Spectroscopy

is the study of emission, absorption and scattering of radiation when atoms or

molecules undertake transitions between levels differ in energy. It is utilized

to predict and study the molecular structure, different aspects of absorption

and emission, electronic transitions and scattering of light. Electromagnetic

radiations are the swaying electric and magnetic field which are spreading perpendicular

to each other. This oscillation frequency divides the electromagnetic radiation

into different regions for example X-rays, visible, ultraviolet, infrared,

microwaves and radio waves.

In

scattering spectroscopy the energy lost by an incident photon is examined after

it undergoes an interaction with the molecule in which energy is exchanged. In

emission spectroscopy, the analysis of the emitted photon of specie as it

undergoes a transition from higher energy level to the lower energy level is

studied. The analysis of the absorbed photon of incident light as specie

undergoes transition from lower to higher level is studied in absorption

spectroscopy. Quantitative as well as qualitative analysis of a substance can

be done whether the substance is in pure or solution form. From the absorbance

value at any wavelength, we can know the concentration of species of interest

as well as quality of substance from a given wavelength. Decision of occurrence

of specific transition is based on choice rule.

For

absorption spectrum analysis in ultraviolet (UV) and visible region the general

interest wavelength differ is extremely small. However, it’s of great interest

because the transitions occurring during this vary according to the electronic

transitions in atoms or molecules and it form to the electronic spectrum

analysis. In visible spectrum analysis the interaction of matter and radiations

is established within the sort of color.

Chromophores

give color to the compound. The unsaturated bonds or groups owing to that

substances particularly have color area unit called as radical. Some groups

themselves don’t give color however increase the colors of different

chromophores per area unit termed as auxochromes. For example C=C, C=O, N?N

etc. area unit the models of chromophores. Whereas, C-Br, C-OH, C-NH2 area unit

the models of auxochrome. The presence of chromophores makes the absorption in

200-800nm wavelength vary. The wavelength maxima differ from radical to radical.

An

electron is excited from the lower energy orbital to the higher energy orbital

when a sample is exposed to photon and energy matches the energy of which

required for excitation. The electronic transition would occur from highest

occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital

(LUMO). Wavelength at the absorption is recorded by photometer.

Electronic Transitions

The absorption of

ultraviolet (UV) or visible radiations are involved with the excitation of

outer electrons of an atom. The electronic transition involves non-bonding n,

sigma ?, pi ? electrons. Typically d and f electrons are also concerned.

Electrons forming single bond are called as the sigma (?) electrons whereas the

electrons forming covalent bond are called as pi (?) electrons. The single or

non-bonding electrons are called as n electrons. Non-bonding electrons are loosely

bound than bonding electrons whereas ? electrons secure a lot of forcefully

than ? electrons. The ?*(anti-bonding orbit) has higher energy than ?* in

excited state.

Transition from bonding to

anti-bonding molecular orbital (N?V)

These

transitions occurring from the bonding orbitals in state to the upper energy

orbitals (anti-bonding orbitals) and called as N?V transitions. These can be

???* or ???* transitions. As an example in parrafins the N?V transitions occur from

???* whereas in olefins these ocuur ???*. The ???* transitions happens in so

much ultraviolet (UV) region whereas ???* transitions are close to ultraviolet

or visible region. ???* transitions occur within the compounds which have double

or triple bonds, aromatic rings, and carbonyl or chemical group. A compound

which have extended conjugation show absorption at higher wavelength. These

transitions typically give molar absorption constant between 1000-10,000

Lmol-1cm-1.

Transitions from Non-bonding to

anti-bonding orbitals (N?Q)

The

transitions occur from nonbonding orbital to anti-bonding are called as N?Q.

These are typically weaker than N?V transition. These may be due to n??* or

n??*. n??* transition mostly happens in the near or far ultraviolet (UV) region

are present in saturated molecules having single bond and unshared electrons

pairs, whereas, n??* transition occurs in the near ultraviolet region or

visible region and are normally present in the compounds where hetero atoms

with unshared electrons are multiply bonded to another atom e.g. C=O, S=O, N=N

etc.

A

chromophore produce two absorption peaks if both lone pair of electrons and pi

electrons are present together. For example in ethanol where oxygen is making

double bond to carbon as well as it has a lone pair of electrons. It means that

there is probability of the n??* transition as well as ???* transition. Molar absorptivity of n??* transition is less

and is from 10-1000 Lmol-1cm-1 whereas that for ???* transition is high and ranges

from 1000-10,000 Lmol-1cm-1.

Application of UV-Visible

Spectroscopy

The

wide application of UV-Visible Spectroscopy is Beer-Lambert Law. Lambert law

states that the amount of light absorbed is directly related to the path length

and is independent of the intensity of incident light.

Mathematically,

Log

I0/I = ? l

Whereas,

I0 = Intensity of incident light

I=

Intensity of transmitted light

l

= Path length

?

= Extinction coefficient

Whereas,

Beer law states that the amount of light absorbed is directly associated with the

concentration of species absorbed or the number of molecules of the species

absorbed within the material.

Log

I0/I = ? c

Whereas,

I0=

Intensity of incident light

I=

Intensity of transmitted light

?=

Molar Extinction constant

c

= concentration of sample

By

combining both the Beer and Lambert law we get,

Log

I0/I= ?cl

In

the above equation the term log I0 /I is replaced by A, which is

called absorbance

A=

?cl

This

law is the basis of the quantitative analysis of the substance. The system

should obey the Beer-Lambert law for analysis by spectrophotometry.

UV-Visible spectroscopy for

antioxidant activity estimation

Antioxidant

capacity of the compounds can be found out by using ultraviolet spectroscopy.

Antioxidants are can scavenge the free radicals and prevent the oxidation

reaction that can successively break the chain reaction which are a cause of

great damage specially in human body. The preventive power of any drug or

substance shows the quantity of drug required to stop any biological process.

In case of antioxidant activity this amount is signified by IC50. It signifies

the amount of antioxidant substance to reduce the concentration of a certain

radical to 50 percent. Therefore, IC50 is the antioxidant index as well as it signifies

that how much amount of antioxidant is needed to reduce the 50 ? of the

radical. In UV-spectroscopy, the spectra of radical is taken with and without

the addition of antioxidant and then antioxidant activity is determined from

the difference.

The

? radical scavenging activity (RSA) is calculated by using the formula

%RSA=

(AO-A)/AO 100

Where

A0 is the absorbance of radical in the absence of antioxidant and A

is the absorbance presence of antioxidant respectively. Percent radical scavenging

activity rest on the concentration of antioxidant. Graph is plotted between

percent radical scavenging activity (%RSA) and concentration of antioxidant added

and 50% consumption of radical is determined from the straight line and that

concentration of antioxidant is called as IC50. The lower the value of the IC50,

greater is the antioxidant activity.

Scavenging

Constant

The reaction expected

to be taking place in radical scavenging is

For all the molecules

reacting with the radicals the scavenging constant can be evaluated by using

well known Benesi- Hilderbrand equation for free radical scavenging,

A0=

Absorbance of radical in absence of antioxidant

A= Absorbance of

radical in presence of antioxidant

? G= extinction

coefficient of radical in absence of antioxidant

? H-G=extinction

coefficient of radical in presence of antioxidant

C?= amount of

antioxidant added

K= Scavenging constant

Plotting a graph

between AO/A-AO vs. 1/ CO results in straight line and scavenging constant

can be found from the ratio of intercept to slope.

Cyclic Voltammetry

Cyclic

voltammetry is a technique used to acquire qualitative information about

electrochemical reactions. It offers a rapid location of redox potentials of

the electroactive species and measures the connection amongst current and

voltage at the electrode surface when it is plunged in the arrangement of

electroactive species yet the setup is kept unstirred. In this way, the current

estimated is just because of the dispersion of electroactive specie at the surface

of electrode. In cyclic voltammetry the potential is sloped straightly

utilizing triangular waveform. The subsequent plot of current against voltage

is called cyclic voltammogram.

Cyclic voltammogram for a single

electron reversible process

In

cyclic voltammetry of a reversible system, the product of initial oxidation or

reduction is then reduced or oxidized individually on reversing the direction

of the scan.

Linear

sweep cyclic voltammetry depends on the analyte which should be electroactive,

the voltage scanrate and the rate of electron transfer (ET) process. Cyclic

voltammetry highly depends on the analyte being used.

Following

are the basic information which we obtained from cyclic voltammetry:

· Anodic

and Cathodic peak potentials (Epa, Epc)

· Peak

potential difference (?Ep)

· Anodic

and cathodic peak currents (Ipa, Ipc)

· Formal

or half wave potential (E1/2)

· Peak

potential at which I= I/2 (Ep/2)

Most

of the time reversible CV wave is displayed by the analyte when all the analyte

is recovered after the scan cycles (forward and reverse). One of the main

criteria of reversible couple is

?EP=

Epa-Epc ?0.058/n

Electron Transfer Process

Electron

transfer is a process which occurs between working electrode and solution

electroactive specie at the electrode surface. It comprises three types which

are as follows

· Reversible

electron transfer process

· Irreversible

electron transfer process

· Quasi-reversible

electron transfer process

Reversible Electron Transfer

Process

in electrochemical reversible process the cell

reverses the reaction by reversing the current and side product will appear or

no new reaction will occur. Following are the characteristics of an electrochemical

reversible electron transfer process.

· The separation of voltage between the current

peak is

?E = Epa – Epa

= 59/n (mV)

· The peak voltage position do not change as a

function of voltage scan rate

· The ratio of the anodic peak current and

cathodic peak current is equal to one

Ipa/Ipa

= 1

·

The peak

currents are directly related to the square root of the scan rate.

Cyclic voltammogram of reversible electron

transfer

Determination of antioxidant

activity by cyclic voltammetry

Cyclic

voltammetry is an important technique that can be operated for the measurement of

antioxidant activity. Primarily cyclic voltammogram of free radical is recorded

and peak current is noted. Upon successive addition of an antioxidant decrease

in the peak current of free radical is observed which shows quenching of free

radical by an antioxidant and concentration of free radicals get decreased in

this approach.

Following formula used

for determination of percent radical scavenging activity (%RSA) through cyclic

voltammetry.

%RSA

=

x

100

Where. Ip0 =

Peak current of a free radical

Ip = Peak

current of free radical in the presence of an antioxidant

Plot of (Ip0?Ip)/Ip0

verses different concentration of an antioxidant gives a straight line and from

the value of slope from the graph, antioxidant activity in term of IC30 and

IC50 is obtained.

Calculation

of scavenging constant (ks)

Scavenging constant

(Ks) is typically calculated using Benesi-Hildebrand equation in order to

measure the scavenging strength between the antioxidant and free radical.

Log

=

logKs + log

Whereas,

C? is concentration of an antioxidant added, Ipo is peak current in

the absence of antioxidant, Ip is peak current in the presence of an

antioxidant, Ks is scavenging constant

By by means of the

value of scavenging constant, an important thermodynamic parameter i.e. Gibb’s free

energy (?G) can be calculated with the formula given below:

?G = ? RT ln Ks

Where,

R is gas constant and T is absolute temperature

Calculation of Diffusion

coefficient (D?)

Diffusion coefficient

is a degree of speed with which ion diffuses towards the electrode in a given

solution. It is also known as diffusivity or diffusion constant. It gives you

the extent of the drift of the ions within the solution towards the electrode.

Randle-Sevick equation is used to calculate diffusion coefficient.

iP = 268, 600 n3/2 AD1/2

C?1/2

iP is the

peak current (µA), n is the number of electrons in Red-ox reaction, F is

Faradays constant (C mol-1), A is an area of an electrode (cm2),

C is the concentration (mol. cm-3), ? is scan rate (V.s-1),

D is diffusion coefficient (cm2.s-1), R is general gas

constant (J K-1 mol-1) and T is the temperature (K).

Basic

Concepts of Theoretical Chemistry

Born

-Oppenheimer Approximation

There are too many variables in the

schrodinger wave equation to be solved for multi- electron system. So many

approximations are proposed and one of the most significant is Born- Oppenheimer

approximation 14. They divide the problem into two parts, in first part the

motion of electron is considered and motion of nuclei is ignored as it is

heavy. The motion of electron is studied in a stationary nucleus charge.

He?e(r,R) =Ee?e(r,R)

He is the electronic Hamiltonian

operator. The Hamiltonian operator becomes now

E=Ee+

All the quantum chemistry methods arises from

this approximation solution. The second problem treats clearly the motion of

the nuclei and reflects electron as an average field. This problem is classical

one not quantum mechanical and it offers to molecular mechanics method.

Basis set

Choice of basis set plays an important role to

increase the accuracy of calculation.

Slater

orbitals function

To solve the schrodinger wave equation, a

mathematical solution to the molecular orbital must be set. In linear

combination of atomic orbitals (LCAO), molecular orbitals ?i necessarily

be written as combination of atomic orbitals ?i.

?i=

Contracted Gaussian function

The slaters type orbitals can also be

contracted into Gaussian type orbitals as a linear function of Gaussian

function,

?m=d1g1+d2g2+d3g3

+……

a normalized Gaussian function for is given

below,

g1s(?,r)=(8?3/?3)1/4e-?r2

Minimal

Basis Sets STO-LG

The original basis set used in Hartee-Fock formulism are STO-LG basis

set, in which each contraction is settled by L primitives (1

). STO-3G is the most

well-known basis set used. This basis set does not produce the

experimental data appropriately and other modifications are needed.

Double-Dzeta Basis Set

Two contractions are needed in mathematical expression of double-dzeta

basis set rather than one. For example, the 6-31G basis set make use one

contraction to define the inner shell atomic orbitals while the valence atomic

orbitals are defined by two contractions. For the inner shell molecular orbitals,

six primitives form a unique contraction. For the valence atomic orbitals, the earlier

contraction is developed by three primitives and the last by only one

primitive. This mathematical description gives more flexibility in the

description of atomic orbitals. There exists the triple-dzeta basis sets form

from following same idea.

Polarized basis set

Polarization functions are also used to

increase the accuracy. It contains adding p-type contractions on H-atom and

d-type on the heavy atoms

Diffuse

function

The addition of diffusion functions improves

the description far from nuclei. Such basis sets are mentioned as + and ++ for

heavy and hydrogen atoms. E.g. 6-311++G

(d,p).

Density functional theory

The

Hartree Fock estimate is surely great however it has its confinements. The

confined HF technique can’t depict the separation of atoms into open-shell

fragments. E.g. for phenol the Hartree Fock bond separation vitality is HFBDE =

49.5kcal/mol while the experimental BDE is around 87kcal/mol! The correlation

energy is absent in Hartree Fock guess.

To consider the electronic correlation many

correlated methods have been developed for molecular calculations. Different

post-HF methods have been created, including those based on the Configuration

Interaction approach and multi-reference methods, the Møller-Plesset

perturbation theory (MP2, MP4…), Multi-configurational self-consistent field

approaches (CASSCF, CASPT2…), coupled-cluster methodes. Most of these post-HF

methods permit reaching a very good accuracy, nonetheless the computational

time is dramatically amplified and only relatively small molecular systems can

be considered.

The density functional theory (DFT) considers

the correlation correction and appears as a good stability between the accuracy

and computational cost, letting treating much larger systems than with post-HF.

In DFT the complicated electronic wave functions

are substituted by the electron density, replacing the ?(x1,x2,……)

with ?(r)

?(r1)=N

(2.24)

The wave function is extremely mind boggling

and actually this is only a mathematical object, without physical reality. The

fact that is composed as a Slater determinant gives a kind of physical meaning,

as is built with one electron orbitals. Nonetheless, is unquestionably not an interpretable.

It can simply give an interpretable as a reaction to an operator. In that

sense, ?(r) is a very interesting variable since it is directly relates toward

a physical meaning.