Principles of Biochemistry (2+1)

6.6.2 Determination of the structure of an unknown acid

Running an unknown fatty acid on a column of known properties can give some information about its structure.

Co-chromatography with authentic standards on different types of columns will serve to identify a fatty acid provisionally and the chain length of an unknown substance can be estimated from identified peaks because there is a simple linear relationship between the log of the retention volume (time) and the carbon number for each particular type of acid on a given column.

Samples of a given component can be collected from the gaseous effluent on the column by allowing the gas stream to pass through a wide glass tube loosely packed with defatted cotton or glass wool wetted with a solvent such as methanol or petroleum ether. Such an isolated component can then be run on another type of column or subjected to specific chemical reactions.

For example, the identity of an unsaturated fatty acid can be confirmed by H 2 reduction to the corresponding saturated acid and by oxidation with permanganate or ozone. With oxidation, the unsaturated acid is split into fragments at its double bonds so that analysis of the products consisting of a 9C dicarboxylic acid and nonanoic (9:0) acid.

Infrared spectroscopy can be used to differentiate between cis and trans double bonds. Instead of collecting a sample of an individual component the gas chromatograph can also be connected directly to a mass spectrometer which allows the identification of many lipid components including most fatty acid. This GCMS combination is increasingly used for fatty acid analysis.

Identification of fatty acids often also involved an initial fractionation of mixtures by AgNO₃ ^- silicic acid chromatography. In this method, either on plates (TLC) or in columns, fatty acids are separated according to the number of double bonds. The mixture, say, unsaturated acids can then is more easily identified by GLC.