Colorimeter

COLORIMETER
INTRODUCTION
• Colorimeter is instrument which is used in the

measurement of
the luminious intensity of light.
• Invented by Louis Jules Duboscq in 1870.
PRINCIPLE
• It is the most common analytical

technique used in
biochemical estimation in clinical
• Color can be produced by any substance when
laboratory.
it binds
with/turns out color forming chromogens.
• The difference in color intensity results in the
difference in the
absorption of light.
• The intensity of color is directly
proportional to the
concentration of the compound being
measured
Relationship wavelength &
between colour
• Wavelength between 400nm to 700nm form
the visible
spectrum of light
• visible band of light in electromagnetic
spectrum
Wavelength Spectrum Colour absorbed Colour
transmitted
(nm) region
400-420 Visible Violet Green-

yellow
420-500 Visible Blue yellow
500-570 Visible Green Red
570-600 Visible yellow Blue
600-630 Visible orange Green-blue
630-700 Visible Red Green

Absorpti & transmittance of
on light
• Light falling on a color solution is either absorbed,
reflected
or transmitted.
Io=It +
I
o
I
t Ia
Ia
Relationship between
absorbance and
transmittance
A = O.D = Log 1/T
= Log( 100/ %T)
= Log100-
Log%T
i.e. O.D. = 2 - Log
(%T)
Transmittance of a solution containing light
absorbing
substance depends upon
1. The nature of light absorbing
substance.
2. Wavelength of light
and
3. Amount of light absorbing ligh
path, which ininturn
substance the depends on the t
concentration of
light absorbing substance and depth of the
solution through which light passes.
%
t
A r
b a
s n
o s
r m
b i
a
n s s
i o
ce
0 0n
0 Concentration 0 Concentration
(a) Relation between absorbance &

concentration.
(b) Percentage transmission &
concentration.
The relationship
between
concentration of the
compound and color
intensity is given by
Beer’s law and
Lamberts law
Basis of colorimetric
techniques
Beer’s law
When monochromatic light passes through a
light absorbing medium, the intensity of the
transmitted light decreases exponentially as the
concentration of the light absorbing material
increases.
AαC
• Where A is light absorbed and C is
concentration of the
solution.
Lamberts law
When monochromatic light passes through a
coloured solution, the amount of light
increas in increases
absorbed ofwith
the layer
the of solutio throug whic
e thickness the n h h
the passes.
light
AαL
• Where L = length of light path
By combini abov equations, ge
ng e we t
A α CL
A= KCL
Where k = constant for coloured

solution
• For standard solution : A =K C L
s s s s
• For unknown solution : A =K C L
u u u u
A =absorbance of unknown
u
C solution
u
A = conc of unknown solution
= con of std of std solution
S
C =absorbance
S c solution
But K =K & L =L
s u s u
A /A = C /C
u s u s
C = A /A X C
u u s s
Parts of
colorimeter
• Light
source
• Monochromator/ wavelength
selector • Filte
• Solution/sample r
holder • Cuvette
• Photosensitive detector
system
• Measuring
device
Flow representation of
colorimeter
Light
source
Common source is a tungsten-
higher powered
filament lamp, –halogen
tungsten (quartz-
iodine) lamp.
Factor of light source are range,

spectral
distribution, stability of radiant
energy and
temperature..
Monochromator/wavelength
selector
• Mono = single. Chromatic- colour
Monochromatic light is the single
colour band of light.
• Monochromator and filters are used to
split the
light from the light source.
• Simple filters are either coloured glass
or
suitably dyed gelatin sandwiched in a
glass.
Complementary filters for coloured
solutions
The selected filters has the color to the complementary to that of the
color of unknown solution
Color
(ROYGBIV)
Wheel
Complementary colors lie diamet on the

wheel and
across the combine to form “white light”,
er so the color
color
of a
compound seen by the eye is the complement of the
color of light absorbed by a colored compound; thus it
completes the color.
λmax
It maximum absorbance by the
is solution at one particular
wavelength .
Solution
holder
Cuvette are rectangular cell ,
square cell or circular one
• Made up of optical glass for visible

wavelength.
Common one is square,
rectangular to avoid
refraction artifacts.
• dimension of cuvette is
1cm.
cuvett
Photo sensitive
•
detector
when light falls on these electric elements electric
current is
generated which deflects a galvanometer needle.
• The meter reading is proportional to the light
intensity ,these
photosensitive detectors are also referred to as
photoelectric
cells.of the common used photo cell is Barrier
• One
layer cell.
Measuring
•
device
Current from detector is fed to a sensitive suitable
measuring
device, usually galvanometer.
• Absorbance scale ranges from 0 to 2 ,

while
• % transmission scale ranges from 0
to 100. absorbance = 100%
• Zero
transmission
• Infinite absorbance =0
transmission.
COLORIMETER
Advantage
✔ It is inexpensive
✔ Very well applicable for quantitative analysis

of colored
compounds.
✔ Easily cartable and
transportable.
COLORIMETER
Disadvantage
✔ Cannot be used for colorless
compounds.
✔ It does not work in UV and IR
regions.
✔ We cannot set specific wavelength, as we have to set a
range as a
parameter.
✔ Similar colors from interfering substances can
produce errors in
results .
Application
• It is widely used in hospital & laboratory for estimation of
biochemical samples , like plasma, serum, cerebrospinal
fluid ( csf )
, urine.
• It is also used to quantitative estimation of serum
components as
well as glucose, proteins and other various biochemical
compound.
• They are used by the food industry and by
manufacturers of paints
and textiles.
• They are used to test for water quality, by screening for
chemicals
such as chlorine, fluoride, cyanide, dissolved oxygen,
iron, molybdenum, zinc and hydrazine.
• They are also used to determine the concentrations of
plant nutrients
(such as phosphorus, nitrate and ammonia) in the soil or
hemoglobin
in the blood and to identify substandard and counterfeit
drugs.
Use of test (T), standard (S) and
blank (B)
In colorimetric estimation , it is
necessary to
prepare a blank (B), a standard (S) &
test: (T).
Test this solution is prepared by treating
a specific
volume of specimen (blood,urine,
CSF…etc)
with reagents.
Use of
blank
• Blank : prepared for rule out color
produced by reagents alone.
A blank solution or reference solution

has
everything except the compound to be
Two types of
•measured
A) Distilled
blank : water as blank
B) reagent blank (reagent used in the is
estimation taken as blank)
Standard : prepared by treating a
pur substan
solution of known conc.
of the reagent
e ce With s.
Standar
d
SECONDARY Standar
PRIMARY substance d
Standard different
taken asfrom the
standard is
substance to be
same substance is used substanceestimated.
taken as standard
as should
standard one which is match the color of final
to be product.
estimated. e.g. methyl red is taken as
e.g. pure glucose is taken standard in estimation of
as serum bilirubin .
Spectrophotometer
A spectrophotometer is an analytical
instrument that can measure the
concentration of a sample via measuring
the light absorption.
This instrument can operate at visible light,

near UV, and near IR lights as well.
We can use this instrument to detect organic
compounds as well.
That is by determining the absorption
maximum. Moreover, we can use it to
determine the colour within a spectral range.
Most importantly, we use it to measure the

concentration of a component in a sample by
determining the amount of light absorbed by
that component.
Applications of spectrophotometer

Colorimeter

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COLORIMETER

• Colorimeter is instrument which is used in the

• It is the most common analytical

400-420 Visible Violet Green-

500-570 Visible Green Red

570-600 Visible yellow Blue

600-630 Visible orange Green-blue

630-700 Visible Red Green

(a) Relation between absorbance &

Where k = constant for coloured

Factor of light source are range,

Complementary colors lie diamet on the

• Made up of optical glass for visible

• Absorbance scale ranges from 0 to 2 ,

✔ Very well applicable for quantitative analysis

A blank solution or reference solution

This instrument can operate at visible light,

Most importantly, we use it to measure the

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