Prince:Ultra High Performance Liquid Chromatography

Brief Introduction

Chromatography separates compounds based on compound properties. For instance, size exclusion chromatography separates compounds based on molecular weight and stokes radius. There are generally two phases in chromatography, the stationary phase and the mobile phase. In column chromatography, the column is made of the stationary phase and the mobile phase pushes molecules through the column. Molecules with a high affinity for the stationary phase are trapped, but molecules with a low affinity for the stationary phase pass through the column in the mobile phase. The gradient, or composition, of the mobile phase during sample loading onto the column has very low column affinity causing the majority of molecules to bind to the column. After loading, one can elute the column in fractions (groups of molecules with similar properties) by increasing the affinity of the mobile phase for the column because the mobile phase competes with the compounds of interest for binding to the stationary phase. Ultra high performance liquid chromatography uses basic chromatography principles, but at a very high pressure and very low flow rate.

UPLC has powerful resolving power and can be attached to nano-esi in consequence of the high pressure and low flow rate. High pressure is required to push mobile phase through the very small C18 beads in the column. Smaller beads means greater total surface area for compound binding improving fraction resolution. The low flow rate allows the chromatography to be connected directly to nano-esi, which ionizes better than conventional esi.

Figure C18 Chemistry: A. C18 column. 18 carbon length chains are bound to a 1.7 μm silica bead. The carbon chain has a high affinity for non-polar molecules. B. The 1.7 μm beads are packed into a column. The beads are uniform in size insuring consistent, reproducible results.