UV – VIS Spectrophotometer

Source

Monochromator

Slit
• Principle
Detector
• Beer’s Lamberts Law
• Operation of UV-VIS Spectroscopic
• Typical Industrial Application
ELECTROMAGNETIC
RADIATION
 SPECTROSCOPY
The study of the interaction of light with matter
The study of interaction of electromagnetic radiation
with matter
• Energy (light or electromagnetic )applied to
matter can be absorbed, emitted, cause a
chemical change, or be transmitted
• The main application of UV-VIS Spectroscopy,
which depends on transitions between electronic
energy levels, is in identifying conjugated pi
electron systems
 Electromagnetic RADIATION
• Light is a form of electromagnetic radiation.

Electromagnetic radiation can be considered as a form of radiant

energy that is propagated as a transverse wave.

It vibrates perpendicular to the direction of propagation, imparts a

wave motion to the radiation
 The
WAVE
Wavelength – the distance of one
complete cycle

WAVE

Frequency – the number of cycles passing

a fixed point per unit time
 Electromagnetic Radiation
• Wave is a way of shifting energy from one place to another
• Electromagnetic radiation consist of discrete packages of energy
which are called as photons.

Frequency (v):
- 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:
- 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 and frequency can be
written as:
• c=
•  = wavelength in centimeters (cm)
• c = velocity of light (3 x 1010 cm/s)
•  = frequency in reciprocal seconds
(s-1)

• The wavelength unit preferred for the

ultraviolet and visible regions of the
spectrum is nanometer (nm)= 10-9 m.
• Wavelength and frequency are
inversely proportional.
 • The laws of quantum mechanics, as photon is
subjected to energy, so
• E = energy of radiation,  = frequency and h =
Planck’s constant (6.63 x 10-34 m2.kg/s or J s)

E = h

• The higher the frequency, the greater the energy of the wave.
• The shorter the wavelength, the greater the energy of the wave.
 Interaction of EMR with
matter
Electronic Energy Levels:
• At room temperature the molecules are in the
lowest energy level Eo
• 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=hv=En-Eo
Where (n=1,2,3,…etc)
 Electromagnetic Spectrum

The diagram shows an approximate to the

spectrum of visible light
 Electromagnetic Spectrum
 We see objects as colored because they transmit or reflect
only a portion of the light in the visible region.

 When white light (which contains the whole spectrum of

wavelengths in the visible region) is passed through an object,
the object will absorb certain of the wavelengths, leaving the
unabsorbed wavelength to be transmitted.

Absorbed Color Transmitted Color

Violet Yellow-green
Blue Orange
Green Violet
Yellow Blue
Orange Green-blue
Red Blue-green
The diagram shows an approximation to the spectrum of electromagnetic

12
 Electromagnetic Spectrum

Why are carrots orange?

Carrots contain the pigment carotene which

absorbs blue light strongly and reflects orange
red and so the carrot appears orange.

13
Absorbance and Complimentary Colour
Absorption
 Absorption
• The process by which energy is transferred
from an electronic wave to an atom or molecule
and causes it to move from ground state to an
excited state.

• Absorption can only occur when an atom or

molecule absorbs a photon of light that has an
energy which exactly corresponds to the
difference between two energy levels.
 Absorption
• Nuclei inside the molecules play an important role
in:
– Determining wavelength of radiation absorbed
– Determine the strength with which the electrons
are bound and thus influence the energy
spacing between ground state and excited state.
Chromophores is refer as the absorbing groups
in a molecule.
A molecule containing a chromophores is called
a chromogen.
Absorbance VS
Emission
 Absorption vs Emission

Energy is emitted by
electrons returning to
lower energy levels
3rd
Excited 2nd
States

1st

Energy is absorbed as
electrons jump to
higher energy levels

Ground State

19
Absorbance VS
Transmittance
 Absorbance VS Transmittance
The absorbance is a logarithm function of
transmittance,

A = - log T
Note:
Since transmittance is often expressed as percentage
and called percent transmittance (%T= T x 100 %), A
also can calculate as,
A= log100- log %T
A = 2 – log %T
Note:
1. Absorbance is zero when transmittance is
100%, ie. No light absorbed by the sample
P = Po
b = 1 cm
T = 1.0 or 100 % and A = 0
P0 P
Transmitted light = P
Incident light = Po

2. Absorbance is infinity when transmittance is

0%, ie. all light absorbed by the sample
If all the light passes through a solution without any
absorption  absorbance is zero and percent
transmittance is 100%.
If all the light is absorbed  percent transmittance is
zero and absorption is infinite.

The relationship between absorbance and transmittance

 Absorbance VS Transmittance
0.10

0.09

0.08

0.07

Coffee Sample
0.06

Abs A
0.05

0.04

0.03

0.02

0.01

0.00
250 300 350 400 450 500 550 600 650 700 750 800 850
NM

100

98

96

94

%T 92

90
%T
88

86

84

82

80
250 300 350 400 450 500 550 600 650 700 750 800 850
NM
 BEER
LAMBERT
LAW
CONCENTRATION of a
substance in solution is
directly proportional to the
ABSORBANCE of the
solution
 Beer’s Lambert Law
 states the relationship between the absorbance of a
solution and the concentration of the absorbing species.
A=abc (1)
Where a = Proportional equation; absorptivity (Lg-1cm-1),
b = optical pathlength (cm)
c = Concentration (g L-1).

A=bc (2)
Where A = absorbance,
 = molar absorptivity (L mol-1cm-1),
b = optical pathlength (cm)
c = Concentration (mol L-1).
It is also known as absorption law or beer-Lambert law
 • This Law tell us quantitatively how the amount
of attenuation depends on the concentration of
the absorbing molecules and the pathlength
over which absorption occur.
• Absorption may be presented as:
Transmittance ( T = P / Po )
or Absorbance ( A = log Po / P)

Po = power of incident radiation

P = power of transmitted radiation
c = concentration
b = pathlength

Absorption of Radiation
 Using the Beer's Law
Beer’s law expressed in equation (1) and (2) , can be used
in several different way:
1. Calculate molar absortivity of species if their
concentrations are known.
2. Can use to measure value of absorbance to obtain
concentration if absorptivity and pathlength are
known.
3. More often , we use a series of standards solution of
the analytes (different concentrations) to construct a
calibration curve, or working curve of A versus c. Then
it can be used to determine the concentration of
unknown.
Calibration Graph
 Limitations of Beer-Lambert
Law
• deviations in absorptivity coefficients at high concentrations
(>0.01M) due to electrostatic interactions between molecules in
close proximity
• scattering of light due to particulates in the sample
• fluoresecence or phosphorescence of the sample
• changes in refractive index at high analyte concentration
• shifts in chemical equilibria as a function of concentration
• non-monochromatic radiation, deviations can be minimized by
using a relatively flat part of the absorption spectrum such as the
maximum of an absorption band
 Experiment: Analysis of Food
Colour
Objectives:

1) To determine  max for food dye sample

(wavelength scan).

2) To prepare a serial dilution and generate a

standard calibration graph for sample
quantitation (photometric scan).
 Material and Method

Chemicals
100 ppm Carmoisine stock (100 ml), Unknown concentration
of Carmoisine sample (two), Distilled water

Apparatus
Perkin – Elmer UV/Vis Spectrophotometer Lambda EZ210,
Sample cuvettes, path length 1 cm, Volumetric flask 50 ml
(five) , Pipette 5 ml, 10 ml and 25 ml (one each),Rubber bulb
(three),Beaker 100 ml (one),Graduated cylinder 50 ml
(one),Dropper (one),Labeling sticker,Tissue paper
 Procedure
1) Prepare a serial dilutions (5 ppm, 15 ppm, 25 ppm, 35 ppm
and 45 ppm) from the 100 ppm food dye stock.
2) Calculate the volume needed, V1 from the 100 ppm food
dye stock for all dilutions by using dilution formula
(M1V1=M2V2).
3) After preparing the serial dilutions, Fill a cuvette with 45
ppm dilution and another cuvette with blank solution, insert
them in the sample compartment. Wipe clean the sides of the
cuvettes.
4) Record the absorbance readings and look at the standard
calibration graph produced.
5) Determine the concentrations of Unknown #1 and
Unknown #2.
Standard Solution
 Result
No Sample Carmoisine Concentration Absorbance (nm)
(ppm)
1 Standard 1 5 0.214
2 Standard 2 15 0.591
3 Standard 3 25 1.025
4 Standard 4 35 1.333

5 Standard 5 45 1.714
6 Unknown 1 ??? 0.775

7 Unknown 2 ??? 1.587

Graph
Standard Preparation
 Prepare the Standards

The concentration and volume of the stock

solution
added should be chosen to increase the
concentration of the unknown by about 30% in
each succeeding flask.
 Example 1
A 7.50 x 10-5 M solution of potassium permanganate
has a transmittance of 36.4% when measured in a 1.05
cm cell at wavelength of 525 nm. Calculate :
(a) The absorbance of this solution Ans:0.439
(b) the molar absortivity of KMnO4 Ans: 5.57 x 103
Answer:
A = - log T = - log 0.364 = 0.439
Using equation 2,
 = A / bc = 0.439 / 1.05 cm x 7.50 x 10-5 mol L-1
= 5.57 x 103 Lmol-1cm-1
 Example 2
At 580nm, the Wavelength of maximum absorption
of complex Fe(SCN)2+ has a molar absorptivity of 7.00
x 10-3 L mol-1 cm-1. Calculate the absorbance of a 2.50
x 10-5 M solution of the complex at 580 nm in 1.00
cm cell.
Answer:
A=bc
= (7.00 x 10-3 L mol-1 cm-1) (1.00 cm) (2.50 x 10-5

mol L-1)
= 1.75 x 10-7
PRACTICE
MAKE
PERFECT!!!
1. A sample in a 1.0 cm cell is determined with
spectrometer to transmit 80% light at a certain
wavelength. If the absorptivity of this substance at this
wavelength is 2.0 Lg-1cm-1, what is the concentration of
the substance? Ans: 0.048 g/l

2. A solution containing 2.5 x 10-5M iron Phenanthroline

complex placed in a 5.00 cm cuvette. Calculate the
transmittance of the solution if the molar absorptivity
is 750 cm-1M-1. Ans: 81%

3. A 20 ppm solution of a DNA molecule isolated from

Escherichia coli was found to give an absorbance of
0.80 in a 2 cm cell. Calculate the absorptivity of the
molecule. Ans: 0.02 ppm-1 cm-1
 Principles of Spectroscopy
• Spectroscopy is the study of how electromagnetic radiation, across a
spectrum of different wavelengths, interacts with molecules - and how
these interactions can be quantified, analyzed, and ultimately
interpreted to gain information about molecular structure
• 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 (v) or wavelength ()
• Spectrometer is an instrument design to measure the spectrum of a
compound.
Absorption spectroscopy
An analytical technique which concerns with the measurement of
absorption or electromagnetic radiation
eg. UV(185-400 nm) /Visible (400-800 nm) Spectroscopy
ELECTRONIC TRANSITIONS
• The possible electronic transitions can
graphically shown as:

The larger the energy jump, the lower the

wavelength of the light absorbed.
• The possible electronic transitions are:
• electron from orbital is excited to
corresponding anti-bonding orbital .
• The energy required is large for this
transition.
• Example: Methane (CH4) has C-H bond only
and can undergo transition and shows
maximum absorbance at 125 nm.
 Molecular hydrogen, H2 transition
• The molecular orbital (MO) picture for the hydrogen molecule consists of one
bonding σ MO, and a higher energy antibonding σ* MO.
• When the molecule is in the ground state, both electrons are paired in the
lower-energy bonding orbital – this is the Highest Occupied Molecular Orbital
(HOMO).
• The antibonding σ* orbital, is the Lowest Unoccupied Molecular Orbital
(LUMO).
• If the molecule is exposed to light of a wavelength with energy equal to ΔE, the
HOMO-LUMO energy gap, this wavelength will be absorbed and the energy
used to bump one of the electrons from the HOMO to the LUMO – in other
words, from the σ to the σ* orbital.
• ΔE for this electronic transition is 258 kcal/mol, corresponding to light with a
wavelength of 111 nm.
• electron in a bonding orbital is excited to
corresponding anti-bonding orbital .
• Compounds containing multiple bonds like
alkenes, alkynes, carbonyl, nitriles, aromatic
compound and etc undergo
• Example: alkenes generally absorb in the
region 165 to 205 nm.
 Ethene transition
• When a double-bonded molecule such as
ethene (common name ethylene) absorbs
light, it undergoes a π - π* transition.
• Because π- π* energy gaps are narrower
than σ - σ* gaps,
• Ethene absorbs light at 165 nm - a longer
wavelength than molecular hydrogen.
• Saturated compounds containing atoms with
lone pair of electrons like O, N, S and
halogens are capable of transition.
• These transition usually requires less
energy than transitions.
• The number of organic functional groups
with peaks in UV region is small (150-250
nm)
• An electron from non-bonding orbital is
promoted to anti-bonding orbital.
• Compounds containing double bond
involving hetero atoms (C=O,CN, N=O)
undergo such transitions.
• transitions require minimum energy and
show absorption at longer wavelength
around 300 nm.
• These electronic transitions are forbidden transition and
only theoretically possible.
• Thus, and electronic transitions show absorption in region
above 200 nm which is accessible to UV-visible
spectrophotometer.
• That means that in order to absorb light in the region from
200 - 800 nm (which is where the spectra are measured),
the molecule must contain either pi bonds or atoms with
non-bonding orbitals
• The UV spectrum is only a few broad of absorption.
 Chromophore
• The part of a molecule responsible for
imparting colour, are called as
chromosphores
• The functional groups containing multiple
bonds capable of absorbing radiation above
200 nm due to and transitions.
• Example: NO2, N=O, C=O, C=N, CN, C=C,
C=S, etc
Chromophore
Chromophore
 Absorbance spectrum
• Below is the absorbance spectrum of an important biological molecule called
nicotinamide adenine dinucleotide, abbreviated NAD+
• This compound absorbs light in the UV range due to the presence of
conjugated pi-bonding systems.
• λmax , which is the wavelength at maximal light absorbance. NAD+ has λmax
=260nm. We also want to record how much light is absorbed at λmax . Here
we use a unitless number called absorbance, abbreviated 'A'
SPECTROMETRIC
instrumentation
UV-VIS spectrometry
well known for its
simplicity, versatility,
speed, accuracy and
cost effectiveness.
UV-Vis Spectrophotometer
UV-Vis Spectrophotometer
 Spectroscopic Techniques and
Chemistry they Probe
UV-vis UV-vis region bonding electrons

Atomic Absorption UV-vis region atomic transitions (val. e-)

FT-IR IR/Microwave vibrations, rotations

Spectroscopic Techniques and

Common Uses
Quantitative
UV-vis UV-vis region analysis/Beer’s Law
Quantitative analysis
Atomic Absorption UV-vis region Beer’s Law

FT-IR IR/Microwave Functional Group Analysis

UV-vis instrumentation

Typical UV VIS
spectrophotometer consists of a
(1) SOURCE, a
(2) MONOCHROMATOR,
(3) SAMPLE CELL,
(4) DETECTOR and
(5) READ OUT
Instrumentation and Basic Components

Block diagram of Spectrometer

Schematic diagram of a double-beam UV-VIS

spectrophotometer
 Components of
UV-VIS
Spectrophotometer
 (1)Sources

All spectrophotometers
require a SOURCE of
continuous radiation
over the wavelengths of
interest.
 Sources of UV radiation
Sources used in UV-Vis Spectrophotometers are continuous sources.
• Continuous sources emit radiation of all wavelengths within the
spectral region for which they are to be used.
• Sources of radiation should also be stable and of high intensity.
• The combined output of these two bulbs is focused on to a diffraction
grating
Continuous
Sources

Visible and
Ultraviolet
near IR
radiation
radiation

Tungsten Deuterium
Lamp Lamp
320-2500 nm 200-400 nm
REQUIREMENTS OF AN IDEAL SOURCE
• It should be stable and should not allow
fluctuations
• It should emit light of continuous spectrum of high
and uniform intensity over the entire wavelength
region in which it’s used
• It should provide incident light of sufficient
intensity for the transmitted energy to be detected
at the end of optic path.
• It should not show fatigue on continued use.
 Source of visible radiation
• The tungsten filament lamp is commonly employed as a source of
visible light.
• This type of lamp is used in the wavelength range of 350 - 2500 nm.
• Tungsten/halogen lamps contain a small amount of iodine in a quartz
"envelope" which also contains the tungsten filament.
• Tungsten/halogen lamps are very efficient, and their output extends
well into the ultra-violet. They are used in many modern
spectrophotometers
 (2) monochromators

All spectrophotometers
require a MONOCHROMATORS
for selecting a narrow band
of wavelengths from the
source spectrum.
 Monochromators
• It is a device used to isolate the radiation of the
desired wavelength from wavelength of the
continuous spectra.
• All monochromator contain the following
component parts:
1) An entrance slit
2) A collimating lens
3) A dispersing device (a prism or a grating)
4) A focusing lens
5) An exit slit
• Polychromatic radiation (radiation
of more than one wavelength)
enters the monochromator
through the entrance slit.
• The beam is collimated, and then
strikes the dispersing element at
an angle.
• The beam is split into its
component wavelengths by the
grating or prism.
• By moving the dispersing element
or the exit slit, radiation of only a
particular wavelength leaves the
monochromator through the exit
slit.
 (3) Sample cells

All spectrophotometers
require a SAMPLE CELL for
holding the sample (usually a
solution) and a reference cells
 Cells
• One of the two divided beams is passed
through the sample solution and second
beam is passed through the reference
solution.
• Both sample and reference solution are
contained in the cells.
 Sample Containers
Sample Container, usually called cells or cuvettes,
must be transparent to the radiation which will pass
through them.
Types of material normally used are:
1. Quartz or fused silica required for the UV region
(wavelength less than 350 nm) and may used in
the visible region and out to about 3000 nm in the
IR region.
2. Silicate Glass ordinary used for the region of 375
to 2000nm because of its low cost compare quartz
3. Plastic cells also used in visible region sample

The most common cell pathlength for studies in

the UV – VIS region is 1 cm.
Quartz Cuvette
Silica Cuvette
Plastic Cuvette
 Cell and Cell Holders

standard cylindrical Long path length

 Handling of Cell
• Clean and Dry Immediately after used
• Avoid scratches on polished cell windows

Cleaning of Cell
• Use the solvent in which the substance was dissolved
• After cleaning rinse with distilled water
• Never use hydrofluoric acid for cleaning – will etch the
surface
• Never use ultrasonic cleaning – may cause cavitation
 (4) detectors

All spectrophotometers
require a DETECTORS for
converting radiant energy
into electrical energy.
 Detector
Usually uses photo-electric devices. It converts the
radiant energy (hv) into an electrical signal.
Examples of detectors are:

 Photomultiplier tube
 Linear photodiode array
 Charge –coupled devices
 Detector
The photomultiplier tube
• The photomultiplier tube is a commonly used detector in
UV-Vis spectroscopy.
• 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.
 Double beam spectrophotometer
detector
• Generally two photocells serve the purpose of
detector in UV spectroscopy.
• One of the photocell receives the beam from
sample cell and second detector receives the
beam from the reference.
• The intensity of the radiation from the reference
cell is stronger than the beam of sample cell.
• This results in the generation of pulsating or
alternating currents in the photocells.
 (5) Read out

All spectrophotometers
require a DEVICE TO READ OUT
the response of the detector.
 Types of Instrument

(A) Single Beam UV – VIS

Spectrophotometer

(B) Double Beam UV – VIS

Spectrophotometer
 (a) Single Beam UV – VIS
Spectrophotometer
The simplest type of spectrometer employs a
single source to supply radiation to the sample
 • In single - beam UV - Vis
Absorption Spectroscopy,
obtaining a spectrum
requires manually
measuring the
transmittance (see the
Beer – Lambert Law) of
the sample and solvent at
each wavelength.

05/21/2024 91
 (b) Double Beam UV – VIS
Spectrophotometer
Has two light paths, one for the sample and one for the
blank or reference.
 • The double-beam design greatly simplifies this
process by measuring the transmittance of the
sample and solvent simultaneously. The
detection electronics can then manipulate the
measurements to give the absorbance.

05/21/2024 93
 Advantage of Single Beam
Instrument
It is well suited for quantitative absorption
measurement at single – wavelength type.
Here, simplicity of the instrumentation, low cost,
and ease of maintenance offer distinct
advantages.
 Advantage of Double beam instrument

Double beam instrument offer the advantage

that they are compensate for all but the
most short term fluctuation in the radiant
output of the sources.
They also compensate for wide variation of
source intensity with wavelength.
Furthermore, the Double Beam design is well
suited for continuous recording of
absorption spectra.
Industrial Application
 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.
 Typical Industrial Application

 Chemical analysis – quality control, quality analysis

 Biochemical analysis – proteins, DNA, enzymes
 Water and environment analysis – EPA & DIN
methods, cation/anion
 Kinetics – enzymes kinetics, substrate kinetic
 Food analysis – food colors (dyes), toxins, additives
(BHA, BHT), contaminants (heavy metals)
 Pharmaceutical – drugs (opium, heroin, cocaine)
THE END