Fonte: Robert M. Rioux & Suprita Jharimune, Pennsylvania State University, University Park, PA
La forza gravitazionale terrestre è in grado di separare miscele eterogenee. Tuttavia, molti sistemi sververa questo tipo di separazione a causa del periodo di tempo solitamente richiesto in questi casi. La centrifugazione è uno degli strumenti più potenti per la separazione di miscele eterogenee1. Comporta l’applicazione della forza centripeta per la sedimentazione della fase più pesante, che porta alla separazione delle due fasi, ed è una tecnica comunemente usata in ambienti industriali e di laboratorio.
In un tipico processo di centrifugazione, le particelle sospese in un fluido vengono filate attorno all’asse di rotazione della centrifuga, causando una forza lontano dall’asse. Questa forza spinge le particelle sul fondo del porta campioni della centrifuga ad una velocità che dipende dalla dimensione e dalla densità delle particelle. La centrifugazione è quindi una tecnica per accelerare il processo di sedimentazione.
Per garantire la sicurezza nell’uso di una centrifuga, l’utente deve essere adeguatamente addestrato e reso consapevole dei pericoli che potrebbero derivare dal suo utilizzo. Le sezioni seguenti presentano una discussione sulla manipolazione e la sicurezza durante il lavoro con le centrifughe.
With the increased usage of centrifuges in laboratories and industry, the risk of hazards for using centrifuges has also increased. However, careful handling of the apparatus and proper knowledge of its use and safety measures can substantially mitigate accidents. A basic guideline for working with centrifuges is provided here. Nonetheless, procedures may vary marginally depending on the particular centrifuge machine used. Hence, it is always recommended to carefully read and understand the user manual of the particular machine before starting to use it.
Centrifugation is one of the most powerful tools used in labs for the separation of heterogeneous mixtures, but it can be hazardous, and therefore safety precautions should be applied.
A centrifuge rapidly spins samples to separate heterogeneous materials, which induces extreme centrifugal forces on the samples and the instrument. Due to the high speed at which a centrifuge operates, it can cause serious injury to the user if mishandled. Therefore, special care must be taken when using a centrifuge to prevent spills and damage to the equipment.
This video will illustrate the mechanism of a centrifuge and how to safely operate and maintain one.
Before going into the details of the safety measures, let’s quickly review the basics of centrifugation. Centrifuges contain a rotor housed on a spindle. Differently sized rotors can usually be fitted on a single spindle to accommodate different sample bottles, tubes, or bags. A motor spins the rotor at a specified RPM or revolutions per minute. This, in turn, imparts the centrifugal force on the heterogeneous mixture directed away from the axis of rotation, which separates particles with different densities.
Different types of centrifuges operate at different RPMs. Low-speed centrifuges run at less than 10,000 RPMs, high-speed up to 21,000, and ultracentrifuges to 30,000 and above. Each type has different rotors that are compatible with it. After the run, a braking system usually slows down the rotor. In any centrifuge, high speed deforms the rotor.
In normal use, it will return to its previous size. However, if the force due to spinning exceeds the maximum force that the rotor can handle, then it might get permanently deformed. This can lead to cracks, which may, in turn, lead to serious failure. Centrifuge failures, on the other hand, may lead to sample leakage, which can be dangerous if hazardous materials are involved and should be handled with care. Now, we will show you how to prepare samples and safely use them in the centrifuge.
First, select appropriate centrifuge tubes that are compatible with the rotating speeds you want to use. Check the documentation to find out the tube’s limits. Tubes should be intact and free of any cracks. Fill each to no more than 3/4 full to prevent spills. Then make sure that the tubes are balanced and cap them tightly.
When centrifuging hazardous chemical or biological samples, take special precautions. Wear appropriate protective equipment, and prepare samples in an appropriate work environment, like a fume hood or laminar flow cabinet. For more information on these safety features, see JoVE’s videos on Fume Hoods.
If necessary, use a secondary containment for hazardous materials, like a glass or plastic insert. Sealed safety tubes providing a contained environment can be used as well. Everyone must be trained before using any centrifuge or rotor.
Select a rotor appropriate to the centrifuge. Check that the rotor and its cover are clean and dry and do not have cracks or deformities. Make extra sure that the O-rings on the rotor or cover are not worn. Also, check the centrifuge spindle to see if it is bent or has any other deformities. Then place the rotor on the spindle making sure it is correctly seated. Insert samples into the rotor, being sure that the tubes fit snugly in the rotor.
Always put balanced tubes opposite each from each other. Cover the rotor, ensuring that the seal is tight. Close the centrifuge lid and start your program. Again, see JoVE’s previous centrifuge video, for more information about running a centrifuge program.
Stay at the centrifuge until it has reached its maximum speed. Familiarize yourself with the location of the power button. Turn off the power button if there are any unusual sounds or vibrations to stop the centrifuge immediately. Then evacuate the immediate area until the rotor has stopped. Once it has stopped, inspect the samples for spillage and the rotor for any abnormalities or imbalances. If there were no problems, wait until the centrifuge has completed its program and completely stopped.
Wait an additional half-hour if aerosol-forming chemicals are present. Otherwise, you may open
the centrifuge immediately. Remove the samples and take them back to a specialized area for opening if necessary. In case of a spill, clean up the centrifuge immediately. Use appropriate PPE to any hazard present, and dispose of contaminated materials according to guidelines. Do not use abrasive brush wires to clean as they may damage the rotor. Alert emergency personnel, and do not attempt to clean the centrifuge if life-threatening hazards have spilled.
Decontaminate the rotor with 10% bleach for any biological samples used in the centrifuge regardless of spill or not. Let the bleach stand for 30 minutes, and then rinse 70% ethanol and air dry. Lastly, remove and inspect the rotor for any damage, and note down your use in the logbook.
You’ve just watched JoVE’s Introduction Into Centrifuge Use. You should now understand how to prepare your samples as well as how to operate and maintain the instrument.
Thanks for watching.
