All natural products begin as mixtures with other compounds from the natural source, often very complex mixtures, from which the product of interest must be isolated and purified. The isolation of a natural product refers, depending on context, either to the isolation of sufficient quantities of pure chemical matter for chemical structure elucidation, derivitzation/degradation chemistry, biological testing, and other research needs (generally milligrams to grams, but historically, often more), or to the isolation of "analytical quantities" of the substance of interest, where the focus is on identification and quantitation of the substance (e.g. in biological tissue or fluid), and where the quantity isolated depends on the analytical method applied (but is generally always sub-microgram in scale). The ease with which the active agent can be isolated and purified depends on the structure, stability, and quantity of the natural product. The methods of isolation applied toward achieving these two distinct scales of product are likewise distinct, but generally involve extraction, precipitation, adsorptions, chromatography, and sometimes crystallizations. In both cases, the isolated substance is purified to chemical homogeneity, i.e. specific combined separation and analytical methods such as LC-MS methods are chosen to be "orthogonal"—achieving their separations based on distinct modes of interaction between substance and isolating matrix—with the goal being repeated detection of only a single species present in the putative pure sample. Early isolation is almost inevitably followed by structure determination, especially if an important pharmacologic activity is associated with the purified natural product.
Structure determination refers to methods applied to determine the chemical structure of an isolated, pure natural product, a process that involves an array of chemical and physical methods that have changed markedly over the history of natural products research; in earliest days, these focused on chemical transformation of unknown substances into known substances, and measurement of physical properties such as melting point and boiling point, and related methods for determining molecular weight. In the modern era, methods focus on mass spectrometry and nuclear magnetic resonance methods, often multidimensional, and, when feasible, small molecule crystallography. For instance, the chemical structure of penicillin was determined by Dorothy Crowfoot Hodgkin in 1945, work for which she later received a Nobel Prize in Chemistry (1964).