Getting the most out of silica hydride columns requires knowledge of how to use them to fully exploit their unique and desirable chromatographic benefits. Examples of such benefits include extremely fast equilibration even between gradient runs, very precise retention for hydrophilic or hydrophobic analytes, and an increased range of solvent compatibility. This article discusses how to successfully use this high performance liquid chromatography approach during method development in an iterative form to clarify decision making processes along the way that are unique to silica hydride and aqueous normal phase retention.
Silica hydride is a type of stationary phase material used in a unique class of high performance liquid chromatography (HPLC) columns. Structurally, the material consists of high purity silica but uses a proprietary manufacturing process to produce a surface containing >95% fewer surface silanols than conventional silica (1). The surface of this material is slightly hydrophobic which can be functionalized if desired with various organic moieties such as cholesterol, phenyl, C8, C18, or very small carbon chains. Stationary phases made from this material are currently marketed as Type-C silica. In addition to ordinary reversed-phase or normal-phase chromatography, these stationary phases can successfully operate in aqueous normal phase (ANP) mode. ANP mode is often used for hydrophilic or polar compounds but differs from hydrophilic interaction liquid chromatography (HILIC), which is sometimes used for retention of these compounds, in that a water-rich environment is not present on the silica hydride stationary phase surface. Because this water layer is believed to play a key role in HILIC retention via analyte partitioning, the mechanism responsible for ANP retention is significantly different and requires different decisions during method development.
The nature of the adsorbed water layer in HILIC methods is thought to contribute to a lack of robustness in many instances of gradient usage. As such, HILIC columns may require lengthy equilibration (2) that consumes both time and solvents. Because the silica hydride surface is slightly hydrophobic, it will adsorb and desorb the mobile phase differently and more quickly. This leads to both faster equilibration and higher precision even when gradients are used. For this reason and others, silica hydride columns are often chosen for analyses of hydrophilic compounds via ANP chromatography.
To demonstrate how an analyst may proceed in developing methods for hydrophilic compounds using silica hydride stationary phases, the separation of the four test solutes (ascorbic acid, pyridoxine, riboflavin, and thiamine) shown in Figure 1 was investigated for the purposes of this article. The polar/ionizable functional groups of these analytes are sufficiently diverse to be representative of other types of hydrophilic compounds that may be encountered during method development or untargeted analysis methods.
Three main goals were set for final methods for the purposes of this article. The first goal was the method could only use LC–MS compatible conditions. The second goal was to keep the analyte retention in a suitable range; methods in which retention is too low often suffer from inadequate separation of peaks while excessive retention lengthens analysis time and wastes solvents. The third goal was that the critical peak pair should be baseline-resolved. A resolution of no less than 1.5 is generally used for baseline separation (7) and therefore was the criterion we chose.