When a suspension of particles is centrifuged, the sedimentation rate of a particle is proportional to the force applied. The physical properties of the solution/medium will also affect the sedimentation rate. At a fixed centrifugal force and liquid viscosity, the sedimentation rate is proportional to the size of the particle and the difference between its density and the density of the surrounding medium.
In this practical, isopycnic density gradient centrifugation has been used, in which the medium employs a gradient to assist the separation. In isopycnic centrifugation, a density gradient which includes all the densities of the sample particles is used. Each particle will sediment through the gradient until it reaches an equilibrium position where the density of the particle is equal to the density of the surrounding medium. In this experiment, a mixture of coloured beads (which have different densities by colour) are placed in this density gradient and centrifuged. These beads separate into a series of coloured layers in the centrifuge tube, each layer corresponds to the density of that particular colour of bead, irrespective of size.
Method:
To pre-form the density gradient, a volume of 9.5 ml of a 60% solution of percoll was added to two polypropylene centrifuge tubes and balanced accurately. (Remember when balancing these tubes the weight of the adapters and tube caps must be taken into account). The tubes were capped and placed in the rotor of the centrifuge in diametrically opposing holes. The rotor was then spun for 45 minutes at a speed of 16,500 rpm. (For this rotor, RPT70T, this speed corresponds to a g-force (RCF) of approximately 21,000g).
To run the experiment:
- A volume of 100µl of density marker beads was gently layered on the top of the density gradient.
- The tube containing the density layer was placed in a bench centrifuge and spun at 2,000 rpm for 10 minutes. The tube was carefully removed and the distance each band of beads had moved from the meniscus was measured. The density of one of the layers or beads is unknown, and the aim of the experiment is to find the density of the 'Unknown' layer.