Centrifugation Techniques Flashcards
Centrifugation
A process used to separate or concentrate material suspended in a liquid medium this method with separate molecules based on their sedimentation rate within a centrifugal field uses the centrifugal force to fact the sedimentation of molecules it is also used to measure the physical properties such as molecular weight shape and density
Type of centrifugation
Preparative of centrifugation which is used to separate one type of material from others
Analytical centrifugation used to measure the physical properties of macromolecules
Preparative and analytical procedures
Preparative procedures can be applied to the separation or purification of biological samples including cells organelles macromolecules Analytical procedures are used to measure the physical characteristics of biological samples the purity size shape and density of macromolecules may be defined by centrifugation
Basic principles of centrifugation
Particles suspended in a solution or pulled downward by the earth’s gravitational force in a solution particles whose mass and density is higher than the solvent sink or sediment while particles that are lighter fluid to the top the greater the difference in mass density the faster the particles sing this sedimentation movement is partially offset by the buoyancy (ability to float) of the particle because the earth’s gravitational field is weak a solution containing particles are very small muscles usually remains suspended due to random thermal motion however the sedimentation of these particles can be enhanced by applying centrifugal forces essential feature does the same thing it increases sedimentation by generating centrifugal force as great as one million times the force of gravity
Centrifugal force equation
If angular velocity is known F = mw^2r Intensity of force Mass of sedimenting particle Angular velocity or rotation in rad/sec Distance of migrating particles from central axis of rotation If velocity is known F= m X v^2/r
Important conclusions from thee equation
The sedimentation velocity of a particle is proportional to its mass
a dense particle moves more rapidly than a less dense one because the opposing buoyant force is smaller for dense particle
shape too is important because it affects the viscous drag the frictional coefficient of a particle is smaller than that of an extended particle of the same mass
The sedimentation velocity also depends on the density of the solution
Relative centrifugal force
Particles suspended in a fluid move under the influence of gravity towards the bottom of the vessel at a rate that depends in general on their size and density centrifugation utilizes force of gravity to increase the sedimentation rate of particles this is achieved by spinning the vessel containing the fluid and particles about an axis of rotation so that the particles expenses centrifugal force acting away from the axis the force measured in multiples of the earth’s gravitational force is known as the relative centrifugal field or more commonly the G-Force for example and rcf of 500g indicates that the centrifugal force applied is 500 times greater than the earth’s gravitational force the rsaf generated by a rotor depends on the speed of the rotor in revolutions per minute and the radius of rotation
RCF equation
RCF = 1.119 X10 ^-5 (rpm)^2 (r)
The force applied to the samples varies according to the size of the centrifuge and largest centrifuge will have a longer radius and a smaller centrifuge will have a shorter radius
Basis of seperation
Size-the size of the particle matters in the application of this principle it works on the basis that the smaller the size the more particles will be towards the base
Shape- the shape of the particle is important in that the X circular particles will settle down more easily than polygon shaped particles
Density- this refers to them mean issue at play in the centrifugation principal the denser than object the lower it will settle
Density gradient centrifugation
Used to separate macromolecules that differ only slightly in size but density two techniques are commonly used in zonal centrifugation the sample being separated example Excel extract the cells is placed on top of the central Asian solution as a thin layer during centrifugation the particles move through the solution due to their greater density the rate of movement depends on their molecular mass centrifugation stops before the particles reach the bottom of the tube drilling a hole into the centrifugation tube is a lying the contents to drip out makes it possible to collect the different particles and support functions during centrifugation the solution to be stabilized in the tube by density gradient is consist of solutions of carbohydrates or colloidal silica gel the concentration of which increases from the surface of the tube to the bottom density gradient prevent the formation of convection currents which would impair The separation of the particles .
Isopycnic centrifugation which takes much longer than zonal centrifugation start with CsCl solution in which the sample material is homogenous lead distributed identity created only form Jones gentrification because of the sedimentation and diffusion processes which particle moves to the region quit responding to its own by intensity certification stops one’s equilibrium is reached the sample obtained by fractionation and their concentration is measured using the appropriate methods
Moving boundary/ zone centrifugation
In moving boundary or differential centrifugation the entire tube is filled with the sample and centrifuge through centrifugation is separation of two particles or cures but any particle in the mixture may end up in the soup on no tent or in the palette or it may be distributed in both fractions depending on its side shaped density and the conditions of centrifugation the palate is a mixture of all the sedimented components and it is contaminated with whatever answer demented particles were initially at the bottom of the shoe the only component which is purified is the slowest sedimenting one but it’s healed is often very low the two fractions are recovered by decanting the supernatant solution from the pallet the supernatant can be centrifuged at high speed to obtain further purification with the formation of a new pellet and supernatant
Rate zonal centrifugation
Particles of the same size but different shape will separate the particle with a greater frictional coefficient will move slowly this technique is called velocity gradient centrifugation in red-zone of centrifugation the sample as applied in the things on at the top of the centrifuge tube on a density gradient under centrifugal force the particles begin sedimenting through the gradient in separate zones according to their size shape and density the run must be terminated before any of the separated particles reach the bottom of the tube
Isopycnic centrifugation
The density gradient column encompasses the whole range of densities of the sample articles the sample is uniformly mixed with a grade and material each particle sediment owners to the position in the centrifuge tube at which the gradient density is equals to its own density and their it remains the iso picnic technique therefore separate particles into separate zone solely on the basis of their density differences independent of time in Manet density-gradient experiments particles of both the great son of the isopycnic principal may enter the final separations for example the gravy and made by officers density range that one component sediments to its density in the tube and remains there were another sediments to the bottom of the through the cells generate ingredient technique often requires long hours of centrifugation
Safety measures
Work surfaces must be level and firm do not use the centrifuge on an uneven or slanted work surface
Balance the tube in the rotor if you want to run a tube with 10 ml of liquid insert another tube with 10 ml of water in the opposing hole on the rotor if the liquid has a higher or lower density than water you must balance the tubes by mass not volume
Do not open the lid while the rotor is moving even though many centrifuges have a safety shut-off if the lid isopen the only thing this does is to stop powering the rotor the rotor will keep spinning due to its own inertia until fiction slows and it eventually stops
If you see the centrifuge wobbling or shaking turn it off or pull the plug a little vibration is normal but excessive amounts can mean danger. first double check that you have correctly balanced the tubes if you have and the wobbling continues contact the manufacturer or dealer and have the unit serviced do not continue to run a centrifuge that wobbles visibly when the rotor is spinning Wear face shield and or safety goggles if you have to work anywhere near as centrifuge that is in use
Do not bump, jar or move the centrifuge while the rotor is spinning make sure you
do not have the cord dangling from the table edge where someone can catch their foot in it and pull down the centrifuge
Precautions working with bio harzadous materials
Examine whether the tubes and bottles are suitable for centrifugation discard tubes with cracks or stress marks
When working with bio hazardous material wipe the outside of the tubes with disinfectant prior to removing them from the biological safety cabinet and before placing them in the safety cups of rotor
Place all tubes in safety buckets or sealed rotor when centrifuging infectious material Inspect the o-ring seal of the safety bucket and the inside of safety buckets or rotors open safety buckets and rotors in a biological safety cabinet If any spills or Leakages are apparent the centrifuge rotor should be cleaned with a mild detergent rinsed thoroughly with distilled water and allowed to air dry completely while in the biosafety cabinet
Clean the rotor and centrifuge well after use
