COLORIMETRY
Shalaka Agarwal
Tutor
Dept. of Biochemistry
BMCRI, Palanpur
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.
• 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.
Principle
• When a monochromatic light passes through a
coloured solution, some specific wavelengths
of light are absorbed maximally by the
chromogen to be measured.
• The absorbance 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.
•Beer's Law: The log of the ratio of intensities of incident light and emergent light is
directly proportional to the concentration of the colouring substance (chromogen)
in the solution provided that the thickness of the solution through which the light
passes is constant.
• Io
•
log _____
C
• I
•
• Io
•log ____ = K1C
• I
Where,
•I0 = Intensity of the incidence light
•I = Intensity of the emergent light
•C = Concentration of the chromogen
•K1 = A constant depending upon the wavelength of the light, the nature of the
chromogen and the thickness of the solution.
• Lambert's law: The log of the ratio of intensities of incident light
and emergent light is directly proportional to the thickness of the
solution through which the light passes provided that the
concentration of the chromogen is constant.
• Io
• log _____ t
• I
•
• Io
• Or log ____
= K2t
• I
• Where,
• t = Thickness of the solution traversed by light
• K2 = A constant depending upon the wavelength of incident
light, the nature of the chromogen and its concentration.
• Beer-Lambert's law is :
• Io
• log _____ Ct
• I
•
• Io
• Or log ____ = KCt
• I
• Here,
• K=A constant depending upon the log of wavelength of the incident
light and the nature of the chromogen.
• Thus the Beer-Lambert's law states that the log of ratio of the intensities of
incident light and emergent light is directly proportional to the concentration
of the chromogen and the thickness of solution through which the light passes.
•
• The ratio of the intensities of emergent light and incident light (I/I o)
is known as transmittance (T). This is a measure of the ability of a
solution to transmit light.
•
• So,
• 1
• log _____ = KCt
• T
• log 1/T is known as the optical density (O.D.) or the absorbance
(A). This is a measure of the ability of a solution to absorb light.
• So, A = KCt
• and A Ct
• In other words, whereas transmittance (T) has a logarithmic
relationship with C and t, absorbance (A) has a direct relationship
with C and t. Therefore, absorbance is used in calculations in all the
colorimetric determinations.
PARTS OF COLORIMETER
Light source
Two kinds of electric lamp.
1. Halogen Deuterium
for measurement in the ultraviolet range
200 – 900 nm
2. Tungsten lamp
for measurement in the visible 400 – 760nm and near-
infrared ranges
• MONOCHROMOTOR :
• 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. Diffraction Grating
• 3. Coloured glass/ dyed gelatin
• CUVETTE (Sample cell ):
• As per lambert – beer's law, path length is fixed to 1 cm.
• Sample cell has 1 cm diameter.
• A container that contains a sample is usually called "cell“
• Two types are available
• 1. Glass
• • wavelength of 340 nm or less hardly passes through a glass cell. It absorbed in
glass cell.
• • Cheap
• 2. Quartz cells
• • It allows passage of light in the entire wavelength in the ultraviolet and visible
ranges.
• • Used for the measurement in the ultraviolet
• range
• • Costly
• 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.
• Potential gradient is created between cathode
and anode.
• GALVANOMETER
• •Readout device/ Measuring device.
• •A galvanometer is used to detect and
measure eletrical current produced by the
photodetecter.
• •It is calibrated to read directly either
transmittance or absorbance or both
Instrument Operation:
Warm-up
Set Monochromator
Set ∞ Absorbance
Set Zero Absorbance w/Blank
Re-adjust as Needed
Preparation of Solutions
• Blank
1. Water Blank
2. Reagent Blank
• Standard
• Test
Wavelength (nm) Region name Colour
observed/Complement
ary colour (absorbed /
reflected)
< 380 U.V. Not visible
380-440 Visible Violet(Green Yellow)
440-500 Visible Blue (Yellow)
500-580 Visible Green (Red)
580-600 Visible Yellow (Blue)
600-620 Visible Orange (Green Blue)
620-750 Visible Red (Green)
> 750 IR Not visible
Verification of Beer-Lambert’s Law
• Beer’s Law states that Optical Density (OD) is
directly proportional to the Concentration of
Colored Particles (Intensity of Color) when the
length of the Light Path is Constant.
• Reagents
• Colored Solution
• Distilled Water
No. Coloured Water OD
solution
1 ------------ 1000 ul
2 100 ul 900ul
3 200 ul 800ul Mix the
Contents
4 500 ul 500 ul and Read at
5 1000ul 0ul 450 nm
Plot a graph of Optical Density against concentration. Check & note down the linearity.
THANK YOU!
