UV-visible spectroscopy

What is Spectroscopy?
• The study of molecular structure and
dynamics through the absorption, emission
and scattering of light.

• 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.
What is Light?
• According to Maxwell,
light is an
electromagnetic field
characterized by a
frequency f, velocity v,
and wavelength λ.
Light obeys the
relationship

f = v / λ.
 Electromagnetic Radiation
• Electromagnetic radiation consist of discrete
packages 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.
The Electromagnetic Spectrum

E = h =c/
 Electromagnetic Radiation
• 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.
 Electromagnetic Radiation

• The relationship between wavelength &

frequency can be written as:
c=νλ
• As photon is subjected to energy, so
E=hν=hc/λ
Electromagnetic Radiation
 Electromagnetic Radiation

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.
 Principles of Spectroscopy
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)
 Principles of Spectroscopy
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

Spectroscopy

Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

WAVELENGTH(nm)
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.
 Interaction of EMR with matter
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

 Transmission and Color

The human eye sees the complementary color to that which is

absorbed
Absorbance and Complementary
Colors
Lambert’s
Law
 Lambert’s Law
• When a monochromatic radiation is passed
through a solution, the decrease in the
intensity of radiation with thickness of the
solution is directly proportional to the
intensity of the incident light.

• Let I be the intensity of incident radiation.

x be the thickness of the solution.
Then
 Lambert’s Law
dI
 I
dx
dI
So,   KI
dx
Integrate equation between limit
I = Io at x = 0 and
I = I at x=l,
We get,
I
ln  Kl
I0
 Lambert’s Law
I
2.303 log  Kl
I0
I K
log  l
I0 2.303
I0
Where, log A Absorbance
I
K
E Absorption coefficient
2.303
A E.l Lambert’s Law
Beer’s
Law
 Beer’s Law
• When a monochromatic radiation is passed
through a solution, the decrease in the
intensity of radiation with thickness of the
solution is directly proportional to the
intensity of the incident light as well as
concentration of the solution.

• Let I be the intensity of incident radiation.

x be the thickness of the solution.
C be the concentration of the
solution.
 Beer’s Law
dI
 C.I
dx
dI
So,  K ' C.I
dx
Integrate equation between limit
I = Io at x = 0 and
I = I at x=l,
We get,
I
ln  K ' C.l
I0
 Beer’s Law
I0
2.303 log K .C.l
I
I0 K
log  C.l
I 2.303
I
Where, log 0  A Absorbance
I
K Molar extinction
E
2.303 coefficient
A E.C.l Beer’s Law
 Beer’s Law
A E.C.l
I I
T  OR  log T log A
I0 I0

From the equation it is seen that the absorbance

which is also called as optical density (OD) of a solution
in a container of fixed path length is directly
proportional to the concentration of a solution.
BEER LAMBERT LAW

Light
I0 I

Glass cell filled with

concentration of solution (C)

As the cell thickness increases, the intensity of I

(transmitted intensity of light ) decreases.
 R- Transmittance
I
R= I0 - original light intensity
I0
I- transmitted light intensity

I
% Transmittance = 100 x
I0
1
Absorbance (A) or optical density (OD) = LogT

I0
= Log = 2 - Log%T
I
I
Log is proportional to C (concentration of solution)
I0
and is also proportional to L (length of light path
through the solution).
A  CL = KCL by definition and it is called
the Beer Lambert Law.
A = KCL
K = Specific Extinction Coefficient ---- 1 g of
solute per liter of solution

A = ECL
E = Molar Extinction Coefficient ----
Extinction Coefficient of a solution containing
1g molecule of solute per 1 liter of solution
 Absorbance x Liter
E =
Moles x cm

E differs from K (Specific extinction Coefficient) by

a factor of molecular weight.

UNITS
A = ECL
A = No unit (numerical number only)
Liter
E =
Cm x Mole
 L = Cm
C = Moles/Liter
Liter Mole
A = ECL = ( )x x Cm
Cm x Mole Liter

A = KCL
A = No unit C = Gram/Liter L = Cm

Liter
K=
Cm  Gram

Liter Gram
A = KLC = ( )x x Cm
Cm x Gram Liter
Transmittance and Concentration
The Bouguer-Lambert Law

T  I / I 0 e  ConstPathlength
Transmittance and Path Length:
Beer’s Law

Concentration

T I / I 0 e  Const Concentration
The Beer-Bouguer-Lambert Law

A  log T  logI / I 0  logI 0 / I   b c

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.
Electronic
Transition
The possible electronic transitions can
graphically shown as:
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.
• 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

• 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
• 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.
• 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

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

 Chromophore
To interpretate UV – visible spectrum following
points should be noted:

1.Non-conjugated alkenes show an intense

absorption below 200 nm & are therefore
inaccessible to UV spectrophotometer.

2.Non-conjugated carbonyl group compound

give a weak absorption band in the 200 - 300 nm
region.
 Chromophore
e.g. O
Acetone which has λmax = 279 nm O
C
H3C CH3

and that cyclohexane has λmax = 291 nm.

When double bonds are conjugated in a

compound λmax is shifted to longer wavelength.
e.g. 1,5 - hexadiene has λmax = 178 nm
2,4 - hexadiene has λmax = 227 nm
CH2 CH3
H2C H3C
 Chromophore
3. Conjugation of C=C and carbonyl group shifts
the λmax of both groups to longer wavelength.
e.g. Ethylene has λmax = 171 nm O
Acetone has λmax = 279 nm C
H2C CH2 H3C CH3

Crotonaldehyde has λmax = 290 nm

O

H2C C
CH3
 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

OH

Phenol λmax = 270 nm

NH2

Aniline λmax = 280 nm

Absorption
& Intensity
Shifts
• 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.
• In alkaline medium, p-nitrophenol shows
red shift. Because negatively charged
oxygen delocalizes more effectively than
the unshared pair of electron.
- -
O + O O + O
N N

-
OH
Alkaline
medium -
OH O

p-nitrophenol
λmax = 255 nm λmax = 265 nm
• 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.
• Aniline shows blue shift in acidic medium, it
loses conjugation.

+ -
NH2 + NH3 Cl
H
Acidic
medium

Aniline
λmax = 280 nm λmax = 265 nm
• 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.

N N CH3

Pyridine 2-methyl
pyridine
• When absorption intensity (ε) of a
compound is decreased, it is known as
hypochromic shift.

CH3

Naphthalene 2-methyl
naphthalene
ε = 19000 ε = 10250
 Shifts and Effects
Hyperchromic shift

Blue Red
Absorbance ( A )

shift shift

Hypochromic shift

λmax
Wavelength ( λ )
BLOCK DIAGRAM OF SPECTROPHOTOMETER
The Spectrophotometer
Single-beam

Double-beam
SPECTROPHOTOMETER
Spectrometer: for producing light of any selected wavelength or color
Photometer: used for measuring the intensity of light.
The two devices are placed at either side of a cuvette filled with a liquid

Exit slit

Entrance slit D etector

R ed I0 I
Readout
device

I0= radiant power arriving at the a = absorptivity of the sample (extinction

Prismthe L = length of the path through the sample
coefficient)
Icuvette
= radiant power leaving
Violet C uvette
c = concentration
C of the absorbing substance
cuvette

Light source M onochrom ator

 COMPONENTS OF SPECTROPHOTOMETER

1. Light source(UV and visible)

2. Optical system/Wavelength selector
(Monochromator)
3. Sample containers
4. Detector
5. Output: Signal processor and readout
 SPECTROPHOTOMETER
Exit slit

Entrance slit Detector

Red I0 I
Readout
device

Prism
Violet Cuvette
I0= radiant power arriving at the cuvette a = absorptivity of the sample (extinction coefficient)
L = length of the path through the sample C
I = radiant power leaving the cuvette c = concentration of the absorbing substance

Light source Monochromator

 WORKING OF SPECTROPHOTOMETER

• White light radiation source that passes through a MONOCHROMATOR ( prism

or a diffraction grating that separates the white light into all colors of the
visible spectrum) .
• After the light is separated, it passes through a FILTER (to block out unwanted
light, sometimes light of a different color) and a SLIT (to narrow the beam of
light).
• Next the beam of light passes through the SAMPLE that is in the sample
holder.(cuvette)
• The light passes through the sample and the unabsorbed portion (reflected)
strikes a PHOTODETECTOR that produces an electrical signal which is
proportional to the intensity of the light.
• The signal is then converted to A READABLE OUTPUT (absorbance )that is
used in the analysis of the sample.
• Calibration curve : generated by measuring the absorbance of several
solutions that contain known concentrations of analyte.

•
 COMPONENTS OF SPECTROPHOTOMETER
1. LIGHT SOURCE

• Deuterium Lamps － Continuous spectrum in the ultraviolet region is

produced by electrical excitation of deuterium at low pressure. (160nm-
375nm)
• Tungsten Filament Lamps － the most common source of visible and
near infrared radiation ( at wavelength 320 to 2500 nm)
• Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths
from 200 to 800 nm)- in UV Spectrophotometer
• Silicon Carbide (SiC) Rod : Radiation at wavelengths:1200 -40000
nm

• NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm) –

for IR Region:
• Laser: Used when high intensity line source is required
 OPTICAL SYSTEM/WAVELENGTH SELECTOR
MONOCHROMATOR

• Optical device
• Disperses a beam of
light into its Exit slit
component
wavelengths Entrance slit Detector
• Allows only a narrow
band of wavelengths to Red I0 I
pass Readout
• Blocks all other device
wavelengths
1. An entrance slit
2.I =Aradiant
collimating
power arrivinglens Prism of the sample (extinction coefficient)
a = absorptivity
0
(concave)
at the cuvette
L = length of the path
Cuvette
Violet through the sample C
I = radiant power leaving the cuvette c = concentration of the absorbing substance
3. A dispersing device
(usually a prism or a
grating) Light source Monochromator
4. A focusing lens
5. An exit slit
 MONOCHROMATOR
•Czerny-Turner setup
• AS A FILTER: It will select a narrow portion of the spectrum
(the bandpass) of a given source.
• IN ANALYSIS: the monochromator will sequentially select
for the detector to record the different components
(spectrum) of any source or sample emitting light.
• Mirror collimates light (parallel rays)
• Gating disperses light ( Prisms were formerly used)
• Light coming through entrance slit is polychromatic
• Light out of exit slit is monochromatic
 CUVETTES ( SAMPLE CONTAINERS)
• The containers for the sample- usually plastic or quartz:
• Reference solution must be transparent to the radiation which will
pass through them.
• Quartz or fused crystalline silica cuvettes for UV spectroscopy .
• Glass cuvettes for Visible Spectrophotometer
• NaCl and KBr Crystals for IR wavelengths
Light Sources
UV Spectrophotometer
1. Hydrogen Gas Lamp
2. Mercury Lamp
Visible Spectrophotometer
1. Tungsten Lamp
InfraRed (IR) Spectrophotometer
1. Carborundum (SIC)
 Dispersion Devices

• Non-linear dispersion
• Temperature sensitive

• Linear Dispersion
• Different orders
Dispersion of
polychromatic light with a
prism
Infrared
monochromatic
Ray
Red
Orange
Yellow SLIT
Polychromatic PRISM
Green
Ray Blue
Violet

Ultraviolet

Polychromatic Monochromatic Ray

Ray
Prism - spray out the spectrum and choose the certain
wavelength (l) that you want by moving the slit.
Photomultiplier Tube Detector

• High sensitivity at
low light levels
• Cathode material
determines spectral
sensitivity
• Good signal/noise
• Shock sensitive

Anode
 The Photodiode Detector

• Wide dynamic range

• Very good
signal/noise at high
light levels
• Solid-state device
Schematic Diagram of a Photodiode
Array

• Same characteristics
as photodiodes
• Solid-state device
• Fast read-out cycles
 Conventional
Spectrophotometer

Schematic of a conventional single-beam spectrophotometer

 Conventional
Spectrophotometer

Optical system of a double-beam spectrophotometer

 Conventional
Spectrophotometer

Optical system of a split-beam spectrophotometer

 Definition of Resolution

Spectral resolution is a measure of the ability of an instrument to

differentiate between two adjacent wavelengths
 Instrumental Spectral
Bandwidth

The SBW is defined as the width, at half the maximum intensity, of

the band of light leaving the monochromator
Natural Spectral Bandwidth

The NBW is the width of the sample absorption band at half the
absorption maximum
Transmission Characteristics of
Cell Materials

Note that all materials exhibit at least approximately 10% loss in

transmittance at all wavelengths
Cells

UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
 Cell Types I

Open-topped rectangular standard cell (a)

and apertured cell (b) for limited sample volume
 Cell Types II

Micro cell (a) for very small volumes and flow-through cell (b)
for automated applications
APPLICATIONS OF
UV / VISIBLE
SPECTROSCOPY
 Applications
• 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.
• Detection of isomers are possible.
• Determination of molecular weight using Beer’s
law.
Two-Component Mixture

Example of a two-component mixture with little spectral

overlap
Two-Component Mixture

Example of a two-component mixture with significant

spectral overlap
Absorption Spectra of Hemoglobin
Derivatives
 Influence of 10% Random Error

Influence on the calculated concentrations

• Little spectral overlap: 10% Error
• Significant spectral overlap: Depends on similarity, can be much higher (e.g. 100%)
 Precision and Accuracy

Precision – Precision + Precision – Precision +

Accuracy – Accuracy – Accuracy + Accuracy +

 STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD

1. Run the sample for

spectrum Absorbance

2. Obtain a monochromatic 2.0

wavelength for the

maximum absorption
wavelength. 0.0

200 250 300 350 400 450

3. Calculate the concentration Wavelength (nm)

of your sample using Beer

Lambert Equation: A = KCL
 A
Slope of Standard Curve =
C

Absorbance at 280 nm

1.0

0.5

1 2 3 4 5
Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where
becomes non-linear.
 SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
Absorbance at 540 nm
1.2

0.8

0.4

1 2 3 4
Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a

standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
• Every instrument has a useful range for a
particular analyte.
• Often, you must determine that range
experimentally.
• This is done by making a dilution series of the
known solution.
• These dilutions are used to make a working
curve.
Make a dilution series of a known quantity
of analyte and measure the Absorbance.
Plot concentrations v. Absorbance.
What concentration do you think the
unknown sample is?
In this graph, values above A=1.0 are not linear. If we
use readings above A=1.0, graph isn’t accurate.
The best range of this spectrophotometer is A=0.1 to
A=1.0, because of lower errors. A=0.4 is best.
 Relating Absorbance and
Transmittance
• Absorbance rises linearly with
concentration. Absorbance is measured
in units.
• Transmittance decreases in a non-linear
fashion.
• Transmittance is measured as a %.
• Absorbance = log10
– (100/% transmittance)