Centrifugation-notes

 CENTRIFUGATION-PRINCIPLE

• Sedimentation is basic principle of centrifugation.
• Sedimentation involves settling molecules due to gravitational force.

Centrifugal force:

 An object moves in circular motion at steady angular velocity where force direct object in outward direction is called centrifugal force. Centrifugation works on centrifugal force here axis of rotation is rotor and object is sample.

BASIC PRINCIPLES OF SEDIMENTATION

 

 RCF DERIVED

Spherical biomolecule-example- protein

If biomolecules are more viscous

SEDIMENTATION COEFFICIENT (OR) SWEDBERG UNIT (OR)S20 

Sedimentation coefficient is defined as sedimentation rate in per unit of centrifugal field . 

S20=v/ω2 r 

Sedimentation coefficient is defined in experimental value to the sedimentation constant at theoretical value in water at 20 degree celcius temperature.

INSTRUMENTATION OF CENTRIFUGE:

 The components of centrifuge were 

1.CENTRIFUGE TUBES 

2.ROTOR 

3.MOTOR

COMPONENTS OF CENTRIFUGE

1.Centrifuge tubes: 

• Centrifuge tubes have conical bottom . 
• It is helpful to hold sample to undergo centrifugation process. 

2.Rotors: 

• The centrifuge rotors are rotating unit with fixed holes with angles 
• It hold centrifuge tubes with sample for sedimentation. 

3.MOTOR : 

• The motor drives the rotor for starting centrifugation process for sedimentation of biological particles at particular centrifugal field.

TYPES OF ROTORS 

• Rotors are rotating unit with fixed angled holes. 
• It holds sample for sedimentation. 
• They are 4 types of rotors:

1.FIXED ANGLE ROTORS 

2.VERTICAL TUBE ROTORS 

3.SWINGING BUCKET ROTORS 

4.ZONAL ROTORS

1.FIXED ANGLE ROTORS:

Angle: Fixed angle rotors are the rotors arranged or inclined at 14 – 40 degree angle . 
Rotor speed : 1,00,000rpm 
Centrifuge tube capacity : The capacity of centrifuge tube is 0.2 -1 L 
Acceleration: 

• During acceleration of centrifugation process the sample biological particles move radially out and travel to short distance before strike slide down on wall and pellet out. After striking wall of test tube by biological particles cause sliding down of particles and make sedimentation quicker is called wall effect. 

DECCELERATION: 

• After deceleration causes wall effect and pelleting out of biological particles. 

ADVANTAGE: 

• Fixed angle rotors are used in isopycnic centrifugation and density gradient centrifugation technique. 

DISADVANTAGE : 

• This rotors lack to sediment organelles of cell due to lack of differential centrifugation activity

2.VERTICAL TUBE ROTORS

Angle 

TRUE 

• These rotors have centrifuge tube arranged at an angle of 90 degree that is parallel to axis of rotation. 

NEAR 

• These rotors have centrifuge tube arranged at an angle of 7 – 10 degree that is parallel to axis of rotation. 

ROTOR SPEED 50,000-65,000 rpm 

CAPACITY The capacity of centrifuge tube is 0.2-1 litre. 

ACCELERATION 

During acceleration of centrifugal process the sample biological particles move along diameter of tube. DECCELERATION 

• During decceleration of centrifuge tube causes reorientation of biological particles perpendicular to axis of rotation. Small amount of wall effect occurs. Shorter time and quicker sedimentaion due to centrifugal field takes place. 

ADVANTAGE 

• It helpful to pellet out DNA,RNA and lipoproteins. 

DISADVANTAGE 

• The pelleting may fallback to solution.

• 3.SWINGING BUCKET ROTORS :

ANGLE: 

• These rotors has bucket swing out in horiontal position during rotor acceleration. During acceleration buckets are perpendicular to axis of rotation. 

CAPACITY: 

• The maximum capacity of testubes equiped in swinging bucket rotors posess 12 litres. 

ROTOR SPEED: 4,000-5,200 rpm 

ACCELERATION 

• During acceleration tubes reorient perpendicular to axis of rotation and parallel to centrifugal field. 

DECCELERATION 

• When the rotor deccelerate the tube fall to original position that is regaining to original orientation. 

ADVANTAGE 

• These rotors are used for density gradient centrifugation . It has cushion effect and lacks wall effect. 

DISADVANTAGE 

• Lack pelleting out process . SWIRLING EFFECT takes which may mix the seperated components .Swirling effect may overcome by slowly accelerating and deccelerating the rotors.

4.ZONAL ROTORS

Zonal rotors are sector shaped. It has rotors of four equal quadrant. 

CAPACITY 

• The maximum capacity of sample holding sector must be 50 - 100 times more than that of swinging bucket rotors. 

ROTOR SPEED 5,000 – 60,000 rpm 

TYPES They are two types 

1.BATCH TYPE 

2.CONTINUOUS FLOW TYPE 

1.BATCH TYPE 

These rotors runs in 2 ways 

1.Low speed -5,000rpm 

2.High speed -60,000 rpm 

• Batch type rotors involves loading of gradients and samples and followed by running of rotors which lead to seperation of bological substances. 

2.CONTINUOUS TYPE ROTORS 

• These rotors has loaded gradients at rest by injecting to rotor and followed by running of tubes. Then rotors came to 2000 rpm followed by injection of sample biological components. Then rotor run to high speed to separate biological components. 

ADVANTAGE 

• It minimize wall effect. It has increased sample size. It helps to harvest cells and viruses. Removes contaminating proteins ,hormones and enzymes

TYPES OF CENTRIFUGES: 

The centrifuges based on their motor capacity classified as 4 types

Centrifuges are generally differs only on following 

1.Speed of sedimentation. 

2.Presence or absence of vaccum system. 

3.Presence or absence of refridgeration system. 

4.Capacity of centrifuge tubes.

1.DESKTOP CENTIFUGES: 

• It is also called as bench top or clinical centrifuges. 

RPM:3,000 rpm. 

• Lack temperature regulatory system. 
• The rotors must not load with odd tubes during acceleration of centrifuge. 

APPLICATION: 

1.Small capacity of sample needed for biochemical application. 

2.Less expensive. 

3.Used for RBC ,yeast and chemical samples. 

2.MICROFUGES: 

• It is used for clinical samples due to need of less samples. 

RPM: 10,000 rpm. 

• Modern microfuges uses refridgeration system to prevent denaturation of samples. Eppendroff tubes are used as centrifuge tubes.

 APPLICATION: It is used for need of less sample for clinical samples. 

3.HIGH SPEED CENTRIFUGES:

• It is used for bulk seperation of industrial biological samples. 

RPM: 1,00,000 rpm. 

Rotors: Zonal rotors. 

• It uses refridgeration system to maitain temperature at 0-4 degree celcius by thermocouple.It help in reduction of heat created by centrifuge due to high speed acceleration. 

APPLICATION: 

1.It help in sedimentation of protein,large intact organelle, cell debris, homogenisation of contractile proteins. 

4.ULTRA CENTRIFUGE: 

• It is used for isolation and purification of biological components. 

RPM: 9,00,000 rpm. 

• It is uses refridgeration system to maitain temperature at 0-4 degree celcius by thermocouple.
• It help in reduction of heat created by centrifuge due to high speed acceleration. 
• It has increased vaccum system of 1-2 µ which help to reduce friction. 
• It has heavy armorous encapsulated chamber. It has flexible drive shaft of 1/16 inches which made of aluminium and titanium.
•  It has speed control device. 

TYPES 

1.PREPARATIVE ULTACENTRIFUGE – 

• It involve in isolation of biological material. 

2.ANALYTICAL ULTRA CENTRIFUGE- 

• It involve in investigation of characteristics of biological compounds. 

APPLICATION 

1.It help to isolate DNA and RNA. 

2.It help to isolate virus.

TYPES OF CENTRIFUGATION [CENTRIFUGAL TECHNIQUES] 

Centrifugation is of two types

1.Preparative centrifugation: 

• It involves isolation of biological material for biochemical investigation. 
• Based on medium of suspension ,preparative centrifugation classified as 2 types: 1.DIFFERENTIAL CENTRIFUGATION. (HOMOGENOUS) 
• 2.DENSITY GRADIENT CENTRIFUGATION. 

2.ANALYTICAL CENTRIFUGATION: 

• It is helpful for characteristics finding of biological material 
• It is helpful in determination of sample purity, confirmational changes, molecular weight and sedimentation coefficient. 
• It needs vaccum system and refridgeration system. It operates at rpm of ultra centrifuges.

1.Preparative Centrifugation:

 A. Differential Centrifugation: 

• The mixture of homogeneous particle seperated by Centrifugation on the basis of density or size.
• Sedimentation of particle are at fixed centrifugal field. 

Technique: 

• We can vary centrifugal field applied for each intracellular organelle and time is required for each sediment completely at various centrifugal field .
•  Before centrifugation initiate all particle of homogenate are homogenously distribute through centrifuge tubes. 
• 1.It lead to sedimentation of particle and till form pellet at bottom of tube. 
• 2.The pellet not made of large particle. 
• 3.Repeated resuspension and recentrifugation pelleted out to form pur pellet of large particle.

DISADVANTAGE: 

• Every pellet washed several times.The pellet obtain not pure lead to contamination with other particle. 

STEPS INVOLVE IN DIFFERENTIAL CENTRIFUGATION: 

1.Homogenisation of specimen,mincing tissue in buffer system which has ph,salt,stabilizing cofactor and chelating agents. 

2.To this 10 % 0.25Mm sucrose and protease inhibitor cocktail was added to minimise proteolytic degradation.(protease inhibitor inhibits cystein protease,serine protease ,aspartate protease and amino peptidase) 

3.Then subcellular fractionation performed . 

SEPERATION OF DIFFERENT CELL COMPONENTS BY CENTRIFUGATION: 

• Centrifugation at 1000 rpm for 5 – 15 minutes lead to pellet out – nuclei and cytoskeleton.
• Centrifugation of about 15 minutes with 10,000rpm pellet out – mitochondria, lysosome.
• Centrifugation of about 50,000 rpm lead to pellet out ribosomes and viruses. 
• Centrifugation of about 1,00,000 rpm for1- 2 hrs lead to pellet out microsome. 
• The pellet and supernatent separate at each step end and recentrifuge to sediment lighter intracellular organelle.

APPLICATION: 

• It is used for cell organelle seperation. It is sedimented based on mass ,shape , particle specific volume of macromolecule. .
•  It is common in microbiology and cytology techniques. 

DISADVANTAGE: 

• Poor resolution. The preparation obtained is not pure.

DENSITY GRADIENT CENTRIFUGATION: 

Principle: 

•  This centrifugation involves as seperation based on density of particle. This Centrifugation has medium with different gradient. 

 INSTRUMENTATION: 

 GRADIENT: 

A good gradient must have following characteristics 

 1. Gradient shouldn’t affect bioactivity of sample.
 2.Gradient shouldn’t be corrosive to rotor. 

3. Gradient must be sterilisable. 

 4.Gradient must be cheap. 

 5.Gradient must be easily removable to form purified sample . 

 6.Gradient must not absorbs uv range. 

 7.Gradient must have increased water soluble

Examples of gradient: 

 1.Sucrose:

 It must have low cost and modest viscosity. 1.29gcm-3 particles are only seperated. 

Disadvantage: 

•  0.25M as low concentration of sucrose cause microbial growth. 
•  Osmotic effect can penetrate cell membrane. 

 2.CAESIUM CHLORIDE: 

•  1.91 g/cm3 of particles in sample can sediment. 
•  It is soluble in water. It has low viscosity. 
•  It is used for RNA and DNA seperation. 

 Disadvantage: 

•  It involves in osmotic effect. It has high cost. 
•  It can absorbs light. 

 3.FICOLL: 

•  It is polymer of sucrose and epichlorohydrin. 
•  It is a non ionic polymer 
•  It has low osmotic effect. 
•  It cause transfusion. 
•  It sediment particle at 418g / cm3. 

 Disadvantages: 

•  It must be highly viscous. 
•  It is costly. 
•  It can be contaminated easily. 

4.PERCOLL: 

•  It is polymer of pyrolisone coated colloidal silica. 
•  It has low viscosity. 
•  It is non toxic. It has 1 – 1.3 g /cm3 compound seperation. 

 PREPARATION OF DENSITY GRADIENT: 

 Density gradient preparation is done by 2 ways 

 1.DISCONTINUOUS. 

 2.CONTINUOUS. 

  1.DISCONTINUOUS DENSITY GRADIENT PREPARATION: 

• Density increases abruptly from one layer to another layer. 
• Layering of solution has continuously decreasing it’s densities. High density solution is at bottom of centrifuge. 
•  Sample was layered at top.Then centrifuged. 
•  It is useful seperation of whole cell and subcellular organism. 
•  It is helpful in seperation of lipoprotein. 

 2.CONTINUOUS DENSITY GRADIENT PREPARATION: 

•  The density decreases from bottom to meniscus. 
• By standing discontinuous layer for long time continuous layer prepared. 
•  A device is used to prepare media of continuous nature
• A device consists of 2 chambers. 

 First chamber:

• It drips to centrifuge tube which has dense solution.

 Second chamber: 

•  It has dense solution. 
•  The tap has first chamber open dense solution to drip to centrifuge tube. 
•  The level of solution in first chamber is compensed by flow of low dense solution of 2 nd chamber to first. The stirring device used to stir the solution of 2 different density.
• Thus first chamber has less dense solution. The solution drip to tube has decreasing density . It is used to separate proteins, ribosomes and enzymes. 

 SAMPLE APPLICATION: 

•  The volume of sample applied depends on cross sectional area of gradient exposed to sample.
• The diameter of centrifuge tube is 2.5 cm will has 1 / cm3 tube height. 
•  Lesser volume of sample not affect resolution difficult to detect after seperation. The sample concentration is less as 10 times than concentration of starting gradient . 
• The sample applied through syringe of 2 -3 mm above gradient inclined at 45 degree angle to wall of tube. For fragile DNA broad mouth pippetes are used

 RECOVERY OF SAMPLES: 

 The recovery of samples takes place by 2 methods 

 1.PUNCTURING TECHNIQUE. 

 2.DISPLACEMENT TECHNIQUE. 

 1.PUNCTURING TECHNIQUE: 

•  Puncturing centrifuge tube are at bottom with needle. 
• Collect dropping medium in separate tubes in fractions of equal volume. 
•  A peristaltic pump used to suck off medium from hole at bottom

2.DISPLACEMENT TECHNIQUE 

• There was a cover without outlet at top of gradient. 
•  Another hole in cover of tube is passed to bottom of tube. 
•  The very dense solution than media pump through this tube. 
•  The dense solution reaches bottom displaced gradient layer from cover and collected in tube

TYPES OF DENSITY GRADIENT CENTRIFUGATION: 

The density gradient centrifugation is of 2 types 

 1.RATE ZONAL CENTRIFUGATION. 

 2.ISOPYCNIC CENTRIFUGATION. 

 1.RATE ZONAL CENTRIFUGATION 

•  The gradient used here has density has less than least dense solution in given sample. 
•  Layering has continuous density gradient. 
•  Centrifugation has low speed at short time. 
•  The sample forms discrete zone based on sedimentation rate. 

Choice of rotors: 

•  It has swinging bucket rotors. 
•  Swinging bucket rotors has longer wavelength and decreased wall effect. 

 Advantage: 

 1.Used to separate particle differ in size but not density . 

 2.Seperate protein differ in molecular weight. 3.Seperation based on RNA – DNA hybrid and ribosomal subunits. 

 DISADVANTAGE: 

• No seperation of organelle like mitochondria, lysosome and peroxisome. 
• It has different density but similar size

2.ISOPYCNIC CENTRIFUGATION: 

•  The medium is with different density will travel sample and separate sample at density of sample and medium became same density . 
•  The gradient density is maximum than most denser centrifugation of sample. 
•  Centrifugation sample at RCF which sediment particle heavier than one desired. 
•  The supernatent layered where density of medium both are same. 
•  The sample move to middle of coloumn after Centrifugation. 

Steps involved: 

 1.Continuous density gradient. 

 2.Sample layered. 

 3.Centrifugation sediment the particle till encounter the gradient greater than density. 

 4.Particles forms zones. 

 Choice of rotors: Fixed angle rotors ,verticle rotors are used . 

 Sample became immobile at or near buoyent density. 

 APPLICATION: 

 1.Seperation based on size. 

 2.Used for subcellular and intracellular organelle seperation. 

3.Used for nucleic acid fractionation

Analytical ultra centrifugation: 

 Principle: 

•  It involvein identification of characteristics of sample. 
•  It operates at high speed . 
•  It has refridgerated and evacuated systems. 

 Instrumentation: 

 1.ROTOR: 

•  It is elliptical in shape. 
•  It has 2 holes holding the cells. 
•  The rotor holds cell vertically in rest or rotating. 

1.ANALYTICAL CELL. 

 2.COUNTER POISE CELL.

1.ANALYTICAL CELL: 

•  It is sector shaped. 
•  It has sample ,holds at 14 mm height and capacity of 1 cm3 of sample.

 2.COUNTERPOISE CELL : 

•  It is involve in balancing analytical cell. 

 3. Window: 

•  The upper and lower planes of analytical cell are transparent with quartz and synthesized sapphire windows. 

4.EVACUATED CHAMBER : 

•  It is produced at 1 -2µ m vaccum. 
•  It involve in reducing friction in sample. 

 5.OPTICAL SYSTEM:

• It involve in Rayleigh interference process with schlieren optical system. 
•  It has passage of light to pass to monitor the progress of centrifugation. 
•  It has xenon flash lamp as source with 230 nm. 
•  The result noticed at beginning of centrifugation at peak and near meniscus

6.REFRIDGERATION: 

• It maintains heat at 0 -4 degree celcius. 
•  It reduces heat created by chamber motor by high speed rpm. 

 7.MOTOR : 

•  The motor has rpm of 2,50,000rpm. 

 8.ROTOR SHAFT: 

•  It is calibrated distance from centre of rotation. 
•  It is of aluminium and titanium. 
•  The shaft is 1/16 inches distance. 

9.CALBRATED GRAPH: 

• The progress moves down the peak of graph shows characteristics of particle. 
•  The optical information photographed

Advantages 

•  It measures sedimentation coefficient. 
•  To know molecular weight of macromolecule. 
•  To know isolated protein ,nucleic acid for purity measurement. 
•  To characteristic change in size ,shape of macromolecule. 
•  Determination of purity of macromolecule. 
•  To detect conformational changes. 
•  Small and larger contaminants recognised as shoulder on main peak or assymetry

Determination of molecular weight : 

• Molecular weight determined from smaller sample size of 20 -120 nm and larger sample concentration of 0.001 – 1 g/dm. 
•  They are 3 methods performed. 
•  1.SEDIMENTATION VELOCITY OR BOUNDARY SEDIMENTATION. 
•  2.SEDIMENTATION EQUILIBRIUM OR BAND SEDIMENTATION. 
•  3.EQUILIBRIUM. 

1.SEDIMENTATION VELOCITY OR BOUNDARY SEDIMENTATION: 

• Speed of moving boundary ultracentrifuge operated randomly distributed particle migrate to solvent radially from centre. 
•  This form velocity boundary between solvent cleared of macromolecule and portion of solvent has macromolecule by optics

2.SEDIMENTATION EQUILIBRIUM OR BAND SEDIMENTATION: 

• The method involves at high speed balance tendency of macromolecule to disperse in opposite directions due to diffusion. 
•  The Centrifugation done till the balance between sedimentation and diffusion of macromolecule has no net movement occurs.
•  It takes long time. 
•  But by using short analytical cell and overspeed techniques time short to few hrs. It is popular than sedimentation velocity

3.EQUILIBRIUM : 

•  It is done all time without Centrifugation. 
•  Based on two places where flow of solute is zero.
• The places are meniscus of solution and bottom of cell . 
•  Because matter not push to either extriemeties is called Archibald method. 

 Disadvantage: 

 1.Lacks of column precise measurements. 

 2.Less accurate

Thank you

References

1.Biophysical chemistry Principles and techniques -Upadhya and Nath

2. Principles and Techniques of Biochemistry and Molecular Biology- Wilson and Walker