Ultra violet visible (UV- Vis) absorption spectroscopy is the measurement of attenuation of the beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurement can be at single wavelength or over an extended spectral range. Principle: The underlying principle behind UV- Vis spectroscopy is, UV absorption spectra arise from transition of electron within the molecule from lower to higher level. The principle is based on the measurement of spectrum of sample containing atoms/ molecules. Based on the Lambert Beer Law, “when beam of light is allowed to pass through transparent medium, intensity of beam of light (decreases exponentially with increase in concentration of absorbing substance) is inversely proportional to concentration and thickness of medium.
A = ƐCL
C = concentration of solution
L = length of solution
A = Absorbance
Ɛ = Molar absorption coefficient
In UV- Vis spectrometry, a beam of light from visible and UV light source is separated into its component wavelength by a prism or diffraction grading. Each monochromatic beam (single wavelength beam) in turn is split into two equal intensity beams by a half mirrored device. One beam, the sample beam passes though cell (cuvette) containing a solution of the compound being studied in transparent solvent. The intensity of beam that have traversed through sample cell is defined as ‘I’.
Other beam passes through reference cell (cuvette), which have suffered little or no light absorption. The intensity of this reference cell is defined as ‘I0’. over short period of time, spectrometer scans all component wavelength normally from 200 – 400 nm (for UV scanning) and 400 – 800 nm (for visible scanning).
Absorbance = log (I0/I)
If beam of monochromatic light is passed through a solution then the absorbance (formerly known as optical density) can be measured by experimental values of original intensity (I0) of beam of light and intensity of beam of light after passing through solution (I).