Tips & Tricks

Tips & Tricks

Transferring centrifugation parameters from a protocol to your own conditions. What do you have to check and possibly adjust?

If you follow a given protocol, make sure to use the same type of rotor and apply the given relative centrifugal force (rcf or g-force) as well as the same temperature and running time. In general, the following major parameters have to be determined to achieve a successful centrifugation run:

  • A: Type of sample
  • B: Type of vessel
  • C: Type of centrifuge
  • D: Type of rotor
  • E: Necessary relative centrifugal force
  • F: Necessary temperature during centrifugation

Relative centrifugal force (rcf) influences running time and the quality of pellets and is determined by the type of sample, tube, rotor, speed, and centrifuge. When using rotors with different radii to spin a particular tube, please use the same rcf instead of the same revolutions per minute (rpm). To convert rpm and rcf you will need the maximum rotor radius and either the rpm value or the rcf value.

  • rpm = speed = revolutions per minute
  • rcf (... × g) = multiples of the Earth’s gravitational force = relative centrifugal force
  • rcm is the rotational radius measured in centimeters (cm)

       (distance from rotor axis to the bottom of the tube in cm)

  • nrpm is the rotating speed measured in revolutions per minute (rpm)

Rotational Speed and centrifugal force

To calculate the relative centrifugal force at the given rotor speed and given rotor radius, please enter the values in the appropriate fields and press the "Calculate RCF" button.

The "Calculate speed" button provides information on the required rotational speed at the given relative centrifugal force and the given rotor radius.

 

Please enter the rotor's radius directly
= Radius      cm
Relative centrifugal force (RCF)

RCF      x g

 
Speed

Speed      rpm

Because this issue is very important, many convenient conversion options now exist. The best, of course, is that the centrifuge itself provides an immediately accessible rpm/rcf conversion button directly on the centrifuge operating panel. This dispenses with having to carry out complex manual calculations.

Further options include calculating nomograms using the formula shown above. To convert rpm and rcf, you will need the maximum rotor radius and either the rpm value or the rcf value. Use a ruler to determine the necessary value. Such nomograms can often be found in operating manuals or on the internet pages of well-known centrifuge suppliers, too. Automatic calculators can be found there as well.

The use of adapters influences rmax to a certain extent as well. The distance from the rotor axis to the bottom of the tube will be shortened, which leads to lower g-force. To create accurate centrifugation conditions, you have to subtract the bottom thickness of the adapter from rmax.

Options for calculating rpm or rcf

You will always need information about the maximum rotor radius rmax, rpm, or rcf to carry out a calculation.

  • Use the rpm/rcf conversion button found directly on a centrifuge.
  • Use the rpm/rcf conversion nomogram.
  • Find a calculator in the internet (homepages of centrifuge suppliers).
  • Use a smartphone app.
  • Manually calculate the rpm or rcf using the standard formula.
  • Consult the operating manual, which provides rotor and adapter specifications.
  • Note the following: When adapters are used, subtract their thickness from rmax or select this at the centrifuge menu or select the appropriate corrected g-force

The optimal g-force for your sample strongly depends on the type of sample. In general, you can state the following for the g-force in decreasing order: virus particles, cell organelles > DNA > RNA > protein > cell culture, blood samples, environmental samples, urine, cell separation. We recommend checking these individually by referring to the scientific literature as there are no general or exact rules available. In general, when using special adapters, you need to decrease the selected radius setting as the tube with adapter is located nearer to the center of the rotor than without an adapter. If you do not reduce the radius setting, you would run your sample with a lower g-force than displayed.

 

 

Adapting the centrifugation time

What can you do if your centrifuge and rotor provide, e.g., only lower g-force than your protocol requires? You can centrifuge for an appropriate longer length of time. But how much longer? And how do you calculate this? The answer: by using the k-factor, a measure of the sedimentation distance. This will tell you how long it will take the particles to settle at the bottom of the test tube.

The k-factor represents the pelleting efficiency of a centrifugation system at maximum rotational speed. The factor is a measure of the sedimentation distance in a test tube; it depends on the difference between the longest and the shortest distance between the sample and rotor axis. The shorter the sedimentation distance, the shorter the centrifugation time. In addition, the angle of the tubes within the rotor influences the sedimentation distance. 

The radius of the rotor and the angle of the bores as well as the type of vessel respectively the adapter you use will all have an impact on the sedimentation distance and thus on the centrifugation time you will need. 

The k-factor is a measure of the sedimentation distance and tells us how long it will take until the particles settle at the bottom of the test tube. 

The smaller the k-factor, the better the pelleting efficiency. 

The formula for calculating the k-factor considers these parameters:

  • Maximum rotor radius
  • Minimum rotor radius
  • Speed

To achieve the same sedimentation result and compensate for not having g-force, you can calculate how much longer to centrifuge using a formula based on the k-factors of both rotors and the centrifugation time of the rotor originally used.

Using a fixed-angle rotor or a swing-bucket rotor

Generally speaking, there are two types of centrifuge rotors available: fixed-angle rotors and swing-bucket rotors. Both types of rotors have advantages and disadvantages. Most fixed-angle rotors have bores at a fixed angle of 45° and the most compact pellet is formed at that angle. Smaller rotor angles result in more diffuse pellet areas.

The advantage of fixed-angle rotors is that they don’t have moving parts like swing-bucket rotors. Fixed-angle rotors are therefore exposed to much lower metal stress than swing-bucket rotors. This results in higher possible g-forces and thus shorter centrifugation times. The disadvantage of these rotors is that they provide only limited vessel capacity.

You will need a fixed-angle rotor when

  • high g-forces are applied,
  • compact pellets are needed, or
  • a rather low number of vessels is used.

The buckets of swing-bucket rotors swing out horizontally at a 90° angle, resulting in the pellets being located directly in the middle of the tube bottom. The strength of these rotors is that they enable high vessel capacity and high vessel flexibility due to the different adapter options available for them. 

Pellets located in the middle of the tube bottom and not on the side of the tube wall as is the case with fixed-angle rotors can be an advantage as well, e.g., when low concentrations are expected and only very low volumes of buffer should be used for resolving the pellet in order to keep the sample concentration as high as possible.

Gradient centrifugation definitely requires a swing-bucket rotor so horizontal layers can form that remain in this position at the end of centrifugation.

A disadvantage of swing-bucket rotors is the high metal stress the moving buckets cause. This results in lower maximum g-forces and possibly requires longer centrifugation times.

You will need a swing-bucket rotor when you

  • have a high sample throughput,
  • need a high vessel flexibility (adapter flexibility),
  • are centrifuging plates,
  • need to have the pellet in the middle of the tube bottom, 
  • or are carrying out gradient centrifugation.