colorimeter
Coloured solutions have the property
of absorbing light of definite
wavelengths.
The amount of light absorbed or
transmitted by a coloured solution
depend on Beer-Lambert law.
Beer’s law:- The intensity of colour
directly proportional to the
concentration of coloured particles in
the solution ( A α C ) i.e light absorbed
is directly proportional to the
concentration.
Lambert’s law:- Amount of light
absorbed by a coloured solution
depends on the length of column or the
depth of the liquid through which light
passes ( A α L ) i.e light absorbed is
directly proportional to the path
length of the cuvette
Combining the two laws
AαC×L
or A =k × C × L ( k= constant)
Let AT= absorbance of the test solution
AS=absorbance of the standard solution
CT= concentration of the test solution
CS= concentration of the standard solution
• So AT = k × CT × L
AS =k × CS × L
• AT k × CT × L
=
AS k × CS × L
• AT CT
AS CS
AT
• CT = × Cs
AS
Concentration of Absorbance of test Conc of
TEST solution Absorbance of standard standard
• In colorimetric estimation it is necessary to
prepare 3 solutions
1) BLANK (B)
2) STANDARD (S)
3) TEST (T)
BLANK ( B) :- To eliminate the effect of light
absorption by the reagent used.
• Water BLANK:
Distilled water taken as blank
• Reagent BLANK:
Reagent used in the estimation is taken
as blank
STANDARD (S):- Solution of known
concentration of the substance
• Both O.D and concentration are known
• So concentration of unknown (TEST) can
be calculated.
TEST (T) :- Test solution is made to know
the concentration of a chemical in the
given sample.
• Prepared by treating a specific volume
of specimen (blood, urine, CSF..etc) with
reagents.
• A coloured derivative of the
compound to be measured is
prepared and its absorbance (or)
Optical Density is measured using
a photoelectric colorimeter. This
value is compared with that of a
standard of known concentration.
Basic components of photoelectric
colorimeter
• Light source
• Filter, used for selecting the
monochromatic light. Filters will
absorb light of unwanted
wavelength and allow only
monochromatic light to pass
through. The colour of the filter
should be complementary to the
colour of the solution.
• Sample holder, called ‘cuvette’.
• Detector ( photocell )
• Display as a digital meter.
• The monochromatic light is
allowed to fall on the coloured sol.
The solution is taken in cuvettes of
fixed diameter to keep the path
length common to the test as well
as standard.
• The solution absorbs part of light
and remaining transmitted light is
allowed to fall on photocells. The
electrical impulse thus generated is
measured and displayed.
end point analysis:-
The serum sample and reagents
are mixed and incubated for a fixed
time to develop the color optimally.
After the incubation period, OD is
estimated and concentration of the
substance is calculated. This is
end point analysis.
kinetic analysis:-
The serum and reagents are
incubated and readings are taken
at 2 and 3 minutes exactly; and
from the difference in OD between
the two values, the concentration is
calculated. This is kinetic analysis.
