A spectrophotometer is an instrument that measures the amount of radiation absorbed by a sample. It works based on Beer-Lambert's law to measure the intensity of radiation as a function of wavelength. The amount of radiation absorbed by a solution is directly proportional to the concentration of the solute. Spectrophotometers are used for quantitative analysis of solutes in solutions. They have components like a light source, monochromator, photometer, and sample holder. Common applications include measuring concentration of solutions, identifying organic compounds, and estimating color properties.

1. Spectrophotometry
Agricultural and Food Engineering Department
Indian Institute of Technology Kharagpur
India -721302
By Amit Kumar

2. Spectrophotometer
• A spectrophotometer is an instrument used to measure the amount
of radiation absorbed by the sample.
• Spectrophotometers work on Beer-Lamberts law. It measure the
intensity of radiation as a function of wavelength.
• The amount of radiation absorbed by the solution is directly
proportional to the concentrate of the solute.
• Spectrophotometer techniques are used for quantitative analysis of
solutes in a solution.

3. Basic schematic diagram

4. Application of spectrophotometers
1. Measurement of concentration of the solution
• Beer-Lambert Law
• Wavelength specific absorption peaks
• Extinction coefficient
2. Identifying the organic compounds
• Wavelength specific absorption maxima for different conformations
fingerprints
3. Colour properties estimation
• Wavelength ranging from 380 – 700 nm.

5. Spectrophotometer
• Single beam spectrophotometer
• Double beam spectrophotometer
• Split beam spectrophotometer

6. Components
1. Light source
2. Monochromator
3. Photometer
4. Sample holder
5. Frame

7. 1. Light source
• The main function is to emit a radiation of required wavelength.
• Selection is based on the required wavelength of radiation.
• Emits polychromatic radiation over a wide range of wavelength.

8. Types of light source
1. Tungsten lamp (330 – 900 nm): 1200 h life
2. Hydrogen/Deuterium lamp (190 – 450 nm): 500 h life
3. Xenon flash lamp (190 – 1000 nm): 1000 h life
H2/D2 lamp: Agilent 8454
Tungsten - halogen lamp Xenon flash lamp

9. 2. Monochromator
• It screens the radiation to block all the other wavelength except the
selected or desired wavelength.
• Converts polychromatic input radiation into monochromatic output
radiation.

10. Types of Monochromators
1. Prism
2. Filters
3. Diffraction gratings

11. 3. Focusing devices
• Lenses + slits + mirrors = Focusing devices
Units Type of radiation Material
Mirrors
X-ray and UV Aluminium
Visible Aluminium
Near Infrared Gold
Infrared Copper or Gold
Lenses
X-ray and UV Silica
Visible Glass
Near Infrared Glass
Cuvettes
X-ray and UV Quartz
Visible Glass/PC
Near Infrared Glass/PC

12. 4. Cuvettes

13. 5. Detectors or photometers
1. Photo diodes array
2. Gallium-Nitride UV detectors
3. Phototubes
2. GaN UV Detectors
1. Photo cells and diodes 3. Phototube

14. Different spectrophotometers
• Visible spectrophotometer (
• UV/Visible (180-800 nm)
• NIR (700-15000 nm)
• NMR
• Atomic absorption spectrophotometer

15. Spectrophotometer in lab
Portable Spectrophotometer (DR 2800, Hach) Spectrophotometer (UV 1700, Shimadzu)

16. Estimates
1. Total phenolic content
2. Antioxidant capacity
3. Protein concentration
4. Enzymatic activity
5. Colour parameters, etc.

17. Thank you.