[Readers Insight] HPLC Column Selection: Core to Method Development (Part I)

Author: Sepuxianyun

HPLC columns can be categorized in various ways. The most common classifications are by base material and by bonded phase. Different combinations of these two factors (especially for silica-based materials) result in a wide range of column types.

More than 90% of commercial HPLC columns use silica as the base material due to its high pressure resistance and excellent surface modifiability for bonding various stationary phases. Silica has evolved significantly over time—from early amorphous silica (which contained many metal ions) to spherical silica, and now to ultrapure silica (purity up to 99.999%) and hybrid silica (which can tolerate pH levels up to 12). These developments have kept silica at the top of the column packing hierarchy.

Core–shell silica columns, which enable fast analysis under lower backpressure, have also become increasingly popular for their efficiency and performance.

Polymeric packings offer excellent chemical stability across a wide pH range (typically pH 0–14) and can even be tailored to create chiral environments for enantiomer separation. However, they generally have lower pressure resistance and less batch-to-batch reproducibility. For this reason, polymer-based columns are less widely used than silica-based ones.

Produced from carbon black through high-temperature graphitization, these materials possess unique physicochemical properties. They can withstand extreme pH conditions (0–14) and high temperatures, providing good retention for highly polar compounds.

In this article, we focus on reversed-phase bonded phase only.

The most commonly used bonded phase, and it can be further subdivided by bonding type, end-capping, and functional modifications:

• Bonding Type: Columns may feature mono-, di-, or tri-functional bonding as well as bidentate bonding, with tri-functional C18 columns offering higher stability but requiring more complex manufacturing processes.

• End-Capping Type: End-capping is intended to mask residual silanol (Si–OH) groups and reduce secondary interactions. However, non-endcapped columns offer different selectivity, so “endcapped” doesn’t necessarily mean “better.” Common types include non-endcapped, TMS (trimethylsilyl) endcapped, and polar endcapped. Columns are typically endcapped once or twice, but due to steric hindrance, complete silanol coverage is impossible—overall masking rates usually remain below 70%.

• Embedded Polar Groups: Incorporating polar functionalities such as amide or carbamate groups can improve peak shape for basic compounds and introduce steric selectivity. These columns are often suitable for 100% aqueous mobile phases. 

• Positively Charged Surface: Some columns modify peak shapes of basic compounds through electrostatic repulsion.

• Side-Chain Protection: Designed to shield residual silanol groups and enhance stability.

C8 columns exhibit weaker hydrophobicity than C18 but offer more uniform bonding density due to shorter carbon chains, sometimes providing spatial selectivity. C4 columns are mainly used for peptides and proteins, where reduced hydrophobicity aids in elution.

These include phenyl, pentafluorophenyl (PFP), and pentabromophenyl phases, which introduce π–π interactions in addition to hydrophobic effects. They excel in separating aromatic isomers that differ in ring substitution positions. Because of their rigidity, these groups cannot be directly bonded to silica and are typically connected via propyl or hexyl linkers. Their unique selectivity has also led to the development of hybrid phases such as biphenyl, perfluorobiphenyl, and mixed C18-aromatic stationary phases.