COLORIMETRY

• It is a most common analytical technique used in biochemical estimation in

clinical laboratory.
•It involves the quantitative estimation of color.
•A substance 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 density.
•The color density will be proportional to the concentration of colored
substance.
•The instruments used in this method are called colorimeter or photometer.
Colorimeter
• Colorimeter is an instrument used for the measurement of colored substance in
solution. Instrument is operative in the visible range of the magnetic spectrum.

PRINCIPLE OF COLORIMETER
•When a monochromatic light passes through a colored solution, some specific
wavelength of light is absorbed which is related to color density.

• The amount of light absorbed or transmitted by a color solution is accordance with

two law, i. e. Beer’s law and Lambert’s law.
 COMPONENT OF COLORIMETER
Light source, Slit Monochromator(filter), Sample holder (Cuvette) , Photocell
(Photosensitive detector system) , Galvanometer (measuring device)
FUNCTION OF EACH COMPONANT
Light source
Two kinds of lamp: -
1. Deuterium : - for measurement in the ultraviolet range 200 – 900 nm.
2. Tungsten lamp: - for measurement in the visible 400 – 760 nm
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: 1. Prism 2. Grating 3. Glass
GLASS FILTER:
• Glass filters selectively transmit light in particular range of wavelength.

GRATINGS :
• GRAPHITE
• Light (Tungsten light) is reflected on graphite. This graft separate light in
different wavelength. 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 ):
A container that contains a sample is usually called cell or cuvette.
Rectangular, square cell or circular
Dimension of cuvette is 1 cm/ 1 cm diameter.
THREE TYPES OF CELL:
• Glass
340 nm wavelength of light absorbed in glass cell, cheap
• Quartz
It allows passage of both type of light, ultraviolet & visible ranges
(used for measurement of both ranges), costly
• Plastic 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 in to 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

DISADVANTAGES:
• Less sensitive.
• It does not work in UV (Cannot be used for colorless compounds )and
IR range
• 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.
Spectrophotometry
• Spectrophotometry is a standard and inexpensive technique to measure light
absorption or the amount of chemicals in a solution.
• It uses a light beam which passes through the sample, and each compound in
the solution absorbs or transmits light over a certain wavelength.

• The instrument used is called a spectrophotometer.

• A spectrophotometer is an analytical instrument used to quantitatively

measure the transmission or reflection of visible light, UV light or infrared light.

• Spectrophotometers are widely used in various disciplines such as physics,

molecular biology, chemistry and biochemistry.
 The spectrophotometer is an instrument which measures an amount of light that a
sample absorbs.
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.
1. In the spectrophotometer, a prism (or) grating is used to split the incident beam into
different wavelengths.
2. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the
test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm.
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 colored solution is directly
proportional to the path of light.
According to Beer's & Lambert's law where,
T=10 -kc. L,
T=transmittance
K=Constant characteristic of the solution
C=concentration of the colored solution
L=Path of light through the colored solution
O. D. =2 – log T%
As per lambert – beer's law path length is fixed to 1 cm.
Differences between Colorimeter & Spectrophotometer
Colorimeter Spectrophotometer
Limited for the visible portion of Ultra violet , visible & infrared region
spectrum (visible light)
Cheap Very costly
Two digit reading after desimal point. Four digits reading after desimal point.
Less sensitive More sensitive
Glass are used Prisms are used
Glass cuvette or test tube is used for Quartz cuvette is used which does not
reading which absorb 340 nm light absorb 340 nm light
Can’t use specific filter Can use specific filter
Tungsten lamps are used Halogen lamps are used
https://www.youtube.com/watch?v=pxC6F7bK8CU Spectrophotometer
https://www.youtube.com/watch?v=gGRMtq7hvHc&list=TLPQMTEwMjIwMjL9Y-WzBK0pWg&index=2