Density Gradient Centrifugation

density gradient centrifugation tube

Density gradient ultracentrifugation (DGUC) is a centrifuge-based technique for providing superior purification of viral vectors (e.g., isolating full AAV particles from partial and empty capsids), along with other materials (such as plasmid DNA) in gene therapy production workflows. DGUC results in a layered gradient based on the density of materials in the original sample.

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The Types of Gradients

To enable the desired separation of different sample materials, different gradients are developed to satisfy specific demands. These types of gradients are differentiated based on chemical structure:

  • Heavy metal salts (e.g., CsCl, Cs2O4)
  • Carbohydrate (e.g., Sucrose, Ficoll)
  • Iodinated compounds (e.g., Iodixanol, Nycodenz)
  • Colloidal silica gels (e.g., Percoll)

Heavy Metal Salts: CsCl and Cs2O4

The commonly used gradient media for isopycnic centrifugation of nucleic acids are CsCl and Cs2O4.

Cs2O4 forms a gradient approximately twice as steep as CsCl with the same rotor speed, allowing nucleic acids of greatly differing buoyant densities to be captured. However, the hydration in both media is distinctly different.

  • In CsCl, the hydration is reduced and the DNA shows an average density of 1.7 g/cm3
  • In Cs2O4, the hydration is greater and results in an average density of 1.4 g/cm

One advantage of CsCl over Cs2O4 is the availability of a linear correlation between guanine and cytosine (G + C) content of the DNA and its density—the greater the G + C content, the higher the DNA density.

However, with CsCl it’s not possible to create sufficiently dense solutions to band RNA. In contrast, RNA can be analyzed in Cs2O4 gradients, although high molecular RNA precipitates. To minimize this, 4 M urea or 5% DMSO is often added.

Carbohydrates: Sucrose and Ficoll

Sucrose is the most important separation medium in zonal centrifugation. It’s also used for isopycnic banding of viruses, organelles and membranes.

3 important things you need to know about sucrose:

  1. One disadvantage is its osmotic properties— solutions with more than 9% m/v sucrose are hypertonic
  2. Highly concentrated solutions are extremely viscous and small molecules may not band at the point corresponding to their density during isopycnic centrifugation
  3. Using it to separate RNA can be problematic due to the potential contamination with ribonucleases

Ficoll is manufactured by copolymerization of sucrose and epichlorohydrin, creating a polysaccharide with an average molecular weight of 400,000 dalton. It’s used for the centrifugation of osmotically sensitive particles—such as cells—as solutions under 20% m/v are practically osmotically inactive.[1]

Iodinated Compounds: Iodixanol, Nycodenz

Iodixanol (commonly sold as a sterile aqueous 60% m/v solution called OptiPrep™) has been increasingly popular in recent years for a wide range of biologics. These two compounds:

  • Show fundamentally lower osmolarity and viscosity compared to sucrose
  • Are stable over a pH range of 2 to 12.5
  • Are non-ionic and highly water soluble

Iodixanol step gradients are used to shorten centrifugation run times for separations that otherwise would use CsCl, such as viral vector separations. [1]

Colloidal Silica Gels: Percoll

Percoll is a colloidal silica gel consisting of particles with an average diameter of 15-30 nm that have been coated with polyvinylpyrrolidone. This coating reduces interaction with biological material and stabilizes the colloid against salt addition as well as freezing and thawing.

One big advantage of Percoll is the low osmolarity of its solutions. Percoll gradients can be either pre-formed or can form self-generated gradients in fixed-angle or vertical rotors. Its use, however, is restricted to isopycnic density gradient centrifugation.

Gradient Profiles

There are several different types of gradient profiles, including:

  • Linear
  • Discontinuous
  • Convex
  • Concave
  • Isokinetic
  • Linear-logarithmic

The profile of a gradient has a great effect on separation and there are many differences when it comes to characteristics and applications. For instance:

  • Linear gradients are used for centrifugation of proteins, hormones, nucleic acids and viruses
  • Discontinuous gradients deliver good results for the separation of cells and viruses
  • Concave gradients are widely used for the separation of lipoproteins

Gradient Formation

For the generation of gradients, we differentiate between continuous and discontinuous.

Discontinuous Gradients

These are created using overlayering and underlayering of the individual gradient materials. 

Discontinuous (or step) gradients often consist of 3 – 6 layered steps. The steps are frequently designed to stop certain components in a sample from proceeding beyond a position, or step. Where the gradient density exceeds that of the component being halted, the component will stop movement down the tube at the step. More dense materials can pass through the step.

A chart depicting the flattening of a discontinuous gradient in centrifugation over time.
Flattening of a discontinuous gradient with increasing time. How quickly the gradient flattens out depends on the interaction between the diffusion speed of the gradient material and its viscosity.

Underlayering is used for low-viscosity media. With it, more exact gradients can be formed, however, care must be taken to ensure that no air bubbles are introduced. Underlaying is especially useful when tube openings are small (e.g., when using Quick-Seal® or OptiSeal tubes) or when using a non-wettable tube material (e.g., when using polypropylene or Ultra-Clear tubes).

Overlaying is when the solution with the highest density is laid down first and then a solution with a lower density is added using a pipette placed against the wall of the tube. It’s used for high-viscosity media.  

Continuous Gradients

These can be generated using different methods, including:

  • Diffusion of discontinuous gradients
  • Gradient mixing chamber
  • Commercial gradient mixer
A graphical representation of a double-chamber gradient mixer.
Double-chamber gradient mixer.

For isopycnic centrifugation with self-generating gradients (e.g., CsCl or Percoll), the gradient profile depends on the initial density of the solution, the rotor geometry, the rotational speed and the temperature. By adjusting each individual parameter, almost any gradient can be formed.

For rate zonal centrifugation, it is necessary to pre-form the gradients. A number of concentrations of a solution can be placed in a tube and immediately spun to form a discontinuous gradient. If the spin is delayed, the gradient flattens out and becomes more linear, convex or concave. 

Gradient mixing chambers can be used to form both discontinuous or continuous gradients. If creating a linear gradient, gradient mixing chambers can be used by connecting two vessels with identical geometry and ensuring thorough mixing of the solutions. By so doing, all gradient profiles can be formed by varying the discharge conditions from each of the chambers.

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.

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