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.

1. Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. •COLORIMETER

3. •Visible spectrum

4. •ColorimeterColorimeter

5. •Photometry

6. 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.

7. • 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.

8. • Colorimeter - PrincipleColorimeter - 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.

9. • The measurement of colour intensity of a
coloured solution by photometry is governed
by two laws

11. 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.

12. Beer’s law
Beer’s law

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14. 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.

15. LAMBERT’S LAW
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16. • 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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18. Relationship between absorbance and
transmittance
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OD %T

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

20. Components of the
colorimeter
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22. 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.

23. 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.

24. 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.

25. 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.

27. COLORIMETER

28. Colorimeter
(1)(1) Wavelength selection,Wavelength selection,
(2)(2) Printer buttonPrinter button
(3)(3) Concentration factor adjustment,Concentration factor adjustment,
(4)(4) UV mode selector (Deuterium lamp)UV mode selector (Deuterium lamp)
(5)(5) ReadoutReadout
(6)(6) Sample compartmentSample compartment
(7)(7) Zero control (100% T),Zero control (100% T),
(8)(8) Sensitivity switchSensitivity switch

29. 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
Relationship between the wavelength and colour

31. Preparation of solution for investigation
• In colorimetric estimation it is necessary to
prepare 3 solutions

36. 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

37. AT
AS
KxCTxL
KxCSxL
=
AT
AS
CT
CS
=
CT =
AT
AS
X CS
AS=KxCSxLAS=KxCSxL
AT=KxCTxLAT=KxCTxL

38. CT =
AT
AS
X CS
Concentration
of TEST
solution
Concentration
of TEST
solution
Absorbance of TEST
Absorbance of STANDARD
Concn of STANDARDX
=
Concentration
of TEST /
100ml
Concentration
of TEST /
100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100X
=
Xml

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Concentration
of TEST /
100ml
Concentration
of TEST /
100ml
Absorbance of TEST
Absorbance of STANDARD
X
=
Xml
Concn of Std X 100
Concentration
of TEST /
100ml
Concentration
of TEST /
100ml
O.D of ‘T’- O.D of ‘B’
O.D of ‘S’- O.D of ‘B’
X
=
Volume of ‘T’
Amount of ‘S’ X 100
Concentration
of TEST /100ml
Concentration
of TEST /100ml
T - B
S - B
X
=
Volume of ‘T’
Amount of ‘S’ X 100

41. VERIFICATION OF BEER’S LAW
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42. B S1 S2 S3 S4 S5 S6 S7 T
Volume of 3% cobalt acetate
[in ml]
- 0.5 1 2 3 4 5 6 -
Volume of 1% HCl [in ml] 6 5.5 5 4 3 2 1 - -
Conc. Of cobalt acetate in
mg/6ml
- 15 30 60 90 120 150 180
O.D at 490nm 0.01 0.03 0.06 0.12 0.18 0.24 0.30 0.36 0.21

43. •By Graphical method
0.17

44. • 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.
Application of colorimeter

45. • 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.

46. Advantage
It is inexpensive .
Very well applicable for quantitative analysis of colored
compounds.
Easily transportable.
•COLORIMETER

47. 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 .
•COLORIMETER

48. 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.

49. 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.

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