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

Abstract

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.

Ion pair partition chromatography of organic ammonium compounds

“…The height of a theoretical plate, H, can according to Karger (23) [cf., Giddings (24)] be expressed by: H = Csv + CV + (A"1 + Cm'V1)"1 (8) where Cs = k'(l + k'y2d2Ds-1 (mass transfer in stationary phase) (9) C*M = /( , k')d2Du~l…”

Method development and validation for the quantitative estimation of Trametinib in API form and marketed tablet dosage form by RP-HPLC

“…The Optimum Chromatographic conditions [7] obtained from experiments can be summarized as below: Observation: The selected and optimized mobile phase [8] was ACN: Methanol: 0.1% OPA = 60:30:10 and conditions optimized were flow rate (1.0 ml/minute), wavelength (235nm), Run time was 06 mins. Here the peaks were separated and showed better resolution, theoretical plate count and symmetry [9] .…”

Chromatography

“…When dry-packing silicic acid, the adsorbent should be added in increments to obtain more efficient columns (656). Heavily-loaded columns (up to 50 wt %) have been studied and applied to steroid separations using stepwise pressure programming (531, 680).…”