Rotors for Density Gradient Centrifugation
Gradients can be centrifuged in fixed-angle, vertical, near-vertical and swinging-bucket rotors. If a larger capacity is required, scaling up to zonal and continuous flow rotors is also possible.
Let’s look at the differences between each type of rotor.
In swinging-bucket rotors, tubes hang vertically while at rest, swing out during centrifugation and are then at a 90° angle with respect to the rotor axis. Because of the physical reorientation of the tubes in the rotor, the gradient always aligns with the direction of gravity. Swinging-bucket rotors are especially useful for pelleting or rate zonal density gradient experiments. Their longer pathlength ensures bands are better distributed when separating particles by size and mass.
Zonal rotors are filled and emptied directly during centrifugation. The conditions for zonal gradient centrifugation are comparable to that of swinging-bucket rotors. The gradient is layered, the sample is loaded from above while the rotor is rotating and there is no gradient reorientation.
In fixed-angle rotors, the centrifuge tubes are located at specific angles to the rotor axis. The angles are different from rotor to rotor, but typically range from 20° to 35°. Compared to swinging-bucket rotors, fixed-angle rotors have a smaller separation distance which typically results in a shorter separation time. Fixed-angle rotors are the most versatile rotors. They will not provide as much separation between bands as a swinging-bucket rotor when running rate zonal experiments, but they may offer faster run times. They also will not provide the high resolution and fast run times of a vertical rotor for isopycnic experiments, but unlike a vertical rotor, fixed-angle rotors can pellet effectively.
Vertical rotors feature the shortest pathlength, and thus fastest run time of any rotor type. For isopycnic density gradients they also provide the highest resolution. The tube slots are fixed vertically and are at a right angle to the centrifugal field during centrifugation. The short pathlength and low k factors of these rotors mean that the particle has only a short distance to travel, which reduces run times. Vertical rotors should not be used for pelleting.
Near-vertical rotors resemble fixed-angle rotors. However, the angle with respect to the axis is clearly smaller, ranging between 7° and 9°. These angles are designed such that a pellet can form (just like in a fixed-angle), but the separation distance and time is similar to that of a vertical rotor. Near-vertical rotors are preferred in some instances over vertical rotors despite their slower run time because the likelihood of contaminating your material of interest is lower, as contaminants will either sediment or float in a near-vertical rotor.
|Rotor Type||Separation Distance||Wall Effects||Reorientation||Centrifugation Time|
|Swinging-Bucket Rotors||Longest separation distance
||No wall effects of the gradients
||Longest centrifugation time
|Fixed-Angle Rotors||Medium separation
||Shorter centrifugation time than swinging-bucket rotors, longer than vertical and near-vertical rotors
|Vertical Rotors||Shortest separation distance
||Wall effects||Gradient reorientation
||Shortest centrifugation time
|Near-vertical Rotors||Close to the shortest separation distance
||Wall effects||Gradient reorientation
||Close to the shortest centrifugation time
A gradient is always aligned with the accelerating field. However, in fixed-angle, near-vertical, and vertical rotors, the gradient’s alignment within the tube changes when the rotor is at rest versus during centrifugation.
At first sight, the reorientation of a gradient might be considered a disadvantage. However, because the separation is spread out during reorientation after centrifugation, the bands are further apart from one another, which could make for easier extraction.
To illustrate with an example, 39 mL tubes are 3.5x taller than they are wide and we are using a vertical rotor.
Because the volume of solution between bands always is the same, the distance between the bands will therefore expand 3.5x when at rest after centrifugation. By contrast, the rotor itself reorients when using a swinging-bucket rotor. The density gradient will therefore not reorient, and the distance between the bands will remain as it was when in the centrifuge.
During centrifugation, the accelerating field is applied at 90° with respect to the axis of rotation, and the walls of the tubes have a different angle to that of the axis of rotation.
The sedimenting particles are first centrifuged against the wall of the tube. Ideally, the particles will run down the tube wall, however, this may not always happen and some of the material moving downward will stick.
- Wall effects are negligibly small with swinging-bucket rotors. Positioning the centrifuge tubes in the direction of the accelerating field has the further advantage of providing better quality separation, as the gradients always remain aligned in the same direction with respect to the accelerating field.
- With zonal rotors, there are no wall effects as long as the sample does not sediment at the outer wall of the rotor.
- Wall effects can be a consequence when using fixed-angle, vertical and near-vertical rotors.
In vertical rotors, it’s not possible to prevent the bands from coming in contact with sedimented or floated material. By contrast, near-vertical and fixed-angle rotors do provide a solution to this because their separation distances are nearly as short as those of vertical rotors, but the geometry ensures that pellets are not distributed all over the tube side and instead collect at the bottom on the outside, with the floated material collecting at the top on the inside.
Swinging-bucket and zonal rotors are suitable for gradient separation where contact with the tube wall is to be avoided.
Choosing the Right Rotor for Your Workflow
With so many unique rotor variations out there, we realize that choosing the right one for your specific needs can sometimes be tricky. Rest assured, we’re here to help. Whether you’re separating via pelleting, flotation or density gradient, our Intellifuge rotor calculator makes it easier to find what you need.