An Introduction to Ultracentrifugation
The Physics Behind the Technology
Centrifugal force influences every particle during centrifugation. How fast particles separate depends on the extent of this force (i.e., how fast the centrifuge is spinning), the physical properties of the particles (e.g., mass, size, shape), and the viscosity and density of the medium.
By manipulating centrifugal speed, medium viscosity, and medium density, centrifugation can accomplish some otherwise extremely challenging separations. Over the decades of centrifugal use, various techniques have been developed to complete specific separations.
Pelleting and Differential CentrifugationPelleting is among the most common centrifugal techniques. Particles sediment out of solution and onto the vessel wall due to centrifugal force. After decanting, the supernatant will be separated from the pellet. Differential centrifugation exploits the fact that different particles pellet at different rates based on their size, shape, and density. By iteratively pelleting and separating sample in multiple centrifugal runs, differential centrifugation can isolate increasingly small materials from a mixture.
Some loss in the pellet can be observed. This is due to the balance between sedimentation and diffusion—the substance is not completely pelletized and is partly diffused in the supernatant.
If separated from the pellet, there can be a loss of the relevant components. To prevent this from happening, centrifugation must be done at the largest-possible g value, lowest-possible temperature, and braked as quickly as possible.
Differential centrifugation produces reliable results if the sedimentation coefficients of the particle are differentiated by at least a factor of three. A typical example of differential centrifugation is the pelletizing of cell membranes or the isolation of a specific EV.
Rate Zonal Centrifugation
Rate zonal centrifugation benefits from the advantages of density gradients—a liquid column whose density increases over the length of the tube. Sample is layered at the top of the tube above a pre-formed gradient.
Like when pelleting, samples are separated by their sedimentation coefficient (which factors size, shape, and buoyant density). However, in rate zonal centrifugation, the centrifuge run is stopped before particles pellet, and the different entities are seen as bands in the tube. The bands closest to the bottom of the tube have a higher sedimentation coefficient than the bands above them.
Particles of similar size and mass migrate at only slightly different rates. To still achieve a good separation, either (1) increase media viscosity or (2) use a longer pathlength tube (similar to size exclusion chromatography or gel electrophoresis); swinging-bucket rotors offer long pathlengths to meet this need.
Finally, rate zonal experiments often start with a concentrated sample in a small volume (generally 10-20% relative to the volume of the total gradient). Using a small sample volume minimizes the differences in starting points at the beginning of the run. Conversely, the larger the sample, the greater the difference in the starting points of the materials being separated. Larger sample loads will have wider bands and less band separation, which can make pure sample recovery more challenging. If available, wider tubes allow for a greater sample load
On the other end of the spectrum, isopycnic centrifugation separates materials based on their apparent (buoyant) density in solution. It’s important to mention that the buoyant density of a material refers to the density of the surrounding fluid at which the material will neither float nor sediment; this value is therefore not an intrinsic particle property and may vary significantly in different gradient media.
Buoyant density also known as a particle’s isodensity or equilibrium density. Isopycnic gradients often benefit from a short pathlength during centrifugation (which minimizes the time required to reach equilibrium) and a longer pathlength at rest after the gradient has reoriented; this is typically best achieved in vertical and near-vertical rotors and is further aided by tubes with a high aspect ratio (length/width).
For isopycnic separations, the commonly used density gradient materials include:
These share the unique property of generating self-forming density gradients in the presence of sufficiently large centrifugal fields, and the g-force applied affects the shape and range of the gradient. Temperature and the starting concentration of the density gradient material also contribute to defining the final gradient.
During centrifugation, these gradients reach equilibrium, which is predominantly stabilized by the balance of centrifugal force and diffusion. Upon reaching equilibrium, the gradient profile remains constant indefinitely while spinning and particles in the solution migrate to reach the point in the gradient that matches their buoyant density, also known as the isopycnic point.
Separation occurs solely due to the density difference of the particles. Since no changes to the gradients formed occur with increasing duration of centrifugation, time is not a factor after equilibrium has been attained.
Because the gradients used in isopycnic separations self-form, there is no inherent need to add the sample at the top or bottom of a gradient. Instead, the gradient-forming material is often mixed with the sample and then loaded uniformly into the centrifuge tube. This may allow for exceptionally large sample loads (greater than 90% of tube volume) and remarkably simple gradient preparation, especially compared to layering gradients for rate zonal.
In some cases, these gradients may still be layered manually to shorten the time it takes for the gradient to reach equilibrium and reduce the total runtime by more than 75% while maintaining ample separation.
Our History in Centrifugation
We introduced the first commercial ultracentrifuge in 1947. Fast forward to today and you’ll discover that we’ve advanced centrifugation technology at a pace unmatched in the industry.
Designed as a complete solution from tube to rotor to centrifuge, our broad centrifugation product portfolio delivers brilliance at every turn.