HPLC Method Development: Practical Aspects of Increasing Analysis Speed While Maintaining Separation Resolution

Abstract

A practical composite of favorable features in column design permits typical separations in one-half the time without loss in resolution compared with commonly used columns. A highly purified, low-acidity 3.5-µm porous silica microsphere support combined with sterically protected, monomeric silane stationary phases provides the basis for the new column type. These units show considerable promise for developing fast, rugged, reproducible methods. Carefully configured columns packed with 3.5-µm particles with a narrow particle size distribution resist the pluggage usually associated with columns packed with traditional 3-µm particles. Extra-column band-broadening and back pressure effects are also minimized so that state-of-the-art HPLC equipment can be used. Columns of highly stable stationary phases have higher operating temperatures that are useful for fast separations, particularly for macromolecules.