COLORIMETRY

In physical and analytical chemistry, colorimetry is a technique used to

determine the concentration of colored compounds in solution. A colorimeter
is a device used to test the concentration of a solution by measuring its
absorbance of a specific wavelength of light or A colorimeter is a device that
measures the intensity and wavelength of light passing through the substance
in a liquid. The colorimeter is also called spectrocolorimeter. The colorimeter
was invented in the year 1870 by Louis J Duboscq.
Visible light is an electromagnetic radiation with wavelength ranging from 400
to 800nm.It is composed of a mixture of seven colors such as violet, indigo,
blue, green, yellow, and orange each and every color of electromagnetic
spectrum falls within a range of wavelength. The energy of light is determined
by wave length.

Energy = plank’s constant × speed of light

Wavelength
Plancks constant=6.62607015*10-34

Because of specific atomic characteristics, each chemical substance absorbs

light of particular range of wavelengths. This is called absorption spectrum.
Even with the absorption spectrum, absorption is maximum at a particular
wavelength, it is called absorption maxima. A light of a particular range of
wavelengths can be generated by passing the light through an appropriate
colour filter. For example: If light is passed through a red filter [600nm] light of
all wavelengths is absorbed except a range around 600nm. Therefore, the light
coming out of the filter would be in red range. Such a beam of light of a
particular colour is called monochromatic light.

PRINCIPLE
Working principle of colorimeter is based on wave properties of light and
absorption of light by substances. 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 I o and It.
So, absorption of monochromatic light by substances can be quantitized based
on two basic laws (Beer’s Lambert Law).

Beer’s law
According to this law the amount of light absorbed is proportional to the solute
concentration present in solution or When a beam of monochromatic light is
passed through an absorbing medium, the intensity of light coming out
decreases exponentially with increase in the concentration of absorbing
substance in the medium this is the Beer’ s law.
The quantity of substance present in the medium can be deduced(conclude,
anumanikkuka) from transmittancy which depends on the specific absorbance
of the substance and intensity of light. If the intensity of incident light is I o and
of transmitted light is I, the transmittancy(T) is equal to I/I o. T is measured in
percentage (%) using the colorimeter.
Absorbance (A) is measured in terms of optical density (OD).
It is calculated from the equation:

A = log Io ÷ I = log 100 ÷ T =2- log T

Transmittancy decrease with increase in the concentration of the absorbing

particle. As the transmittancy decreases, the OD increases simultaneously.

Lambert’s law:
According to this law the amount of light absorbed is proportional to the
length as well as thickness of the solution taken for analysis or the intensity of
light coming out decreases exponentially with increase in the thickness of
medium through which the incident light passes, this is the Lambert’s law.
Absorbance(A) = log10 Io/It = asb
Where,
Io is the intensity of incident light
It is the transmitted light
A is the test absorbance of test
as is the standard absorbance
b is the length / thickness of the solution

COMPONENTS AND WORKING

A colorimeter consists of a light source, a lens system, a filter selector, a
cuvette holder, a photocell and a galvanometer. All these components, except
galvanometer are fixed in a straight line inside a cabinet. Galvanometer OD
adjusting knob and switches for galvanometer and power supply are fixed on
the cabinet.

 The light source is a tungsten lamp. The white light from the lamp is
passed through a slit to a condenser less.
 This lens condenses the incident light into a narrow beam of light which
falls on the glass filter.
  The glass filter filters off all rays & allow the monochromatic light to pass
through the filter.
 The monochromatic light coming from the filter pass through the
solution kept in the cuvette & reaches the photocell.
 The OD adjustor between the filter and cuvette holder helps to adjust
the OD to zero.
 The photocell generates an electric signals indirect proportion to the
intensity of light falling on it. These electric signals are amplified by an
amplifier and then read with the galvanometer.

APPLICATION

 It is used in laboratories and hospitals to estimate biochemical samples

such as urine, cerebrospinal fluid, plasma, serum, etc.
 It is used in the manufacturing of paints.
 It is used in textile and food industry.
 It is used in the quantitative analysis of proteins, glucose, and other
biochemical compounds.
 It is used to test water quality.
 It is used to determine the concentration of haemoglobin in the blood
 The colorimeter is also used by diamond merchants to measure the
optical properties of precious stones.
 In the printing industry, this device is a basic element in a colour
management system, in addition to checking the electronic components
and quality of pulp paper and measuring the quality of the printing ink.
ADVANTAGES AND DISADVANTAGES
 It is an inexpensive method, widely used in the quantitative analysis of
coloured samples, easy to carry, and transport.

Some disadvantages are as follows:

 Analysis of colourless compounds is not possible, does not work in IR

and UV regions.

SPECTROPHOTOMETRY
The qualitative and quantitative estimations of substances using
spectrophotometer is called spectrophotometry. The spectrophotometer is an
instrument which measures an amount of light that a sample absorbs or
Spectrophotometer is device that produces a light of a particular wavelength &
measures the intensity of outcoming waves passing through the substance in a
liquid. A spectrophotometer is made up of two instruments: a spectrometer
and a photometer. The spectrometer is to produce light of any wavelength,
while the photometer is to measure the intensity of light. The
spectrophotometer is designed in a way that the liquid or a sample is placed
between spectrometer and photometer. The photometer measures the
amount of light that passes through the sample and delivers a voltage signal to
the display. If the absorbing of light change, the voltage signal also changes.
Spectrophotometers come in a variety of shapes and sizes and have
multipurpose uses to them. The different types of spectrophotometers
available are all different from one another, based on their application and
desired functionality. The most popular spectrophotometers are 45 degrees,
sphere and multi-angle spectrophotometers. Spectrophotometers can also be
classified into 2 types based on the range of light source wavelengths like IR
spectrophotometer & UV-visible spectrophotometer.
PRINCIPLE
 The principle of spectrophotometer is based on Beer’s Lambert’s law.
 According to Beer’s Lambert’s law, when a monochromatic light passess
through an absorbing medium, the intensity of the transmitting light
decreases exponentially as the concentration of the absorbing medium
increases.
 The light absorbed by a solution depends on the amount of material
dissolved in it.
 When the solution is less concentrated less light is absorbed & more
light is transmitted, When the solution is more concentrated more light
is absorbed & less light is transmitted.
 The concentration of the substance is calculated by measuring the
optical density of the solution. The OD increases proportionately to the
increases in the concentration of the solution.

COMPONENTS AND WORKING

Spectrophotometer consists of a light source, a lens, a prism, a diaphragm, a
sample holder, a photocell & a galvanometer or digital display.

 The light source is a tungsten lamp, the white light coming from the
lamp is focused onto the prism by lens.
 The prism splits the light into a spectrum of seven colours each of which
is composed of waves of a particular wavelength.
  The diaphragm filters off all other waves except the waves of desired
wavelength. Hence waves of the desired wavelength alone fall on the
sample.
 The sample holder holds the cuvette. The cuvette is made of pure glass.
The thickness of the wall of the cuvette and the diameter are uniform.
The sample is taken in the cuvette & inserted in the holder of the
spectrophotometer.
 The OD adjusting knob is used to set the OD to zero before measuring
the OD of the sample.
 The photocell measures the intensity of the outcoming light waves.
 The digital display converts the light signals into digital numbers &
display the OD value in number.

APPLICATION
 Detection of concentration of substances
 Detection of impurities
 Structure elucidation of organic compounds
 Monitoring dissolved oxygen content in freshwater and marine
ecosystems
 Characterization of proteins
 Detection of functional groups
 Respiratory gas analysis in hospitals
 Molecular weight determination of compounds
 The visible and UV spectrophotometer may be used to identify classes
of compounds in both the pure state and in biological preparations.

Following are the types of spectrophotometers,

 Single beam spectrophotometer: A single beam
spectrophotometer utilizes one beam of light that passes through
the sample and the intensity of the light reflected from a reference
is measured without the sample.
 Double beam spectrophotometer: A double beam
spectrophotometer utilizes two beams of light: a reference beam
and a sampling beam that passes through the sample. Some
 double beam spectrophotometers have two detectors that allow
the two beams to be measured at one time.
 Split beam spectrophotometer: A split beam spectrophotometer
contains a beam splitter which channels light along a reference
path and a sample path simultaneously to two separate detectors.

➨Advantages of single beam type: cheaper due to less parts, high throughput,
high sensitivity, less complicated
➨Advantages of Double beam type: High stability due to simultaneous
measurements of reference and sample.
➨Advantages of Split beam type: Good stability, Good noise

➨Disadvantages of single beam type: There is significant amount of time

needed between two events (taking the reference and making sample
measurement) and hence drift problems arises. With modern electronics and
design, this problem is not seen any more in most of the applications.
➨Disadvantages of Double beam type: High cost, low sensitivity due to poor
light throughput, low reliability due to more complexity.
➨Disadvantages of Split beam type: stability poorer than double beam type as
two detectors can drift independently, noise not as good as single beam type
as light is splitted and hence less than 100% passes through the sample.