UV Visible spectroscopy

Mrs. Poonam Sunil Aher (M.Pharm, PhD)
Assistant Professor
Sanjivani College of Pharmaceutical Education
and Research (Autonomous),
Kopargaon, Ahmednagar-423603 (M.S.), INDIA
Mobile: +91-9689942854
UV / Visible Spectroscopy

Spectroscopy
 It is the branch of science that deals with the study of
interaction of matter with light.
OR
 It is the branch of science that deals with the study of
interaction of electromagnetic radiation with matter.

Electromagnetic Radiation
 Electromagnetic radiation consist of
discrete packets of energy which are
called as photons.
 A photon consists of an oscillating electric
field (E) & an oscillating magnetic field
(M) which are perpendicular to each
other.

 Frequency (ν):
 It is defined as the number of times electrical
field radiation oscillates in one second.
 The unit for frequency is Hertz (Hz).
1 Hz = 1 cycle per second
 Wavelength (λ):
 It is the distance between two nearest parts of
the wave in the same phase i.e. distance
between two nearest crest or troughs.

 The relationship between wavelength &
frequency can be written as:
c = ν λ
 As photon is subjected to energy, so
E = h ν = h c / λ

Violet 400 - 420
nm
Yellow 570 - 585
nm
Indigo 420 - 440
nm
Orange 585 - 620
nm
Blue 440 - 490
nm
Red 620 - 780
nm
Green 490 - 570
nm

Principles of Spectroscopy
 The principle is based on the
measurement of spectrum of a sample
containing atoms / molecules.
 Spectrum is a graph of intensity of
absorbed or emitted radiation by sample
verses frequency (ν) or wavelength (λ).
 Spectrometer is an instrument design to
measure the spectrum of a compound.

1. Absorption Spectroscopy:
 An analytical technique which concerns
with the measurement of absorption of
electromagnetic radiation.
 e.g. UV (185 - 400 nm) / Visible (400 - 800
nm) Spectroscopy, IR Spectroscopy (0.76 -
15 μm)

2. Emission Spectroscopy:
 An analytical technique in which
emission (of a particle or radiation) is
dispersed according to some property of
the emission & the amount of dispersion
is measured.
 e.g. Mass Spectroscopy

Interaction of EMR
with
Matter

Interaction of EMR with matter
1.Electronic Energy Levels:
 At room temperature the molecules are in the lowest
energy levels E0.
 When the molecules absorb UV-visible light from EMR,
one of the outermost bond / lone pair electron is
promoted to higher energy state such as E1, E2, …En,
etc is called as electronic transition and the difference
is as:
∆E = h ν = En - E0 where (n = 1, 2, 3, … etc)
∆E = 35 to 71 kcal/mole

Interaction of EMR with matter
2.Vibrational Energy Levels:
 These are less energy level than electronic energy levels.
 The spacing between energy levels are relatively small i.e.
0.01 to 10 kcal/mole.
e.g. when IR radiation is absorbed, molecules are excited from
one vibrational level to another or it vibrates with higher
amplitude.
3. Rotational Energy Levels:
 These energy levels are quantized & discrete.
 The spacing between energy levels are even smaller than
vibrational energy levels.
∆Erotational < ∆Evibrational < ∆Eelectronic

Beer Lamberts Law:
A = ε b c
A=absorbance
ε =molar absorbtivity with units of L /mol.cm
b=path length of the sample (cuvette)
c =Concentration of the compound in solution, expressed in mol /L

• σ → σ* transition
1
• π → π* transition
2
• n → σ* transition
3
• n → π* transition
4
• σ → π* transition
5
• π → σ* transition
6
The possible electronic transitions are

• σ electron from orbital is excited to
corresponding anti-bonding orbital σ*.
• The energy required is large for this
transition.
• e.g. Methane (CH4) has C-H bond only
and can undergo σ → σ* transition and
shows absorbance maxima at 125 nm.
• σ → σ* transition
1

• π electron in a bonding orbital is excited
to corresponding anti-bonding orbital π*.
• Compounds containing multiple bonds
like alkenes, alkynes, carbonyl, nitriles,
aromatic compounds, etc undergo π →
π* transitions.
e.g. Alkenes generally absorb in the
region 170 to 205 nm.
• π → π* transition
2

• Saturated compounds containing atoms
with lone pair of electrons like O, N, S
and halogens are capable of n → σ*
transition.
• These transitions usually requires less
energy than σ → σ* transitions.
• The number of organic functional groups
with n → σ* peaks in UV region is small
• n → σ* transition
3

• An electron from non-bonding orbital is
promoted to anti-bonding π* orbital.
• Compounds containing double bond
involving hetero atoms (C=O, C≡N, N=O)
undergo such transitions.
• n → π* transitions require minimum
energy and show absorption at longer
wavelength around 300 nm.
• n → π* transition
4

•These electronic transitions are forbidden
transitions & are only theoretically
possible.
•Thus, n → π* & π → π* electronic
transitions show absorption in region
above 200 nm which is accessible to UV-
visible spectrophotometer.
•The UV spectrum is of only a few broad of
• σ → π* transition
5
• π → σ* transition 6
&

The possible electronic transitions can
graphically shown as:

Terms used
in
UV / Visible
Spectroscopy

Chromophore
The part of a molecule responsible for imparting color, are called as
chromospheres.
OR
The functional groups containing multiple bonds capable of absorbing
radiations above 200 nm due to n → π* & π → π* transitions.
e.g. NO2, N=O, C=O, C=N, C≡N, C=C, C=S, etc

Auxochrome
The functional groups attached to a chromophore which modifies the ability of
the chromophore to absorb light , altering the wavelength or intensity of
absorption.
OR
The functional group with non-bonding electrons that does not absorb
radiation in near UV region but when attached to a chromophore alters the
wavelength & intensity of absorption.

Auxochrome
e.g. Benzene λmax = 255 nm
Phenol λmax = 270 nm
Aniline λmax = 280 nm
OH
NH2

• When absorption maxima (λmax) of a
compound shifts to longer wavelength, it
is known as bathochromic shift or red
shift.
• The effect is due to presence of an
auxochrome or by the change of solvent.
• e.g. An auxochrome group like –OH, -OCH3
causes absorption of compound at longer
wavelength.
• Bathochromic Shift (Red Shift)
1

• In alkaline medium, p-nitrophenol shows
red shift. Because negatively charged
oxygen delocalizes more effectively than
the unshared pair of electron.
p-nitrophenol
λmax = 255 nm λmax = 265 nm
• Bathochromic Shift (Red Shift)
1
OH
N
+ O
-
O
OH
-
Alkaline
medium
O
-
N
+ O
-
O

• When absorption maxima (λmax) of a
compound shifts to shorter wavelength, it
is known as hypsochromic shift or blue
shift.
• The effect is due to presence of an group
causes removal of conjugation or by the
change of solvent.
• Hypsochromic Shift (Blue Shift)
2

• Aniline shows blue shift in acidic medium,
it loses conjugation.
Aniline
• Hypsochromic Shift (Blue Shift)
2
NH2
H
+
Acidic
medium
NH3
+
Cl
-

• When absorption intensity (ε) of a
compound is increased, it is known as
hyperchromic shift.
• If auxochrome introduces to the
compound, the intensity of absorption
increases.
Pyridine 2methylpyridine
• Hyperchromic Effect
3
N N CH3

• When absorption intensity (ε) of a
compound is decreased, it is known as
hypochromic shift.
Naphthalene 2-methyl naphthalene
ε = 19000 ε = 10250
CH3
• Hypochromic Effect
4

Wavelength ( λ )
Absorbance
(
A
)
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λmax

PRINCIPLES OF
UV - VISIBLE
SPECTROSCOPY

Principle
 The UV radiation region extends from 10 nm to 400 nm and the visible
radiation region extends from 400 nm to 800 nm.
Near UV Region: 200 nm to 400 nm
Far UV Region: below 200 nm
 Far UV spectroscopy is studied under vacuum condition.
 The common solvent used for preparing sample to be analyzed is either
ethyl alcohol or hexane.

Instrumentation
Components of UV-Visible
spectrophotometer
 Source
 Filters & Monochromator
 Sample compartment
 Detector
 Recorder

Five Basic Optical Instrument Components
 1) Source – A stable source of radiant energy at the desired wavelength (or
range).
 2) Wavelength Selector – A device that isolates a restricted region of the
EM spectrum used for measurement (monochromators, prisms & filters).
 3) Sample Container – A transparent container used to hold the sample
(cells, cuvettes, etc).
 4) Detector/Photoelectric Transducer – Converts the radiant energy into a
useable signal (usually electrical).
 5) Signal Processor & Readout – Amplifies or attenuates the transduced
signal and sends it to a readout device as a meter, digital readout, chart
recorder, computer, etc.

LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follow
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp

Wavelength Selectors
 Wavelength selectors output a limited, narrow, continuous group of
wavelengths called a band.
Two types of wavelength selectors:
A) Filters
B) Monochromators
A)Filters –
Two types of filters:
a) Interference Filters
b) Absorption Filters

Cont..
B. Monochromators
 Wavelength selector that can continuously scan a broad range of
wavelengths.
 Used in most scanning spectrometers including UV, visible, and IR
instruments.
Refractive type
PRISM TYPE
Reflective type
Diffraction type
GRATING TYPE
Transmission Type

SAMPLE COMPARTMENT
 Spectroscopy requires all materials in the beam path other than the analyte should
be as transparent to the radiation as possible.
 The geometries of all components in the system should be such as to maximize the
signal and minimize the scattered light.
 The material from which a sample cuvette is fabricated controls the optical window
that can be used.
 Some typical materials are:
 Optical Glass - 335 - 2500 nm
 Special Optical Glass – 320 - 2500 nm
 Quartz (Infrared) – 220 - 3800 nm
 Quartz (Far-UV) – 170 - 2700 nm

Detectors
 After the light has passed through the sample, we want to be able to detect and
measure the resulting light.
 These types of detectors come in the form of transducers that are able to take
energy from light and convert it into an electrical signal that can be recorded, and if
necessary, amplified.
 Three common types of detectors are used
 Barrier layer cells
 Photo emissive cell detector
 Photomultiplier

SUMMARY
 Types of source, sample holder and detector for various EM region
REGION SOURCE SAMPLE
HOLDER
DETECTOR
Ultraviolet Deuterium lamp Quartz/Fused
silica
Phototube, PM
tube, diode
array
Visible Tungsten lamp Glass/Quartz Phototube, PM
tube, diode
array

Types of dosage form
 Two types
 Single component dosage form
 Multi component dosage form

Method for single component dosage form:
 1. Calibration method or beer lamberts law method 2.Method of least
Square
 3. Single point standardization method
 4. Double point standardization method

 Beer law method or Calibration curve method:
 1. prepare standard stock
 2. prepare dilution of standard stock
 3. calculate range
 4. calculate wavelength
 5. Perform the assay
 6. Draw the graph
 7. Calculate concentration by using regression line equation Y = m x + c
 8. calculate the unknown concentration by graph
 9. Calculate the purity of drug by using Beer law A=abc

 This method is modified in new form
 Method of least squares:

 4. Double point standardization method:

 C TEST= ( A test- A STD1)( C STD 1- C STD2) + CSTD1(
ASTD1-ASTD2)
 ______________________________________
(ASTD1- ASTD2)

DIFFERENT UV-VISIBLE SPECTROPHOTOMETRIC METHODS
FOR MULTICOMPONENT ANALYSIS
 (a) Simultaneous equation method
 (b) Absorbance ratio method
 (c) Geometric correction method
 (d) Orthogonal polynomial method
 (e) Derivative spectrophotometry
 (f)Difference spectrophotometry

(a) Simultaneous equation method:
 If a sample contains two absorbing drugs (X and Y)
each of which absorbs at the λ-max of the other (λ1
and λ2), it may be possible to determine both the drugs
by the simultaneous equations method.

The information required is
 The absorptivities of X at λ1 and λ2, aX1 and aX2.
 The absorptivities of Y at λ1 and λ2, aY1 and aY2.
 The absorbances of the diluted sample at λ1 and λ2, A1
and A2.
Let, Cx and Cy be the concentration of X and Y
respectively in the sample.
 The absorbance of the mixture is the sum of the
individual absorbances of X and Y

At λ1 A1 = aX1* Cx + aY1* Cy …………..(1)
At λ2 A2 = aX2* Cx + aY2* Cy …………..(2)
Multiply the equation (1) with aX2 and (2) with aX1
A1 aX2 = aX1 Cx aX2 + aY1 Cy aX2 …………(3)
A2 aX1 = aX2 Cx aX1+ aY2 Cy aX1 ………….(4)
A1 aX2 - A2 aX1 = aY1 Cy aX2 - aY2 Cy aX1
A1 aX2 - A2 aX1 = Cy (aY1 aX2 - aY2 aX1)
Cy = (A1 aX2 - A2 aX1) / (aY1 aX2 - aY2 aX1) ……….(5)
Same way we can derive
Cx = (A2 aY1 – A1 aY2) / (aY1 aX2 - aY2 aX1)………... (6)
These equations are known as simultaneous equations and by solving
these simultaneous equations we can determine the concentration
of X and Y in the sample.

(b) Absorbance ratio method:
The absorbance ratio method is a
modification of the simultaneous equations procedure.
In the quantitative assay of two components in
admixture by the absorbance ratio method,
absorbances are measured at two wavelengths, one
being the λ-max of one of the components (λ2) and
other being a wavelength of equal absorptivity of two
components (λ1), i.e. an iso-absorptive point.

 At λ1 A1 = aX1* Cx + aY1* Cy …………… (1)
 At λ2 A2 = aX2* Cx + aY2* Cy…………....(2)
Now divide (2) with (1)
A2/A1 = (aX2* Cx + aY2* Cy)/(aX1* Cx + aY1* Cy)
 Divide each term with (Cx + Cy)
A2/A1 = (aX2* Cx + aY2* Cy) / (Cx + Cy) (aX1* Cx + aY1* Cy) / (Cx + Cy)
Put Fx = Cx / (Cx + Cy) and Fy = Cy / (Cx + Cy)
A2/A1 = [aX2 Fx + aY2 Fy] / [aX1 Fx + aY1Fy]
Where Fx is the fraction of X and Fy is the fraction of Y i.e. Fy = 1-Fx
Therefore,
A2/A1 = [aX2 Fx + aY2 (1-Fx)] / [aX1 Fx + aY1(1-Fx)]
= [aX2 Fx + aY2 – aY2Fx] / [aX1 Fx + aY1 – aY1Fx]

At iso-absorptive point
aX1 = aY1 and Cx = Cy
There fore A2/A1 = [aX2 Fx + aY2 – aY2Fx] / aX1
= (aX2 Fx/ aX1) + (aY2/ aX1) –( aY2Fx/ aX1)
Let Qx = aX2/aX1 , Qy = aY2/aY1 and absorption ratio Qm = A2/A1
Qm = Fx (Qx-Qy) + Qy
Fx = (Qm – Qy) / (Qx – Qy) ………………………..(3)
From the equations (1) A1 = aX1 (Cx + Cy)
there fore Cx + Cy = A1 / aX1
There fore Cx = (A1/aX1) – Cy ……………………(4)
From the equation (3)
Cx / (Cx + Cy) = (Qm – Qy) / (Qx – Qy)
There fore Cx / (A1 / aX1) = (Qm – Qy) / (Qx – Qy)
There fore Cx = [(Qm – Qy) / (Qx – Qy)] X (A1 / aX1) …………(5)

(c) Geometric correction method:
 A number of mathematical correction procedures have been
developed which reduce or eliminate the background irrelevant
absorption that may be present in samples of biological origin.
 The simplest of this procedure is the three point geometric
procedure, which may be applied if the irrelevant absorption is
linear at the three wavelengths selected.

If the wavelengths λ 1, λ 2 and λ 3 are selected to that
the background absorbances B 1 , B 2 and B 3 are
linear, then the corrected absorbance D of the drug
may be calculated from the three absorbances A 1 , A 2
and A 3 of the sample solution at λ 1, λ 2 and λ 3
respectively as follows,
Let v D and w D be the absorbance of the drug alone in
the sample solution at λ 1 and λ 3 respectively, i.e. v
and w are the absorbance ratios vD/D and wD/D
respectively.
B 1 = A 1 – vD, B 2 = A 2 –D and B 3 = A 3 –wD

Let y and z be the wavelengths intervals (λ 2 – λ 1 ) and (λ 3 - λ 2 )
respectively
D= y(A 2 -A 3 ) + z(A 2 – A 1 ) / y (1-w) + z(1-v)
 This is a general equation which may be applied in any situation where A
1, A 2 and A 3 of the sample, the wavelength intervals y and z and the
absorbance ratio v and w are known.

(d) Orthogonal polynomial method:
The technique of orthogonal polynomials is another mathematical
correction procedure, which involves more complex calculations than the
three-point correction procedure. The basis of the method is that an
absorption spectrum may be represented in terms of orthogonal functions
as follows
A(λ ) = p P (λ ) + p1 P1 (λ ) + p2 P2 (λ ) ….. pn Pn (λ )
 Where A denotes the absorbance at wavelength λ belonging to a set
of n+1 equally spaced wavelengths at which the orthogonal
polynomials, P (λ ) , P1 (λ ), P2 (λ ) ….. Pn (λ ) are each defined.

(e)Derivative Spectroscopy:
 For the purpose of spectral analysis in order to relate
chemical structure to electronic transitions, and for
analytical situations in which mixture contribute
interfering absorption, a method of manipulating the
spectral data is called derivative spectroscopy.
 Derivative spectrophotometry involves the conversions
of a normal spectrum to its first, second or higher
derivative spectrum. In the context of derivative
spectrophotometry, the normal absorption spectrum is
referred to as the fundamental, zero order, or D 0
spectrum.

INVENTION
 This technique was first described by
Hammond and Price in 1953, followed by the
work of Morrison and French et al.
 Theoretical aspects have been discussed by
several authors and a number of reviews
concerning these aspects and the performance
of the technique have been published.

 A first-order derivative is
the rate of change of
absorbance with respect to
wavelength. A firstorder
derivative starts and
finishes at zero.
 It also passes through zero
at the some wavelength as
max of the absorbance
band.
 Either side of this point are
positive and negative bands
with maximum and
minimum at the some
wavelengths as the
inflection points in the
absorbance band.
 This bipolar function is
characteristic of all odd-
order derivatives.

 The first derivative D 1 spectrum is a plot of the rate of change of
absorbance with wavelength against wavelength i.e. a plot of the
slope of the fundamental spectrum against wavelength or a plot of
dA/dλ vs. λ. . The maximum positive and maximum negative slope
respectively in the D spectrum correspond with a maximum and a
minimum respectively in the D 1 spectrum. The λmax in D
spectrum is a wavelength of zero slope and gives dA/dλ = 0 in the
D 1 spectrum.
 The second derivative D 2 spectrum is a plot of the curvature of
the D spectrum against wavelength or a plot of d 2 A/ dλ 2 vs. λ.
The maximum negative curvature in the D spectrum gives a
minimum in the D 2 spectrum, and the maximum positive
curvature in the D spectrum gives two small maxima called
satellite bands in the D 2 spectrum. The wavelength of maximum
slope and zero curvature in the D spectrum correspond with cross-
over points in the D 2 spectrum.

OBTAINING DERIVATIVE SPECTRA
• Derivative spectra can be obtained by optical, electronic, or
mathematical methods.
• Optical and electronic techniques were used on early UV-
Visible spectrophotometers but have largely been superseded
by mathematical techniques.
• The advantages of the mathematical techniques are that
derivative spectra may be easily calculated and recalculated
with different parameters, and smoothing techniques may be
used to improve the signal-to-noise ratio.

OPTICALAND ELECTRONIC TECHNIQUES
The main optical technique is wavelength modulation,where
the wavelength of incident light is rapidly modulated over
a narrow wavelength range by an electromechanical
device. The first and second derivatives may be generated
using this technique
The electronic method suffers from the disadvantage that the
amplitude and wavelength shift of the derivatives varies
with scan speed, slit width, and resistance-capacitance
gain factor.

MATHEMATICAL TECHNIQUES
To use mathematical techniques the spectrum is first
digitized with a sampling interval of wavelenth. The size
depends on the natural bandwidth (NBW) of the bands
being processed and bandwidth of the instrument used to
generate the data. Typically, for UV-Visible spectra, the
NBW is in the range 10 to 50 nm.
Firstderivative spectra may be calculated simply by taking
the difference in absorbance between two closely
spaced wavelengths for all wavelengths

Where the derivative amplitude, D, is calculated for a
wavelength intermediate between the two absorbance
wavelengths.
For the second-derivative determination three closely-spaced
wavelength values are used
Savitzky and Golay developed a very efficient method to
perform the calculations and this is the basis of the
derivatization algorithm in most commercial instruments.
Other techniques for calculating derivatives, for example,
using Fourier Transforms, are available but not
commercially popular.

QUANTIFICATION  If we assume that the zero-
order spectrum obeys Beer’s
law, there is a similar linear
relationship between
concentration and amplitude
for all orders of derivative
 For single component
quantification the selection of
wavelengths for derivative
spectra is not as simple as for
absorbance spectra because
there are both positive and
negative peaks.
 For the even order derivatives
there is a peak maximum or
minimum at the same
wavelength as the absorbance
spectrum but for the odd-
order derivatives this
wavelength is a zero crossing
point.

Spectral Discrimination as a qualitative fingerprinting
technique to accentuate small structural differences between
nearly identical spectra
Spectral resolution enhancement as a technique for increasing
the apparent resolution of overlapping spectral bands in order
to more easily determine the number of bands and their
wavelengths
Quantitative Analysis as a technique for the correction for
irrelevant background absorption and as a way to facilitate
multicomponent analysis.
USES

ADVANTAGES
•An effective enhancement of resolution, which can be useful
to separate two or more components with overlapping spectra.
• A discrimination in favour of the sharpest features of a
spectrum, used to eliminate interferences by broad band
constituents.

(f)Difference Spectroscopy:
 Difference spectroscopy provides a sensitive method for detecting
small changes in the environment of a chromophore or it can be
used to demonstrate ionization of a chromophore leading to
identification and quantitation of various components in a mixture.
The essential feature of a difference
spectrophotometric assay is that the measured value is the
difference absorbance (Δ A) between two equimolar solutions of
the analyte in different forms which exhibit different spectral
characteristics.
 The criteria for applying difference spectrophotometry to the
assay of a substance in the presence of other absorbing substances
are that:
A)Reproducible changes may be induced in the spectrum of the
analyte by the addition of one or more reagents.
B) The absorbance of the interfering substances is not altered by the
reagents.

 The simplest and most commonly employed technique for altering the
spectral properties of the analyte properties of the analyte is the
adjustment of the pH by means of aqueous solutions of acid, alkali or
buffers
A B
A)The Spectrum of compound in A(acid) and B(Base)
B) The difference spectrum of B relative to A

Application of UV spectroscopy:
 Qualitative & Quantitative Analysis:
 It is used for characterizing aromatic compounds and conjugated olefins.
 It can be used to find out molar concentration of the solute under study.
 Detection of impurities:
 It is one of the important method to detect impurities in organic solvents.
 Eg. In cyclohexane, benzene is impurity and it is checked at 255 nm
 Eg detection of impurity in starting material of nylon
 Detection of isomers are possible.

 Quantitative analysis:
 UV absorption spectroscopy can be used for the quantitative
determination of compounds that absorb UV radiation. This determination
is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length
of the cell that is used in UV spectrophotometer.
Other methods for quantitative analysis are as follows.
a. calibration curve method
b. simultaneous multicomponent method
c. difference spectrophotometric method
d. derivative spectrophotometric method

 Qualitative analysis
UV absorption spectroscopy can characterize those types of compounds
which absorbs UV radiation. Identification is done by comparing the
absorption spectrum with the spectra of known compounds.
UV absorption spectroscopy is generally used for characterizing aromatic
compounds and aromatic olefins.
 Quantitative analysis of pharmaceutical substances
Many drugs are either in the form of raw material or in the form of
formulation. They can be assayed by making a suitable solution of the
drug in a solvent and measuring the absorbance at specific wavelength.
Diazepam tablet can be analyzed by 0.5% H2SO4 in methanol at the
wavelength 284 nm.

 Chemical kinetics:
 To study the chemical reaction and kinetic study of reactant and product
 Charge transfer transition reaction:
 To study the charge transfer transition reaction of product by using total
wave function
 Tautomeric equilibrium:
 to study the tautomeric equilibrium of keto and enol forms.
 Eg: ethyl acetoacetate in this keto forms show absorption band at 275 nm
 Structure of charcoal:
 To study the structure of charcoal

 Molecular weight determination
Molecular weights of compounds can be measured spectrophotometrically
by preparing the suitable derivatives of these compounds.
For example, if we want to determine the molecular weight of amine then
it is converted in to amine picrate. Then known concentration of amine
picrate is dissolved in a litre of solution and its optical density is measured
at λmax 380 nm. After this the concentration of the solution in gm moles
per litre can be calculated by using the following formula.
 "c" can be calculated using above equation, the weight "w" of amine
picrate is known. From "c" and "w", molecular weight of amine picrate can
be calculated. And the molecular weight of picrate can be calculated
using the molecular weight of amine picrate.

 Dissociation constants of acids and bases.
PH = PKa + log [A-] / [HA]
From the above equation, the PKa value can be
calculated if the ratio of [A-] / [HA] is known at a
particular PH. and the ratio of [A-] / [HA] can be
determined spectrophotometrically from the graph
plotted between absorbance and wavelength at
different PH values.

Reference Books
 Introduction to Spectroscopy
 Donald A. Pavia
 Elementary Organic Spectroscopy
 Y. R. Sharma
 Practical Pharmaceutical Chemistry
 A.H. Beckett, J.B. Stenlake

RESOURCES
 http://www2.chemistry.msu.edu/faculty/reusch/VirtTx
tJml/Spectrpy/UV-Vis/spectrum.htm
 http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visi
ble_spectroscopy
 http://teaching.shu.ac.uk/hwb/chemistry/tutorials/mo
lspec/uvvisab1.htm

UV-VISIBLE
SPECTROPHOTOMETER
INSTRUMENTATION

Components of UV-Visible
spectrophotometer
Source
Filters & Monochromator
Sample compartment
Detector
Recorder( Read out system)

LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Xenon discharge lamp
Various Visible radiation sources are as follow
a. Tungsten filament lamp
b. Mercury arc lamp
c. Carbonone lamp

For ultra violet region
1 Hydrogen discharge lamp
 Consist of two electrode contain in Hydrogen filled silica glass or quartz envelop
 Gives continuous spectrum in region 185-380nm. above 380nm emission is not
continuous

 Working:
 In HDL the a continuous spectrum is produced by electrical
excitation of hydrogen gas at low pressure( 0.2-0.5 torr)
 In this continuous spectrum first initial formation of excited
hydrogen species by ionization of hydrogen.
 H2(gas) ionization H2* dissociation H2’ + H2’’

Deuterium lamps:
 Deuterium arc lamps measure in the UV region
190 - 370 nm
 As Deuterium lamps operate at high
temperatures, normal glass housings cannot be
used for the casing. Instead, a fused quartz, UV
glass, or magnesium fluoride envelope is used.
 When run continuously typical lamp life for a
Deuterium lamp is approximately 1000 hours
 Deuterium lamps are always used with a
Tungsten halogen lamp to allow measurements
to be performed in both the UV and visible
regions.
 Working is same like hydrogen lamp. Deuterium
gas is used.

 3. xenon discharge lamp:
 It is also called xenon arc lamp
 In this lamp xenon gas is filled under the pressure 20-30 atm
 This gas is passed through 2 electrodes which are separated
by 8 mm distance
 The intense arc is formed between two electrodes by
supplying high voltage
 This lamp give spectrum in the 200-1000 nm therefore it can
work in uv and visible region

 Tungsten filament lamp:
 This lamp is used in visible region
 It gives continuous spectrum in 350-2000 nm region
 It is also work in IR region
 It can not used in UV region
 The lamp consist of tungsten filament which act as
electrode
 This electrode is evacuated in quartz envelop
 The tungsten gas is filled in the envelop
 The filament is heated upto 3000k and creates plasma or
light in the visible region

 Mercury arc lamp:
 In this lamp inert mercury gas is filled in the form of vapor
 This lamp is used in UV and Visible region
 Excitation of mercury atoms done by high electrical voltage
and it gives high intensity of continuous lines

Filters &Monochromator
The Monochromator/Filter will select a narrow portion of the spectrum (the band
pass) of a given source
 FILTERS ARE OF TWO TYPES:
Absorption Filters
Interference Filters
MONOCHROMATORS ARE OF TWO TYPES:
Refractive type
PRISM TYPE
Reflective type
Diffraction type
GRATING TYPE
Transmission Type

FILTERS
1.ABSORPTION FILTERS(Gelatin filter)
Absorption filters, commonly manufactured from dyed glass or pigmented gelatine
resins is and sandwiched between two glass plate
Band widths are extremely large {30 – 250 nm}
Combining two absorbance filters of different λmax

 The most common type of gelatine filter is constructed by sandwiching a thin layer
of dyed gelatine of the desired colour between two thin glass plates.

INTERFERENCE FILTERS:
 These are used to select wavelengths more
accurately by providing a narrow band
pass typically of around 10nm
 These filters rely on optical interference
(destructive wave addition) to provide
narrow bands of radiation.

 Interference filter consists of a dielectric spacer film made
up of CaF2, MgF2 between two parallel reflecting films.
 As light passes from one medium to the other the direction
and wavelength of light can be changed based on the index
of refraction of both mediums involved and the angle of the
incident and exiting light
 Due to this behaviour, constructive and destructive
interference can be controlled by varying the thickness (d)
of a transparent dielectric material between two semi-
reflective sheets
 As light hits the first semi-reflective sheet, a portion is
reflected, while the rest travels through the dielectric to be
bent and reflected by the second semi-reflective sheet

MONOCHROMATORS
 PRISM MONOCHROMATOR:

 Material used in prism monochromator:
 1. Glass – visible region
 2. Quartz: uv and visible
 3. Borosilicate glass: both region
 4.Sodium chloride: IR
 5.Potassium bromide: IR

 Three types of prism monochromator:
 1. Refractive type monochromator: Cornu
Prism
 2. Reflective Type monochromator: Littrow
prism
 3. Dispersive power type monochromator:
Bunsen Prism

 1. Cornu prism:
 It is also called refractive prism
 It consist of 30˚ two prism
 So the total optical angle of prism is 60˚
 Here two right handed quartz prism 30˚ and left handed 30˚
quartz prism are fabricated together
 In this prism incident light strike on the prism gives minimum
deviation but it gives maximum dispersion
 Here refracted light is directly focused on exit slit

 2. Littrow Prism :
 It is also called reflective type prism
 It is 30˚ optical prism where one of coated by reflective film
like aluminum, copper or gold material
 The reflective light is back to pass through the prism and
immerse on the same direction of the light source
 The incident light is can not cross the second surface of
prism because the surface is coated with reflective material

Cornu prism Litrrow prism
Incident light
Reflected light

 3. Bunsen prism:
 It is simple 60 prism
 It is dispersive type prism

GRATING MONOCHROMATOR
 Gratings are rulings made on glass, Quartz or alkyl halides
 Depending upon the instrument no. of rulings per mm defers
 If it is UV-Visible no. of gratings per mm are more than 3600.

 Types of grating monochromator
 Two types
 1. Diffraction or reflective type grating
 2. transmission type grating

 The mechanism is that diffraction produces reinforcement.
 The rays which are incident on the grating gets reinforced with the reflected rays
and hence resulting radiation has wavelength governed by equation
m λ =b(sin I + sin r)
m – order
λ – desired wavelength
b – grating spacing
i – angle of incidence
r - angle of diffraction

Eg: Czerny turner grating monochromator:
It is reflective type monochromator

TRANSMISSION TYPE GRATING:
 It is similar to diffraction grating, but refraction produces instead of reflection
 Refraction produces reinforcement
 The wavelength of radiation produced by transmission grating can be expressed by
d sin Φ
λ = d = 1/lines per cm
m

Eg: Litrrow Blazed grating monochromator:
It is also called Claised Monochromator.
The configuration of monochromator in a such way that the
entrance slits are positioned at the 90 degree angle to
focusing and collimated lens. And the entrance slit and exit
slit are in the same direction
The diffraction angle is calculated by following formula
d( sin α +sin β )= n λ
α is angle of incident light
β is angle of diffracted light
λ is wavelength

SAMPLE COMPARTMENT
 Spectroscopy requires all materials in the beam path other than the
analyte should be as transparent to the radiation as possible.
 The geometries of all components in the system should be such as
to maximize the signal and minimize the scattered light.
 The material from which a sample cuvette is fabricated controls the
optical window that can be used.
 Some typical materials are:
 Optical Glass - 335 - 2500 nm
 Special Optical Glass – 320 - 2500 nm
 Quartz (Infrared) – 220 - 3800 nm
 Quartz (Far-UV) – 170 - 2700 nm •

 Ideal characteristics of sample holder:
 1. minimizes reflection
 2. path length 1 cm
 3 calibrated in sixe
 4 should be 0.1 to 10 cm long
 5 use square holders mostly in spectrometric
experiment
 6 use cylindrical holders in colorimetric experiment

 Precaution while handling to holders:
 1 don’t touch holder surface
 Always clean surface by soft tissue paper
before use
 Always calibrate the cuvette before use
 Don’t dry the cuvette in hot air oven or direct
on flame
 When corrosive solution used in experiment
then clean the cuvette by using methanol and
dip the cuvette in methanol for 15 min

DETECTORS
 After the light has passed through the sample, we want to be able to detect and
measure the resulting light.
 These types of detectors come in the form of transducers that are able to take
energy from light and convert it into an electrical signal that can be recorded, and if
necessary, amplified.
 Three common types of detectors are used
 Barrier layer cells
 Photo emissive cell detector
 Photomultiplier

BARRIER LAYER CELL or PHOTO VOLTAIC CELL
 It constitutes
 The barrier layer cell consist of semiconductor material such as Selenium, which is
deposited on a strong metal base of iron.
 Thin layer of silver or gold is sputtered over the surface of semiconductor which act as
a second electron collector
 metallic base plate like iron or aluminium which acts as one electrode( cathode) and
silver layer act as anode
 This total assembly is fitted in glass or plastic window

 Principle:
 When the radiation is incident upon the surface of selenium, electrons are generated
at the selenium- silver surface and the electrons are accumulated on silver and they
collected by the silver semiconductor material .
 This accumulation at the silver surface creates an electric voltage difference is
between the silver surface and the base of the cell.
 A photocurrent will flow which is directly proportional to the intensity of incident
radiation beam.
 This detector does not require external power supply.
 It is directly connected to galvanometer.

 Advantage
 1. very robust in construction
 2. it is cheap
 3. rugged
 4. no require external power supply
 5 good for portable instrument
 6 It shows linear relationship
 Disadvantages:
 1. It is not very sensitive to reading
 2. It shows fatigue

Photo emissive cells Detector
/Photocell
 Phototubes are also known as photo emissive cells.
 A phototube consists of an evacuated glass bulb or tube
 It contains two electrodes
 There is light sensitive cathode inside it.
 The inner surface of cathode is coated with light sensitive layer such as potassium
oxide and silver oxide or selenium.
 When radiation is incident upon a cathode, photoelectrons are emitted.
 These are collected by an metal wire anode.
 The current which is created between anode and cathode is measured as radiation
falling on the detector.
 Then these are returned via external circuit. And by this process current is amplified
and recorded.

 Advantages:
 It is sensitive
 The signal is easily amplified
 Disadvantages:
 It shows some dark current (means it shows no signal
after some time)

The photomultiplier tube
 The photomultiplier tube is a most commonly used detector in
UV spectroscopy.
 A photomultiplier tube is an evacuated glass tube which
contains one photocathode and 9-16 electrodes known as
dynodes.
 The surface each dynode is Be-Cu, Cs-Sb.
 A photon of radiation entering the tube strikes the cathode,
causing the emission of several electrons.

 Principle:
 When radiation falls on metal surface of the photocathode, it emits electrons.
 These electrons are accelerated towards the first dynode which is kept as 90V at
positive voltage. Which is more positive than the cathode.
 The electrons strike the first dynode, causing the emission of several electrons for
each incident electron.
 These electrons are then accelerated towards the second dynode, to produce more
electrons which are accelerated towards dynode three and so on.
 Eventually all electrons are finally collected at the anode.

 By this time, each original photon has produced 106 - 107 electrons.
 The final current is amplified or recorded in the terms of signal and response
 Photomultipliers are very sensitive to UV and visible radiation.
 They have fast response times.
 Intense light damages photomultipliers;
 they are limited to measuring low power radiation
 The transit time between absorption of photon and arrival of the shower of electrons
is typically 10-100 sec.

 Advantages:
 It is extreme sensitive than other detector
 The emission of photon in less time so this process require 10-100 sec
 Each photon produce 106-10 7 electron
 Disadvantages:
 Due to intense light it damages the PMT
 It require high voltage power supply
 Instrument may damage if excessive current drawn from final anode
 This detector can not response to low DC current

 Spectrophotometric titration. Definition: The process
of determining the quantity of a sample by adding
measured increments of a titrant until the end-point, at
which essentially all of the sample has reacted, is
reached.
 Principle is a method to measure how much a chemical
substance absorbs light by measuring the intensity of
light as a beam of light passes through sample solution.
The basic principle is that each compound absorbs or
transmits light over a certain range of wavelength.

 one species in a complexation titration absorbs electromagnetic radiation,
we can identify the end point by monitoring the titrand’s absorbance at a
carefully selected wavelength.
 For example, we can identify the end point for a titration of Cu2+ with
EDTA, in the presence of NH3 by monitoring the titrand’s absorbance at a
wavelength of 745 nm, where the Cu(NH3)4
2+ complex absorbs strongly.
 At the beginning of the titration the absorbance is at a maximum. As we
add EDTA the concentration of Cu(NH3)4
2+, and thus the
absorbance, decreases as EDTA displaces NH3. After the equivalence point
the absorbance remains essentially unchanged.

 The resulting spectrophotometric titration is shown
below in panel (a).
 Note that the titration curve’s y-axis is not the actual
absorbance, A, but a corrected absorbance, Acorr
Acorr = A × (VEDTA + VCu)/VCu
 where VEDTA and VCVCuu are, respectively, the volumes
of EDTA and Cu.
 Correcting the absorbance for the titrand’s dilution
ensures that the spectrophotometric titration curve
consists of linear segments that we can extrapolate to
find the end point.

 Examples of spectrophotometric titration curves:
 (a) only the titrand absorbs;
 (b) only the titrant absorbs;
 (c) only the product of the titration reaction absorbs;
 (d) both the titrand and the titrant absorb;
 (e) both the titration reaction’s product and the titrant absorb;
 (f) only the indicator absorbs. The red arrows indicate the end points for
each titration curve.

UV Visible spectroscopy

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