Use of High-Efficiency Nonpolar Columns in Systematic Separation and Identification of Steroids by Combined TLC-GLC Method of Analysis

Abstract

In our attempts to develop a general, systematic method of analysis for steroid hormones and metabolites serious limitations to the exclusive use of gas-liquid chromatography (GLC) for this purpose soon became apparent. Natural mixtures of steroids often contain so many isomers that difficultly separable pairs, though not necessarily the same ones, arise no matter what type of GLC column is used for their separation. In addition, steroids extracted from biological materials contain relatively large amounts of extraneous, interfering substances which must be removed without inducing qualitative or quantitative alterations of steroids. In 1963, I observed the ability of thin-layer chromatography (TLC) to provide both the required cleanup and quantitative preliminary separation of total steroids into simpler mixtures(1). It turned out that TLC in a chloroform:methanol system, and GLC on a nonpolar column made an excellent combination since separations achieved by the two methods were complementary(2–5).