6 centrifugation (1)
- 1. Centrifugation Centrifugation is a process used to separate particles or concentrate materials suspended in a liquid medium. In biology, the particles are usually cells, subcellular organelles, viruses, large molecules such as proteins and nucleic acids. The theoretical basis of this technique is the effect of gravity on particles (including macromolecules) in suspension. Two particles of different masses will settle in a tube at different rates in response to gravity. Centrifugal force (measured as xg, gravity) is used to increase this settling rate in the centrifuge. The two common types of centrifugation are analytical and preparative; the distinction between the two is based on the purpose of centrifugation. Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Optimal methods are used in analytical ultracentrifugation. Molecules are observed by optical system during centrifugation, to allow observation of macromolecules in solution as they move in gravitational field. The samples are centrifuged in cells having windows that lie paralleled to the plan of rotation of the rotor head. As the rotor turns, the images of the cell (proteins) are projected by an optical system on to film or a computer. The concentration of the solution at various points in the cell is determined by absorption of a light of the appropriate wavelength (Beer's law is followed). The other form of centrifugation is called preparative and the objective is to isolate specific particles which can be reused. There are four basic types of centrifuges that you are likely to encounter in the laboratory: 1. Clinical centrifuges - inexpensive tabletop centrifuges that can run at a speed of up to 3000 rpm. These can pellet cells, but not organelles or biomolecules. 1
- 2. 2. Microfuge - these can typically run at a speed of up to 14,000 rpm, sufficient speed to pellet nucleic acids and denatured proteins. Microfuges are specially adapted for small volumes of sample. 3. High speed centrifuges - can run at speeds up to 25,000 rpm, sufficient to pellet cell nuclei and most biomolecules. These are often refrigerated. 4. Ultracentrifuges - The ultracentrifuge is capable of reaching even greater velocities and requires a vacuum to reduce friction and heating of the rotor. It can run at speeds up to 75,000 rpm, sufficient to allow fractionation of biomolecules, for example: plasmid DNA, chromosomal DNA, and RNA. Ultracentrifuges are usually refrigerated and are very expensive and delicate pieces of machinery. Rotors Rotors for a centrifuge are either fixed angles, swinging buckets, continuous flow, or zonal. Fixed angles generally work faster; substances precipitate faster in a given rotational environment. The most common is a rotor holding 8 centrifuge tubes at an angle of 34 属C from the vertical (such as the Sorvall SS-34 rotor or the Beckman JA- 20). Fixed angle rotors: In a fixed angle rotor, the materials are forced against the side of the centrifuge tube, and then slide down the wall of the tube. This action is the primary reason for their apparent faster separation, but also leads to abrasion of the particles along the wall of the centrifuge tube. Swinging bucket (horizontal rotors): For swinging bucket rotors, the materials must travel down the entire length of the centrifuge tube and always through the media 2
- 3. within the tube. Since the media is usually a viscous substance, the swinging bucket appears to have a lower relative centrifugal force, which is it takes longer to precipitate anything contained within. If, however, the point of centrifugation is to separate a molecules or organelles on the basis of their movements through viscous field, then the swinging bucket is the rotor of choice. Moreover, if there is a danger or scraping off an outer shell of a particle (such as the outer membrane of a chloroplast), then the swinging bucket is the rotor of choice. Most common clinical centrifuges have swinging buckets. Since the buckets are easy to interchange, this type of rotor is extremely versatile. Cell biologists employ zonal rotors for the large scale separation of particles on density gradients. Zonal rotors can contain up to 2 liters of solution and can work with tissue samples measured in ounces (or even pounds). Rotor tubes: For biological work, the tubes are divided into functional groups: made of regular glass, Corex glass, or nitrocellulose. Regular glass centrifuge tubes can be used at speeds below 3,000 RPM that is in a standard clinical centrifuge. For work in the higher speed ranges, centrifuge tubes are made of plastic or nitrocellulose. Preparative centrifuge tubes are made of polypropylene (sometimes polyethylene) and can withstand speeds up to 20,000 RPM. These tubes should be carefully examined for stress fractures before use. A tube with a fracture will hold fluids before centrifugation, but the cracks will open under centrifugal force. 3
- 4. Relative Centrifugal Force (RCF) The centrifugal force generated by a centrifuge is determined by the speed of rotation and the distance of the sample from the axis of rotation. The centrifugal force is usually compared to the force of gravity and is reported as the relative centrifugal force (RCF) in g units. (A force of 1 g is equal to the force of gravity at the earth's surface) The following formula relates the RCF to the rotor speed and diameter: RCF = (1.119 x 10-5) * (rpm)2 * r where r is the radius of rotation (the radius of the rotor) in cm and rpm is the centrifuge speed in revolutions per minute. The number "1.119 x 10-5" is a conversion factor that allows us to use "rpm" and "g" units. The RCF is dependent upon the radius of the rotation of the rotor, the speed at which it rotates, and the design of the rotor itself (fixed angle or swinging bucket). Rotor speed and design can be held constant, but the radius will vary from the top of a centrifuge tube to the bottom. If a measurement for the radius is taken as the mid-point, or as an average radius, and all forces are mathematically related to gravity, then one obtains relative centrifugal force, labeled as xg. Three r-values given (by the manufacturer) for a rotor: the maximum, from the center of rotation to the bottom, top and middle of minimum and average r. These correspond to the distances for the sample tube. Practical pointers 1. Centrifuge sample containers come in a variety of shapes, sizes, and materials. Be careful to use the correct tubes. Glass tubes always require an adapter to cushion them because direct contact between the glass and a metal rotor will result in a 4
- 5. cracked or shattered tube. Adapters may slide over the tube like a sleeve or may be cushions that sit in the bottom of the rotor slot. (The "cushion" adapters may be found in clinical centrifuges but are not used in high speed centrifuges.) 2. Tubes that are over-filled can spill their contents inside the rotor. For fixed-angle rotors it is especially important not to fill the tubes more than 2/3 to 3/4 full. Spills need to be wiped up immediately to prevent corrosion of the rotor. It is always a good practice to wash out a rotor when you are finished with it; even when there's no apparent spill, liquid from the tubes often spatters. Rinse the rotor with distilled water, dry with paper s to let any moisture drain. Wipe up any liquid in the chamber towels, and store upside down on top of paper towel of the centrifuge, then use a paper towel (dampened with distilled water) to wipe down the chamber. Dry the chamber well; if the refrigeration has been used, don't leave the door open any longer than necessary because condensation will build up on the chamber walls. 3. Most important of all, a rotor must be balanced: tubes in opposite positions should be equal in weight. The tubes can be balanced by adding or removing liquid. An unbalanced centrifuge can vibrate violently, and horror stories abound about unbalanced ultracentrifuges crashing through walls. 4. Finally, note that it is important to keep your samples cold during the lab by using chilled tools and glassware and by keeping the samples on ice whenever possible. Procedures that disrupt cell structure release degradative enzymes, which are less active at colder temperatures. 5