Tapeshwar Yadav

(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry
•COLORIMETER
•Visible spectrum
•Colorimeter
•Photometry
 Colorimetry
• It is the most common analytical technique
used in biochemical estimation in clinical
laboratory.
• It involves the quantitative estimation of color.

• A substance to be estimated colorimetrically,

must be colored or it should be capable of
forming chromogens (colored complexes)
through the addition of reagents.
• Colored substance absorb light in relation to their
color intensity.
• The color intensity will be proportional to the
conc. of colored substance.
• The instruments used in this method are
colorimeter or photometer or absorptiometers.
• Colorimeter - Principle
• When a monochromatic light passes through a
coloured solution, some specific wavelengths of light
are absorbed which is related to colour intensity.
• The amount of light absorbed or transmitted by a
colour solution is in accordance with two law i.e.
Beer’s & Lambert’s Law.
• Themeasurement of colour intensity of a
coloured solution by photometry is governed
by two laws
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.
• 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

Beer’s law
09/12/15 08:51
Lambert’s law :
• The amount of light transmitted decreases
exponentially with increase in pathlength (diameter)
of the cuvette or thickness of colored solution
through which light passes.
• i.e. the amount of light absorbed by a colored
solution depends on pathlength of cuvette or
thickness or dept of the colored solution.
 LAMBERT’S LAW

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• 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 conc. of
colored solution & increase in the path length of
cuvette or thickness of the colored solution
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Relationship between absorbance and
transmittance
OD %T

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 Absorption & transmittance of light
• Light falling on a color solution is either absorbed, reflected or
transmitted.
Io=It + Ia

Io It

Ia
Components of the
colorimeter

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 Parts of the colorimeter

Light source : tungsten filament lamp

Slit : it is adjustable which allows only a beam of
light to pass through. it prevents unwanted or
stray light
Condensing lenses: light after passing through slit
falls on condenser lense which gives a parllel
beam of light.
Filter :
• made of colored glass. Filters are used for selecting light of
narrow wavelength.
• filters will absorb light of unwanted wavelength and allow only
monochromatic light to pass through.
For ex: a green filter absorbs all color, except green light which
is allowed to pass through. Light transmitted through a green
filter has a wavelength from 500-560 nm.
• Filter used is always complimentary in color to the color of
solution.
Cuvette(sample holder) : the monochromatic light from the
filter passes through the colored solution placed in a cuvette.
• it is made up of special glass/plastic/quartz material.
• it may be square/rectangular/round shape with fixed diameter
(usually 1 cm)& having uniform surface. the colored solution in
the cuvette absorbs part of light & remaining is allowed to fall
on detector.
• For ex : a solution of red color transmits red light & absorbs
the complimentary color green.
Detector (photocell):
• Detector are photosensitive elements which converts light
energy into electrical energy.
• The electrical signal generated is directly proportional to
intensity of light falling on the detector.
Output : the electrical signal generated in photocell is
measured by galvanometer, which displays percent
transmission & optical density.
COLORIMETER
 (1) Wavelength selection,
(2) Printer button
(3) Concentration factor adjustment,
UV mode selector (Deuterium lamp)
Colorimeter (4)
(5) Readout
(6) Sample compartment
(7) Zero control (100% T),
(8) Sensitivity switch
Relationship between the wavelength and colour
Approx. wavelength Colour absorbed (Filter) Colour of solution
<4oonm Ultra violet (UV-rays) Not visible
400-420nm Violet Green-Yellow
420-500nm Blue Yellow
500-570nm Green Red
570-600nm Yellow Blue
600-630nm Orange Green-Blue
630-700nm Red Green
>700nm Infrared (IR-rays) Not visible
Preparation of solution for investigation
• In
colorimetric estimation it is necessary to
prepare 3 solutions
Derivation of the Formula
• Combining the two laws
• AαCxL
• OR A=KxCxL
• Let AT=absorbance of the test solution
• CT=concentration of the test solution
• AS=absorbance of the standard solution
• CS=concentration of the standard solution
 AASS=KxC
=KxCSSxL
xL
AATT=KxC
=KxCTTxL
xL

AT KxCTxL
=
AS KxCSxL
AT CT
=
AS CS
AT C
CT = X S
AS
 AT C
CT = X S
AS

Concentration
Concentration Absorbance of TEST X Concn of STANDARD
of
ofTEST
TEST =
solution
solution Absorbance of STANDARD

Concentration
Concentration Absorbance of TEST X Concn of Std X 100
of
ofTEST
TEST// =
100ml
100ml Absorbance of STANDARD Xml
Concentration
Concentration Absorbance of TEST X Concn of Std X 100
of
ofTEST
TEST// =
100ml
100ml Absorbance of STANDARD Xml

Concentration
Concentration O.D of ‘T’- O.D of ‘B’ X Amount of ‘S’ X 100
of
ofTEST
TEST// =
100ml
100ml O.D of ‘S’- O.D of ‘B’ Volume of ‘T’

T-B Amount of ‘S’ X 100

Concentration
Concentration X
of
=
ofTEST
TEST/100ml
/100ml S-B Volume of ‘T’

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 VERIFICATION OF BEER’S LAW

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 B S1 S2 S3 S4 S5 S6 S7 T

Volume of 3% cobalt acetate - 0.5 1 2 3 4 5 6 -

[in ml]

Volume of 1% HCl [in ml] 6 5.5 5 4 3 2 1 - -

Conc. Of cobalt acetate in - 15 30 60 90 120 150 180

mg/6ml

O.D at 490nm 0.01 0.03 0.06 0.12 0.18 0.24 0.30 0.36 0.21
•By Graphical method

0.17
 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.

• 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.
 •COLORIMETER

Advantage
It is inexpensive .

Verywell applicable for quantitative analysis of colored

compounds.

Easily 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 .
Use, care and preventive maintenance of a
Colorimeter:
• Read the user manual carefully.
• Use the correct type of cuvette in the colorimeter as
recommended by the manufacturer.
• Make sure that the cuvette is clean and it’s optical surfaces are
dry and free from finger marks and scratches.
• Bring filter in to place before switching on the colorimeter.
• Before reading the absorbance of a solution, check that it is
clear, there are no air bubbles in it.
• Remove the cuvettes from the instrument when not in use.
 CONTD…
• Clean the outside of the cuvette with tissue paper to remove any
marks from the optical surfaces.
• To prolong the life of the lamp, switch off the colorimeter after
use.
• At the end of the day, disconnect It from the main switch and
cover the colorimeter with its protective cover.
• At regular intervals check the mains power adapter and cable
for wear and tear and replace if damaged.
• Keep in cool place away from corrosive chemicals or fumes.
 THANK YOU

12-09-15 08:51 AM