Fractionation of samples typically starts with centrifugation. Using a centrifuge, one can remove cell debris, and fractionate organelles, and cytoplasm. For example, nuclei, being relatively large, can be spun down at fairly low speeds. Once nuclei have been sedimented, the remaining solution, or supernatant, can be centrifuged at higher speeds to obtain the smaller organelles, like mitochondria. Each of these fractions will contain a subset of the molecules in the cell. Although every subset contains fewer molecules than does the crude lysate, there are still many hundreds of molecules in each. Separating the molecule of interest from the others is where chromatography comes into play. We will consider several separation techniques.
Figure 9.2.1: Fractionation by centrifugation
Many chromatographic techniques are performed in “columns." These are tubes containing the material (called the “support") used to perform the separation . Supports are designed to exploit the chemical, or size, differences of the many molecules in a mixture. Columns are “packed" (filled) with the support and a buffer or solvent carries the mixture of compounds to be separated through the support. Molecules in the sample interact differentially with the support and consequently, will travel through it with different speeds.