Spectrophotometer

Dr . S. Jayakumar
Objectives of this lecture
Spectrophotometer
Introduction
Components
1. Sources of light
2. Monochromators
3. Slit
4. Sample containers
5. Detectors
6. Readout devices
Principles
Beer’s –Lambert’s Law
Types
Single and double beam instruments
Applications
Qualitative &Quantitative analyses
Introduction
 Photometry – Study of the measurement of intensity of light.

 Colorimetry- Study the color of specific wavelength of visible

light (400 -750 nm).

 Spectrophotometry- Study of color of a very narrow range of

wavelength in UV (10-200 nm), visible (400-750 nm) and IR
(2.5-100 µm)

 Concentration of a biochemical compound can be

determined.

 Used to estimate the compounds in a complex mixture.

 Though the instruments different the basic principles of both

are however the same.
When a narrow
beam of light is
allowed to pass
through a
prism/grating, it is
dispersed into
seven colours
from red to violet
and the band is
called “Spectrum”.
 The spectrum obtained by white light -
continuous spectrum.
 The spectrum is formed by electromagnetic
waves and the wavelength of each color
varies.
 The different types of electromagnetic
radiation with increasing order of wavelength
is : Cosmic rays < γ rays < X rays < Ultraviolet
rays < Visible light< Infrared rays <
Microwaves < Radio waves
Regions of electro magnetic
spectrum and their wavelength range

Region Wavelength range

X- rays 10-2 to 102 Ao
Far Ultraviolet 10 – 200 nm
Near Ultraviolet 200 – 400 nm
Visible 400 – 750 nm

Near Infra red 0.75 – 2.2µm

Mid Infra red 2.5 – 50µm
Far Infra red 50 - 100µm
Microwaves 0.1 – 100 cm
Radio waves 1 – 1000 m
A beam of radiation from an electric bulb
consist of several wavelengths and is
known as polychromatic

A beam in which all the rays have

the same wavelength is known
as monochromatic
Components of Spectrophotometer
Radiation Source

A stable continuous radiation

is required

Hydrogen gas lamps or

Deuterium lamps - employed
to provide an Ultra violet
radiation.

Deuterium lamps have the

advantage of producing
continuous radiation of
higher intensity.
 Lamps used in UV-Vis Spectrophotometer

Deuterium Lamp (deuterium gas)

- UV Region
- Wavelength Range : 190~420nm

Tungsten Lamp (halogen- iodine and bromide)

- Wavelength Range : Part of the UV and
Visible

Xenon Lamp (xenon gas)

-Wavelength Range : 190~800nm
 Monochromator
It is a device that breaks the polychromatic radiation into
component wavelengths.

The monochromator unit consists of :

• Entrance slit – defines narrow beam of radiation from source.

• Collimating lens (polished surface) - collimates the lights.

• Prism (make-quartz)- disperses the light into specific

wavelength.

• Focusing lens -captures the dispersed light & sharpens the

same to the sample cuvette via exit slit

• Exit slit- allows the corrected wavelength of light to the

sample cuvette.
 Wavelength Selectors (Slit)
-It limit the radiation to be absorbed by a sample.

-Sensitivity of the equipments (UVS & AAS) are improved when the
bandwidths are narrow -transmission is high.

Types
Absorption Filters

Cutoff Filters

Interference Filters
 Sample container (Cuvette)
 For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.

 Glass is suitable for visible but not for UV spectroscopy because it

absorbs UV radiation.

 Quartz can be used in UV as well as in visible

spectroscopy Long pathlength 1 cm pathlength cuvet
Opaque
Face

Transparent
Face

1 cm 1 cm
Short pathlength (b)
Holding capacity 1.5 mL to 3.5 mL
 Photosensitive Detectors

 The detectors are devices that convert radiant energy

into electrical signal.

 A Detector should be sensitive, and has a fast

response over a considerable range of wavelengths.

 In addition, the electrical signal produced by the

detector must be directly proportionate to the
transmitted intensity.
 Principles
Beer’s Law
The amount of light absorbed is proportional
to the concentration of the absorbing
substance

100% light 40% light 100% light 70% light

60% light absorbed 30% light absorbed

Rate of Absorption
 Lambert ’s Law

The amount of light absorbed is proportional to the

thickness (length) of the absorbing material
(Cuvette)

100% light 40% light 100% light 70% light

Larger path length

Smaller path length

60% light absorbed 30% light absorbed

Rate of Absorption
 Types The components of a single
beam spectrophotometer
Light source - white light of constant intensity

slits

Grating
slits Separates white light
Phototube
into various colors
detects light &
measures intensity Rotating the grating
Sample
changes the wavelength
When blank is the sample going through the sample
Po is determined,
17
otherwise P is measured
 Double Beam Spectrophotometer

Beam Chopper Semi-transparent

Mirror
Sample

Tungsten Slit Quartz Photo-

Grating
Lamp Cuvette multiplier
Reference
(Blank)
Mirror Mirror
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Applications
The applications of UV/Vis Spectrometer are quite
vast.
Mainly it is used for qualitative and quantitative
determinations such as enzyme assays, molecular weight
determination.
 Ts routinely used in analytical chemistry for
the quantitative determination of different analytes, such
as metal ions, highly conjugated organic compounds, and
biological macromolecules.
Spectroscopic analysis is commonly carried out in
solutions but solids and gases may also be studied.