Colorimetry:

• Photometry broadly deals with the study of the phenomenon of light absorption by molecules in
solution.
• The specificity of a compound to absorb light at a particular wavelength (monochromatic light) is
exploited in the laboratory for quantitative measurements.
• From the biochemist’s perspective, photometry forms an important laboratory tool for accurate
estimation of a wide variety of compounds in biological samples.
• Colorimeter and spectrophotometer are the laboratory instruments used for this purpose.
• They work on the principles discussed below.
• When a light at a particular wavelength is passed through a solution (incident light), some
amount of it is absorbed.
• Therefore, the light that comes out (transmitted light) is diminished.
• The nature of light absorption in a solution is governed by Beer-Lambert law.
• Beer’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 molecules).
• Or,
• When a parallel beam of monochromatic light passed through a solution, the absorbance (A) of
the solution is directly proportional to the concentration (c) of the substance in the solution.
• i.e. A ∝ c
• According to Lambert’s law, the transmitted light decreases exponentially with increase in the
thickness of the absorbing molecules (i.e. the amount of light absorbed is dependent on the
thickness of the medium).
• Or, the absorbance (A) of the solution is directly proportional to the path length of light (t) which
is the thickness of the solution.
• i.e. A ∝ t

Combination of these 2 laws (Beer-Lambert Law) gives 2 equations:

1) A=εct and
2) I/I0 = e-Kct = 10 –εct

(see next slide)

• By combining the two laws (Beer-Lambert law), the following mathematical
derivation can be obtained:
• I/I0 = e-Kct = 10 -εct
Where,
• I = Intensity of the transmitted light
• I0 = Intensity of the incident light
• K= Molar co-efficient or proportionality constant of the absorbing solution (characteristic of the
substance being investigated)
• c = Concentration of the absorbing substance (moles/l or g/dl)
• t = Thickness of medium through which light passes.
• ε=Molar extinction co-efficient of the absorbing solution = K/2.303 (2.303 is the factor when
converting log e to log 10)
• e (Euler’s number)= the base of natural logarithm (=2.718)

• When the thickness of the absorbing medium is kept constant (i.e. Lambert’s law), the
intensity of the transmitted light depends only on concentration of the absorbing
material. In other words, only the Beer’s law is operative.
• In chemistry, the molar absorption coefficient or molar attenuation coefficient (ε) is a measurement of how
strongly a chemical species absorbs, and thereby attenuates, light at a given wavelength.
• It is an intrinsic property of the species.
• The SI unit of molar absorption coefficient is the square metre per mole (m2/mol)
• But in practice, quantities are usually expressed in terms of M−1⋅cm−1 or L⋅mol−1⋅cm−1 (the latter two units are
both equal to 0.1 m2/mol).
• In older literature, the cm2/mol is sometimes used; 1 M−1⋅cm−1 equals 1000 cm2/mol.

• The molar absorption coefficient is also known as the molar extinction coefficient and molar absorptivity

• ε=Molar extinction co-efficient or molar absorption coefficient or molar absorptivity of the absorbing
solution =
• The ratio of transmitted light (I) to that of incident light (I0) is referred to as
transmittance (T).
• T= I/ I0 or T% = (I/I0 ) x 100
• Absorbance (A) or optical density (OD) is very commonly used in laboratories.
• The relation between absorbance and transmittance is expressed by the following equation.
• Absorbance is the negative logarithm of transmittance to the base ten (10).
• A= -log10 (T)= log10 (1/T) = log 10 (100/T%) =2-log T%
• A colorimeter is an essential laboratory instrument used to measure the concentration of colored
compounds in a solution, based on absorbance of light. It's widely used in clinical biochemistry
for estimating blood glucose, urea, creatinine, proteins, etc.
• Components of a Colorimeter:
• Light source – Usually tungsten lamp (visible range)
• Filter – Selects specific wavelength (complementary to colour of solution)
• Cuvette – Holds the sample (glass or plastic)
• Photodetector – Detects transmitted light
• Display – Shows absorbance or optical density
Procedure (Simplified):
• Prepare blank (B), standard (S), and test (T) solutions.
• Measure absorbance of blank, standard and test.
• Calculate concentration of unknown using:
• Concentration of test=(AT/ AS​​)×Concentration of standard
• i.e. (OD T-ODB/ODS-ODB) x (Amount of Standard/Volume if sample) X 100
(unit= mg/dl for e.g. Plasma glucose or serum creatinine)
Uses of colorimetere in Biochemistry:

Uses of colorimetere in Biochemistry:

Tests Principle
--------------------- ---------------------------------
Glucose estimation : Glucose oxidase-peroxidase method
Urea estimation : Diacetyl monoxime reaction
Total protein : Biuret method
Creatinine estimation: Jaffe’s reaction
Adv & Dis adv of Colorimeter:
Advantages:
• Simple and low-cost
• Quick results
• Accurate for colored compounds

Limitations:
• Only works for colored solutions
• Less sensitive than spectrophotometers
• Prone to interference from turbidity
• Colorimeter is limited to visible light only with wavelength of 400-700 nm.
Notes:
1) Single beam spectrophotometer:
• It operates between 325 nm to 1000 nm wavelength using the single beam of light.
• The light travels in one direction and the test solution and blank are read in the same.

2) Double beam spectrophotometer:

• It operates between 185 nm to 1000 nm wavelength.
• It has two photocells.
• This instrument splits the light from the Monochromator into two beams.
• One beam is used for reference and the other for sample reading. It eliminates the error which
occurs due to fluctuations in the light output and the sensitivity of the detector
Notes:

1) Infrared (IR) radiation spans from 700 nanometers (nm) to 1 millimeter (mm) in the electromagnetic spectrum.
• This corresponds to a frequency range of roughly 300 gigahertz to 400 terahertz.
• Infrared radiation is often categorized into near-, mid-, and far-infrared, with the boundaries between these sub-
bands not always precisely defined.

2) Ultraviolet (UV) radiation falls within the wavelength range of 100 to 400 nanometers (nm) on the
electromagnetic spectrum.
• This range is shorter than visible light but longer than X-rays.
• UV radiation is further divided into three bands: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm).

3) X-rays typically have a wavelength range of 0.01 to 10 nanometers (nm).