How to compare different mobile phases

[pipettemonkey]

A recent paper showed that 1-propanol : EtOH (5:2) was as good, perhaps better than ACN at resolving protein/peptide peaks. There is also a cost and toxicity advantage with the alcohol mix as the organic.

The propanol/EtOH is a stronger eluent than acetonitrile (in system with 0.1% TFA on a Vydac C4 column). The most hydrophobic protein in my standards mix is BSA. Propanol/EtOH elutes BSA at around 28% organic, while ACN elutes BSA near 40% organic.

I ask because you simply cannot do a 4-50% organic grad for both, because the alcohol mix is a stronger eluent.

My initial thought was to keep the % change in organic/time as a constant at 0.4 %/min. However, I don't think this is fair either, as ACN has a much broader composition range to elute these standards compared to the alcohol mix.


What is the best way to put these two solvent systems head to head to see how well each mobile phase separates a peptide/protein standard mixture? And then, how can I put a number on it?

[tom jupille]

As a vastly oversimplified first approximation, you could cut the gradient range in the PrOH/EtOH system to 0 - 35% instead of 0 - 50% (since your last peak elutes at 70% of the original time. Then:
There are two things to consider: efficiency and selectivity. The former is best characterized by the peak capacity: divide the total time by the average peak width; that tells you how many peaks will "fit". I suspect that the PrOH/EtOH system will be significantly worse (viscosity effects), but the only way to know for sure is to try.
There is no way to generalize selectivity effects; what works better for one sample can work worse for another. If your samples are consistent, you can compare the resolution between key pairs of peaks. I like to use the "critical" (i.e., worst resolved pair of peaks of interest) as a benchmark in this regard.