Experiment # 1

Colorimetry
 Introduction
 Colorimetry is a technique widely used in
biochemical estimations. It involves
quantitative estimation of colour. A substance
estimated by colorimetry, must be either
coloured or more commonly, capable of
forming chromogens, through interaction with
suitable reagents.
 The instrument known as colorimeter or
photometer is in fact, an absorptiometer, since
it is the amount of light absorbed, which is
measured.
 Principle
The colorimetry is based on the combination of
following two laws which may be stated as follows:
 Beer’s Law
According to Beer’s law, when light passes through a
coloured solution, amount of light transmitted
decreases with the increase in concentration of the
coloured substance.
KC= log10 I0/I
Where,
K= constant
C= concentration
I0= Intensity of incident light
I= Intensity of transmitted light
 Lambert’s Law
This law states that the amount of light transmitted
decreases with increase in thickness of the layer of
coloured solution.
Kl= log10 I0/I
Where,
K= Constant
l= Thickness of layer of solution
I0= Intensity of incident light
I= Intensity of transmitted light

l α 1/I
 The thickness of layer of solution is inversely
proportional to the intensity of light transmitted.
 Colorimeter
 The name colorimeter refers to an instrument
used in colorimetry. It is a device that
measures the absorbance of particular
wavelengths of light by a specific solution.
This device is most commonly used to
determine the concentration of a known
solute in a given solution by the application
of the Beer-Lambert law, which states that
the concentration of a solute is proportional
to the absorbance.
 COMPONENTS OF
COLORIMETER
The major components of colorimeter are:
1. Light source
2. An adjustable slit
3. Condensing lens
4. A set of colored filters
5. Sample chamber (cuvette) to hold the working solution
6. A detector to measure the transmitted light
7. A meter to display the output from the detector
 Light source
For visible region measurement> tungsten
lamp> 380-750 nm
In colorimetry the light with wavelength of
visible region is used for the determination
of the solution. Wavelength of light is
defined as “the distance between two
peaks as the light travels in wave-like
manner.” This distance is expressed in
nanometer (nm). Other units may be used
are Angstrom (Aº) and millimicron (mμ).
1 nm = 1 mμ =10 Aº = 10-9
 An adjustable aperture/ slit
Colorimeter includes an adjustable slit through which the
beam of selected wavelength passes which prevents
stray light.
 Condensing lens
Light after passing through slit falls on condenser lens
which gives a parallel beam of light.
 Filters
Common colorimeters contain a set of filters, which allow
selection of light of narrow wavelengths. These filters
are changeable in order to maximize accuracy.
For example, a green filter absorbs all the component
colors of white light, except green light which is allowed
to pass through. Light transmitted through a green filter
has a wavelength from 500-560 nm. Similarly, other
suitable filters can be used to select light of narrow
wavelengths in the range of 400-700 nm.
 Cuvette
The monochromatic light from the filter passes through the
colored solution placed in a cuvette. Cuvette is made up of
special glass/ plastic/ quartz material. Cuvettes may be square,
rectangular or round shaped with a fixed diameter usually 1cm
and having a uniform surface. The solution absorbs some light
and the residual light falls on a detector.
 Detector
Detectors are photosensitive elements which converts light into
an electrical signal.
 Meter
Electrical energy from a detector is displayed on some type of
meter or galvanometer as transmittance or absorbance.
 Colored solutions have the property
of absorbing light of certain
wavelength and transmitting others.
Color of a solution depends on
transmitted light. e.g. the
hemoglobin solution absorbs blue-
green light and transmits the
complementary colors and the
solution appears red. In colorimetry,
filters are chosen appropriately, so
that absorption is maximum at the
selected wavelength region.
Color of Color Wavelength
solution absorbed of absorption
(nm)

Yellow/ Green Violet 400- 435

Yellow/ Orange Blue 435- 490

Red Blue/ Green 490- 500

Purple Green 500- 560
Violet Yellow/ Green 560- 580
Blue/ Green Yellow/ Orange 580- 650

Bluish green Red 650- 700

 In colorimetric measurements, cuvettes of same diameter
are used. Ratio of intensity of emerging light (I) to that of
incident light (I0) is know as transmittance (T).
 Absorbance is directly proportional to the concentration of
colored substance, over a wide range. i.e. a more
concentrated solution gives a higher absorbance reading.
 Steps in colorimetry
In colorimetric estimations, it is necessary to prepare a test, a
standard and a blank. The test solution is prepared by
treating a specific volume of the specimen with reagents
indicated in the procedure. A standard solution is prepared
by similarly treating a solution of the pure substance of
known concentration. A blank is always run by treating a
volume of water equal to the specimen with the reagent.
This is to correct for the color given by reagents alone in the
procedure.
An appropriate filter is inserted into the photometer. The cuvette
is filled with water to about three fourth and placed in position.
Light is allowed to pass through the cuvette. Absorbance is
adjusted to zero.
Blank solution is taken in another cuvette and is placed in the
cuvette compartment. Absorbance is noted (B). Similarly,
absorbance of test (T) and standard (S) are measured.
Satisfactory results are usually obtained when absorbance
values (optical density) of T and S are in the range of 0.1-0.7.
Concentration of the compound in the specimen=
(T-B)
_____ * Concentration of solution
(S-B)