The increased availability of sub-2-micron (STM) columns and increased demand for methods friendly to mass spectrometers has led to a strong trend toward conversion of existing HPLC methods to smaller diameter and smaller particle size columns. While the conversion is a simple mathematical exercise requiring the scaling flow rates, gradient times, and injection volumes, many users observe less than perfect results. Here we look closely at the problem and propose calculations that improve the speed and/or resolution in a more predictable and beneficial way.
This document is a technical application note authored by Michael Woodman of Agilent Technologies. It addresses the challenges of converting existing High-Performance Liquid Chromatography (HPLC) methods to modern sub-2-micron columns, which offer reduced analysis time and better compatibility with mass spectrometry.
The text details mathematical approaches to scaling flow rates, gradient times, and injection volumes. It specifically discusses the relationship between column efficiency and linear velocity using van Deemter plots, and highlights the impact of extracolumn dispersion on small particle size columns. The document includes experimental results demonstrating these principles using Agilent 1200 Series Rapid Resolution LC systems and ZORBAX SB-C18 columns.
| System | Agilent 1200 Series Rapid Resolution LC |
|---|---|
| Software | ChemStation 32-bit version B.02.01 |
| Column 2 | Agilent ZORBAX SB-C18, 3.0 mm x 150 mm, 3.5 um |
| Aqueous Solvent | 25 mM phosphoric acid in Milli-Q water |
| Organic Solvent | Acetonitrile |
| Sample | Standard mixture of chlorinated phenoxy acid herbicides |
| Column Temperature | 25 °C |
Benefits include reduced analysis time, reduced solvent consumption, improved sensitivity, and greater compatibility with mass spectrometer ionization sources.
It is more useful to express gradient slope as % change per column volume rather than % change per minute to accurately render new gradient conditions.