centrifugation of suspensions



In cell and molecular biology, microbiology, medicine, biochemistry as well as other fields of science, centrifugation has been adopted as a vastly used technique in the separation of mixtures. In order to carry out diagnoses which depend on the separation of molecules, isolation of cells, the use of subcellular organelles, centrifugation has been thought to be priceless because these substances are required in high yield and it saves time. (Ford and Graham, 1991)

Centrifugation therefore presents a process whereby separation or even concentration of materials is done in a liquid source. The underlying principle of this technique is the effect gravity possesses on molecules present in suspension. In response to gravity, particles of different masses settle at different rates in the same tube. The centrifuge which is the device used to carry out centrifugation when switched on, possesses a centrifugal force that facilitates the settling of particles in a medium. This could be carried out on a continuous flow basis or on a batch.

Particles migrate radially from the axis of rotation when centrifugal forces are applied at particular revolutions per minutes or speed. The Relative Centrifugal Force or RCF in centrifugation is known to be the force exerted on the particles as compared to gravity. Various molecules e.g. viruses, membrane fractions, bacteria, nuclei, ribosomes, animal and plant cells all separate differently and at various speeds

There are various types of centrifugation to be adopted. Differential centrifugation is adopted based on the particle size. The most common type of this application is in basic pelleting and where somewhat not totally pure preparation of subcellular organelles. Density gradient centrifugation is used to purify macromolecules and also organelles. Here, one layer of gradient media is placed upon another in that sequence, with the lighter being on top of the heavier. The cell to be investigated is put on the top layer and spun in the centrifuge. This is most often classified into isopycnic or density and size or rate - zonal separation. (Rickwood and Graham, 2001)

The tubes to be are a very important aspect of centrifugation. Chemical compactibility of the sample to be investigated as well as the tube should be taken into consideration. Easy recovery of materials from tubes and tubes that prevent the sample from leaking out should be used.

To carry out centrifugation, the material to be separated is put in a tube, the tube is put in the rotor and balanced to avoid damage to the tube. The rotor usually has two, four to sixteen wells in which the tube is placed into. The rotor is a fast moving dense metal that gives off heat and the weight gives it great advantage in that it has great momentum. The lid is covered to protect and serves to protect the user from injury caused by a spinning rotor (Rickwood et al, 1994)

Laboratory centrifuges are of two major types; the small ones called microcentrifuges and larger ones called centrifuges. The difference is that they differ in capacity and speed.

Generally for particles to separate, factors such as viscosity, centrifugal acceleration, their shape and size, density in-between the liquid and the particle.(Rickwood and Graham, 2001) Because a lot of centrifuges did not possess the settings for RCF( relative centrifugal force) , a formula exists to convert RCF to RPM( Revolutions per minute) ;

g= (1.118 x 10-5) RS2

with S being the centrifuges speed (in RPM), R represents the rotor of the centrifuge radius (cm) and g is the RCF.


Reagents and Equipment

Unknown Suspension of Samples A and B, Centrifuge.


Into three different 1.5mL microcentrifuge Eppendorf tubes, 1000l of the unknown sample A was transferred. The Eppendorf tubes were then placed and balanced in the centrifuge. The centrifuge was set to 5000rpm and samples spun for one minute. The mixture was seen to separate into two distinct regions. The darker colour remained at the top while the green colour settled at the bottom. The equivalent of RCF was calculated in terms of g. By the use of careful and accurate pippetting, the supernatant was removed with the aid of a 200L pipette.

Again to two different microcentrifuges, 1500 L of Sample B was added and then spun in the well balanced centrifuge. This was done for one minute at 5000 RPM. The samples were further subjected to 10000 RPM for one minute. Records were noted and the supernatant discarded.


A lot of cells present in a liquid medium and even particles, when left to stand would separate on their own as a result of gravity but it consumes a lot of time waiting to use them when they have been separated. Some other particles which are small in size would not at all separate if not aided with a centrifuge where a very high centrifugal force is applied. Numerous applications for centrifugation exist which range from isolating macromolecules such as deoxyribonucleic acid, ribonucleic acid, lipids, proteins etc. Studies of these molecules can be used in the detection of diseases like cancer and also treatment.

g= (1.118 x 10-5) RS2

Where R is 5cm, S speed of centrifuge is 5000 RPM

g= (1.118 x 10-5) RS2

g= 1397.50

Where R is 5cm, S speed of centrifuge is 10000 RPM

g= (1.118 x 10-5) RS2

g= 5590.00

For incomplete centrifugation, solutions should be subjected to further spinning by increasing the rotation speed. The time could also be increased.

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