Spectrophotometry and its types helps us to understand the phenomenon of Spectrophotometry

1. Spectrophotometry

3. • Every chemical compound absorbs, transmits or reflects light over a certain range of
wavelength, thus amount of light detected after passing through compound is analyzed for
quantitative estimation
• Spectrophotometry is a method to measure how much a chemical substance absorbs light by
measuring the intensity of light as a beam of light passes through sample solution.
• Basic principle is that each compound absorbs / transmit light over a certain range of
wavelength
• Most useful methods of quantitative analysis in various fields
• A Spectrophotometer is a device designed for evaluation of spectrophotometric
measurements

4. Components of Spectrophotometer
Light Source:
Tungsten lamp for Visible spectrum
Deuterium or Hydrogen Lamp for UV-light
Collimator:
Transmits a beam of light/ Produces a parallel beam of rays
Monochromator/ Prism:
Splits the light into its component wavelength. Helps to select a range of
wavelength for measurements
Wavelength selector:
Blocks unwanted wavelengths & allows selected wavelength to pass
Cuvette:
To hold sample in liquid state
Glass cuvette for Visible light
Quartz cuvette for Ultra-violet light
AgCl or KBr cuvette for Infrared light
Detector:
Converts light signal into an electrical signal

5. Principle of Spectrophotometry

6. Principles of light absorption
• Two fundamental principles govern the absorption of light by a
solution:
1. The absorption of light passing through a solution is exponentially
related to the number of molecules of the absorbing solute, i.e. the
solute concentration [ C ].
2. The absorption of light passing through a solution is exponentially
related to the length of the absorbing solution, l.

7. Beer-Lambert Law
• The absorbance of light by a material in a solution is directly proportional to its concentration in
that solution.
• The linear relationship between absorbance and concentration of an absorbing species.
• Absorbance (A) depends upon followings,
• A = εlc
• Where,
• A = Absorbance
• ε = Molecular absorptivity
• l = Path length of the light absorbing sample (cm) / width of the cuvette
• c = Concentration of the absorbing species (mol/L)

9. A spectrum of electromagnetic radiation transmitted through a substance, showing dark lines or
bands due to absorption at specific wavelengths.
The Absorption Spectrum
• Spectrometer records the degree of absorption by a sample at different wavelengths and the resulting
plot of absorbance (A) vs wavelength (λ) is known as spectrum
λmax = (wavelength at which there is a maximum absorption)
εmax = (the intensity of max absorption)

10. Types of Spectrophotometry
Ultra Violet Spectrophotometry
Visible Spectrophotometry

11. UV Spectrophotometry
Ø Uses light over the UV range (180-400nm)
Ø A prism of suitable material & geometry will provide a continuous spectrum in
which the component wavelengths are separated in space.
Ø In addition to prisms, Diffraction Gratings are also employed for producing
monochromatic light.
Ø A diffraction grating comprises of a series of parallel grooves on a reflecting
surface. Grooves can be considered as separate mirrors from which the
reflected light interacts with light reflected from neighbouring grooves to
produce interference.
Ø Quartz cuvettes used to hold samples

12. Visible Spectrophotometry
• Uses visible range (400-700nm) of electromagnetic radiation
spectrum
• Plastic or Glass cuvettes can be used for visible
spectrophotometry

13. Application of UV-visible Spectrophotometry
Clinical Diagnosis:
To study enzyme kinetics. Enzyme activity can be studied by analyzing speed of reaction.
Reagents are attached to the substrates which can be monitored for enzyme activity.
Widest use is in the field of clinical diagnostics as an indicator of tissue damage. Entry of
enzymes into the blood stream upon tissue damage indicates the extent of tissue damage
depending upon the enzyme activity assessed through spectrophotometric analysis, such as
liver, pancreas or other tissue/organ damage may be diagnosed.
Dissolution / In Vitro Release Assay of Drugs:
Drug formulation design depends largely on the drug dissolution profile and also in vitro
release assay of a pharmaceutical agent / molecule is assessed spectrophotometrically.
Quantification of DNA, RNA & Proteins:
Based on the absorption profile / properties of biomolecules, they are quantified with
greater precisions
Dye, Ink & Paint Industries: Spectrophotometry is used in the quality control in the
development of the dyeing reagents, inks and paints
Heavy Metals & Organic Matter from Environmental / Agricultural Samples: Field
samples are used to monitor / assess the health and nutritional status of the field