CENTRIFUGATIONREPRESENTED TO: DR. IU KHAN

REPRESENTED BY: FARZEEN JAVAID 0188-BH-CHEM-11

COURSE TITLE: SEPARATION TECHNIQUES (COMPULSORY)

COURSE CODE: CHEM-4201

GOVT. COLLEGE UNIVERSITY LAHORE

HistoryEnglish military engineer Benjamin Robins (1707-1751) invented a whirling arm apparatus to determine drag.In 1864, Antonin Prandl invented the first dairy centrifuge in order to separate cream from milk.In 1879, Gustaf de Laval demonstrated the first continuous centrifugal separator, making its commercial application feasible.

Introduction

WHAT IS CENTRIFUGE?Centrifuge is device for separating particles from a solution according to there size, shape, density, viscosity of the medium.

WHAT IS CENTRIFUGATION?Centrifuge uses centrifugal force to separate phases of different densities.

DefinitionCentrifugation is a procedure that involves the use of centrifugal force for the sedimentation of mixture with a centrifuge used in industry and in laboratory settings. More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis.

Principle of centrifugationA particle whether it is a precipitate a macromolecule or a cell organelle is subjected to a centrifugal force when it is rotated at a high rate of speed. The centrifugal force F is denoted by equation F=mω2 rWhere

F= intensity of the centrifugal forcem= effective mass of the sedimenting particleω= angular velocity of rotation r= distance of the migrating particles from the central axis of

rotation A more common measurement of F in terms of the earths gravitation force , g, is relative centrifugal force, RCF its defined by RCF = 1.119 x 10-5 (rpm)2 (r)This equation relates RCF to revolutions per minute of the sample . Equation dictates that the RCF on a sample will vary with r, the distance of the sedimenting particles from the axis of rotation . The RCF value is reported as “ a number times gravity ,g .”

A centrifuge is a piece of equipment, generally driven by an electric motor, that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis to separate substances of different densities.Tubes in the centrifuge are tilted so centrifugal force can pull denser substances towards the bottom of the tube.

Relative Centrifugal Force (RCF) measures acceleration applied to the sample

RCF=(r*ω2)/g r=distance from center or motor to sampleω= angular velocity (radians/ second)g=9.8m/s2

General IdeaFeed added to spinning bowlSedimentation of particles occurs in centrifugal fieldFlow is upwards at a particular rate which determines residence time in deviceSeparation happens if sedimentation velocity is high enough for particle to reach side of bowl within residence timeLarge particles have higher settling velocities than small particlesBoth large and small are still particles, have small Reynolds no.s (<1) and obey Stokes’ Law

Components of the centrifugationOn and off control, operation time control (timer), rotation speed control, temperature control (in refrigerated centrifuges), vibration control (safety mechanism) and brake systemRefrigeration system (in refrigerated centrifuges).BaseLid/coverCasing Electric motorRotor.

There are different types of rotors. The most common are the fixed angle, the swinging buckets, the vertical tube and the almost vertical tube types.

Instrumentation of centrifugationThe basic centrifuge consists of two components one is Electric motor with drive shaft to spin the sample and a Rotor to hold tubes or other containers of the sample. A wide variety of centrifuges are available ranging from a low speed centrifuge used for routine pelleting of relatively heavy particles. There are three types of centrifuges

1. Low –speed centrifuges .Its also called as clinical centrifuge.The common centrifuge has a maximum speed in the range of 4000 to 5000 rpm with RCF value approx. up to 3000Xg. These instruments usually operate at low temperature with no means of temperature control of the samples.Two types of rotors fixed angle and swinging bucket may be used in this instruments.

Low speed centrifuges are especially useful for the rapid sedimentation of coarse precipitates or red blood cells.The sample is centrifuged until the particles are packed into a pellet at the bottom of the tube. The upper liquid portion, the supernatant, is then separated by decantation.

2. High Speed Centrifuges:High speed centrifuges for more sophisticated biochemical applications. Higher speeds and temperature control of the rotor chambers are essential.Rotor chambers in most instruments are maintained at or near 40 C. Three types of rotors are available for high speed centrifugation.

a. Fixed Angel rotor.b. Swinging –bucket rotorsc. vertical rotor

High speed centrifuges are used to sedimenta. Cell debris after cell homogenization.b. Ammonium sulfate precipitates of proteins and cellular organelles such

as chloroplasts, mitochondria and nuclei.

3. Ultracentrifuges:The most sophisticated of the centrifuges are the ultracentrifuges because of the high speeds attainable intense heat is generated in the rotor so the spin chamber must be refrigerated and placed under high vacuum to reduce friction.it is a high speed centrifuge that has fixed head rotors .It is mainly used in separation of lipoproteins .since the separation is long process there is generation of heat and thus are provided with internal cooling system.Ultracentrifuges can be used both for preparative work as well as for analytical. Preparative models its primarily used for separations and purification of samples for further analysis and analytical models which are designed for performing physical measurements on the sample during sedimentation.

Types of Centrifugationa) Preparative 1. Differential Centrifugation 2. Density gradient Centrifugation 2a. Rate-Zonal 2b. Isopycnicb) Analytical

a) Preparative CentrifugationIt is used to separate organelles and moleculesIt can handle larger liquid volumesno optical read-outSeparation methods used in preparative ultracentrifugation:

1. Differential Centrifugation- pelleting, 2. Density Gradient Centrifugation

1.Differential Centrifugationbased on the size of the particlesused for simple pelleting, for the separation of subcellular organelles and macromoleculesfirst, sample must be homogenisedultra centrifugationsedimentation depends on mass, shape and partial specific volume of a macromolecule, as well as solvent density, rotor size, rate of rotation.Usually uses a fixed angle rotor

2.Density Gradient Centrifugation

method to purify subcellular organelles and macromolecules.

density gradients generated by placing layer after layer of gradient mediaDensity gradient centrifugation classified into two:

2a. Rate-Zonal separation (size) 2b. Isopycnic Separation (Density)

2a.Rate- Zonal Centrifugationuse of continuous density gradient of solvent such as sucrose.density increases towards the bottom of the tubesample layered on the topmolecules form discrete bands after centrifugationseparation based on size of the moleculesSwinging bucket rotors

2b. Isopycnic Separationbased on the density of the moleculesMix gradient material with the sample molecule (CsCl)molecules move to the position where their density is same as the gradient material (isopycnic position)inorder to generate a gradient, we select a CsCl

concentration that will give a range of densities that includes the range of molecules that have to be separated.

used for the separation of DNASwinging bucket or fixed angle rotor

b) Analytical Centrifugation

uses small size samplesbuilt-in optical systemuses relatively pure sample

Types of CentrifugesTypes centrifuges depends upon

Maximum speed of sedimentationPresence /absence of vacuumTemperature control refrigeration.Volume of sample and capacity of centrifugation tubesDepending on the particular application, centrifuges differ in their overall design and size.a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated

1. Small BenchtopWith or without refrigeration

Slow speed (eg up to 4000 RPM)

Common in clinical lab (blood/plasma/serum

separation)can take approx (up to) 100 tubes, depending on diameter

2. Microcentrifuge (“microfuge”, “eppendorf”)

Sample volume is small in tubes Refrigerated with or withoutcan generate forces up to ~15,000 x gTake tube of small volume up to 2ml.Commonly used of concentration proteinvery common in biochemistry/molecular biology/biological labs

3. High Speed centrifuges (15,000 – 20,000 RPM)

Refrigerateduse for protein precipitates, large intact organelles cellular debris from tissue homogenisation and micoorganismThey operate maximal centrifugal force of approx 10000g Use for research applicationsdifferntial sepration of nucleus, mitrochondrial, protein precipitate,etc.

4. Ultracentrifuge (65,000 RPM)

Refrigerated and evacuatedThe detail biochmestry analysis of subcellular

structures and isolate biomolecules.

Operate at upto 90000 glimited lifetimeexpensiverequire special rotorscare in use – balance critical!research applications

5. Large-capacity preparative centrifuge

Centrifugal fields of 3000 to 7000g.Efficient separation of coarse precipitates or whole cells.

Types of rotorsFixed angleSwinging bucket rotors (Vertical/ Horizontal)

1. Fixed angle rotorThe rotor (mainly made of aluminium) is very compact.There are boreholes with a specific angle (like 45°) within the rotor. These boreholes are used for the sample tubes.

2. Swinging bucket rotors a. Vertical rotors

Vertical rotors Sample tubes are held in vertical position during rotationThis type of rotor is not suitable for pelleting applications but is most efficient for isopycnic (density) separations due to the short pathlength . Applications include plasmid DNA, RNA, and lipoprotein isolations.

b. horizontal rotorsThe rotor looks like a cross with bucket. Within these buckets, different tubes can be centrifuged. For a safe centrifugation, a specific adadpter for every tube shape is mandatory.

Centrifuge tubesCentrifuge tubes or centrifuge tips are tapered tubes of various sizes made of glass or plastic. They may vary in capacity from tens of mm, to much smaller capacities used in micro-centrifuges used extensively in molecular biology laboratories. The most commonly encountered tubes are of about the size and shape of a normal test tube (~ 10 cm long). Micro-centrifuges typically accommodate micro-centrifuge tubes with capacities from 250 μl to 2.0 ml These are exclusively made of plastic.

Glass centrifuge tubes can be used with most solvents, but tend to be more expensive. They can be cleaned like other laboratory glassware, and can be sterilized by autoclaving.

Plastic centrifuge tubes, especially micro-centrifuge tubes tend to be less expensive. Water is preferred when plastic centrifuge tubes are used. They are more difficult to clean thoroughly, and are usually inexpensive enough to be considered disposable

Three microcentrifuge tubes: 2 mL, 1.5 mL and 200 μL (for PCR).

Four screw-top micro-centrifuge tubes Four screw-top micro-centrifuge tubes.

Calculation of Centrifugal field

Centrifugal field from the center of rotation may be calculated using the equation G=ω2 r

Calculation of Angular velocityAngular velocity ω may be calculated by using this equation ω= 2 π rev min-1

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Applications of CentrifugationSeparate solids from solution Used during urinalysis to separate disease identifying componentsSeparate blood into plasma Purification of mammalian cellsFractionation of subcellular organelles (including membranes / membrane fractions)Fractionation of membrane vesicles

Application in Water Treatment

Commercial applications

• Centrifuges with a batch weight of up to 2,200 kg per charge are used in the sugar industry to separate the sugar crystals from the mother liquor.

• Standalone centrifuges for drying (hand-washed) clothes – usually with a water outlet.

• Large industrial centrifuges are also used in the oil industry to remove solids from the drilling fluid.

Preventive MaintenanceLubricate and clean motor.Clean case.Inspect power cords and plugs.Inspect controls and switches.Ensure appropriate menu settings for proper use.Ensure tightness of rotor.Check lights and indicators.Verify that alarms are operating properly. Ensure safety switch is functioning.If refrigerated, ensure temperature reading is working.Replace/repair gaskets, seals, and vacuum pump (if applicable).

Common Failures

Machine will not start.Power supply is faulty. Safety switch or timer not working properly.Motor not working properly.Motor making noise.Rotor is broken. Lid will not open or close.

Thank you!