Density Gradient Centrifugation – Principle, Protocol, Uses

Density gradient centrifugation is a mechanical separation technique used to separate and concentrate particles present in a liquid medium. It is carried out by using centrifugal force and a density gradient medium in a centrifuge tube.

In this method, the sample is placed in a tube containing fluid whose density gradually increases from top to bottom. When the tube is rotated at high speed, the particles move through the gradient. They are separated into different bands according to their size, shape, mass and density.

The gradient medium is generally made by sucrose, cesium chloride or Percoll. It gives stability to the separated bands and prevents their mixing by convection currents. The increasing density and viscosity of the medium helps in clear separation of particles.

Density gradient centrifugation is mainly of two types. These are rate-zonal centrifugation and isopycnic centrifugation. In rate-zonal centrifugation, particles are separated mainly on the basis of size and mass. In isopycnic centrifugation, particles move until their density becomes equal to the density of the surrounding gradient medium.

Principle of Density gradient centrifugation

Density gradient centrifugation is based on the separation of suspended particles in a liquid medium having increasing density from top to bottom of the centrifuge tube. When centrifugal force is applied, the particles move through the gradient and separated according to their size, shape, mass and density.

In rate-zonal centrifugation, the sample is placed as a thin layer on the top of the density gradient. The maximum density of the gradient is lower than the density of the particles. During centrifugation, particles move downward at different speed and form separate bands in the tube. The gradient medium also prevents the mixing of these bands due to convection current.

In isopycnic centrifugation, the separation is based on the buoyant density of the particles. Here, the density gradient covers the whole density range of the particles present in the sample. Each particle moves in the gradient until it reaches a position where its own density become equal to the density of the surrounding medium.

At this point, the particle does not move further. The centrifugal force is balanced by the buoyant force. Thus, particles are separated as stable bands at different position in the tube. This method is used for separation of DNA, viral particles, cell organelles and other macromolecules.

Parts/Components of Density Gradient Centrifugation

The following are the important parts/components of density gradient centrifugation-

1. Centrifuge machine- It is the main instrument used to rotate the sample tubes at high speed and produce centrifugal force.
2. Refrigerated chamber- It holds the rotor and maintains required temperature during centrifugation.
3. Electric motor- It gives high speed rotational power to the rotor.
4. Drive spindle or hub- It is the central part on which the rotor is attached and rotated.
5. Swinging-bucket rotor- It contains metal buckets that swing outward and become horizontal during rotation. It is commonly used for good band separation.
6. Fixed-angle rotor- It holds the centrifuge tubes at a fixed angle during centrifugation. It is used for many isopycnic separations.
7. Vertical rotor- It holds the tubes in vertical position during the run. It shortens the sedimentation path of particles.
8. Zonal rotor- It is a large volume rotor having core and radial septa. It is used for loading and unloading of large sample during centrifugation.
9. Centrifuge tubes and bottles- These are used to hold the density gradient medium and sample. They may be made of plastic, glass, polyallomer or cellulose nitrate.
10. Adapters- These are used inside buckets to support different size of centrifuge tubes.
11. Rotor lids and bucket caps- These are used to close the rotor or buckets tightly. They also help in aerosol-tight sealing for safety.
12. O-rings and vacuum grease- These are used to seal the rotor and bucket parts and prevent leakage.
13. Syringes and cannulas- These are used for careful layering of different density solutions during preparation of step gradient.
14. Pipettes- These are used to add sample or gradient solution slowly without disturbing the layers.
15. Two-chamber gradient maker- It is used to prepare smooth continuous density gradient by mixing light and dense solutions.
16. Automated gradient former- It is used to prepare density gradient automatically by tilted rotation of the tube.
17. Gradient pump- It is used to pump different concentration of gradient medium into tube or zonal rotor.
18. Hollow needle- It is used to puncture the bottom of plastic tube and collect gradient fractions drop by drop.
19. Piston fractionator- It pushes the gradient layers out of the tube in controlled manner and helps in clean fraction collection.
20. Fraction collector- It collects separated fractions in different tubes or vials in fixed volume.
21. UV-Vis spectrophotometer- It is used to detect protein or nucleic acid bands in the collected fractions.
22. Refractometer- It is used to measure refractive index of fractions. This helps to calculate density of each gradient layer.

Density gradient centrifugation Protocols

The following are the steps involved in density gradient centrifugation-

1. Gradient preparation

The density gradient medium is first prepared in a centrifuge tube. The medium may be prepared as discontinuous gradient or continuous gradient.

In discontinuous gradient, solutions of different density are layered one over another. The dense solution is kept at the bottom and lighter solution is kept toward the top. It is usually done carefully by using syringe or cannula.

In continuous gradient, the density changes gradually from top to bottom of the tube. It can be prepared by using gradient maker, gradient master, diffusion method or freeze-thawing method. In self-generating gradient, Percoll or cesium chloride is mixed with the sample and gradient is formed during centrifugation.

2. Sample loading and balancing

The sample is then loaded into the prepared gradient. In rate-zonal centrifugation, the sample is placed as a thin layer on the top of the gradient. The volume of sample is kept small so that it forms a narrow zone.

In isopycnic centrifugation, the sample may be placed on the top, bottom or mixed with the gradient medium. The particles then move until they reach their own buoyant density position.

Before centrifugation, all tubes are balanced properly. Opposite tubes and buckets should have equal weight. This prevents vibration and damage of the centrifuge.

3. Centrifugation

The tubes are placed in a suitable rotor. Swinging bucket rotor is generally used for rate-zonal centrifugation because it gives better band separation. Fixed angle rotor or vertical rotor can be used for isopycnic centrifugation.

The centrifuge is then set with required speed, time and temperature. The condition depends on the type of particle, gradient medium and method used. The acceleration and braking should be slow to prevent disturbance of the gradient and separated bands.

4. Fractionation

After centrifugation, the separated bands are collected from the gradient. This process is called fractionation.

In bottom puncturing method, the bottom of the tube is punctured with a needle and the gradient is collected drop by drop. In top unloading method, a dense inert liquid is injected from bottom and the gradient comes upward and collected from the top.

In piston fractionation, a piston is inserted from the top of the tube. It pushes the gradient downward and different layers are collected separately.

5. Analysis

The collected fractions are then analysed for the presence of desired particles. The fractions may be checked by UV spectrophotometry at 254 nm or 280 nm.

The density of each fraction can be measured by refractometer. Further analysis can be done by SDS-PAGE, mass spectrometry or biological activity test. This helps to identify the purity and position of the target particle in the gradient.

Uses of Density gradient centrifugation

The following are the uses of density gradient centrifugation-

• Cell separation- It is used to separate specific cell types such as lymphocytes, monocytes, Leydig cells, stem cells and bone marrow cells.
• Organelle isolation- It is used to isolate subcellular organelles such as mitochondria, nuclei, chloroplasts, lysosomes and peroxisomes from tissue homogenate.
• Membrane separation- It is used for separation of plasma membrane fragments and cellular vesicles.
• Nucleic acid purification- It is used for purification of genomic DNA, plasmid DNA and RNA.
• DNA density separation- It is used to separate DNA molecules having different densities such as N14 DNA and N15 DNA. It is also used for isolation of Okazaki fragments.
• Protein complex isolation- It is used to isolate endogenous protein complexes, enzymes and plasma lipoproteins.
• Ribosome separation- It is used for separation of ribosomal subunits such as 30S, 50S and 70S. It is also used to separate polysomes.
• Study of macromolecules- It is used to determine density, molecular weight and sedimentation coefficient of macromolecules.
• DNA form detection- It is used to detect conformational changes in DNA such as linear DNA, relaxed circular DNA and supercoiled DNA.
• Virus purification- It is used for purification of viruses such as Influenza, Polio, Dengue, HIV-1 and Herpes virus for research and vaccine production.
• Viral capsid separation- It is used to separate empty viral capsids from full viral capsids. This is important for recombinant Adeno-Associated Virus (rAAV) vector purification.
• Virus study- It is used to study virus assembly and to identify sub-viral particles.
• Nanoparticle purification- It is used to separate and purify synthetic nanoparticles according to their size, shape and density.
• Carbon nanotube sorting- It is used to sort single-walled carbon nanotubes (SWNTs) on the basis of chirality and electronic properties.
• Material particle separation- It is used for separation of metal nano-colloids such as Germanium particles and nanometer-sized pigment particles found in inks.

Advantages of Density Gradient Centrifugation

The following are the advantages of density gradient centrifugation-

• High resolution separation- It gives high resolving power and can separate particles having very small difference in sedimentation coefficient.
• Less cross-contamination- It prevents contamination between slow moving and fast moving particles. The separated particles form bands instead of mixed pellet.
• Stable separation- The gradient medium has increasing density and viscosity. It stabilizes the moving bands and prevents disturbance by convection currents.
• Sample preservation- The particles remain suspended in the gradient medium. They are not forced into a hard pellet, so delicate particles are preserved better.
• Analytical use- It is used to measure sedimentation coefficient of macromolecules. This also helps to estimate molecular weight and detect conformational changes.
• Viral capsid separation- It is useful for separation of full viral capsids from empty viral capsids on the basis of their buoyant density.
• Serotype independent method- It can be used for purification of different viral serotypes by same method. It does not require specific affinity resin for each serotype.
• Cost effective in small scale- It is more economical for research work and small-scale clinical trial. It does not need costly specialized resins like affinity chromatography.

Limitations of Density Gradient Centrifugation

The following are the limitations of density gradient centrifugation-

• Limited capacity- It can separate only small amount of sample at one time. It is difficult for large-scale and commercial purification.
• Droplet sedimentation- Small molecules of gradient medium may diffuse into the sample layer very fast. This forms an unstable dense zone and reduces resolution.
• Osmotic damage- High osmotic gradient media can remove water from sensitive cells, organelles and vesicles. This causes shrinkage and changes their natural density.
• Wall effect- Particles may strike the wall of centrifuge tube during sedimentation. This produces abnormal sedimentation and disturbed bands.
• High viscosity- Some gradient media become highly viscous at high concentration. This slows down particle movement and increases centrifugation time.
• Hydrostatic pressure damage- High speed centrifugation produces high pressure inside the tube. This may damage delicate biological particles.
• Mechanical sensitivity- The gradient layers are very fragile. They may be disturbed by vibration, sudden acceleration and sudden braking.
• Low yield- High purity separation may reduce the total yield. Some desired particles may be lost during removal of contaminants.
• High cost- It needs costly gradient media, suitable centrifuge and special rotors. Manual preparation and fraction collection also makes the process labor intensive.

Properties of density gradient 

A perfect density gradient media includes the following properties:

• Enough solubility to provide the necessary range of densities
• Does not create solutions with high viscosity in the required density range.
• Does not have to be hypoosmotic or hyperosmotic when the particles that are to be separated are highly osmotically sensitive
• Solutions for the gradient must be adjusted to the pH and Ionic strengths that are compatible with the particles that are being separated.
• Does not affect the biological activities of the sample
• Nontoxic and not processed by cells
• Do not disrupt assay protocols or cause a reaction with the tubes for centrifuge.
• The property can be used to determine the level of concentration
• Easy to get rid of the pure product
• Autoclavable
• Reasonable price

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