COLORIMETER

Dr. Gangadhar Chatterjee

MBBS;MD
Assistant Professor
RCSM Govt. Medical college, Kolhapur, MH, India
 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 laboratory.

• Color can be produced by any substance when 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 between wavelength & 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

 Absorption & transmittance of light

• Light falling on a color solution is either absorbed, reflected

or transmitted.

Io=It + Ia
Io It

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 substance in the light

path, which in turn depends on the 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 s
n s
c i
o
e
n
0 0
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 absorbed increases with the
increase in thickness of the layer of the solution through which
the light passes.

AαL
• Where L = length of light path
 By combining above equations, we get

A α CL

A= KCL

Where k = constant for coloured solution

• For standard solution : As =Ks Cs L s
• For unknown solution : Au =Ku Cu Lu

Au =absorbance of unknown solution

Cu = conc of unknown solution
AS =absorbance of std solution
CS = conc of std solution

But Ks =Ku & L s =Lu

Au/As = Cu/Cs

Cu= Au/As X Cs
 Parts of colorimeter

• Light source

• Monochromator/ wavelength selector

• Filter
• Solution/sample holder
• Cuvette
• Photosensitive detector system

• Measuring device
Flow representation of colorimeter
 Light source
Common source is a tungsten-filament lamp,
higher powered tungsten –halogen (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.
• filters range is 400-680 nm
Complementary filters for coloured solutions
The selected filters has the color to the complementary to that of the color of unknown
solution
 Color Wheel
(ROYGBIV)

Complementary colors lie across the diameter on the color

wheel and combine to form “white light”, so the 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 is maximum absorbance by the 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.

cuvettes
 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.

• One of the common used photo cell is Barrier 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.

• Zero absorbance = 100% 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 measured

• Two types of blank :

A) Distilled water as blank
B) reagent blank (reagent used in the estimation is
taken as blank)
Standard : prepared by treating a solution of the
pure substance of known conc. With reagents.

Standard

PRIMARY Standard
same substance is used as
standard one which is to be
estimated.
e.g. pure glucose is taken as
standard in estimation of blood
glucose.
SECONDARY Standard
substance taken as standard is
different from the substance to be
estimated.
substance taken as standard should
match the color of final product.
e.g. methyl red is taken as standard in
estimation of serum bilirubin.
 Reach me @
ganga.chatterjee@gmail.com
ganga.chatterjee@outlook.com
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