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I only ever need C18… for reversed phase chromatography don’t I ?

We’re probably all guilty at some point of starting all new method development projects with our favourite and most trusted C18 column installed on the system. Whilst are old favourite C18 may perform an adequate job it may lead to a lot more work during method development or a method that it is non-optimal.

Not all C18 columns are the same either, ever taken a look at the literature supplied with your column and wondered what pore-size, carbon loading, end-capping to name but a few mean or, more importantly, what effect they have on your separation.

The main driver behind resolution, the primary function of nearly all chromatographic separations, is separation factor – sometime referred to as selectivity or α. This has been largely forgotten in recent times due to the advances in efficiency that UHPLC instruments, sub 2µm porous and core-shell particle have brought us. Stationary phase is the largest single factor that affects separation factor but is largely ignored due to the focus and attention given to increasing efficiency.

 

different phases

Column manufacturers provide lots of different phases – either variation on a theme (various types of C18) or increasingly more diverse phases.

 
 

 
Reverse Phase Stationary Phases

Reverse phase separations are characterised by having a stationary phase that is less polar than the mobile phase.

Several popular reverse phase, bonded stationary phases are shown. Octadecylsily (or C18) is commonly used, as it is a highly robust hydrophobic phase, which produces good retention with hydrophobic (non-polar) analyte molecules. This phase can also be used for the separation of polar compounds when used with mobile phase additives, which will be discussed later.

In general, shortening the alkyl chain will shorten the retention time. There are only very slight selectivity differences between, for example, C18 and C8 columns.

The use of more polar phases such as cyano, phenyl or amino phases show altered selectivity compared to the alkyl phases. These phases are able to interact with polar analyte functional groups, via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions.

  Reverse Phase Stationary Phases
 

Aq Type Stationary Phases

One method of producing a water wettable phase involves close control of the spacing between the bonded phase ligands on the silica surface. Knowing the surface area of the silica and adjusting the carbon loading allows the ‘inter ligand distance’ to be controlled. This gives more (or less) access to the silica (silanol) surface, allowing the polar influence of low energy surface silanol groups to alter the selectivity of the separation.

Stability in highly aqueous mobile phases is achieved via the adsorption of a layer of water at the silica surface. The vicinal (low energy) silanol groups become hydrated and so the driving force towards ligand self-association is lost as the layer of adsorbed water at the silica surface effectively repels the ligands and they remain ‘activated’.

The alternative method of introducing stability in 100% aqueous mobile phases is to use polar end-capping reagents. These reagents are chosen to retain good peak shape with almost all applications whilst again introducing a polar characteristic to the separation and hence altering the column selectivity. The mechanism of high aqueous stability is similar to the previous case where an adsorbed layer of water at the surface prevents phase collapse

 

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Stationary Phases
 

 
Polar Embedded Phases

Polar embedded ligands contain a modification to the alkyl chain which is usually an amide, carbamate or other suitable polar functional group.

The phase allows several distinct advantages including:

  • Use with 100% aqueous mobile phases.
  • Polar group that gives an alternative selectivity.
  • The ability to separate polar, ionisable and especially highly basic compounds with excellent efficiency and peak shape
  • Enhanced robustness over shorter chain and polar bonded phases.

The proposed mechanisms of interaction are shown along with some useful applications.

  1. Here the analyte polar functional group is shown interacting with the amide ‘spacer’ – this is analogous to the interaction with the silanol surface to produce alternative selectivity.
  2. A layer of water attracted to the polar embedded moiety acts to stop phase collapse.
  3. The polar moiety interacts with a lone silanol group to shield the surface and reduce secondary interactions (peak tailing

 

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Phenyl :

The use of more polar phases such as cyano, phenyl or amino phases show altered selectivity compared to the alkyl phases. These phases are able to interact with polar analyte functional groups, via dipole-dipole interactions and the phenyl column can interact with analyte aromatic moieties via π-π electron interactions.

Phenyl
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