Spectrophotometer Working principle and Application

1. SPECTROPHOTOMET
ER
M.Sc Zoology (1st Semester)
Roll. No. 2313177450003
Saraswati Vidya Mandir Mahila PG College, Gorakhpur
Aastha Singh

2. Index
 Meaning
 Spectrophotometer
 Types of Spectrophotometer
 Principle of Spectrophotometer
 Lembert’s Law
 Beer’s Law
 Working of Spectrophototmeter
 Application of Spectrophotometer
 Reference

3. Meaning of spectrophotometer
Colorimetry
Spectrum
Photometry
Spectrophotometer

4. Spectrophotometer
 Spectrophotometer is an instrument that measures the
amount of light that passes through (is transmitted
through) a sample.
 Uses a type of light to detect molecules in a solution.
 Light is a type of energy, and the energy is reported as
wavelengths in nanometers (nm).

5. Sample Holder
Display
Knobs or buttons used to calibrate the spec to
measure the designated molecule
Wavelength
Selection

6. Two different types of spectrophotometer
 Ultraviolet (UV) Spectrophotometers
Uses ultraviolet light of wave lengths from 200 nm to
350 nm.
 Visible (VLS) Light Spectrum
Spectrophotometers
Uses visible light (white light) of wave lengths from
350 nm to 700 nm.

7. The Visible Light Spectrum
R O Y G B I V

8. Working principle of spectrophotometer
 When a beam of monochromatic light passed through a
solution it may transmitted as a such or some of may be absorbed.
 Proportional the transmitted light can be represented by intensity
of the incident radiation.
T=I/Io
Absorbance(A) of light through a solution in inversely
proportional to log10 of %T.

9. A = Log (1/T)
= Log (I/Io)
Where I = Intensity of transmitted light
Io = Intensity of incident light
 The quantitative determination of compounds by
spectrometeric technique is based on two law.
1. Lamberts Law
2. Beers Law

10. Lambert’s Law
 Its state that light absorbed by solution is directly proportional
to length of the light through the solution.
Thus, A = Log (Io/I) = E.C.l
Where, A = Absorbance
E = Molar absorptivity coefficient
l = Path length of the sample
C = Concentration of solution

11.  Calculating the absorbance of a sample using the equation
depends on the absorbance is directly proportional to the path
length of the sample.

12. Beer’s Law
 Its state that amount of light absorbed is directly proportional
to concentration of absorbing solute in the solution.
Thus, A = Log (Io/I) = E.C.l
Where, C = Concentration of solution mole per liter
E = Molar absorptivity coefficient
l = Path length of the sample

13.  Calculating the absorbance of a sample using the equation
depends on the absorbance is directly proportional to the
concentration of the sample.

14. How a spectrophotometer works

15. How a spectrophotometer works
1. White light hits the prism or grating, it is split into the colors of
the rainbow (Visible Spectrum).
2. The wavelength knob rotates the prism/grating, directing
different color of light toward the sample.
3. The wavelength of light produced by the tungsten lamp range
from about 350 nm (Violet light) to 700 nm (red light).

16. 3. The molecules in the sample either absorb or Transmit the light
energy of one wavelength or another.
4. The detector measures the amount of light being transmitted by
the sample and reports that value directly (% transmittance) or
converts it to the amount of light absorbed in absorbance units
(au) using Beers Law.

17. Applications of a spectrophotometer
 Determines the presence and concentration of samples.
 Determines the purity of a sample.
 Look at the change of samples over time.
 Molecular weight determination of compounds.
 Respiratory gas analysis in hospitals.
 The visible and UV spectrophotometer may be used to identify
classes of compounds in both the pure state and in biological
preparations.

18. Reference
 Spectrophotometer- Principle, Instrumentation, Applications
January 8, 2022 by Sagar Aryal.
 By Anne Marie Helmenstine, Ph.D. Updated on February 2020.
 https://www.slideshare.net/ssmvjunwani/spectrophotometer
 https://byjus.com/chemistry/spectrophotometer-principle
 Spectrophotometer Dictionary, Thesaurus, Acronyms,
Encyclopedia, Wikipedia.

19. Thank You