Cell Fractionation by Centrifugation, Essay Example

Introduction

Centrifugation is a separation technique used to separate a mixture of components on the basis of difference in density and size. The technique involves two stages of separation .The first stage employs the terminal velocity difference of different particles and according to stokes law

V=2R (p-p1)a? (9µ) where R represents the radius of the particle, P is the particles  density, µ the  velocity of the medium,P1 the density of medium and a the centrifuges centrifugal acceleration and thus terminal velocity is a function of density of particle and its radius. This therefore means that larger and heavier particles move through the medium at a higher speed and settle at the bottom. The second stage utilises difference in sedimentation speed of organelles because a cell homogenate has organelles of different shapes sizes and density. The sedimentation rate is affected by force applied (proportionately) and also the property of the solution .In order to study cell organelles separation of fractions of sub cellular particles through centrifugation is essential especially density  gradient centrifugation which ensures purified cellular components are obtained. In such biochemical studies two centrifugal approaches are employed: isopycnic centrifugation and rate- zonal centrifugation. In rate zonal centrifugation the sample particles are separated by exploiting the difference in their sizes while in isopycnic centrifugation particles are separated on the basis of the difference in their densities; the separation gradient medium has a density range which incorporates densities of all sample particles and these particles sediment at a position in the medium where their density equates that of the gradient medium.

Materials and Methods

The following materials were used in this experiment: fresh spinach leaves ,miracloth, funnel, homogenizer, Beckman high speed centrifuge,12ml Beckman centrifuge tubes, 20 conical centrifuge tubes  ,20 test tubes ,a test-tube rack, spectronic 20 colorimeter, spectronic cuvette, phase contrast microscope, microscope slides and cover slips, organelle isolation medium made of 50Mm tricine at a P.H of 7.8,30mM sorbitol,0.1% bovine serum albumin,2mM EDTA, 95% ethanol and percolli in organelle isolation medium.

Percolli density gradient were prepared by pippeting 4 ml of 70% percoll into a 12ml Beckman centrifuge tube.  A layer of 6ml of 35% percoll was then put on top of the 70 % layer while taking care not to disturb it.100g 0f fresh spinach leaves was then chopped into 1×1 cm pieces and placed in 250ml of cold organelle isolation medium and then homogenized. The homogenate was then filtered through 1 layer of miracloth into a beaker that was chilled in ice. 2ml of the filtrate was gently layered on top of the gradient percoll using a 5ml pipette. The loaded gradient tube was then placed in the centrifuge and centrifugation done at 5,000 x g for 10minutes.After centrifugation 1ml fractions were then put into 12 labelled and punctured test tubes. one drop of selected gradient fraction was placed on the dark phase contrast microscope slide, a cover slip mounted and observed at 400x.further step to analyse chlorophyll was conducted in which 0.5 aliquots from each fraction tube was taken and  mixed with 4.5 ml ethanol  in conical centrifuge tubes then centrifuged at maximum speed for three minutes. blank made of 0.5 ml water and 4ml ethanol was  used to zero the 20 spectronic using an absorbance of 654nm and then absorbance the chlorophyll extract measured at 654 nm .Two graphs one showing chlorophyll absorbance and another showing the concentration of chlorophyll in each fraction were plotted.

Table of Results

Concentration of Chlorophyll in the Different Layers

There were two clear layers of supernatant and one pellet. This formed three clear layers. The top layer of the supernatant is labelled as layer 1 while the pellet is layer three.

Discussion

The speed and distance of migration of the various cell organelles on the centrifugation media is dependent on the size and the molecular weight of the particle in question. The chloroplasts therefore settled more at the bottom (pellet due to the speed at which they migrated under the influence of the centrifugal force. The organelles are larger as the concentration increases. In keeping with the beer lamberts law on absorbance, the organelles absorb more incident light when the concentration is high and decreases when the concentration reduces in a proportionate manner.

Conclusion

Most of the chloroplasts were concentrated at the bottom of the tube due to the migration as it was influenced by the centrifugal force. Different settling patterns are normally seen depending on the medium used for the experiment. The chloroplasts are organelles that are large in size. During extraction several the organelles suffered several breakages and hence different sizes were obtained. Spectrophotometers use the absorbance to measure the concentration of a substance in liquid medium. The higher the concentration, the more the absorbance and conversely the lesser the transmittance.

References

Hames B (2000). Biochemistry. Washington: BIOS Scientific Publishers Limited

Lenniger A. (2005). Principles of Biochemistry. New York: W H Freeman publishers

Stryer L. (2005). Biochemistry. New York: W H Freeman