanced when EB is specifically intercalated into the

adjacent base pairs of double stranded DNA [61]. The increased fluorescence can be

150

quenched upon the addition of the second molecule which could replace the bound EB from

the DNA. On addition of charge transfer complex to Ct-DNA pretreated with EB, there is

quenching of fluorescence intensity of EB-Ct-DNA complex was observed (Fig. 4.12 A).

The extent of quenching of the emission intensity gives a measure of the binding propensity

of the interacting molecule to Ct-DNA. The Stern–Volmer quenching constant value, Ksv,

obtained as a slope of F0/F vs Q ([complex]/[DNA]) was evaluated for complex was found

to be 4.9 x 103 M−1 (Fig. 4.12 B). The high Ksv value of the charge transfer complex

indicated their significant binding with Ct-DNA. The sites where CT complex bind with Ct-

DNA is further confirmed by another experiment in which Hoechst 33258 dye was taken as

a marker for groove binding [74]. It was observed that, there is no fluorescence intensity

quenching of Hoechst-Ct-DNA complex with increasing concentration of CT complex

(0−30 μM) (Fig. 4.13). So, from this observation, it can be confer that there is intercalative

types of interaction of CT complex to Ct-DNA.

151

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