Principles of Centrifugation

 CENTRIFUGATION

• Centrifugation is a separation process which uses the action of centrifugal force (a force which acts on a body moving in a circular path and is directed towards the centre around which the body is moving) to promote accelerated settling of particles in a solid-liquid mixture.
• Centrifugation is the process where a mixture is separated through spinning. During Centrifugation, the solid particles were separated from the liquid particles.
• In 1864, Antonin Prandl invented the first dairy centrifuge in order to separate cream from milk.
• In 1879, Gustaf de Laval demonstrated the first continuous centrifugal separator, making its commercial application feasible.

CENTRIFUGE

• The Centrifugation process was carried out in conical tubes (centrifuge tubes) with the help of an instrument named “Centrifuge”.
• The centrifuge separates the particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
• A centrifuge is used to separate the particles or macromolecules from Cells, Cellular components, Proteins and Nucleic acids.

RELATIVE CENTRIFUGAL FORCE (RCF)

• The Relative Centrifugal Force (RCF) is the product of rotational radius (r) and the square of angular velocity (ω), and the gravitational acceleration relative (g).

Relative Centrifugal Force (RCF) = r ω²/g

where

      r – rotational radius;

ω – angular velocity;

g – gravitational acceleration 

REVOLUTIONS PER MINUTES (rpm)

• The speed of the rotor during centrifugation was expressed as Revolutions per minute (rpm).
• The Revolutions per minute (rpm) is a measure of the frequency of rotation, specifically the number of rotations around a fixed axis in one minute.

SEDIMENTATION RATE (ᴠ)

• The centrifuge works under the principle of Sedimentation (the process of depositing sediment).
• Sedimentation is the deposition of particles suspended in a liquid medium to the bottom of the container, according to their density and size.
• In centrifugation process, the particles will sediment progressively with time towards the bottom of the sample tube.
• The sedimentation rate of the particles depends on (a) Density of the particles (⍴_p), (b) Radius of the particle (r_p), (c) Density of the medium (⍴_m) and (d) Viscosity (η).
• The Sedimentation rate of the spherical particle can be calculated by

ᴠ = 2/9 × r²_p (⍴_p - ⍴_m)/η × g

where

        ᴠ  - Rate of sedimentation

        2/9 -  Shape factor constant for spherical particles

        r_p  - Radius of the particle

        ⍴_p  - Density of the particle

        ⍴_m - Density of the medium

        η - Viscosity

        g - Gravitational field

 SVEDBERG UNIT (or) SEDIMENTATION COEFFICIENT

• The Sedimentation rate (v) of the particle can be expressed in terms of its sedimentation rate per unit of centrifugal field. It is commonly referred as “Sedimentation coefficient” or Svedberg Unit (S or Sv).
• Svedberg Unit (S or Sv) is a non-SI unit (International system of units) for sedimentation rate.
• The basic unit of Sedimentation coefficient is 1 × 10^-13 sec.
• The Svedberg Unit (S or Sv) is named after the Swedish Chemist Theodor Svedberg (1884 – 1971), winner of the 1926 Nobel Prize in Chemistry for his work on disperse systems, colloids and his invention of the ultracentrifuge.
• Svedberg unit for Viruses are 40 to 1000 S, for Lysosomes 4000 S, for Bacteria 70 S, for Fungi 80 S and Mitochondria 20 × 10³ S to 70 × 10³ S.
• The equation for standard Sedimentation coefficient (S_{20 w}) is

Sedimentation coefficient (S_{20 w}) = S_obs (1 - ῡ⍴_{20 w}/1 - ῡ⍴_T) × η_T/ η₂₀ × η/ η₀

where

         S_{20 w}  - Standard sedimentation coefficient

        S_obs - Experimentally measured sedimentation coefficient

        η_T/ η₂₀ - Relative viscosity of water at temperature compared with that at 20 °C

        η/ η₀ - Relative viscosity of the solvent to that of water

        ⍴_{20 w} - Density of water at 20 °C

        ⍴_T  - Density of the solvent at temperature (°C)

        ῡ  - Partial specific volume of the solute