COLORIMETER

COLORIMETER
•What is colorimeter ?
•Colorimetry.
•Principle of colorimeter.
•Beer's and Lambert's law.
•Components of colorimeter.
•Functions of components.
•Advantages and Disadvantages.
 What is
colorimeter ?

• A colorimeter (or photometer) is an instrument used to

measure the color intensity of a solution by measuring the
amount of light transmitted or absorbed by the color solution.

• The absorbance of light (monochromatic light of particular

wave length in visible range 400-760 nm) is expressed as
optical density, light transmitted is expressed as
transmittance (T).
Visible spectrum
 COLORIMETR
Y
•It is a most common analytical technique used
in biochemical estimation in clinical laboratory.

•It involves the quantitative estimation of colour.

•A substrate must be estimated colorimetrically,

must be coloured or it should be capable
of forming chromogens (coloured
complexes) through the addition of reagents.
•Coloured substance absorb light in relation
to their colour density.

•The colour density will be proportional to

the concentration of coloured substance.

•The instruments used in this method are

called colorimeter or photometer.
 PRINCIPLE OF COLORIMETER

•When a monochromatic light passes through a

coloured solution, some specific wavelength of light is
absorbed which is related to colour density.

•The amount of light absorbed or transmitted by a

colour solution is accordance with two law, i.e. Beer’s
law and Lambert’s law.
 BEER’S LAW
When a monochromatic light passes through a
colored solution, amount of light transmitted
decreases exponentially with increase in concentration
of colored substance when the length of the light path is
constant.
• i.e. the amount of light absorbed by a colored solution is
directly proportion to the conc. of substance in the colored
solution.
BEER’S LAW
Lambert’s law :
• The amount of light transmitted decreases exponentially with
increase in path length (diameter) of the cuvette or thickness of
colored solution through which light passes, when the
concentration of chromogen is constant.
• i.e. the amount of light absorbed by a colored solution
depends on path length of cuvette or thickness of the
colored solution.
• Combined beer’s- lambert’s law is thus expressed as amount
of light transmitted through a colored solution decreases
exponentially with increases in conc. of colored solution &
increase in the path length of cuvette or thickness of the colored
solution.
• Combining the two Laws
•

k
• where k is extinction coefficient or absorption coefficient and
is fixed for given substance at specific concentration and
wavelength.
 Relatioships between absorbance and transmitance
• When a monochromatic light with an original intensity Io passes
through a solution that can absorb radiant energy of certain
wavelength, the intensity of transmitted light Ie will be less than Io

OD %T

Io Ie
Where, Io = Intensity of incident Light
Ie = Intensity of transmitted Light
Transmittance (T) = Ie/Io
Transmittance in percentage = Ie/Io x 100

Absorbance (A) or optical density (OD) which is directly proportional

to concentration.
A = -log Ie/Io
= - log T
= log 1/T
To convert T to %T both denominator ad numerator are multiplied
by 100.
A= log 1 x 100 = log(100)
T 100 ( T% )
A = log 100 - log %T
A = 2 – log%T

OD and tranmittance are related reciprocally

Where Io= Ie= absorbance = 0%
Ie= 0 = absorbance = 100 %
• It is a photometric technique which states that when a beam of
incident light of intensity Io passes through a solution, the
following occur:
• A part of it is reflected which is denoted as Ir
• A part of it is absorbed which is denoted as Ia
• Rest of the light is transmitted and is denoted as It
• Therefore, Io = Ir + Ia + It
• To determine Ia the measurement of Io and It is sufficient
therefore, Ir is eliminated. The amount of light reflected is kept
constant to measure Io and It.
 COMPONENT OF COLORIMETER
➢Light source
➢Slit
➢Monochromator(filter)
➢Cuvette
➢Photocell
➢Galvanometer
 FUNCTION OF EACH COMPONENT

Light source

Two kinds of lamp:-

[Link] Deuterium :- for measurement in
the ultraviolet range 200 – 900 nm.
2. Tungsten lamp:- for measurement in the
visible 400
– 760 nm and near-infrared ranges.
 MONOCHROMATOR(FILTER) :

FILTER:

• Used for selecting the monochromatic light.

• Filters will absorb light of unwanted wavelength and allow
only monochromatic light to pass through.

Three Types:
[Link]
[Link]
[Link]
 PRISM
•When light travels from one medium to
another medium , it is refracted and enters in
the new medium at a different angle.
Prism wavelength spectrum
 GLASS FILTER:-

•Glass filters are selectively transmit light in

particular range of wavelength.
GRATINGS :
•GRAPHITE

•Light (Tungsten light) is

reflected on graphite. This
graft separate light in
different wave length . By
rotation of slit, desirable
wave length of light come out
from slit. And Beam of that
wave length is generated.
•Desired wavelength selected
by the adjustment of an exit
slit.
 CUVETTE (Sample cell ):

➢As per lambert – beer's

law path length is fixed
to 1 cm.
➢Sample cell has 1 cm
diameter.
➢A container that
contains a sample is
usually called
cell.
THREE TYPES OF CELL:-

[Link]

•340nm wavelength of light absorbed in

glass cell.
• c h e ap
 2 . Q u a rtz
• It allows p a s s a g e both type of light, ultraviolet &
visible r a n g e s .
•S o used for m e a s ur e m e nt of both r a n g e s .
• costly.

[Link] cuvette
• Shorter Life Span
• Easily get Scratches
• Low Cost
PHOTOCELL (PHOTODETECTOR)
•These are the devices to measure the
intensity of light by converting light energy into
electric energy.
•They are made up of light sensitive
material such as selenium.

GALVANOMETER
•Readout device.
•A galvanometer is used to detect and measure
electrical current produced by the
photodetector.
ADVANTAGE:-

• It is very easy to operate.

• It is inexpensive .
• Very well applicable for quantitative analysis of colored
compounds.
• Easily transportable.
DISADVANTAGES:-
• It does not work in UV and IR regions.
• Limited range of filters are available.
• If the light source is not stable ,there is a
possibility of errors due to a change from the
initial light intensity during a measurement.
•The manual operation are limited.
 Application of colorimeter
- 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.
 Preparation of Solution for Investigation
• In colorimeter estimation, it is necessary to prepare three solutions:
1. Blank (B)
2. Standard (S)
3. Test (T)

4. Blank:- Two types of blank are used:

a. Water blank:- It is used to adjust the OD to zero and 1% T to 100.
b. Reagent blank:- It is prepared by adding all reagents except the
substance to be estimated.
2. Standard:- It is a solution of known concentration of the substance
in pure form to be estimated.
3. Test
Spectrophotomet
er
 Principle
The working of colorimeter & Spectrometers is
based on Beer's & Lambert's law.

Beer's Law:-It states that the optical density

of a solution is directly proportional to the
concentration of the solution .

Lambert's law:-It states that the optical density

of a coloured solution is directly proportional to
the path of light.
 Differences: Colorimeter &
Spectrophotometer
•Spectrophotometer •Colorimeter
●
Ultra violet & infrared ●
Limited for the visible
region also visible portion of spectrum
e.g.340nm (visible light)
●
Very costly ●
Cheap
●
Four digits reading after ●
Two digit reading after
desimal point . desimal point.
●
More sensitive ●
Less sensitive
●
Prism are used. ●
Glass are used.
 ●
Glass cuvette or test
●
Quartz cuvette is used
tube is used for reading
which does not absorb
which absorb 340nm
340nm light.
light.
●
Halogen lamps are used. ●
Tungsten lamps are
• Can do kinetic method. used.
●
Can’t do kinetic
method.
 Experiment No-6
Photometry:- Photometry is a branch of science that deals with
measurement of the intensity of light.
Photometer:- Photometer is any of several instruments used to
measures various aspects of the intensity of light.
• In this, the intensity of absorbed, transmitted and reflected light is
measured and is related to the concentration of the test substance.
• Photometric principle are applied to the several kind of analytical
techniques.
1. Colorimetry, spectrophotometry, atomic absorption and
turbidometry- where absorbed or transmitted light is measured.
2. Flame emission photometry – where emitted light is measured.
• Principle:- When a monochromatic light with an original
intensity Io passes through a solution that can absorb radiant
energy of certain wavelength, the intensity of transmitted light I e
will be less than Io

OD %T

Io Ie
Where, Io = Intensity of incident Light
Ie = Intensity of transmitted Light
Transmittance (T) = Ie/Io
Transmittance in percentage = Ie/Io x 100

Absorbance (A) or optical density (OD) which is directly

proportional to concentration.
A = -log Ie/Io
= - log T
= log 1/T
To convert T to %T both denominator ad numerator are multiplied
by 100.
A= log 1 x 100 = log(100)
T 100 ( T% )
A = log 100 - log %T
A = 2 – log%T

OD and tranmittance are related reciprocally

Where Io= Ie= absorbance = 0%
Ie= 0 = absorbance = 100 %
 Colorimeter
• A colorimeter (or photometer) is an instrument used to measure
the color intensity of a solution by measuring the amount of light
transmitted or absorbed by the color solution.

Principle:- When a monochromatic light ( Light of specific wave

length ) falls on a colored solution, a proportion of incident light is
absorbed by the colored solution and remaining light is
transmitted. The amount of transmitted light depends upon
concentration of color in solution, which depends upon
concentration of colored producing substances.
• The measurement of color intensity of a colored solution by
photometry is governed by two laws:-
1. Beer’s Law
2. Lambert’s Law
3. Beer’s Law:- The amount of light transmitted through a
colored solution decreases exponentially with increase
in concentration of colored substance when the length of
the light path is constant.

4.
Lambert’s law :
• The amount of light transmitted decreases exponentially with
increase in path length (diameter) of the cuvette or thickness of
colored solution through which light passes, when the
concentration of chromogen is constant.
• Combined beer’s- lambert’s law is thus expressed as amount
of light transmitted through a colored solution decreases
exponentially with increases in conc. of colored solution &
increase in the path length of cuvette or thickness of the colored
solution.
• Combining the two Laws
•

k
• where k is extinction coefficient or absorption coefficient and
is fixed for given substance at specific concentration and
wavelength.
Components of colorimeter

➢ Light source
➢ Slit
➢ Monochromator(filter)
➢ Cuvette
➢ Photocell
➢ Galvanometer
Application of Colorimeter
Aim:- Verification of Beer’s Law

Procedure

[Link]. KMNO4 Distilled Concentrat OD

Solution water ion of
KMNO4
S1 -- 5ml -- 0

S2 1ml 4ml 0.005

S3 2ml 3ml 0.01

S4 3ml 2ml 0.015

S5 4ml 1ml 0.02

S6 5ml -- 0.025
 Fully Auto Analyzer
The auto analyzer perform all the
function of semi auto analyzer.

[Link] dispensing of reagent (by reagent

probe).

[Link] dispensing of samples .

[Link] mixing of reaction mixtures.

[Link] of reacting mixture .

 Advantages:-
• Many samples with different parameter can analyzed at
time.
• Good precision
• Less reagent required.
• Less sample required.
• • Less
For Sample
man & For Reagent
power required .
• For incubation period.
• Maintain the temperature
• Can stored result in memory.
• It have facility to accommodate various samples, standards, calibrations &
Q.C. Sera.
• Automated L-J Chart is visible
• Programmable wash cycles between samples & tests for minimum carry over.
• Auto dilution is also possible
 Two types of fully auto
analyzer:-
Batch analyzer
Random analyzer

Random Access analyzer
- Perform Any number of Parameter from any number of sample.
- More sample in the short period of time .
- Facility of continuous loading of sample
- Facility of “stat” analysis - Urgent sample.
- Facility of autodilution.
- Plotting of daily & monthly Q.C. Chart (L-Jchart).
- Capability to perform a test with 2 to 3 reagents.
- Some of the analyzer has separate assembly
to wash cuvette so very less chance of
contamination.

Example : ERBA XL 640

