Liquid Chromatography (LC): Columns in LC/HPLC – Bonded Phase Columns (C18)

Columns in LC/HPLC are considered the “heart of the separation”. Controlling a separation process means understanding and controlling the physics and the chemistry inside the column. To do so, it is necessary to understand how columns are packed and how packings are prepared.

Amongst, the most popular columns in liquid chromatography are the so called C18 columns. These are silica bonded phase columns that are used for the separation of nonpolar compounds.

How these bonded phase columns are prepared?

The first step called silylation involves the reaction of fully hydroxylated silica with chlorodimethylalkylsilane and heating to drive off  HCl (Fig. 1). Variations in the chain length and functional groups on the alkyl group produce the wide variety of bonded-phase columns. If we would stop at this point we would have a column with 10%  free silanol sites (Si-OH sites) because of steric hindrance. The column would work well for the separation of acidic or neutral compounds but it would give poor separations for amines and bases.

Another reaction step is therefore required so that the column would be fully functional (end-capping reaction). This involves bonding of the remaining silanols with a smaller compound, chlorotrimethyl silane. After this treatment, free silanols are 1% and the column can be used for amine separations without peak broadening. The process by which these bonded groups are attached is reversible in the presence of water at either low or high pH.

Fig. 1: Bonded-phase column and silylation and end-capping reaction

There are an increasing number of bonded phase choices with some specific to high aqueous mobile phases or other applications. Popular choices are the C8 and C4, phenyl, diphenyl, CN and PFP phases that offer significant differences in selectivity from the straight-chain alkyl phases and may provide successful separations.

In general, larger solutes, such as proteins, are best separated on short-chain reversed phase columns (C3, CN) bonded to wide pore silica gels (pore size: 300 A). Peptides and small molecules are separated on longer-chain columns (C8, C18). There are many cases, however, where this convention does not apply. Therefore, it is a good idea initially to select a phase in the middle of the hydrophobic spectrum (i.e. C8), then change to a more hydrophobic phase or more hydrophilic phase depending on initial results and the solubility of the sample.

REFERENCES

L.R. Snyder. J.J. Kirkland, “Introduction to Modern Liquid Chromatography”, 2^nd edition, Wiley, 1979

A. Weston and P. Brown, “HPLC and CE Principles and Practice”, Academic Press, 1997

A. Braithwaite and F.J. Smith, “Chromatographic Methods”, 4^th Ed., Chapman & Hall, 1990