Density Gradient Centrifugation


22 févr. 2014 (il y a 3 années et 1 mois)

74 vue(s)

In your lab notebook,

short write

of the experiment as if you had carried it
out in the lab
. I think the highlighted yellow text is
the short write up I have to do
so I have to write that in my own words.

Density Gradient Centrifugation


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
between its density and the density of the surrounding medium.

In this practical,
isopycnic density gradient centrifugation

has be
en used, in which the
medium employs a gradient to assist the separation. In

centrifugation, a density
gradient which includes all the densities of the sample particles is used. Each
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.


To pre
form the density gradient, a

volume of 9.5 ml of a 60% solution of perco
ll 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
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 menisc
us was measured. The
density of one of the layers or beads is unknown, and the
aim of the

is to find the density of the ‘Unknown’ layer.