What are C18 HPLC columns?

C18 has been functionalized on many different kinds of silica gels. A wide variety of HPLC columns have been developed for the analysis of many different analytes. Pore design of these columns vary significantly in terms surface area, pore volume, pore diameter, and other parameters. We discuss different types of C18 columns here.

The most common application of C18 phase is the analysis of small particles which have <5,000 Da molecular weight. These columns are called small or narrow pore C18 columns. These columns feature a pore diameter that is usually around 8-12 nm which leads to exclusion of large particles from the pore volume. Column performance is dependent on how well the bonded phase is functionalized to the silica gel - the amount of C18 groups on the phase or carbon load, the bonding type of the C18 group, and how well residual silanols have been end-capped. 

Samples analyzed using mobile phases with higher aqueous content are usually difficult to separate using a normal C18 column. The usual cause is the C18 groups preventing access of the water molecules into the pore volume in a phenomenon called phase dewetting. Aqueous C18 columns are designed to tackle this problem by having some phase modifications such as a slightly lower carbon load than normal C18 columns.

For faster chromatographic runs, C18 columns with larger pore sizes and lower carbon load are employed. These fast or rapid C18 columns allow for shorter analyte paths through the column for quicker runs. The sacrifice is that peaks are closer in results, making it crucial to have a well-bonded phase in order to preserve comparable peak shapes with non-rapid C18 columns.

For large molecules such as proteins and peptides, wide pore C18 columns are used. The pore sizes of these columns are even larger than fast columns, usually around 30 nm. Unlike fast columns, however, the large pore size exists not for a quicker run time but for large molecules to be able to access the pore volume. Good wide pore columns will feature good phase definition in order to deliver excellent analyte resolution.

There are other C18 phases that exist with modified phases for specialized applications such C18 modified with additional functional groups for enhanced steric selectivity or have interspersed ionizable groups for mixed-mode interaction. For more, check out GL Sciences’ full selection of C18 columns.

Here are a few applications for several compounds for C18 columns:

See the rest of C18 applications in our Applications section.