low-speed centrifuge: Principles, RCF Conversion, Rotor Types & Practical Usage Tips

I. Working Principle of a low-speed centrifuge

The physical basis of sedimentation separation in alow-speed centrifugeis the effect of centrifugal force on the sample. When a centrifuge tube containing the sample is placed in the centrifuge, the centrifuge starts working, and the sample undergoes uniform circular motion, generating an outward centrifugal force. The larger the radius of rotation and the faster the rotation speed, the greater the centrifugal force, and the faster the particles settle. Because different particles have different masses, densities, sizes, and shapes, their sedimentation velocities differ in the same fixed centrifugal field.

II. Centrifugal Force and Relative Centrifugal Force

1. Centrifugal Force

F = mw²r (where F is the centrifugal force intensity, m is the effective mass of the sedimenting particles, W is the angular velocity of the centrifugal rotor in rad/s, and r is the centrifugal radius in cm, i.e., the distance from the center of the centrifuge shaft to the bottom of the test tube in a horizontal centrifuge, or the distance from the center of the test tube opening in a vertical centrifuge)

2. Relative Centrifugal Force (RCF)

Centrifugal force is usually expressed as relative centrifugal force (RCF), which is the ratio of the magnitude of the centrifugal force F to the Earth's gravitational force, measured in grams (g). RCF is a parameter only related to the centrifuge and has no direct relationship with the sample.

Relative centrifugal force (RCF) is the true standard for measuring centrifugation effectiveness because it directly reflects the magnitude of the centrifugal force exerted on the sample, thus determining the efficiency and speed of particle separation. Regardless of the size or model of the centrifuge rotor, as long as the RCF value is the same, the centrifugal force exerted on the sample is the same. This makes experimental conditions comparable between different laboratories and different models of centrifuges.

III. Rotational Speed (RPM)

RPM, short for Revolutions Per Minute, is a unit describing the pure rotational speed of a centrifuge rotor, indicating how many revolutions the rotor makes per unit time (one minute). For example, when a centrifuge displays a speed of 10.000 RPM, it means that its rotor rotates 10.000 times per minute.

It only describes the speed of the machine's rotor rotation. This parameter only reflects the rotor's rotational frequency.

IV. RPM to RCF Conversion

The conversion formula between RCF and RPM is as follows:

RCF = 1.118 × 10^(-5) × r × (RPM)^2

Or:

g = (RPM / 1000)^2 × r × 11.18

The meaning and units of each term in the formula:

RCF (g): Relative centrifugal force, unit is "g" (a multiple of the Earth's gravitational acceleration).

r: Centrifugal radius, unit is "cm". This refers to the effective radius of rotation from the rotor axis to the bottom of the centrifuge tube (or the center of the sample).

RPM: Revolutions per minute, unit is "revolutions per minute".

1.118 × 10^(-5): This is a constant used to balance units and express the result in "g".

V. Selection of Centrifuge Rotors

The rotor is an integral part of the centrifuge and can be divided into horizontal rotors and angle rotors, each with its own application range.

1. Horizontal Rotor

The basket of a horizontal rotor can swing horizontally at a 90° angle, allowing the sample precipitate to be located directly in the middle of the bottom of the tube. The advantage of these rotors is the availability of different adapter options, enabling high capacity and high flexibility.

Sample separation effect:

The sample precipitate is located in the middle of the tube bottom, rather than on the side of the tube wall as in a fixed-angle rotor. The advantage is that when the concentration of the sample to be separated is low, as long as the sample is concentrated as possible at the bottom of the tube, and then resuspended with a small volume of buffer, the sample concentration can be maximized. Additionally, gradient centrifugation also requires a horizontal rotor to form multiple horizontal gradient layers and ensure that the positions of each horizontal gradient are maintained after centrifugation.

Scenarios requiring a horizontal rotor:

① High sample throughput;

② Gradient centrifugation required;

③ Sample needs to be centrifuged to the middle of the tube bottom;

2. Angle Rotor

(1) Fixed-Angle Rotor

In a fixed-angle rotor, the sample tube is centrifuged at a fixed angle, and the sample usually precipitates on the tube wall. Unlike a horizontal rotor, a fixed-angle rotor does not have movable parts, and the rotor experiences less metal pressure. Therefore, a fixed-angle rotor can achieve higher relative centrifugal force and thus shorter centrifugation time (Eppendorf 5430 R can achieve a relative centrifugal force of up to 30.000 x g). The disadvantage is that the centrifugal throughput is limited (the maximum centrifugal throughput of the Eppendorf benchtop centrifuge is 6 x 250 mL, and the maximum centrifugal throughput of the floor-standing high-speed centrifuge is 6 x 1 L).

Sample Separation Performance

Most fixed-angle rotors achieve the tightest sample precipitate at a 45° angle. Fixed-angle rotors less than 45° result in a more dispersed sample precipitate area, potentially leading to resuspension of the precipitate during supernatant transfer and affecting separation performance.

Fixed-angle rotor is required:

① Requires greater relative centrifugal force for complete separation;

② Requires more compact particles;

③ Smaller sample volume;

(2) Vertical-angle rotor:

The centrifuge tube is placed vertically, with the centrifugal force direction parallel to the tube axis. The sample settles rapidly along the length of the centrifuge tube, resulting in the shortest sedimentation path among the three types of rotors. Advantages: Fastest separation speed, reduces sample diffusion, significantly improves separation precision, and is suitable for demanding ultracentrifugation experiments.

Scenarios requiring vertical angle rotors:

Primarily used in ultracentrifuges, enabling precise experiments such as the separation of subcellular components (e.g., mitochondria, chloroplasts) and rapid preparation via density gradient centrifugation. It is a key component for precise separation in scientific research.

Summary on rotor selection:

VI. Relationship between FCF (g-value), rpm, and r-value for different rotors

This can be read from the following two graphs:

Relative centrifugal force (FCF) nomogram for a high-speed rotor

To determine an unknown value in a particular column, use a ruler to arrange the known values in the other two columns, ensuring they fall at the intersection of the ruler and the third column. For example, at a rotor speed of 80.000 rpm and a rotation radius of 20 mm, the relative centrifugal force (FCF) is approximately 150.000 g.

To determine an unknown value in a column, use a ruler to arrange the known values in the other two columns, ensuring they fall at the intersection of the ruler and the third column. For example, when the rotor speed is 5000 rpm and the rotation radius is 40 mm, the relative centrifugal force (FCF) is approximately 1100 g.

Centrifuge usage and precautions

I. Classification of Centrifuges

(1) According to speed, centrifuges can be classified as: low-speed centrifuges, high-speed centrifuges, and ultra-high-speed centrifuges.

(2) According to capacity, centrifuges can be divided into: microcentrifuges (mini centrifuges or micro-centrifuges), small-capacity centrifuges, large-capacity centrifuges, and ultra-large-capacity centrifuges.

(3) According to whether or not they are refrigerated, centrifuges can be divided into: refrigerated centrifuges and ambient-temperature centrifuges.

II. Centrifuge Use

1. Preparation Before Start-up

Ensure the centrifuge is placed on a stable and firm surface and connected to the power supply.

Check that the rotor is installed correctly and securely.

Select the appropriate rotor and centrifuge tubes according to the experimental requirements, and ensure the caps of the centrifuge tubes are tightly closed.

Clean the centrifuge chamber of any debris to ensure that no foreign objects affect the centrifugation process.

2. Setting Parameters

Turn on the centrifuge power switch and set the rotation speed (RPM), time (minutes), and temperature (if applicable) according to the experimental requirements.

Confirm that the set parameters are within the centrifuge's allowable range to avoid overloading.

3. Loading Samples:

Place the centrifuge tubes symmetrically in the centrifuge chamber to ensure rotational balance.

Use specialized tools or gloves to load and unload centrifuge tubes and rotors to prevent hand injuries.

Close the centrifuge lid and ensure the locking mechanism is in place.

4. Start-up and Monitoring:

After confirming all settings are correct, press the "Start" button to start the centrifuge.

During centrifugation, carefully observe the centrifuge's operating status. If any abnormal sounds or vibrations are heard, stop the centrifuge immediately and check.

5. End-of-Life and Unloading:

The centrifuge will automatically stop after the preset time. Wait until the rotor has completely stopped rotating before opening the centrifuge lid.

III. Precautions

1. Balancing Issues

Starting a centrifuge without balancing can result in minor damage to the machine, or even serious injury or death!

Centrifuging at 1.000.000 x g results in 1 g of sample producing 1.000 kg. If this is accidentally ejected, it's equivalent to a high-speed bullet, extremely dangerous.

The principle of balancing is: the solutions in corresponding centrifuge tubes should have equal density and volume—meaning they have the same center of gravity during centrifugation.

For weight balancing, a precision balancing deviation of no more than 0.1g is generally required, while for routine applications, the deviation should generally not exceed 1g. The weight deviation must not exceed the range specified in the centrifuge instruction manual.

Avoid loading an odd number of centrifuge tubes into the rotor. When the rotor is only partially loaded, the tubes should be evenly distributed to ensure uniform load distribution across the rotor body. When there is an odd number of centrifuge tubes, the balancing tubes must be filled with a material of similar density; for example, water can be used for balancing bacteria from a culture medium.

(1) Balancing of 12-hole fixed angle rotor:

(2) Rotor Balancing

Not only must the symmetry of the centrifuge tubes within a single basket be considered, but the balance of the centrifuge tubes in the opposite basket must also be taken into account. In this case, two principles must be remembered during balancing:

When placing centrifuge tubes in a single basket, ensure that the basket's center of gravity is at its center point;

When placing centrifuge tubes in the opposite basket, use the placement position of the first basket as a reference, strictly adhering to the principle of rotor center point symmetry, and then place them in appropriate positions.

(3) Balancing odd numbers: If you find it troublesome to use a blank balancing tube (filled with water) for balancing, you can use the following method:

3x balancing method: When there are odd numbers of centrifuge tubes that are multiples of 3. such as 3. 6. 9. 15. 21:

The 2x balancing plus 3x balancing method: When there are 5. 7. 11. 13. 17. or 19 tubes, you can follow the principle of central symmetry, first keep 3 tubes in balance, and then keep the other 2 tubes in balance. This way, the result is still balanced.

2. Safety Issues

(1) Never open the centrifuge lid during centrifugation to prevent injury from flying objects.

(2) Centrifuge within the recommended speed range to prevent overexposure.

(3) For volatile, toxic, or corrosive samples, additional protective measures should be taken, and operations should be performed in a fume hood.

(4) Check the chemical resistance of all centrifuge tube materials. The material selection must be compatible with the experiment. Do not use counterfeit or substandard centrifuge tubes, or aged, deformed, or cracked centrifuge tubes.

(5) For refrigerated centrifuges: When the centrifuge is pre-cooled, the centrifuge lid must be closed. After centrifugation, remove the rotor and place it upside down on the lab bench, wipe off any remaining water in the chamber, and leave the centrifuge lid open. During pre-cooling, the rotor cap can be placed on the centrifuge platform or the lab bench. Never leave it loosely on the rotor, as it could fly off and cause an accident if the centrifuge is accidentally started!

(6) If the electric centrifuge makes noise or vibrates, immediately disconnect the power and troubleshoot the problem.

(7) After separation, turn off the centrifuge first. Only after the centrifuge has stopped rotating can you open the centrifuge cap and remove the sample. Do not force it to stop.