Colorie metry & Spectrophotometry

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Clinical Biochemistry LaboratoryI Year MBBCh

KumarPradeep.G 1

Photo Electric Colorimetry & Spectrophotometry

Introduction:

Colorimetry and Spectrophotometry, collectively termed as photometry is an important

biochemical laboratory tool used for the quantitative estimation of various componentsin the biological samples, based on their specificity to absorb Monochromatic Light(light at a particular wavelength).

Both Colorimeter and spectrophotometer are the laboratory equipment used for this

purpose.

Principle:

When Light at particular wavelength is passed through a solution (Incident Light) some

amount of it is absorbed and therefore, the light that comes out (transmitted Light) is

decreased. This phenomenon is explained by Beer-Lambert Law.

Beers's Law:

Beers's law states that the amount of transmitted light decreases exponentially with an

increase in the concentration of absorbing material.

i.e the amount of light absorbed depends on the concentration of the absorbing

molecule.

Lambert's law:

The transmitted light decreases exponentially with the increase in the path length (b) of

the absorbing media (molecule).

I.e the amount of light absorbed depends on the path length of the medium.

By combining two laws (Beer-Lambert law) the following mathematical derivation can

be obtained.


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I = I0 cb

Where

I = Intensity of the transmitted light

I0= Intensity of the incident light

= Molar absorption (extinction) coefficient of the absorbing moleculeC= concentration of the absorbing molecule

b= Path length through which light passes

When path length (b) is kept constant (Lambert's law) then the intensity of the

transmitted light depends only on concentration of the absorbing material

i.e only Beer's law is applicable.

Transmittance (T):

T = Transmitted Light intensity (I) / Incident Light Intensity (I0)

T= I / I0

Absorbance (A): Is expressed in relation with the transmittance. Absorbance is also

termed as Optical Density (O.D).

A = log10 I0 / I

A = log10 1 / T

A = log10 100/ %T

A = 2- log10 %T

Absorbance is the generally used quantity in the laboratories because it gives a linear

relation with concentration.

Photoelectric Colorimeter (Colorimeter): Is the instrument used for the measurement

of colored substances. This instrument is operative in the visible range (400-800 nm) ofthe electromagnetic spectrum of light.

The working of the colorimeter is based on the principle of Beer-Lambert law.

Instrumentation:

Light Source ------ Filter ------Cuvette / Sample holder ------ Detector ----- Display

LightSource: A lamp usually serves as a light source

Filter: Allows the passage of a small range of wavelength as incident light

SampleHolder (Cuvette): Is a glass holder with a fixed thickness.


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Detector: Detects the intensity of the transmitted light

Display: An electronic digital display that displays the values of % T or O.D.

Spectrophotometer:

This is operative both in visible (400-800 nm) as well as in ultraviolet region of the

electromagnetic spectrum.

This is more sensitive equipment than colorimeter, also works based on Beer-Lambert

law.

The basic instrumentation of this is as that of colorimeter only.

But the filter in spectrophotometer is more sophisticated and can allow only a particular

wavelength rather than allowing a range of wavelength like in colorimeter.

And the sample holder (cuvette) is made up of quartz instead of glass.

Calibration of Colorimeter & Spectrophotometer:

Switch on the Equipment at least 15 minutes before you use.

Adjust the % T to 0 by keeping a black body (Black opaque plastic cuvette which dose

not allow light to pass through) at the desired wavelength

Adjust % T to 100 and absorbance (O.D) to 0 by keeping a cuvette filled with distilled

water at the desired wavelength.

Beer-Lambert law better works when the % T of the absorbing material is in the range

of 28% - 90 % and the absorbance (O.D) in the range of 0.050 - 0.550 hence dilute the

solutions appropriately to get the values in the desired range.

Procedure:

Standard: A known concentration of the molecule in study taken is termed a standard.

Standard is mixed with the fixed volume of the reaction mixture ( reagents), and diluted

with distilled water if necessary.


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Reference ( Control): All the constituents of the reaction mixture as taken above

except the standard ( the molecule in study) are taken and in place of standard distilled

water for the same volume is added.

Unknown: Approximately diluted biological sample in which we have to analyze the

concentration of the biomolecule is termed as unknown.

Calculation: Estimation of the concentration of the molecule in study in the unknown

sample is carried by the following equation.

Concentration of Unknown ( mg / dL or m.mol/L) =

O.D of Unknown

X Concentration of the Standard (mg / dL / m.mol/L)

O.D of Standard

Result:

The concentration of t he molecule in study is expressed in mg/ dL or in m.mol/L.

Colorie metry & Spectrophotometry

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