2. 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
Objectives of this lecture
3. 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.
Introduction
4. 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”.
5. 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
6. 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
7. 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
9. 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.
10. Deuterium Lamp (deuterium gas)
- UV Region
- Wavelength Range : 190~420nm
Lamps used in UV-Vis Spectrophotometer
Tungsten Lamp (halogen- iodine and bromide)
- Wavelength Range : Part of the UV and
Visible
Xenon Lamp (xenon gas)
-Wavelength Range : 190~800nm
11. 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.
Monochromator
12. 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.
Absorption Filters
Cutoff Filters
Interference Filters
Types
13. 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
1 cm 1 cm
Opaque
Face
Transparent
Face
Long pathlength
Short pathlength (b)
1 cm pathlength cuvet
Sample container (Cuvette)
Holding capacity 1.5 mL to 3.5 mL
14. 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.
15. Beer’s Law
The amount of light absorbed is proportional
to the concentration of the absorbing
substance
100% light 40% light
60% light absorbed 30% light absorbed
100% light 70% light
Rate of Absorption
Principles
16. Lambert ’s Law
The amount of light absorbed is proportional to the
thickness (length) of the absorbing material
(Cuvette)
100% light 40% light
60% light absorbed 30% light absorbed
100% light 70% light
Larger path length
Smaller path length
Rate of Absorption
17. 17
Light source
Grating
Rotating the grating
changes the wavelength
going through the sample
slits
slits
Sample
Phototube
The components of a single
beam spectrophotometer
When blank is the sample
Po is determined,
otherwise P is measured
Separates white light
into various colors
detects light &
measures intensity
- white light of constant intensity
Types
20. 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.
