Washing microparticles may be done via centrifugation. This procedure must be performed carefully. Excess centrifugation will result in resuspension difficulties. For the purposes of pelletizing, it is important to understand the settling velocities of particles. For spherical particles, settling velocity can be calculated using Stokes’ Law:
V = 2ga2 (p1 - p2) / 9n
- V = Velocity in cm/sec
- g = g force in cm/sec2
- p1 = density of particle in g/cm3
- p2 = density of suspending media in g/cm3
- n = coefficient of viscosity in poises (g/cm-sec)
- a = radius of spherical particle in cm
For calculating the settling velocity of polystyrene spheres at 1 G in 20 oC water, Stokes Law can be expressed in the following formula, where d = diameter in micron, p1 = 1.05 g/cm3, ir2 = 1.00g/cm3 and n = 1.002 cp.
V= 2.77 x 10-6d2
To estimate appropriate times for centrifugation, settling velocity is multiplied by the G forces generated by the centrifuge.The resultant velocity is then compared to the height of the centrifuge tube. For example: A 1.0m particle placed in a microcentrifuge generating 10,000 G will settle at a velocity of 2.77 x 10-2 cm/sec. Pelletizing the particle in a 4 cm high tube would require a 144 second (minimum) centrifuge run. The actual time required to form an acceptable pellet could possibly be 50% longer. These calculations are intended to be used as guidelines to assist in determining centrifugation time. Different size particles yield dramatically different settling velocities. A 10.0m particle could settle in 2 seconds under the conditions mentioned earlier, whereas a 0.01m particle could take at least 4 hours to settle. Brownian motion and particle concentration also affect settling rate.
