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.
The analysis of hydrophilic compounds has presented many challenges to chromatographers. Reversed-phase chromatography was
commonly used for these applications because of the high solubility of hydrophilic compounds in aqueous-based solvents (3).
However, reversed-phase chromatography is poorly suited to the retention of these types of compounds. To obtain adequate retention
and selectivity, ion-pair reagents are often added to the mobile phase. In this mode, the ion-pair reagent contributes to
analyte retention either by neutralizing an opposite charge on the analyte in the bulk eluent or interacting with the analyte
while adsorbed onto the stationary phase surface (4). There are numerous examples of ion-pair reversed-phase chromatography
successfully used in an analysis of hydrophilic compounds (5,6). The approach works for UV-based analyses, but the ion pair
agents used are not compatible with liquid chromatography–mass spectrometry (LC–MS) and are known for other nondesirable issues.
Furthermore, very high water content in the mobile phase is often required for retention, which is less preferable in an LC–MS
method. Because retention in the ANP mode is based on an analyte's polarity, ion pair agents are not necessary to obtain retention
of these types of compounds.
Figure 1: Structures of (a) ascorbic acid, (b) riboflavin, (c) pyridoxine, and (d) thiamine.
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
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.