When radiant energy is incident on matter transitions such as electronic, vibrational, rotational, and translational, take place in the molecules of the substance. By monitoring these changes it is possible to derive valuable information regarding the structure of molecules (qualitative analysis) and also their concentration (quantitative analysis).

Spectroscopy is classified into various branches depending on the type of the radiant energy employed. For instance,

• UV Spectroscopy
• IR Spectroscopy

• The radiant energy is characterized by its wavelength (λ ) and the frequency (ν). They are related by the following equation.
• C = λ ν, ν in Hz (cps) and λ in cms, C = speed of light = 3 x 10¹⁰ cm s ^-1.
• Thus ν and λ are inversely related. The energy content of the radiation is given by, E = hν where E is energy in ergs (or J) and h is the Planck's constant equal to 6.63 x 10-27 erg sec.
• Also in Infra-red spectroscopy the term wave number () is used, expressed in cm ^-1 and is related to wavelength as () = 1 /λ

• UV SPECROSCOPY: Utilizes electromagnetic radiation of range 200 to 400 nm and is generally employed in organic analysis.
• Vacuum UV is between 160 - 210 nm and Visible range is 400 - 750 nm. 1 nm = 10^-9m.
• Applications: a) quantitative estimations b) Study of conjugated systems, gives indication of arrangement of some functional groups (chromophores)
• Source: UV- H₂ or D₂ discharge lamp.
• Equipment: Usually a double beam recording spectrophotometer with quartz optics and a photoelectric detector
• Sample handling: (for UV) Very dilute solution of the compound using an alkane (hexane) or an alcohol (ethanol) as solvents.
• Solvents by themselves should not be UV absorbing.
• Both absorption wavelength (λ) and intensity of absorption are measured (A).

Quantitative analysis

• I₀ = Intensity of reference beam that is intensity of incident light. I_t = Intensity of sample beam intensity of transmitted light.
• Absorbance A = log I₀ / I_t
• Absorbance is directly related to the concentration and the path length of the cell cuvette or sample container, normally it is 1 cm, however cuvettes of extremely small path lengths are commonly used in biochemical analysis.
• A = ξcl (epsilon) = molar absorptivity, characteristic of the compound c = concentration of the compound in mol lit^-1 l = cell path length in cms.
• ξ is defined as the absorbance of the solution when the concentration is 1M with a path length of 1 cm. For example ξ_max is 13000 for mesityl oxide.
• Problem: Calculate the ξ_max of a 1.0 x 10-4 M solution of a compound in ethanol that shows an absorbance of 1.05 at 237 nm in a 1 cm cell.
• Method 1: If ξ_max is known for the compound, measure Absorbance of the solution at its λ_max of the given compound and use the relation A = ξcl
• Method 2: Make standard solutions of the compound of different concentrations, measure Absorbance for the different standards at that λ_max wavelength. Plot a graph with absorbance in the Y-axis and concentration in the X-axis. Measure the absorbance of the same sample of unknown concentration and read the corresponding concentration from the graph.

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