1. A centrifuge is a device that separates particles from a solution through use of a rotor. In biology, the particles are usually cells, subcellular organelles, or large molecules, all of which are referred to here as particles. Centrifugation

2. A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In a solution, particles whose density is higher than that of the solvent sink (sediment), and particles that are lighter than it float to the top. The greater the difference in density, the faster they move. If there is no difference in density (isopycnic conditions), the particles stay steady. Centrifugation

3. A centrifuge is used to separate particles or macromolecules: -Cells -Sub-cellular components -Proteins-Proteins -Nucleic acids Basis of separation: -Size -Shape -Density Methodology: Utilizes density difference between the particles/macromolecules and the medium in which these are dispersed. Dispersed systems are subjected to artificially induced gravitational fields.

4. Representative example of preparation of granulocytes by a one-step double density centrifugation method. left-hand panel, a whole blood sample diluted 1 ∶ 3 in PBS is layered on a double density Ficoll gradient: poly (density 1113 g/L) and H (density 1077 g/L). Right-hand panel, after centrifugation (500 g for 35 minutes at 22°C), erythrocytes (RBC), granulocytes (PMN), mononuclear cells (MNC) and plasma fractions are clearly separated.

5. BASIC PRINCIPLES OF SEDIMENTATION the effect of sedimentation due to the influence of the Earth’s gravitational field (g¼981 cm s–2) versus the increased rate of sedimentation in a centrifugal field (g>981 cm s–2) is apparent. To give a simple but illustrative example, crude sand particles added to a bucket of water travel slowly to the bottom of the bucket by gravitation, but sediment much faster when the bucket is swung around in a circle. Similarly, biological structures exhibit a drastic increase in sedimentation when they undergo acceleration in a centrifugal field.

6. When designing a centrifugation protocol, it is important to keep in mind that More dense biological structure is, faster it sediments in centrifugal field More massive a biological particle is, the faster it moves in a centrifugal field; Denser the biological buffer system is, the slower the particle will move in a centrifugal field; Greater the frictional coefficient is, the slower a particle will move; Greater the centrifugal force is, the faster the particle sediments; Sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal.

7. Rate of Sedimentation dependent upon applied centrifugal field(cms-2) G, 1.Determined by radial distance (r) of particle from axis of rotation (in cm) 2.sqaure of angular velocity ( ω ) of rotor G=rω2

8. Calculation of centrifugal field: Angular velocity of rotor expressed in terms of rotor speed in RPM as 2π rad ω

9. CALCULAT ON OF ANGULAR VELOC TY ntr fuga fie d s genera y expressed n mu t p es of the grav tat ona f e d cms–2) The re at ve centr fuga fie d (g) RCF wh ch s the rat o of the centr fuga at on at a specified rad us and the speed to the standard acce erat on of grav ty ca cu ated from the fol ow ng equat on: The ce g (981 acce er can be II illilillili l, iilil,.iill,iiiil,l, lilil ilil lii lii

10. One radian usua y abbrev ated as 1 rad, represents the ang e subtended at the centre of a c rcle by an arc w th a ength equa to the radius of the c rc e S nce 360o equa s 2 radians, one revolution of the rotor can be expressed as 2 rad. According y the angu ar ve oc ty n rads per second of the rotor can be expressed n terms of rotor speed s as:,l llllll i ii.i. ll ilil l,llii i

11. CALCULATION OF CENTRIFUGAL F ELD d therefore the centr fuga f e d can be expressed as: I anil i l

12. CALCULAT ON OF ANGULAR VELOC TY ntr fuga fie d s genera y expressed n mu t p es of the grav tat ona f e d cms–2) The re at ve centr fuga fie d (g) RCF wh ch s the rat o of the centr fuga at on at a specified rad us and the speed to the standard acce erat on of grav ty ca cu ated from the fol ow ng equat on: The ce g (981 acce er can be II illilillili l, iilil,.iill,iiiil,l, lilil ilil lii lii

13. elative centrifugal fo ce and speed o the centr fuge at d fe ent adii o the cent ifugat on spindle to a poin along he centr fuge tube Although the relative centrifugal force can easily be calculated, centrifugation manualsrrmanualsrr usuallycontain r anomograph for f theconvenient conversion between iififrrf itti.

14. Types of centrifuges Centrifugationtechniquestaketakeacentral positionininmodern biochemical, cellular and molecular biological studies. Depending on the particular application, centrifuges differ in their overall design and size. However, a common feature in all centrifuges is the central motor that spins a rotor containing the samples to be separated. Particles of biochemical interest are usually suspended in a liquid buffer system contained in specific tubes or separation chambers that are located in specialised rotors. Thebiologicalmediumis chosenfor thespecificcentrifugal application and may differ considerably between preparative and analytical approaches. The optimum pH value, salt concentration, stabilising cofactors and protective ingredients such as protease inhibitors have to be carefully evaluated in order to preserve biological function.

15. The most obvious differences between centrifuges are: The maximum speed at which biological specimens are subjected to increased sedimentation; The presence The potential manipulation or absence of a vacuum; for refrigeration or general of the temperature during a centrifugation run; and The maximum volume of samples and capacity individual centrifugation tubes. for

16. Many different types of centrifuges are commercially available including: large-capacity low-speed preparative centrifuges; refrigerated high-speed preparative analytical ultracentrifuges; preparative ultracentrifuges; large-scale clinical centrifuges; and small-scale laboratory microfuges. centrifuges;

17. Typesof rotors Swingin g Bucke t Roto r FixedAngl e Roto r u Supernatant J M~M~, \ - " ~~ellet Longer distanceof travel may allow Sedimenting particles have only short distance to travel before better separation, such as in density gradient centrifugation. Easier to withdraw supernatant without disturbing pellet. pelleting. Shorter run time. The most widely used rotor typ~. 2.

18. _..__.._ Ce"'rilugol field.VerticalTubeRotor Cccd'" r-;.;~-- - c..1rifuO.1 "tid Swinging Bucket Rotor........ _ 3.