The technique used to separate organelles is referred to as Ultracentrifugation or Subcellular Fractionation. It is used to extract undamaged organelles from tissue and separate them so they structure and Biochemistry can be studied in a greater detail under an electron microscope. The process starts with tissue, in this case leaf tissue first being sliced into fine pieces with scissors or a scalpel to decrease size. Fine strips of tissue will then be placed in a test tube.
Cells contain enzymes (proteins) which are static in numerous parts of the cell, if the cell is to be burst these enzymes will start digesting the cell or organelles that they do not normally interact with, so an ice cold environment is used and an ice cold chemical solution called a buffer is added to the tissue, these in term decrease enzyme activity (enzymes become rigid at low temperatures). With the risk of digestion by means of enzymes gone a homogeniser, a fine brush that fits the test tube perfectly and spins (has a slight resemblance to a kitchen blender), is used to burst the cells and create a mixture of organelles. Before continuing the process the mixture is filtered to remove impurities, leaving only the organelles.
The mixture will be placed in an ultracentrifuge; a machine that spins at rapid speeds, thus increasing the gravitational field, this causes the organelles to separate according to size and density. The heavier organelles for example the nuclei will ‘sink’ to the bottom of the test tube at lower speeds (around 800 rpm) while ribosomes require speeds of around 3,000 rpm and upwards. The collection of organelles at the bottom of the test tube is known as a pellet, this will be separated from the supernatant (mixture of organelles still in the test tube). This step will be repeated numerous times until the organelle wanted is separated.
Although the process in the ultracentrifuge is quite accurate some impurities, i.e. Other Organelles of similar size and density can be mixed with in the pellet, so a process known as density sucrose gradient separation is used. A solution of sucrose is placed in the ultracentrifuge with the impure pellet and by diffusion of sucrose the impurities and wanted sample are separated according to density and mass.
Source by Carl S. Richardson