Photometry, colorimetry, spectophotometry

1. PHOTOMETRY , COLORIMETRY AND
SPECTROPHOTOMETRY , THEIR APPLICATION.
Presented by :-
Gadhavi Shital N.
Msc.Sem -2 (Botany)
Paper :- 406
Department of life sciences
H.N.G.U Patan

2. CONTENT
 Photomtery principle
 Principle of operation
 Uses of photometry
 Colorimetry principle
 Principle of operation
 spectrophotometry application
 Principle of operation
 Limitations
 Uses of colorimetry and spectrophotometry

3. PHOTOMETRY
Principle
• The word is composed of the Greek word photo-
“light” and metry – “measure”
• The basis of photometric working is that , the species
of alkali metals and alkaline earth metals are
dissociated due to the thermal energy provided by the
flame source.
• Due to this thermal excitation, some of the atoms are
exited to a higher energy level where they are not
stable.
• The absorbance of light due to the electrons excitation
can be measured by using the direct absorption
techniques .

4. • The subsequent loss of energy will result in the
movement of exited atoms to the low energy ground
state with emission of some radiations, which can be
visualized in the visible region of the spectrum.
• The absorbance due to the electron excitation can be
measured by using the direct absorption technique
while the emititing radiation intensity is measured
using the emission techniques .
• The wavelength of emitted light is specific for specific
elements .

6. Principle of operation

7. Uses of photometry
• Photometry is used in various industries like
chemicals, soils , agriculture, pharmaceuticals, glass,
and ceramics, in plant materials and water , and in
biological and microbiological laboratories .
• It is used in determination of potassium, sodium,
magnesium and calcium in biological fluids like serum,
plasma, urine etc , is routinely carried out by
photometer .
• Analysis of industrial water natural water for
determining elements responsible for hard water is
standard procedure in many laboratories.

8. COLORIMETRY
• Colorimetry is the use of the human eye to
determine the concentration of colored species.
• Colorimetric methods are applicable to dilute
solutions.
• For a colorimetric method to be quantitative , it must
from a compound with definite color
characterisitics.
• Color amount must be directly propotional to the
concentration .
• Colored compound must obey beer’s law and
lambert’s law.

9. PRINCIPLE
Beer’s Law: The amount of light absorbed by a
substance in a solution is proportional to a number
of absorbing molecules… i.e. concentration of the
absorbing solution
 Log e
𝐼˳
𝐼
=kc
 I=I˳𝑒−𝑘𝑐
k = constant for particular solution
c = concentration

10. • Lambert’s law :The amount of light absorbed by a
substance in a solution is proportional to the
thickness of the absorbing molecules …i.e . path
length
Log e
𝐼˳
𝐼
=kd
 I=I˳ 𝑒−𝑘𝑑
d= length of absorbing layer
k = constant for particular solution

11. • Visual observations :- because colorimetry is
based on inspection of materials with the
human eye, it is necessary to review aspects
of visible light.
• Visible light is the narrow range of
electromagnetic waves with the wavelength
of 400-700 nm.
• Visible light is only small portion of the
electromagnetic spectrum.

12. COLORIMETER

13. Principle of operation

14. SPETROPHOTOMETRY
Applications
Qualitative analysis : used to identify classes of
compound in both pure state and in biological
preparations. This is based on the fact that
absorption spectra are specific for a class of
compound.
Denatration of double standard DNA : DNA absorbed
at 260 nm. This absorbance increase with rise in
temperature , as double standard DNA become
single stranded.

15. Continues….
• Enzyme assay and kinetic studies : the
quantitative assay of enzyme activity is carried
out when substrate or product is colored
absorbs light in UV range.
• Molecular weight determination : the
molecular weight of amines , , sugars and
many aldehyde and ketone compounds can be
determined .

16. Continues…..
• Control purity : impurity in compound can be
detected easily spectrometric studies
• 1, carbon disulfide impurity in carbon
tetrachloride can be detected easily by
measuring absorbance at 318 nm where only
carbon disulfide absorb.
• Protein folding: protein or protein– nucleic
acid interaction can be studies

17. SPECTROPHOTOMETER

18. INSTRUMENTATION
Basic component :
• Light sources
• Monochromatic and filter
• Sample holder
• Photo detector

19. Principle of operation

20. Limitation
• Applicable for monochromatic light.
• Applicable lower concentration.
• Applicable only clear solution.

21. • Colorimetry and spectophotometry are of prime
importance in biochemical research .
 If the absorbancy index at a specific wavelength is known,
the concentration of a compound can be readily
determind by measuring optical density at that
wavelength . As the value of absorbancy index is very large
in nucleotides, very small quantities of the absorbing
material can be accurately measured.
 The course of a reaction can be determind by measuring
the rate of formation or disappearance of a light absorbing
substance .thus NADH absorbs strongly at 340 nm–
whereas the oxidised form NAD—has no absorption at this
wavelength . Therefore, reactions involving the production
or utilisation of NADP can be assayed by the technique .

22. Compound can be frequently identified by
determining the characteristic absorption
spectra in the ultraviolet and visible region of
the spectrum.

23. REFERENCE:
• Biochemistry
C. B. Power
G. R . Chatwal

24. THANK YOU …