Describe the principles and illustrate the methodology of Colorimetry and
Spectrometry
BC 14.6.1, BC 14.6.2
Definition:
Colorimetry is a technique of measuring the concentrations of colored substances in the solution
based on light absorption by molecules in a solution
The colour intensity of a solution is proportional to the concentration of the reacting substances
and it is possible to obtain a measure of the concentration of the substance by determining the
depth of the colour.
It is commonly used to measure substances like Glucose, Urea, Protein, Creatinine after
converting them into Colored compounds.
Colored solutions have the property of absorbing certain wavelengths of transmitting others.
This property is based on Beer-Lambert’s law.
Beer Lambert’s Law:
It states that when a ray of monochromatic light passes through an absorbing medium, the
amount of light, which is absorbed by a given solution, is directly proportional to the
concentration of the absorbing substance in the solution and the optical path through which the
light travels
Transmission is defined as the ratio of the intensity of transmitted light to that of incident light.
𝑰𝒕
T= 𝑰𝒐
Optical density / Absorbance is the logarithmic ratio of the incident light to that of emergent light
A= -Log T
A= Log Io/It
𝟏𝟎𝟎
A= log ( )
%𝑻
A= 2-log%T
Beer- Lambert’s law can be expressed as :
A= abc
A= Absorbance
a=constant
b= path length
c= concentration of compound in solution
When the length of column through which the light passes is kept constant by using cuvettes of
same diameter for both test and standard ,the intensity of transmitted light depends only on the
concentration of substances.
OD/ Absorbance α Conc of the substance
Instrumentation of Colorimeter
1. A light source (often a low voltage filament lamp)
2. An adjustable aperture
3. A set of colored filters
4. A cuvette to hold the working solution
5. A detector to measure the transmitted light
6. A meter to display the output from the detector (GALVANOMETER)
Applications of Colorimeter
1. Measure the Hemoglobin content of blood
2. Used for Basic research and estimations in chemical laboratories.
3. Estimation of Lead in urine and faeces.
4. To test water quality by screening chemicals.
5. To monitor growth and culture of bacteria, yeast
6. Estimate Chlorophyll in samples.
Steps In The Operation Of The Photoelectric Colorimeter
1. Place the glass filter recommended in the procedure in the filter slot. Fill the colorimeter tube
(cuvette) to about three fourth with the distilled water and place in cuvette slot.
2. Switch ‘on’ the instrument and allow it to warm up for 4 – 5 minutes.
3. Adjust to zero optical density. Take ‘blank’ solution in another tube and with this placed in
the cuvette slot, read the optical density (B).
4. Take ‘test’ solution in the cuvette, and as with ‘blank’ read the O.D. (T). Finally take
‘standard’ solution in the cuvette and record O.D. (S).
5. Satisfactory results are obtained only when the O.D. values are in the range 0.1 – 0.7.
6. The reading should be taken with the lighter solution first (lower conc.) followed by the
darker solutions (more conc.) in order to avoid erroneous readings due to carryover effect.
Calculation by using the formula:
𝐀𝟏 𝐛𝟏𝐜𝟏
=
𝐀𝟐 𝐛𝟐𝐜𝟐
(b= path length is same for both solutions) so,
𝑨𝟏 𝒄𝟏
=
𝑨𝟐 𝒄𝟐
Concentration of unknown substance present in 100ml sample
OD of test −OD of Blank
=
OD of standard−OD of blank
x Concentration of the standard
volume of the sample
x 100
ODT, ODS and ODB represents optical density of test, standard and blank solutions respectively
