UV-Visible

Spectrophotometers

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 Fundamentals of
spectroscopy
Spectroscopy

Spectroscopy pertains to the dispersion of an
object's light into its component colors i.e.energies

It is the study of the interaction of electromagnetic
radiation with matter

By performing this dissection and analysis of an
object's light, astronomers can infer the physical
properties of that object (such as temperature, mass,
luminosity and composition).

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 Electro Magnetic Radiation
•

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Electromagnetic spectrum

1021 1018 1015 1012 109 106

Hz
10-3 1 200 500 106 109 1012

Ultraviolet

Radio
Microwave
Visible

Infrared
Cosmic

X-ray

λmax

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Types of electromagnetic
radiation

Absorption :
EM energy transferred to absorbing molecule
(transition from low energy to high energy state)

Emission :
EM energy transferred from emitting molecule to
space (transition from high energy to low energy
state)

Scattering :
Redirection of light with no energy transfer

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Absorption vs. Emission
hν
ν

En

En
hν
ν hν
ν

Eo Eo
Absorption Emission

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Transitions and Spectra of Atoms

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Transitions and Spectra of
molecules

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 Beer Lamberts law

In the optics the beer –lamberts law is the empirical
relation ship that related the absorption of the light
to the properties of the material through which the
light is traveling.
Equation:
There are several ways in which the law can be
expressed,
A ∝ CL = KCL by definition it is called the Beer
Lambert Law
A = KCL

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 Beer Lamberts law
. 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
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 Beer Lamberts law

E differs from K (Specific extinction

Coefficient)
by a factor of molecular weight.
Io IT Io IT

path length b Path length b

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 Explanation of the 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.

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 R- Transmittance
R= I0I - original light intensity
I-I0transmitted light intensity
% Transmittance = 100 x I
I0
Absorbance (A) or optical density (OD) = Log 1
T
I
= Log = I0 2 - Log%T
I0 I
Log is proportional to C (concentration of
solution) and is also proportional to L (length of
light path through the solution).
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Absorbance & Beer’s Law

As the concentration of the sample increases

the absorbance increases.
Intensity α Concentration 14
 Transmission and Color

The human eye sees the complementary color to

that which is absorbed
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Absorbance & Complementary
colors

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 Instrumentation of
spectrophotometer

In spectrophotometric analysis a source of radiation
is used that extends into the ultraviolet region of the
spectrum.

From this, definite wavelength of radiation are
chosen possessing a bandwidth of less than 1nm .

This process necessitates the use of more
complicated and consequently more expensive
instrument . The instrument employed is a
spectrophotometer .

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 Components of
spectrophotometer

Instruments for measuring the absorption of U.V. or
visible radiation are made up of the following
components
1. Sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout

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Schematic diagram of the
instrument
Single beam UV-Visible spectrophotometer

Double beam UV-Visible spectrophotometer

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 Instrumental components
1.Source of UV radiation

Itis important that the power of the

radiation source does not change
abruptly over it's wavelength range. Deuterium lamp

The electrical excitation of deuterium or
hydrogen at low pressure produces a
continuous UV spectrum.

The mechanism for this involves formation of
an excited molecular species, which breaks
up to give two atomic species and an
ultraviolet photon.

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This can be shown as;
D2 + electrical energy ® D2* ® D' + D'' + hv

Both deuterium and hydrogen lamps emit radiation
in the range 160 - 375 nm. Quartz windows must be
used in these lamps, and quartz cuvettes must be
used, because glass absorbs radiation of
wavelengths less than 350 nm.

The tungsten filament lamp is commonly employed
as a source of visible light

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Sources of the visible radiation

This type of lamp is used in the wavelength range
of 350 - 2500 nm.

The energy emitted by a tungsten filament lamp is
proportional to the fourth power of the operating
voltage.

This means that for the energy output to be
stable, the voltage to the lamp must be very
stable indeed. Electronic voltage regulators or
constant-voltage transformers are used to ensure
this stability.

Tungsten/halogen lamps contain a small amount
of iodine in a quartz "envelope" which also
contains the tungsten filament
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The iodine reacts with gaseous tungsten, formed
by sublimation, producing the volatile compound
WI2.

When molecules of WI2 hit the filament they
decompose, redepositing tungsten back on the
filament.

The lifetime of a tungsten/halogen lamp is
approximately double that of an ordinary tungsten
filament lamp. Tungsten/halogen lamps are very
efficient, and their output extends well into the
ultra-violet.

They are used in many modern
spectrophotometers.

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 Wavelength
selector(monochromator)
All monochromators contain the following component
parts;

An entrance slit

A collimating lens

A dispersing device (usually a prism or a grating)

A focusing lens

An exit slit

Polychromatic radiation (radiation of more than one
wavelength) enters the monochromator through the
entrance slit

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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.

Czerney-Turner grating monochromator

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 Cuvettes

The containers for the sample and reference
solution must be transparent to the radiation
which will pass through them.

Quartz or fused silica cuvettes are required for
spectroscopy in the UV region.

These cells are also transparent
in the visible region.

Silicate glasses can be used

for the manufacture of cuvettes for use between
350 and 2000

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 Detectors

The photomultiplier tube is a commonly used detector in
UV-Vis spectroscopy.

It consists of a photoemissive cathode (a cathode which
emits electrons when struck by photons of radiation),
several dynodes (which emit several electrons for each
electron striking them) and an anode.

A photon of radiation entering the tube strikes the
cathode, causing the emission of several electrons.

These electrons are accelerated towards the first dynode
(which is 90V more positive than the cathode).

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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, the electrons are collected at the anode.
By this time, each original photon has produced 106 -
107 electrons.

The resulting current is amplified and measured.

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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.

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UV VIS Spectros will have following
Features

This system is simple to operate and control and
customized to accommodate the protocols.

With the modes like Scan, multiwavelength, Time
scan , Concentration as a Standalone and the
software features like overlay , compare spectra,
Spectra zooming and smoothing, peak picking
etc
the instrument has the flexibility to suit any
application.

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UV Vis Spectro photometer general
Specifications
SPECTRAL Range : 190 to 1100 nm
Bandwidth : 2 nm
Accuracy : + 0.5 nm
Readability : 0.1 nm
Repeatability : + 0.2
PHOTOMETRIC System : Double beam
optics
Range : +/- 2.5 Abs.
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 Applications

In the determinations of the
 Nutrients like N, P, K, Ca, Mg, Zn, Cu, B,Mo,etc in the
agricultural soil, plants etc.
 Organic compounds in the biological matter.
 Glucose, fructose, carbohydrates, proteins etc in the
foods.
 Edible dyes, alcohols, etc in the beverages
 Purity of the constituents in the pharmaceuticals.
 Toxic elements like Cadmium, lead, mercury etc.in
the effluents.

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 Constituents in compositions used in the metallurgy,
fertilizer , pesticides, chemical, petro chemical, steel
, cement, glass and other industries.

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 THANK YOU

Presented by B.Srinivas. MSC

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