Centrifugation Principle, RCF and Rotor Types Explained (Video)

0:02 Introduction

Today we'll learn about centrifugation. Centrifugation separates the components of a liquid sample by spinning it, so that particles move outward and settle according to their size, shape and density. The video has nine parts.

0:19 Where the centrifuge came from

First, we will see where the centrifuge came from. Centrifugation began outside the laboratory, in dairies. Milk left to stand separates slowly, because the fat globules are slightly less dense than the surrounding milk and rise under gravity over many hours. In 1878, the Swedish engineer Gustaf de Laval patented a continuous cream separator that spun the milk and separated the cream in minutes. Laboratory centrifuges apply the same principle to cells, organelles and large molecules. In the 1920s Theodor Svedberg built the ultracentrifuge to measure the size of protein molecules.

1:00 Why particles sediment

Second, we will learn why particles sediment. Sedimentation is the movement of particles through a liquid under gravity or centrifugal force until they settle. Under gravity alone, grains of sand settle in seconds and cells settle over minutes to hours, while ribosomes and proteins settle so slowly that random diffusion keeps them spread through the liquid. In a spinning rotor, the particles move outward as if gravity were thousands of times stronger, so cells and large organelles settle in minutes. Three forces act on a particle in the tube. The centrifugal force pushes the particle outward, the buoyant force of the displaced liquid acts in the opposite direction, and friction with the liquid grows as the particle speeds up. The particle soon reaches a steady speed, its sedimentation velocity. The sedimentation velocity rises with the size of the particle, roughly with the square of its radius, and with the difference between the density of the particle and the density of the liquid. The velocity falls as the viscosity of the liquid rises, and it falls further when the particle is elongated rather than compact. A particle with the same density as the liquid does not sediment at all, however fast you spin the rotor. Sedimentation also competes with diffusion, which moves particles from crowded regions to emptier regions and so spreads them back through the tube. For a very small particle, the random motion of diffusion can exceed the movement produced by the rotor. For these reasons intact cells pellet at low force, while nuclei, mitochondria, ribosomes and proteins need progressively higher forces.

2:47 Relative centrifugal force

Third, we will define relative centrifugal force. The force from spinning depends on more than how fast the rotor turns, so protocols use one measure of force that any rotor can reproduce. Relative centrifugal force, or RCF, is the force on the sample expressed as a multiple of Earth's gravity, and you write it as times g. A force of one thousand times g means that the sample experiences one thousand times the pull of Earth's gravity. To reach a given force, you set the speed of the rotor in revolutions per minute, or rpm. The formula is RCF equals one point one one eight times ten to the minus five, times the radius in centimeters, times the speed in rpm squared. The constant converts the radius in centimeters and the speed in rpm into a multiple of g. The radius is the distance from the axis of the rotor to the point in the tube you care about. Because the tube spans a range of radii, the force at a fixed speed increases from the top of the liquid to the bottom of the tube. For each rotor, manufacturers publish a minimum radius at the top of the liquid, a maximum radius at the bottom of the tube, and an average radius between the minimum and the maximum. The speed enters the formula squared, so doubling the rpm raises the force four times, while doubling the radius raises the force only twofold. For the same reason, the same rpm in two rotors with different radii gives different forces. A protocol should therefore state the force in times g, and you convert that value to rpm for your own rotor.

4:26 Worked example

Fourth, we will work through two examples with the formula. You can find the force from the speed, or find the speed from the force. In the first example, a rotor with a radius of ten centimeters turns at three thousand rpm. You square three thousand and get nine million. You multiply one point one one eight times ten to the minus five by ten and get one point one one eight times ten to the minus four. You then multiply that result by nine million. The force is about one thousand six times g. At the same three thousand rpm, a radius of five centimeters gives about five hundred three times g, half the force at ten centimeters. The second example runs in reverse. A protocol asks for five thousand times g, and your rotor has a radius of eight centimeters. You multiply one point one one eight times ten to the minus five by eight and get eight point nine four four times ten to the minus five. You divide five thousand by that value and get about fifty-five million nine hundred thousand. You take the square root of that number, and you set the centrifuge to about seven thousand four hundred eighty rpm.

5:38 Rotor types and centrifuge classes

Fifth, we will compare rotor types and centrifuge classes. The rotor type sets where the pellet forms and how the layers of liquid behave, so you choose the rotor before you set the speed. A fixed-angle rotor holds the tubes at a set angle to the axis, often between twenty-five and forty-five degrees. Particles travel a short distance to the outer wall and slide down it, so the pellet forms on the outer side of the tube near the bottom. Fixed-angle rotors pellet cells and debris quickly. In a swinging-bucket rotor, the buckets hang vertically at rest and swing out to the horizontal as the rotor speeds up. The pellet forms flat at the bottom of the tube and the layers of liquid stay level, so you use a swinging-bucket rotor to separate blood and to run gradients. A vertical rotor holds the tubes parallel to the axis and suits short gradient runs. Centrifuges also fall into classes by the force they reach. A microcentrifuge spins tubes of one point five to two milliliters at up to about fifteen to twenty-one thousand times g, and a benchtop or clinical centrifuge reaches a few thousand times g with larger tubes. High-speed refrigerated floor models reach tens of thousands of times g, and the fastest approach one hundred thousand times g, where the range of the ultracentrifuge begins. A refrigerated centrifuge holds the chamber at a set temperature, often four degrees Celsius, so proteins and cells stay cold during the run.

7:14 Pellets and differential centrifugation

Sixth, we will look at pellets and differential centrifugation. When the run ends, the sample has separated into a pellet and a supernatant. The pellet is the material packed at the bottom or against the outer wall of the tube. The supernatant is the liquid above the pellet, and you remove the supernatant by pipetting or pouring without disturbing the pellet. Which part you keep depends on the experiment. Typical settings show how widely the required force varies. You separate plasma or serum from blood cells at about one thousand to two thousand times g for ten minutes, and you harvest bacteria from a culture at about four thousand to six thousand times g for ten to fifteen minutes. You collect mammalian cells gently, at about two hundred to three hundred times g for five minutes, so that the cells stay alive. Differential centrifugation uses a series of runs at increasing force to separate the organelles of a tissue homogenate, which is tissue or cells broken open in buffer so that the organelles are released. You spin the homogenate at about six hundred to one thousand times g for ten minutes to pellet the nuclei and the unbroken cells. You move the supernatant to a new tube and spin it at about ten thousand to twenty thousand times g for twenty minutes to pellet the mitochondria. A final spin of the supernatant at about one hundred thousand times g for sixty minutes pellets the microsomes, small vesicles of membrane, and this step already needs an ultracentrifuge. Each pellet is enriched for one kind of organelle but is never pure, because particles start at all heights in the tube. A small particle that starts near the bottom reaches the pellet before a large particle that starts at the top.

9:08 Running a centrifuge safely

Seventh, we will learn how to run a centrifuge safely. A rotor stores a large amount of energy at speed, so the way you load and run the centrifuge matters for the sample and for your safety. You balance the rotor by placing tubes of equal mass directly opposite each other. If you have an odd number of samples, you fill a balance tube with water to the same mass. An unbalanced load makes the rotor vibrate, and the vibration can damage the drive shaft or the rotor itself. You use tubes rated for the force you plan to apply, and you close the lids before the run. For infectious samples you use sealed buckets or rotors with aerosol-tight lids, and you open these rotors in a biological safety cabinet. Before you start, you set the force in times g, the time, the temperature, and the acceleration and the braking. The acceleration setting controls how quickly the rotor reaches its set speed, and the braking setting controls how quickly the rotor slows. A slow brake keeps loose pellets and liquid layers from mixing as the rotor stops. You never open the lid until the rotor has stopped completely, and you stop the run if you hear unusual noise or feel vibration.

10:24 Centrifugation in everyday lab work

Eighth, we will use these settings in everyday laboratory work. We will look at serum tubes, urine sediment and silica spin columns, and then convert an old protocol to a new rotor. Blood collection tubes for serum often contain a gel whose density lies between the density of the serum and that of the clot, the solid mass of blood cells and fibrin. The gel is thixotropic, so it flows under the force of the spin and sets again when the rotor stops. During the spin it moves up and forms a barrier between the two layers, and you can then pour or pipette the serum off cleanly. Clinical laboratories concentrate the cells and crystals in urine before microscopy. You spin a fixed volume at about four hundred times g for five minutes, the cells and crystals collect at the bottom of the tube, and you examine the sediment under the microscope. In molecular biology you purify DNA or RNA on small silica spin columns, each a silica membrane held inside a microcentrifuge tube. You load the sample in a chaotropic salt solution, which denatures proteins and lets the silica bind the nucleic acid, and a microcentrifuge drives the liquid through the membrane at about ten thousand times g or more. The nucleic acid stays bound to the silica while proteins and other contaminants pass into the collection tube. An ethanol wash removes the salt, and a final spin with a low-salt buffer releases the nucleic acid into a clean tube. Protocols often state rpm for a rotor you do not own, so you convert the old setting to times g first and then convert times g back to rpm for your rotor. For this conversion, use the maximum radius, the distance from the axis to the bottom of the tube. For a third worked example, a protocol written for a rotor with a radius of eight point four centimeters specifies thirteen thousand rpm. The force is one point one one eight times ten to the minus five, multiplied by eight point four, multiplied by thirteen thousand squared, which gives about fifteen thousand nine hundred times g. Your rotor has a radius of six centimeters. You divide fifteen thousand nine hundred by one point one one eight times ten to the minus five times six, take the square root, and set about fifteen thousand four hundred rpm. Force and time can also trade against each other. In the same rotor, the time a particle needs to reach the pellet is inversely proportional to the force, so ten minutes at four thousand times g pellets about as well as twenty minutes at two thousand times g. The trade-off has limits, because a gentler spin protects fragile cells, and a stronger spin compacts the pellet and can damage the cells packed inside the pellet.

13:18 Troubleshooting

In the final section, we will learn how to troubleshoot a failed run. Four common failures each have a sign that you can recognize during or after the spin. If you see no pellet, the force or the time was too low, or the sample held very little material. Check whether you entered the protocol value as a force in times g or as a speed in rpm, and convert the protocol value for your rotor. If the pellet is loose or smeared up the wall, the brake was too hard or you poured off the supernatant too quickly. Use a gentler brake and remove the supernatant with a pipette. When the centrifuge vibrates or shows an imbalance error, the tubes differ in mass or do not sit opposite each other, so you stop the run and rebalance the rotor before you start again. Lysed cells or damaged organelles point to a force that was too high or to a sample that warmed during the run. After a blood spin, for example, a pink or red supernatant shows that red cells have broken open, so you lower the force for the cell type and use a cooled rotor.

14:25 Recap and quiz

Here is a summary of what you learned. Centrifugation began in the dairy, where spinning separated cream from milk. Particles sediment because the spinning rotor drives them outward, and their sedimentation velocity rises with the square of the particle radius and with the density difference from the liquid. Relative centrifugal force gives the force as a multiple of Earth's gravity, and the formula uses the radius in centimeters and the square of the rpm, so the same rpm in rotors of different radius gives different forces. Fixed-angle rotors pellet material on the outer wall, and swinging-bucket rotors pellet it flat at the bottom. Differential centrifugation uses increasing force to collect nuclei, mitochondria and microsomes in turn, and you balance the rotor before each run. Now try a question on what you learned. A microcentrifuge rotor has a radius of seven centimeters, and a protocol asks for twelve thousand times g. Work out the speed in rpm that you would set on the centrifuge. Pause here and try it before the answer appears. You multiply one point one one eight times ten to the minus five by seven, and you divide twelve thousand by the result to get about one hundred fifty-three million three hundred thousand. You take the square root of that number, and you set the centrifuge to about twelve thousand three hundred eighty rpm.