BS2011 Practical & Employability Skills

Density Gradient Centrifugation

Introduction

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,isopycnicdensity gradient centrifugation has been used, in which the medium employs a gradient to assist the separation. In isopycniccentrifugation, 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 layersin 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 removedand 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.

What you are required to do

Please note carefully below what needs to go into your lab notebook or into UELPlus [as a checklist, the steps required have been underlined below]

  1. In your lab notebook, do a short write-up of the experiment as if you had carried it out in the lab. You also need to do various calculationsas listed below: write these calculations into your notebook, which should also contain either a drawn graph, or else a printout of a graph done in Excel (see below). You also need to input some results from the experiment into UEL Plus (see below).
  2. There is a Word file (centrifugation practical data.doc) on UEL Plus. This contains the results from the experiment,giving the distance in cm that each band of beads has moved from the meniscus.
  3. Calculate the mean and standard deviation for the distance moved (in cm) for each of the different coloured beads in turn and also for the ‘Unknown’. This should give you 8 sets of means and standard deviations.
  4. You are also given the total distance in cm in the data file; calculate the Rf ratio for each band of beads using the mean distance travelledfor each band of beads. Calculate the Rf ratio for each coloured band using the following formula: (mean distance migrated by the coloured band/total distance).
  5. Plot a graph of the Rf ratio (mean distancemigrated by the coloured band/total distance) for each coloured band against the density of that coloured band. You can either draw the graph directly into your lab notebookand draw the line of best fit through the points, or if you prefer to use Excel to plot the graph, you must then print out a copy of the Excel graphand include the line of best fit of the points and fix it into your lab notebook.
  6. Use the graph to determine and record in your notebook the density of the unknown (the orange band) from the graph. Make sure you show on your graph how you have estimated the density of the ‘Unknown’.
  7. ON UEL PLUS follow the instructions for the data which you should enter

DENSITIES OF COLOURED BANDS

These are the densities of the various coloured bands in gm/cm3, which you will need in making the graph. The density should be plotted on the graph as the horizontal (x) axis.

BANDDENSITY

Yellow 1.033

Green 1.051

Red 1.063

Orange 1.086

Brown 1.098

Pink 1.119

Violet 1.138

BlueUnknown

Resources:

The series of books entitled ‘Practical Skills in …’ (‘…Biomolecular Sciences, .. Forensic Science’ etc) contain short sections on drawing graphs (including calibration graphs) – such graphs were included in the first year module BS1000.

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