Reverse phase gradient by increasing bufer conc.

[Arkcon]

This is in some ways a general question on reverse phase HPLC gradients of a type I'd seen from time to time. The gradient goes something like this:

Time %A %B %C
0 min 2 20 78
10 min 20 20 60


Where A is a buffer -- 35 mM ammonium acetate, B is acetonitrile, and C is HPLC water. The column is a typical C18, and the first analyte to come off is potassium sorbate, and the second is phenoxyethanol, the second one being a more non-polar molecule of these two typical preservatives.

My question is: how does this work? The solvent concentration is the same. The water concentration is essentially the same. There's greater salt concentration, but how does that drive a reverse phase separation? Diminished water activity because of greater ionic strength? What problem does methods such as these fix? And what sort of pitfalls are there in making methods such as these?

[danko]

How does this work? Elementary Dr. Watson

It’s just not a gradient elution – Reversed Phase Chromatography wise.
Rather it is isocratic elution. The buffer could have other roles but it’s not the question in this context.

Best Regards

[kubowicz.tomasz]

Hello

I've never seen gradient method with buffer:water

Tom

[Arkcon]

Thanks, for a while, I was suspecting that this was hydrophilic interaction chromatography. But I don't see an analyte that needs it. Maybe another common ingredient needs it, or the developer just did it for no reason. We'll see if we can look into it at some time.

[HPLCaddict]

This is in some ways a general question on reverse phase HPLC gradients of a type I'd seen from time to time. The gradient goes something like this:

Time %A %B %C
0 min 2 20 78
10 min 20 20 60

Where A is a buffer -- 35 mM ammonium acetate, B is acetonitrile, and C is HPLC water.

Are you sure you got this right? It would make sense if A: acetonitrile, B: buffer, C: water. This is something that can be seen sometimes - the sense behind it is to hold the buffer concentration constant while running the gradient.