When C18 is Not Quite Enough in HPLC...

By: Denise Wallworth, Reporter EU Vol 29

a Phenyl Stationary Phase may do the Job

Denise Wallworth denise.wallworth@sial.com

The automatic column choice in reversed phase HPLC is traditionally a C18 or perhaps a C8, but this does not always provide the optimum selectivity desired – it might not always have the best selectivity or the elution order that is required. Whether the column is struggling to retain polar molecules or requires a gradient to control a long retention, the alternative selectivity provided by a simple switch to Ascentis Phenyl may provide the ideal solution. This changes the mechanism from the predominantly hydrophobic interactions used in C18 π-π to ones, taking advantage of π-accepting capabilities in polar aromatic and heterocyclic solutes. The result can be just the change in selectivity needed through enhanced retention and selectivity.

Because of the poorer wetting characteristics of hydrophobic phases, method reproducibility may also suffer on a C18 column if a highly aqueous mobile phase is in use. Ascentis Phenyl columns use pure phenyl chemistry so are highly compatible with 100% aqueous mobile phases.

The secret of Ascentis Phenyl

  • Low bleed for LC/MS and UV
  • Greater selectivity for polar molecules
  • Excellent peak symmetry and efficiency
  • Compatible with 100% aqueous mobile phases
  • Can be used in RP and HILIC (ANP) modes
  • Proprietary trifunctional bonding chemistry for high column stability

Figure1: Separation of nucleosides in 100% aqueous mobile phases

When using Ascentis Phenyl columns, it is also important to evaluate the use of methanol over the more typical acetonitrile as this can give additional benefi ts of increased retention. It is thought that ACN forms a multiple solvation layer over the stationary phase while MeOH forms a monolayer, increasing the π-π effect1. Mixes of MeOH with ACN can also be explored.

Extensive studies2 have shown that the butyl spacer used in Ascentis Phenyl is optimum for effi cient interactions to occur between the solute and the phenyl moiety – allowing freedom of movement in the phenyl ring for interaction. This, combined with highly effective, proprietary end capping and a tightly controlled surface area specifi cation, provides excellent performance and separation efficiency.

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  1. M. Yang, S. Fazio, D. Munch, P. Drumm, Journal of Chromatography A. 1097 (2005), 124 –129.
  2. M. R. Euerby, P. Petersson, W. Campbell and W. Roe, Journal of Chromatography A (2007), 138 –151.

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