1. Spectrophotometer
Dr . S. Jayakumar

2. Objectives of this lecture
Spectrophotometer
Introduction
Components
Sources of light
Monochromators
Slit
Sample containers
Detectors
Readout devices
Principles
Beer’s –Lambert’s Law
Types
Single and double beam instruments
Applications
Qualitative &Quantitative analyses

3. Introduction
Photometry – Study of the measurement of intensity of light.
Colorimetry- Study the color of specific wavelength of visible light ( nm).
Spectrophotometry- Study of color of a very narrow range of wavelength in UV ( nm), visible ( nm) and IR ( 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.

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
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 µm
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

8. Components of Spectrophotometer

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. 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

11. 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.

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.
Types
Absorption Filters
Cutoff Filters
Interference Filters

13. 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
Short pathlength (b)
1 cm pathlength cuvet
1 cm
Opaque
Face
Transparent
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. Principles
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
70% light
Rate of Absorption

16. Lambert ’s Law
The amount of light absorbed is proportional to the thickness (length) of the absorbing material (Cuvette)
40% light
60% light absorbed
30% light absorbed
100% light
70% light
Larger path length
Smaller path length
Rate of Absorption
100% light

17. Types The components of a single beam spectrophotometer Light source
- white light of constant intensity
slits
Grating
slits
Separates white light
into various colors
Phototube
detects light &
measures intensity
Rotating the grating
changes the wavelength going through the sample
Sample
When blank is the sample
Po is determined,
otherwise P is measured

18. Double Beam Spectrophotometer
Beam Chopper
Semi-transparent
Mirror
Tungsten
Lamp
Grating
Photo-
multiplier
Quartz
Cuvette
Sample
Slit
Reference
(Blank)
Mirror

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.