It is a analytical spectroscopic technique which involves the promotion of electrons from ground state to excited state or higher energy state when the molecules absorbs the light of ultraviolet region that is why it is also called as absorption spectroscopy.
The instrument used to measure the absorbance of sample is called as UV Visible spectrophotometer. Generally the absorbance of liquid sample can be measured in the UV Visible spectrophotometer solid and gases sample can also be measured. Homogeneous and diluted sample solution shall be prepared for the measurement. Samples for the measurement is placed in transparent rectangular shaped cell known as cuvettes. Cuvettes are made up of fused silica and quartz glass because these are transparent in UV and visible region and not of glass as it absorbs light in UV region.
Ranges of UV and visible region
UV (Ultra violet) : 200 to 400 nm
Visible : 400 to 800 nm
Principle of UV Spectroscopy
Principle of UV spectroscopy based on the Beer Lambert law. It states that when a beam of monochromatic light is passes through the solution of homogeneous absorbing medium then the absorbance is directly proportional to the concentration of the solution and to the length of the light path i.e. width of the cuvette (1 cm). Mathematically:
This proportionality can be converted into an equality by including a proportionality constant (ϵ).
A= ϵ cl
Absorbance (A) : It is defined as the ratio of intensity of incident radiation to the intensity of transmitted radiation.
A= log10 (Io/ I)
Hence the Beer Lambert law in terms of intensities is expressed as:
A= log10 (Io/ I) = ϵ cl
ϵ = molar absorption coefficient (epsilon)
l = Length of light path
c = Concentration of solution
Instrumentation and Working of UV spectroscopy :
UV Spectrophotometer generally consist of following components:
- Light Source : Light sources employed in UV visible spectrophotometer is i) Tungsten filament lamp which emits radiation of 375 nm. ii) Hydrogen deuterium lamp which emits radiation below 375 nm So both deuterium and hydrogen lamp emit radiation in the range of 160 to 375 nm.
Light source should be ideal as following :
- It should be stable
- It should not fatigue on continuous use
- Should emit light of continuous spectrum
- Monochromator :Monochromator is consist of
- Entrance slit
- Collimating lens
- Prism/ grating
- Exit slit
Monochromator is also called as wavelength selector. Polychromatic radiations enters the monochromator through entrance slit and then strikes to prism which disperse the radiation into its components and the radiations of particular wavelength leaves the monochromator through exit slit.
- Sample compartment : Sample compartment is made up of fused silica and quartz which is Transparent and rectangular in shape with flat ends and its thickness is generally 1 cm it is known as cuvettes. Sample under measurement is placed in the cuvettes and the beam of monochromatic light passes through sample and reference solution placed in cuvettes.
- Detector : Detector will receives the radiation from sample cell and reference cell and convert into signals which is recorded into the read out device.
Detector should be ideal as following:
- It should provide quantitative response
- It should have low noise level
- It should have shorter response time
- It should detect the radiation of wide spectrum
Types of detector used in UV visible spectrophotometer
- Barrier layer cell
- Phototubes or photoemessive tubes
- Photomultiplier tubes
- Amplifier : Amplifier will amplify the signals from detector and generate recordable signals.
- Read out device : Read out device is generally a computer system which stores or record the data and generate the spectrum of sample under investigation.
Application of UV Spectroscopy :
- Detection of functional group.
- Identification of unknown compound.
- Detection of impurities.
- Used in the Quantitative and Qualitative analysis.
- Determine the extent of conjugation.
- To determine the configuration of geometrical isomer.