Introduction of Centrifugation and Principle of Sedimentation

Centrifugation

Introduction of Centrifugation and Principle of Sedimentation

INTRODUCTION

Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium.

It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and isolated macromolecules such as proteins or nucleic acids.

The development of the first analytical ultracentrifuge by Svedberg in the late 1920s and the technical refinement of the preparative centrifugation technique by Claude and colleagues in the 1940s positioned centrifugation technology at the centre of biological and biomedical research for many decades.

Today, centrifugation techniques represent a critical tool for modern biochemistry and are employed in almost all invasive subcellular studies.

While analytical centrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies, preparative centrifugation methodology is devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest.

Most undergraduate students will be exposed to preparative centrifugation protocols during practical classes and might also experience a demonstration of analytical centrifugation techniques.

BASIC PRINCIPLE OF SEDIMENTATION

From everyday experience, the effect of sedimentation due to the influence of the Earth’s gravitational field (g=981 cm s-2) versus the increased rate of sedimentation in a centrifugal field (g>981 cm s–2) is apparent.

To give a simple but illustrative example, crude sand particles added to a bucket of water travel slowly to the bottom of the bucket by gravitation, but sediment much faster when the bucket is swung around in a circle.

Similarly, biological structures exhibit a drastic increase in sedimentation when they undergo acceleration in a centrifugal field.

The relative centrifugal field is usually expressed as a multiple of the acceleration due to gravity. Below is a short description of equations used in practical centrifugation classes.

When designing a centrifugation protocol, it is important to keep in mind that:

  • The more dense a biological structure is, the faster it sediments in a centrifugal field.
  • The more massive a biological particle is, the faster it moves in a centrifugal field.
  • The denser the biological buffer system is, the slower the particle will move in a centrifugal field.
  • The greater the frictional coefficient is, the slower a particle will move.
  • The greater the centrifugal force is, the faster the particle sediments.
  • The sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal.

Biological particles moving through a viscous medium experience a frictional drag, whereby the frictional force acts in the opposite direction to sedimentation and equals the velocity of the particle multiplied by the frictional coefficient.

The frictional coefficient depends on the size and shape of the biological particle. As the sample moves towards the bottom of a centrifuge tube in swing-out or fixed-angle rotors, its velocity will increase due to the increase in radial distance.

At the same time the particles also encounter a frictional drag that is proportional to their velocity. The frictional force of a
particle moving through a viscous fluid is the product of its velocity and its frictional coefficient, and acts in the opposite direction to sedimentation.

Introduction of Centrifugation and Principle of Sedimentation

 

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