In daily work, it is common for teachers to inquire about the silane bonding methods on the surface of silicone monomers, whether it is single-bonded, double-bonded, or triple-bonded. They also ask whether there are active silicon hydroxyl groups on the filler surface, and whether they are end-capped. They may also ask if there are any polar functional groups embedded in the carbon chain, and what kind of functional groups they are. In some project standards, the stationary phase is required to be deactivated silica gel. Familiarity with the stationary phase bonding methods and modifications can help understand the conditions of column usage and select the appropriate chromatographic column.
The commonly used method for preparing the packing material of a reverse phase chromatography column is to perform a covalent reaction on the organic silane and the silicon hydroxyl groups on the surface of the silica gel to “bond” and generate a stationary phase or a coordinating group R.
X3-Si-R+≡Si-OH→≡Si-O-Si（X2）-R+HX
(silane) (silanol) (final stationary phase)
Preparation of HPLC columns can be achieved through several different silane-silica gel reactions. The monomeric approach is the most widely used technique in reverse phase columns, which involves a one-to-one bonding reaction between a silane molecule and a silanol group. This type of bonding results in a relatively perfect and reproducible stationary phase. The resulting packing material typically has the highest column efficiency because the stationary phase layer is relatively uncrowded, allowing solute molecules to quickly diffuse in and out of the stationary phase. Conversely, stationary phases with multiple functional groups and high degrees of polymerization exhibit lower solute diffusion rates and lower column efficiencies, especially at higher flow rates.
01 Bonding Method
Currently, there are four main types of silane-silica gel bonding technologies for monomers: single-bond bonding (Ultisil® XB-C18), double-bond bonding (Sunfire C18), triple-bond bonding (Ultisil® PAH), and double-tooth bonding (ZORBAX Extend C18). Single-bond bonding is advantageous for improving mass transfer rates and accelerating column equilibration. However, when used for a long time under low pH conditions, the single-bonded stationary phase is prone to loss, resulting in a shortened retention time in the chromatogram. On the other hand, using multiple bonding methods (double-bond, triple-bond, and double-tooth bonding) can improve the stability of the stationary phase under low pH conditions.
02 Silica End-Capping
Due to steric hindrance, the silanol groups on the surface of silica gel cannot fully bond with silane molecules, and the residual silanol groups may undergo secondary ion exchange with basic compounds, leading to poor peak shape. Therefore, smaller volume TMS (such as trimethylsilyl) or other end-capping techniques are often used to minimize residual silanol groups on the surface of the packing material. However, TMS is susceptible to hydrolysis and degradation at low pH, which can cause changes in retention time and selectivity. On the other hand, end-capped packing materials improve the stability of the chromatographic column under alkaline conditions, reduce column bleeding, and facilitate the separation of polar and basic compounds while providing good peak shapes.
03 Steric protection – side chain alkyl group
Introducing methyl, isopropyl, or isobutyl groups to the side chain of the octadecylsilane on the surface of silica gel can greatly shield the effect of the silicon hydroxyl group on the surface of silica gel. The spatial protection is very helpful for low-pH separation experiments, which can avoid the breakage of the O-Si bond under low-pH conditions but cannot be used under high-pH conditions. Fixed phases with spatial protection function can exist in various coordination forms (such as C8, C18, cyanide, and benzene), and each coordinating group can maintain high stability under low-pH conditions of the mobile phase (as low as pH 0.8). Common chromatographic columns of this type include Welch Ultisil® LP series columns.
04 Embedding polar functional groups
Typically, reverse phase columns have poor separation for strongly polar and strongly basic substances. Embedding polar functional groups (such as urea, amide, ether, or formate ester groups) into the carbon chain of the reverse phase stationary phase can increase the selectivity for these types of substances. At the same time, the polar functional groups shield the silanol groups on the silica surface, greatly improving peak shape and avoiding tailing in the analysis of basic substances. A typical example is the Ultisil® Polar-RP column that utilizes an amide functional group.
In addition, the presence of polar functional groups in the carbon chain can increase the wettability and hydrophilicity of the stationary phase, which prevents phase collapse when using high aqueous mobile phases of 95% or more. Such columns have different selectivity than traditional reverse phase C18 and C8 columns, especially for polar substances such as phenols.
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