Chromatography and isotopes

The different solubilities of substances in aqueous and organic solvents provide another basis for analysis. In its earlier form, a separation was conducted in complex apparatus by partition of substances in various solvents. A simplified form of the same principle evolved as ‘‘paper chromatography,” in which small amounts of substances could be separated on filter paper and identified by appropriate colour reactions. In contrast to electrophoresis, this method has been applied to a wide variety of biological compounds and has contributed enormously to research in biochemistry.

What Is the Difference Between a Peptide and a Protein?

The general principle has been extended from filter paper strips to columns of other relatively inert media, permitting larger scale separation and identification of closely related biological substances. Particularly noteworthy has been the separation of amino acids by chromatography in columns of ion-exchange resins, permitting the determination of exact amino acid composition of proteins. Following such determination, other techniques of organic chemistry have been used to elucidate the actual sequence of amino acids in complex proteins. Another technique of column chromatography is based on the relative rates of penetration of molecules into beads of a complex carbohydrate according to size of the molecules. Larger molecules are excluded relative to smaller molecules and emerge first from a column of such beads. This technique not only permits separation of biological substances but also provides estimates of molecular weights.

Perhaps the single most important technique in unravelling the complexities of metabolism has been the use of isotopes (heavy or radioactive elements) in labelling biological compounds and “tracing” their fate in metabolism. Measurement of the isotope-labelled compounds has required considerable technology in mass spectroscopy and radioactive detection devices.

A variety of other physical techniques, such as nuclear magnetic resonance, electron spin spectroscopy, circular dichroism, and X-ray crystallography, have become prominent tools in revealing the relation of chemical structure to biological function.