Selectivity Contribution: Column vs. MP/gradient Debate

[Rob Burgess]

Hi All,

In general what would contribute more to the "selectivity" of a fairly complex typical pharmaceutical RP-HPLC type impurity analysis - would it be contributions from the column or mobile phase contributions combined with gradient settings? My gut feeling says the selectivity contribution from the mobile phase and gradient settings far outweighs the contribution from the column alone. If this is the case, is there any real value in multiple column screening for early phase method development work? Would it make more sense to just stick with a common and garden C18 column and look at which organic mobile phase gives the greatest indication of peak selectivity? Then you would just develop and model your gradient separation using DryLab or other chromatography modelling software.

Also as an aside, is it possible to measure the contribution to selectivty of the mobile phase and column separately? I suppose this type of investigation would need to be run under fixed conditions which removes the possibility of enhancing selectivity by altering the gradient conditions.

[zokitano]

This is my simplified point of view for this issue:

The column (the stationary phase more precise) has the bigger selectivity effect because its chemistry mainly contribute whether one substance will remain longer or shorter in the column. Proper selection of the stationary phase is crucial for the separation of different compounds that chemically differ from the other compounds in the sample (additives etc).
For example, chiral stationary phases provide better selectivity for chiral molecules compared with the achiral phases. Here the mobile phase can slightly change the selectivity of the chiral compounds which selectivity is mainly determined by the chiral stationary phase used for their separation.

When an appropriate stationary phase is chosen for separation of similar types of analytes that can unambiguously separate them from other compounds (unwanted and with different chemistries), then one can plays with the mobile phase composition, temperature etc for achieving the desired separation (and selectivity) among the analytes with similar chemistries or interactions with the stationary phase.

That's why when one begins to develop a separation method firstly thinks about the stationary phases suitable, chooses stationary phase and then chooses the mobile phase solvents. As you can conclude from the latter, stationary phase plays bigger role in the analyte selectivity than the mobile phase does.

Regards

[Uwe Neue]

You raised some good questions, and I believe to have answered some of them already in a set of recent publications. Specifically I am referring to the following two publications:
1. U. D. Neue, J. E. O’Gara, A. Méndez, “Selectivity in Reversed-Phase Separations: Influence of the Stationary Phaseâ€

[Mattias]

I think Uwe summed it up well.

For me, working mainly with peptides, the pH of the mobile phase is by far the most important selectivity parameter. The column is just there to provide a nice plate count and not too much tailing. Then it doesn't matter if it is a phenyl, C18 , C8 and so on...

The only time where it can make a selectivity difference is when I have to separate stereoisomers of the same peptide (with L and D amino acids). But nowadays I go to the UPLC instead and get baseline separation....