Ultraviolet-visible spectroscopy involves measuring the absorption or emission of electromagnetic radiation as a function of wavelength. It can be used to qualitatively and quantitatively analyze organic molecules and biomolecules. The technique utilizes a light source, wavelength selector such as a monochromator, sample container, and detector. It has various applications including enzyme assays, molecular weight determination, and studying cis-trans isomerisms.

1. Ultra-visible spectroscopy
T. HARI MURALI KRISHNAA

2. PRINCIPLE
To study the absorption or emission of electromagnetic radiation as a function of wavelength are
called spectrometers. The absorption of light by any absorbing material is governed by two laws.
First is Lambert law – ( amount of light absorbed is proportional to the thickness of the
absorbing material and is independent of the intensity of the incident light). second one is Beer’
s law this law states that the amount of light absorbed by a material is proportional to the
number of absorbing molecules i.e., the concentration of absorbing solution.
Ultraviolet and visible light, when incident on an organic molecules having pielectron and or non
bonded electrons would excist them and results in absorption if light.
When the energy difference between the highest occupied molecular orbital(HOMO) and lowest
occupied molecular orbital (LUMO) of molecule matches the energy of the incident light
maximum absorption occurs.
The wavelength at which maximum absorption occurs is referred to as the ^max. this position is
highly influenced by the extended of conjugation in the molecule. Higher the conjugation higher
would be the ^max value

3. Instrumentation

5. Light source: hydrogen, deuterium lamb, and tungsten lamb are most commonly used. The
glass tube fill with hydrogen and deuterium gas at low pressure. Xenon lamb used for ultra
violet radiation but it not stable as the hydrogen lamb.
Wavelength selector:
 the selectors are of two types, filter and monochromotors.
 FILTERS:
 it absorbing light in all other region except for one, which they reflect. Mainly gelatin filters are
used. Glass filters is their have low transmittance.(5.20%).
MONOCHROMATORS:
the monochromotor resolves polychromatic radiation into its individual wavelengths and isolate
these wavelengths into very narrow bands.
PRISM:
 A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of
its ability to refract different wavelength to a different extent. Two types of prisms namely 600 cornu
quarts prism and 300 littrow prism are usually employed in commercial instrument. It depends upon
the angle and the material of which it is made

6. GRATINGS:
◦ gratings are often used in the mnochromoter of spectrophotometers operating operating in UV. The
gratings possesses a highly aluminized surface etched with a large number of parallel grooves which
are equally spaced. These groove are also known as lines. A grating way have anywhere between
600 to 2000 lines per mm on the surface depending on the region of the spectrum in which it is
indented to operate it.
SAMPLE CONTAINER:
◦ Cuvettes are mainly used, cuvettes meant for the visible rejoin are made up of either ordinary glass
or sometimes quartz. Since glass absorbs in the UV region, quartz or fused silica cells are used in this
region. Path length – 1cm. Capacity 3ml.
DETECTORS:
• depends on the photoelectric effect. That collects these electrons from photons and measure the
their number of current. This current directly proportional to the light intensity.

7. APPLICATION:
 qualitative analysis
Quantitative analysis
Enzyme assay
Molecular weight determination
Study od cis trans isomerism's
Physicochemical studies
Turbidimetry

8. Qualitative analysis:
 It may be used to identify the classes of components in pure state and in biological preparation.
 Identification of substance in biological milieu.
 It provides information about absorption and functional groups of biomolecules.
 Identification of an unknown components or structure basis of its absorption spectrum.
 given compound, sufficient chemical information about the substance such as elements of present
in should be known.
 Thus compounds which do not absorb in 220 – 280nm -- aliphatic or alicyclic hydrocarbons or their
derivatives.
 220-250 – tow unsaturated linkages in conjugation.
 220-230—double bonds.
Vitamin K1 is – 450- 500. nucleotides – GMP- 255nm, UMP- 262nm, CMP-271nm, AMP-259nm.

9. Quantitative analysis:
 determine the unknown components.
Chemical specie can be determined.
 nucleic acid – 254nm
Protein-280nm
Enzyme Assay:
 Quantitative analysis of enzyme activity.
 mainly based on absorption and scattering of light on substance.
 example: lactate dehydrogenase, which is engaged in the transfer of electrons from lactate to
NAD+. The products of the reaction are pyruvate, NADH, and a proton. NADH Absorb The
Radiation In 340nm.

10. Molecular weight:
 M = awb/OD
M = 10a/E%
1cm
 M- molecular weight
 w – weight of compound in grams per liter
 b- path length
Study of cis – trans isomerism:
Physiochemical studies:
 determine the empirical formulas,
 determine the weak acids and bases in organic acids
 protein dye interaction
 chlorophyll protein complex
 determination of labile intermediates.

11. Control of purification:
 impurities in a compound can be detected.
 e.g.: carbon disulfide impurity in carbon tetrachloride can be detected – 318nm carbon
disulfide was absorbed.
 benzene impurity detected by 280nm.
Difference spectroscopy:
 developed by chance and Williams.
 it mainly used foe toxicology laboratory to check the toxic drugs.
 to identify the environment chromophore.
Turbidimetry and nephelometry:
 to measure the scattering of light. And to absorb the colloidal particles in the solution.