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All HPLC C18s Are the Same Aren’t They?

It’s just a silica support with a C18 bonded on – surely there can’t be much difference between them? Wrong. There are huge differences between standard C18 phases and simply swapping one C18 for another, without accounting for these differences, will produce differences in retention time, resolution and peak elution order.
Most of us are aware of the difference between endcapped / non-endcapped columns or the idea of carbon loading. Although this is a start, differences in selectivity are caused by many complex factors and none of the manufacturers have established a standardized system of quantifying the effects of these differences.
The following is an overlay of 4 modern standard C18 columns by popular manufacturers:


  Column 1 Column 2 Column 3 Column 4
Column 279 252 178 130
Manufacturer Zorbax Waters Thermo Phenomenex
Brand Eclipse Sunfire Hypersil Gold Kinetex
Style XDB-C18 C18 C18 C18 100A
H 1.077 1.031 0.881 0.963
H/10 0.1077 0.1031 0.0881 0.0963
S 0.024 0.034 0.002 0.009
A -0.063 0.044 -0.017 -0.137
B -0.033 -0.014 0.036 -0.011
C (2.8) 0.055 -0.186 0.162 0.007
C (7.0) / 10 0.0088 -0.0099 0.0479 0.0125
C( 7.0) 0.088 -0.099 0.479 0.125

 

These show significant differences in retention and selectivity behavior between the test probe compounds and similar differences would be expected in most analyses.

There are several independent databases which assess the parameters deemed most likely to establish the selectivity ‘character’ of a column, including factors such as hydrophobic retention, shape selectivity, acid and base retention mechanisms. One example is the PQRI database being adopted by the United States Pharmacopoeia (USP). For more explanation on the parameters assessed in this database :

H   Hydrophobicity. A measure of the ability of the stationary phase to retain hydrophobic compounds. Traditionally ‘carbon loading’ was used as a measure of this parameter, but this took no account of several key effects (e.g. efficiency of ligand bonding process, ligand accessibility / performance and the degree / type of endcapping used. The PQRI ‘H’ value provides a far more quantifiable practical value to this critical feature.
‘H’ gives a measure of retention, but has only a minor effect on selectivity.
S   Steric or shape effect. The ability of the stationary phase to discriminate between molecules of similar size / LogP, but with a different 3D shape. This can have a significant effect on selectivity. In some cases, this parameter can be exploited to allow resolution of isomers.
A   Hydrogen Bond Acidity. Generally a desirable feature that gives a level of secondary interaction via low activity silanol groups on the support surface. This influences selectivity, particularly with retention of weakly basic molecules without excessive peak tailing.
Many phases with high A values provide the ability to use 100% aqueous mobile phases.
B   Hydrogen Bond Basicity. Desirable feature built into certain stationary phases (e.g. polar embedded phases) to provide significantly modified selectivity. Larger ‘B’ values can have considerable effects in enhancing retention of weak acids.
C (2.8)   Silanol Ionisation at pH 2.8. At this pH, all residual silanol groups should be in the less active (un-ionized , vicinal) form. Any acidic silanols that have been activated (e.g. by metal ions in stationary phase support) remain charged and can have significant detrimental effect, particularly with excessive tailing of bases. More traditional phases tend to have high C (2.8) values, with resulting poor peak shape.
C (7.0)   Silanol Ionisation at pH 7.0. At this pH, all residual silanol groups should be fully ionized and the C (7.0) value gives a measure of the total amount of ionized silanol groups available for secondary interaction with analytes.
For optimal alternative selectivity, ideally we should have a large C(7.0) – giving large amounts of silanols available for secondary interactions, but a low C (2.8) – low amounts of activated silanols that cause peak tailing.
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