Isopycnic centrifugation

Isopycnic centrifugation, also known as density gradient centrifugation or equilibrium sedimentation is a technique used to separate molecules on the basis of buoyant density. (The word "isopycnic" means "equal density".) Typically, a "self-generating" density gradient is established via equilibrium sedimentation, and then analyte molecules concentrate as bands where the molecule density matches that of the surrounding solution. To illustrate the process, consider the fractionation of nucleic acids such as DNA. To begin the analysis, a mixture of caesium chloride and DNA is placed in a centrifuge for several hours at high speed to generate a force of about 10^5 x g (earth's gravity). Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA. After some time a gradient of the caesium ions is formed, caused by two opposing forces: diffusion and centrifugal force. The sedimenting particles (caesium ions) will sediment away from the rotor, and become more concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of solvated caesium chloride and is always directed towards the center of the rotor. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is more dense near the bottom of the tube, and less dense near the top.

The DNA molecules will then be separated based on the relative proportions of AT (adenine and thymine base pairs) to GC (guanine and cytosine base pairs). An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than supercoiled plasmid DNA, which is denser than linear chromosomal DNA.

References

• Gerald Karp, Cell and molecular biology: Concepts and experiments, fourth edition, 2005, Von Hoffman press

Isopycnic gradient ultracentrifugation is a process used frequently to separate plasma LPs (lipoproteins). Since lipids have lower buoyant densities than proteins, LPs containing higher % of lipid component relative to protein component will float to upper layers.

References

• Roger A. Davis and Jean E. Vance, Structure, assembly and secretion of lipoproteins, 1996, Elsevier Science B. V.