UV-VISIBLE

SPECTROPHOTOMETER
By :
Najiya Ibrahim N k
M-pharm I
Department of Quality Assurance
CONTENTS
➢ Introduction
➢ Theory and law
➢ Instrumentation
➢ Choice of solvent and solvent effect
➢ Applications
PHARMACEUTICAL ANALYSIS
o It is a branch of science which derives its principles.
o It is a branch of chemistry, which involves a series of processes for
the identification, determination, quantitation and purification.
o This is mainly used for the separation of the components from the
mixture and for the determination of the structure of the compound.
o Analytical techniques are based on the measurement of one of the
properties of sample (physical or chemical or electrical properties)
o These properties of the sample help in characterisation and
determination of the compound.
Based upon the determination there are two types of analytical methods.
➢ Qualitative analysis
➢ Quantitative analysis

Qualitative analysis is used for the identification of the chemical

compound.
Quantitative analysis is used for the determination of the amount of the
sample.
 ANALYTICAL TECHNIQUES
PHYSICAL /
CHEMICAL /
INSTRUMENTAL RADIOACTIVE BIOLOGICAL
CLASSICAL
METHOD METHODS METHODS
METHODS

OPTICAL METHODS
VOLUMETRIC BIOLOGICAL
(SPECTROSCOPIC
METHOD RADIO ASSAY
METHODS)
IMMUNE
ASSAY
ELECTRO
CHEMICAL GRAVIMETRIC MICROBIOLOGY
METHODS METHOD ASSAY

SEPERATIONAL
METHODS

THERMO-
ANALYTICAL
METHODS
 OPTICAL METHOD /
SPECTROSCOPIC METHOD

Absorption of Emission of Scattering of Refraction: Rotation of EMR: Other methods

EMR: UV, IR, EMR: EMR: Refractometry Polarimetry, Spectro- : mass
NMR, AAS Fluorimetry, Nephelometry,
polarimetry spectroscopy
Flame photometry Turbidimetry,
ELECTROCHEMICAL METHODS
Conductometry

Polarography SEPERATIONAL METHODS

(Chromatography)
Potentiometry Paper
Column
pH meter
HPLC

TLC
RADIOACTIVE METHODS
HPTLC
Radioimmune assay
GC

Electrophoresis
THERMAL ANALYTICAL METHODS
Thermogravimetric
Analysis

Differential scanning
calorimetry BIOLOGICAL METHODS
Biological Assay

CHEMICAL / CLASSICAL METHODS

Microbiology Assay
Volumetric methods

Gravimetric methods
 SPECTROSCOPIC METHOD

• Spectroscopy is the field of study that measures and interprets the

electromagnetic spectra that result from
the interaction between electromagnetic radiation and matter as a
function of the wavelength or frequency of the radiation
• Interaction of electromagnetic radiations with matter results in
Absorption, Emission, or scattering of electromagnetic radiation.
• Spectroscopy is the study of interaction of electromagnetic
radiation with matter based on the Bohr-Einstein frequency
relationship E = hv, where h is the proportionality constant called
Planck's constant and v is the frequency
 DEFINITIONS
• Electromagnetic Radiations :- Electromagnetic radiation can be defined
as a form of energy that is generated when electrically charged particles
move through matter or a vacuum.
• Electromagnetic Spectrum :- The entire distribution of electromagnetic
radiation. It comprises the span of all electromagnetic radiation and consists
of all the subranges, commonly referred to as portions, such as visible
light or ultra-light radiation.
• Electromagnetic waves :- These are transverse waves
composed of two oscillating wave fields that vibrate perpendicular
to each other and perpendicular to the direction of propagation of
radiation.

• Wavelength :- It is the distance between two

successive maxima on an electromagnetic wave. It is denoted by the
Greek word Lambda (λ). It is expressed in terms of m, cm, µm,
nm, Å.
• Frequency :- The number of wavelength units passing through a
given point in unit time is called the frequency of radiation. It
is denoted by the letter ν. It is expressed in cycles per second or
in Hertz (Hz) or in Fresnel.
• Wave number :- it is defined as number of waves per centimetre in
vacuum. It is the reciprocal of wavelength and is expressed in cm.
• Electronic spectra :- It arises due to electronic transitions in a molecule
by absorption of radiations falling in the visible and ultra violet regions.
While the electronic spectra in the visible region span 12,500 – 25,000 cm⁻¹,
those in ultraviolet region span 25000 – 70000 cm⁻¹
 UV-VISIBLE SPECTROSCOPY
DEFINITION :-

Ultraviolet-visible (UV-Vis) spectrophotometry is a

technique used to measure light absorbance across the
ultraviolet and visible ranges of the electromagnetic
spectrum. When incident light strikes matter it can either
be absorbed, reflected, or transmitted.
INTRODUCTION

Most of the organic molecules and functional groups are transparent in the
portions of the electromagnetic spectrum which we call them here as
Ultraviolet (UV) and Visible regions. Consequently, absorption
spectroscopy is of limited utility in this range of wavelengths. However, in
some cases we can derive useful information from these regions of the
spectrum. That information, when combined with the detail provided by
infrared and nuclear magnetic resonance spectra, can lead to valuable
structural proposals. UV-Visible spectrophotometry is mainly used for
quantitative analysis and serves as an useful tool for structural elucidation.
 The UV and visible spectroscopy deal with the recording of
absorption of radiations in the ultraviolet and visible region of the
electromagnetic spectrum.
The UV region extends from 100-400 nm.
The visible region extends from 400-800 nm.

Range of UV-Visible region

➢Near UV : 200 – 400 nm

➢Far UV : 10 – 200 nm
➢Visible region : 400 – 800 nm

Vacuum UV region is possible only if the instrument is evacuated of air

because oxygen and other air particles absorbs UV light in this region.
 THEORY
In the case of UV and visible spectroscopy, the transition occurs between electronic
energy levels i.e., it involves the excitation of electrons from the ground state to the
higher energy state, hence it is also known as Electronic spectroscopy.
The absorption of electromagnetic radiations causes the atoms or molecules to excite from a
state of lower energy (ground state) to a state of higher energy (excited state). This is known
as transition.

The electromagnetic radiations that is absorbed has the energy equal to the
energy difference between the excited and the ground state.
➢ Molecular orbitals are formed as a result of the overlapping of two atomic
orbitals (s-s, p-p or s-p). If the formation of molecular orbital increases
the stability of the molecule as compared to the separated atoms, then the
orbital is called bonding molecular orbital.
➢ If the formation of the molecular orbital decreases the stability of the
molecule, then the orbital is an antibonding molecular orbital.
➢ Bonding and antibonding molecular orbitals are formed by the overlap of
two s (σ) atomic orbitals, or an s (σ) orbital with a p (π) orbital or by head
to head or side by side overlap of two p (π) orbital. A bonding orbital is
represented as σ and π while antibonding orbitals are designated as σ*
and π*
➢ In addition to bonding and antibonding orbitals, there exists a
nonbonding molecular orbital which neither contributes to the stability
nor decreases the stability of a molecule. It consist of unshared pair of
electrons.
ELECTRONS ASSOCIATED WITH MOLECULAR ORBITALS
The electrons that are associated with these molecular orbitals that undergo
transition and contribute to the absorption characteristics of organic and
inorganic molecules are of the following three types.
• Sigma (σ) electrons
• Pi (π) electrons
• Non-bonding (n) electrons

Sigma (σ) electrons

➢ These are associated with the saturated (single) bonds in molecules.
➢ These electrons are tightly held because the σ bonds are strong hence the energy of the
UV or visible region is not sufficient to overcome this attraction.
➢ Compounds containing single σ bonds such as C-H, C-C, O-H do not absorb UV-Visible
radiation and appear colourless in UV region.
➢ This can be used as solvents in UV spectroscopy.
Pi (π) electrons
➢ These are involved in unsaturated compound such as alkenes, alkynes
and aromatic compounds.
➢ Π bonds are weak , hence they are easier to excite to the higher energy
level.
➢ The transition of π electrons give absorption bands in the ultraviolet
region.

Non-bonding (n) electrons

➢ These are not involved in bonding between the atoms in molecules.
➢ They exist as lone pair on atoms like Nitrogen, Oxygen, Sulphur and
Halogen.
➢ N electrons are less firmly held as compared to σ electrons hence are
easily excited by ultraviolet radiation.
ELECTRONIC TRANSITION
The transition involved are :
• σ to σ* transition
• n to σ* transition
• π to π* transition
• n to π* transition
σ to σ* transition
• Sigma bonds are strong
• The energy required to bring this transition is very high
• These transitions are studied in far UV region below 250nm

n to σ* transition
• Requires less energy that σ to σ* transition and are observed in ordinary UV-Visible
spectrophotometers.
• These occurs in organic molecules that contain a heteroatom like N, O, S or halogen.
• These are characteristics of alkyl halides, amines, ethers and sulphides.
 π to π* transition
• These type of transition occurs at longer wavelength, as excitation of π electrons
requires lesser energy that n to π* transition.
• Compounds containing double or triple bonds such as alkenes, alkynes, cyanides,
azo compounds, carbonyl compounds, aromatic etc., undergo π to π* transition.
• The band corresponding to this type are called K bands.

n to π* transition
• These type of transition is characteristic of molecules like C=O, NO2, NO, N=N etc.
• This involves the excitation of electrons of unshared electron pair on heteroatom such as
O, N, or S to π* antibonding orbitals associated with a double or triple bond in the
molecule.
• This involves least amount of energy hence it gives rise to an absorption band at
longer wavelengths.
• These bands are called R bands.
ABSORPTION LAW
The two laws are related to the absorption of radiation are :
1) Beer's law – related to the concentration of absorbing species
2) Lambert's law – related to the path length / thickness of the absorbing species

I0 = IA + I

Where,
I0 = intensity of incident light
IA = intensity of absorbed light
I = intensity of transmitted light
TRANSMITTANCE (T)
It is defines as the amount of radiation that passes through sample unchanged.
It is defined as the ratio of intensity of light emerging from the sample (I) to
that of incident light (I0)

Hence, T = I/I0 It is expressed as percentage transmittance

%T = I/I0 × 100

ABSORBANCE (A)
Absorbance is the amount radiation absorbed by a sample. It is a logarithmic
function of T and is expressed as:
Absorbance = log (1/T)
A = log (1/ I/I0)
A = log ( I/I0) ------------ (1)
BEER'S LAW
Beer's law states that " The intensity of a beam of monochromatic light decreases
exponentially with increase in the concentration of absorbing species arithmetically ".

-dI/dc α I
-dI/dc = KI
Where, -dI/dc = rate of increase in the intensity (I) with respect to concentration (c)
K = Proportionality constant
-dI/I = Kdc

Integrating on both sides

∫dI/I = k ∫dc
-ln I = Kc + b ---------- (2)
Where, b = constant of integration
If the concentration of absorbing species is zero, there is no absorbance. Hence the
intensity of the incident light (I0) at zero concentration is equal to the intensity of the
transmitted light (I)

i.e., I0 = I, when c = 0
Substituting the above values in the equation (2)
-ln I0 = K(0) + b
-ln I0 = b
Substituting the value of b in the equation (2)
-ln I = Kc – ln I0
ln I0 - ln I = Kc
Since (ln a – ln b = ln a/b)
Hence, ln I0/I = Kc
I0/I = eKc
Inversing both the sides
I/ I0 = e-Kc
I = I0 e-Kc -------------- (3)
(3) is the equation for beer's law
LAMBERT'S LAW
Lambert's law states that " when a beam of light is allowed to pass through a transparent
medium, the rate of intensity of monochromatic light decreases with thickness of medium and is
directly proportional to the intensity of incident light "

-dI/dt α I
-dI/dt = KI
-dI/I = Kdt
Integrating on both sides
∫dI/I = K ∫dt
-ln I = Kt + b -------------(4)
Substituting value of b from the beer's law i.e., b = -ln I0
ln I = Kt – ln I0
ln I0 – ln I = Kt
ln I0/I = Kt
I0/I = e Kt
I/I0 = e –Kt
I = I0 e –Kt ---------------- (5)
(5) is the equation for Lambert's law
Equation (3) and (5) can be combined to get
I = I0 e –Kct
[Converting natural logarithm to base 10 and K = K × 0.4343 ]
I = I0 10 -kct
I/I0 = 10 -Kct
Inversing on both sides
I0/I = 10 kct
Taking log on both sides
Log I0/I = Kct ----------- (6)
According to Equation (1)
A = Log I0/I
Hence equation (6) can be
A = Kct or A = ct ------------ (7)
The above equation is the mathematical equation for Beer-Lambert's law
Where,
A = absorbance or optical density or extinction co-efficient
C = concentration of sample solution (mmol/lit)
t = path length (normally 10 mm or 1 cm )

Hence Beer-Lambert's law states that the quality of light or radiation absorbed by
a substance dissolved in a fully transmitting solvent is directly proportional to the
concentration of the substance and the path length of the ligth through the
solution.
Equation 7 can be rewritten as,
ε = A/ct
Where, ε = molar extinction co-efficient or absorption co-efficient
A = absorbance
c = concentration
t = path length
If c = 1 mmol/lit and t = 1 cm, Then
ε=A
Hence, molar absorption co-efficient is the specific absorption coefficient for a
concentration of 1 mmol/lit and pathlength of 1 cm.
 DEVIATION OF BEER'S LAW
According to beer's law, there exist a direct relationship between the absorbance and
concentration. Hence a graph is plotted between absorbance on Y-axis and concentration
on X-axis, a straight line is obtained. But at times, deviations may occur from the linear
relationship, indicating an apparent failure of beer's law. This deviation maybe
described as positive or negative deviations depending upon the shape of the curve.
If the curve between absorbance and concentration is curved upward, it is termed as positive
deviation and if the curve is downwards, it is termed as negative deviation.
Positive deviation occurs when a small change in concentration leads to a large change in
absorbance. Negative deviation occurs when a large change in concentration produces small
change in absorbance.
Deviation is classified into :
• True deviation or real deviation
• Chemical deviation
• Instrumental deviation
REAL DEVIATIONS
It is related to the concentration of the absorbing substance. At higher concentration,
molecules of the absorbing species undergo collisions and interact with each other. Due to
this the average distance gets reduced between the molecules, which affect the charge
distribution of the neighbouring molecules. Hence the molecules will not absorb radiation
in same manner as dilute solutions.
CHEMICAL DEVIATION

This occurs when the absorbing species undergo chemical reactions such as association,
complex formation, dissociation, hydrogen bonding, hydrolysis, ionization or
polymerization.
Example : benzyl alcohol in chloroform exists as a polymer. Upon dilution it dissociates into
its monomers. The polymeric form absorbs radiation at 3.0004 µ showing positive deviation,
while the monomeric form of benzyl alcohol absorbs maximum radiation at 2.750 to 2.765 µ
showing negative deviation from beer's law.
This can be corrected by using appropriate wavelength, buffers and suitable solvents.
 INSTRUMENTAL DEVIATIONS
• Beer's law is obeyed only when a monochromatic radiation is used.
Polychromatic radiations leads to negative deviations from Beer's law
• Any fluctuation in the intensity of the radiation source may also cause
deviations from the beer's law.
• Changes in the sensitivity of the detector employed and defects in the
amplification of the radiation deviations.
• Improper slit width also contributes towards deviation from Beer's law.
Improper slit width allows stray radiation to reach the detector. These stray
radiations are absorbed by the impurities present in the sample solution and
leads to changes in the absorbance value of sample.
LIMITATION OF BEER'S LAW

It is applicable only at a low concentrations

1. It is not applicable to suspensions
2. It is not valid, if the absorbing particles undergoes coagulation because
coagulated particles cause scattering of the radiations which may leads to
either decrease or increase in the absorbance value.
3. Only monochromatic light can be used.
4. The measured absorbance value should lie between 0.7 - 0.2 units (20 –
65%) for better results