Clinical Chemistry, Vol 17, 757-764, Copyright © 1971 by the American Association for Clinical Chemistry

Rapid Liquid-Chromatographic Separation of Steroids on Columns Heavily Loaded with Stationary Phase

¹ Department of Chemistry, Northeastern University, Boston, Mass. 02115.

Properties of heavily loaded columns are illustrated, especially as applied to steroid separation. By "heavily loaded" we mean that the column contains an amount of stationary phase such that the pores of the solid support are essentially filled. The solid support can be loaded with as much as 35-50% by weight of stationary liquid phase, depending on the pore volume and the activity of the solid support. Using heptane/dioxane/formamide, we studied several heavily loaded columns with different liquid loadings and solid support particles of various diameters. Porasil C (10-25 µm particle size) gave markedly improved speed and efficiency characteristics over Porasil A (44-53 µm). A separation efficiency as great as 2.5 effective plates per second was achieved with support material having the smaller particle diameter. Qualitative reasons for this good efficiency are discussed. Column efficiency was independent of quantity (from 1 µg to 1 mg) of injected steroid sample. Finally, applicability of heavily loaded columns to separation of steroids is illustrated. With stepwise pressure programming, 10 steroids can be separated in 35 min.