Laboratory Centrifuges

1. Laboratory Centrifuges

2. Definition • A laboratory centrifuge • is laboratory equipment, • driven by a motor, • spins liquid samples at high speed. • There are various types of centrifuges, depending on the size and the sample capacity. • They vary widely in speed and capacity • work by the sedimentation principle, where the centripetal acceleration is used to separate substances of greater and lesser density. • comprise a rotor containing two, four, six, or many more numbered wells within which the samples containing centrifuge tips may be placed. • Centrifuges are used in • chemistry, biology, and biochemistry for isolating and separating suspensions.

3. Operation • Increasing the effective gravitational force will more rapidly and completely cause the precipitate to gather on the bottom of the tube as a "pellet". • The remaining solution is called the "supernate" or "supernatant". • The supernatant liquid is then either • decanted from the tube without disturbing the pellet, or • withdrawn with a Pasteur pipette. • The rate of centrifugation is specified by the acceleration applied to the sample, typically measured in revolutions per minute (RPM) or relative centrifugal force (RCF).

4. Operation • The particles' settling velocity in centrifugation is a function of their • size and shape, • centrifugal acceleration, • the volume fraction of solids present, • the density difference between the particle and the liquid, and the viscosity.

5. Rotor objectives Generally spoken, there are two main types of rotors: • Fixed-angle rotor • The 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.

6. Rotor objectives • Swing-out rotor (= horizontal rotor) • The 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.

7. DesignRotor objectives • The rotor is closed by a rotor lid. • The rotor is located in a rotor chamber which is covered by a metal centrifuge lid. • The open lid prevents the motor from turning the rotor when the rotor chamber is open. • During the run, the lid is locked. • The lid protects the user from being injured by touching a rapidly spinning rotor. • The rotor chamber and the lid of high quality centrifuges are robust enough to survive a rotor failure at full speed.

8. DesignRotor objectives • After a rotor crash, a centrifuge should not be reused as the enormous forces during a crash may have damaged essential parts of the device. • The rotor must be balanced by placing samples or blanks of equal mass opposite each other. Since most of the mass is derived from the solvent, it is usually sufficient to place blanks or other samples of equal volume. • As a safety feature, some centrifuges may stop turning when wobbling is detected (automatic imbalance detection, see Safety).

10. Theory • During circular motion • the acceleration is the product of the radius and the square of the angular velocity and it is traditionally named "relative centrifugal force" (RCF).

11. Theory • The acceleration is measured in multiples of "g“, the standard acceleration due to gravity at the Earth's surface, or × "g", and it is given by where g = acceleration r = rotational radius (centimeter, cm) N = rotating speed (revolutions per minute, r/min)

12. Calc. of RCF: • To calculate the RCF value at any point along the tube or bottle: • measure the radius, in mm, from the center of the centrifuge spindle to the particular point. • draw a line from the radius value on the right hand column to the appropriate centrifuge speed on the left hand column. • the RCF value is the point where the line crosses the center column. • The formula: R = Radius in mm from centrifuge spindle to point in tubeN = Speed of spindle in RPMRCF = (11.17 x 10-7) RN2

14. Types of centrifuge There are at least five types of centrifuge: • preparative centrifuge • analytical centrifuge • angle fixed centrifuge • swing head centrifuge • haematocrit centrifuge

15. Centrifuge tubes • Centrifuge 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.

16. Centrifuge tubes • 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

17. Centrifuge tubes Three microcentrifuge tubes: 2 mL, 1.5 mL and 200 μL (for PCR). Four screw-top micro-centrifuge tubes.

18. Centrifuge at NASA Ames Research Center. Featured in movie “Space Cowboys,” 2000

19. Mechanical Failure • Is caused by age and by improper use or inadequate care of centrifuge or rotor. Especially the rotor.

20. Care and Attention • Safe high-speed spin requires nearly perfectly balanced load. • Age, use, and misuse contribute to rotor flaws. • A rotor which comes apart at high speed can be deadly.

21. Tiny flaws are …NOT so tiny at 80,000 rpm These are micro-fissures and cracks caused by stress and corrosion in the bottom of a tube cavity.

22. (Purdue 2003) This rotor came apart while coming up to speed (not yet spinning at full speed) The 6 inch (15 cm) long chunk damaged (ruined) but did not completely penetrate the centrifuge lid and housing. The motor and spindle were also destroyed.

23. Follow instructions in manual and rotor care guide Use only compatible rotors Check routinely for rotor damage Don't overfill tubes Cap tubes Balance load carefully Check that rotor correctly seated on drive spindle Run at < max safe speed continued…… To reduce wear and chance of failure

24. Stay right there until full speed is reached. Stop the centrifuge if anything seems unusual Never open until stopped (never ‘manual brake’) Clean rotor gently Dry completely Store rotor upside down (why?) Maintain a careful rotor log Derate and retire rotors for age/use To reduce wear and chance of failure, continued

25. (MIT 1999) This rotor split in half at 55,000 rpm after 3 h of what was supposed to be a much longer spin. Grad student report “Rotor was manufactured in 1986 and was covered for 10,000 hours of operation or 5 years, whichever comes first.” No surprise that it flew apart 13 years later. Fortunately the centrifuge housing contained it.

26. (Cornell, 1998) The rotor failure was not contained. Much other damage done, equipment ruined, chemicals spilled. Nobody was near, or someone would have been badly hurt or killed.