Centrifugation Basics

By: Mark Frei, BioFiles v6 n5, 4–5

BioFiles Volume 6, Number 5 — Centrifugation

Download BioFiles v6 n5 (4.66 Mb PDF)

The earth's gravitational force is sufficient to separate many types of particles over time. A tube of anticoagulated whole blood left standing on a bench top will eventually separate into plasma, red blood cell and white blood cell fractions. However, the length of time required precludes this manner of separation for most applications. In practice, centrifugal force is necessary to separate most particles. In addition, the potential degradation of biological compounds during prolonged storage means faster separation techniques are needed.

The rate of separation in a suspension of particles by way of gravitational force mainly depends on the particle size and density. Particles of higher density or larger size typically travel at a faster rate and at some point will be separated from particles less dense or smaller. This sedimentation of particles, including cells, can be explained by the Stokes equation, which describes the movement of a sphere in a gravitational field.1 The equation calculates the velocity of sedimentation utilizing five parameters (see Figure 1).

The Stokes equation

Figure 1. The Stokes equation.

From the Stokes equation five important behaviors of particles can be explained:

  1. The rate of particle sedimentation is proportional to the particle size.
  2. The sedimentation rate is proportional to the difference in density between the particle and the medium.
  3. The sedimentation rate is zero when the particle density is the same as the medium density.
  4. The sedimentation rate decreases as the medium viscosity increases.
  5. The sedimentation rate increases as the gravitational force increases.

Most particles are so small that gravitational force is insufficient to overcome the random molecular forces of particles to influence separation. Centrifugation, the name given to separation applications which involve spinning around an axis to produce a centrifugal force, is a way to increase the magnitude of the gravitational field. The particles in suspension experience a radial centrifugal force moving them away from the axis of rotation.2 The radial force generated by the spinning rotor is expressed relative to the earth's gravitational force and therefore is known as the relative centrifugal force (RCF) or the "g force." The g force acting on particles is exponential to the speed of rotation (defined as revolutions per minute; rpm). Doubling the speed of rotation increases the centrifugal force by a factor of four. The centrifugal force also increases with the distance from the axis of rotation. These two parameters are of considerable significance when selecting the appropriate centrifuge. Table 1 summarizes the applications that can be classified by the relative centrifugal force.3

Table 1. Classes of centrifuges and their applications.

Parameters Low speed High speed Ultracentrifuge
Speed ranges (r.p.m. x 103) 2–6 18–22 35–120
Maximum RCF (x 103) 8 60 700
Pelleting Applications      
   Bacteria Yes Yes*
   Animal and plant cells Yes Yes Yes*
   Nuclei Yes Yes Yes*
   Precipitates Some Most Yes*
   Membrane organelles Some Yes Yes
   Membrane fractions Some Some Yes
   Ribosomes/polysomes Yes
   Macromolecules Yes
   Viruses Most Yes

* Can be done but not usually used for this purpose.

RCF is dependent on the speed of rotation in rpm and the distance of the particles from the center of rotation. When the speed of rotation is given in rpm (Q) and the distance (r) is expressed in centimeters, RCF can be calculated by using the formula in Figure 2.

RCF Formula

Figure 2. Formula for relative centrifugal force (RCF).

A nomogram can also be used to obtain the speed of a centrifuge rotor necessary for a desired RCF (see Figure 3). This quick estimate is useful for low speed centrifugation applications. However, it is more accurate to use the RCF calculation for speeds in excess of 10,000 rpm.

Nomogram for estimation of centrifuge rpm.

Figure 3. Nomogram for estimation of centrifuge rpm setting.

Nomogram instructions

  1. Measure the radius (cm) from the center of the centrifuge rotor to the end of test
    tube carrier.
  2. Obtain the relative centrifugal force necessary for the application.
  3. A straight line connecting the value of the radius with the relative centrifugal force (g) value will enable the speed of the rotor (rpm) to be read off of the right column.

References

  1. Laboratory Techniques in Biochemistry and Molecular Biology, P.T. Sharpe, p.18.
  2. Biological Centrifugation, J. Graham, p. 3.
  3. Centrifugation, Essential Data, D. Rickwood, p.12.

 

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