SPECTROSCOPY
Spectroscopy is the study of the interaction between matter and electromagnetic radiation
as a function of the wavelength or frequency of the radiation
Principle of spectroscopy includes the measurement of transmittance of light beam
intensity after it passes through a sample or after transmittance.
A SPECTROPHOTOMETER is an instrument for measuring the intensity of light in a
part of the spectrum, especially as transmitted or emitted by particular substances.
TYPES OF SPECTROSCOPIC TECHNIQUES
Spectroscopic techniques are classified on the basis of the radiative energy involved.
Atomic absorption spectroscopy (AAS)
Ultraviolet and visible spectroscopy (UVS)
Fourier Transform Infrared (FT-IR) Spectroscopy
Nuclear Magnetic Resonance (NMR) Spectroscopy
Diagram showing range of different electromagnetic radiations
UV-Visible Spectroscopy
Ultraviolet and visible (UV-Vis) is the most common analytical technique.
Basically it is an absorption spectroscopy deals with the measurement of the intensity of a
beam of light after it passes through a sample or after transmittance from a sample
surface.
Absorption measurements can be at a single wavelength or over an extended spectral
range.
UV light extends from 200-400nm range.
Visible light extends from 400-800nm range.
The best UV-Vis absorption is by molecules that contain unsaturation in its structure.
For molecules with absorption in the visible region, the compounds will often appear
colored.
Absorption follows Beer's Law, A=εbC where ε is the molar absorptivity coefficient, b is
path length, and C is concentration.
UV-Visible spectroscopic technique is applicable on those molecules that contains
chromophores in them.
CHROMOPHORES is the moiety that causes a conformational change of the molecule when
hit by light. Chromophoric compounds are called UV active compounds.
INSTRUMENTATION:
Samples are typically placed in a transparent cell, known as a cuvette. Cuvettes are
typically rectangular in shape, commonly with an internal width of 1 cm. (This width
becomes the path length, L in the Beer’s–Lambert law.) The most widely applicable
cuvettes are made of high quality fused silica or quartz glass because these are
transparent throughout the UV, visible and near infrared regions.
LIGHT SOURCE: Tungsten filament lamps, hydrogen deuterium lamps
MONOCHROMATOR: Prisms modifies the direction of light
DETECTOR: Phototubes containing a photocathode which emits electrons when
illuminated, such that the tube conducts a current proportional to the light intensity.
AMPLIFIER
The alternating current generated in the photocells is transferred to the amplifier.
Generally current generated in the photocells is of very low intensity, the main purpose of
amplifier is to amplify the signals many times so we can get clear and recordable signals.
Flow diagram of instrumentation of UV Visible spectroscopy
APPLICATIONS OF UV VISIBLE SPECTROSCOPY
Detection of Impurities in organic molecules.
Structure elucidation of organic compounds such as in detecting the presence or absence
of unsaturation, the presence of hetero atoms.
Quantitative and qualitative determination of compounds.
Many drugs are either in the form of raw material or in the form of formulation. They can
be assayed by making a suitable solution of the drug in a solvent and measuring the
absorbance at specific wavelength.
REFERENCES
1. Camel, V., et al., Open digital educational resources for self-training chemistry lab safety rules.
2020, ACS Publications.
2. Crockett, J.M., Laboratory safety for undergraduates. Journal of Chemical Health & Safety, 2011.
18(4): p. 16-25.
3. Gandhi, K., et al., Calibration and Standardization, in Chemical Quality Assurance of Milk and
Milk Products. 2020, Springer. p. 147-173.