Separation

Different separation techniques can be used to enrich certain particles (DNA, RNA, proteins, organelles, vesicles, micelles, cells etc.) specifically from complex biological mixtures such as cell and tissue homogenates, blood, urine and other body fluids, so that they can then be selectively investigated. Separation of these types of particles can be based either on the different sedimentation rates of different particles in a fluid, or on their different densities. Density gradient centrifugation (also referred to as band, equilibrium or isopycnic centrifugation), exploits the principle that particles of a certain density migrate into a density gradient until they reach an equilibrium density layer. The first applications of density gradient centrifugation were reported in the early 1950s. Back then, cell organelles were enriched with the aid of buffered saccharose gradients and it is uncontested that the knowledge gained with these enriched materials made a contribution to modern molecular biology. Soon it was discovered that the enrichment of mammalian cells requires more complex separation media, particularly due to their sensitivity towards osmotic fluctuation. Noble and Boyum described methods for separating mononuclear cells from whole blood and bone marrow as early as 1967 and 1968. Based on this pioneering scientific work, numerous applications in today‘s biomedical research and routine diagnostics require highly enriched, viable and functionally intact cell populations as the starting material. The separation of such cells by density gradient centrifugation has proven to be the most often used method due to its uncomplicated and robust nature.