Chromatography Forum

Just a simple question about glycerol retention in reverse phase HPLC.

So glycerol is highly polar and to retain it you should have a mobile phase with a high organic content because that makes it alot more non polar.

So does this mean that since there's a non polar column and a non polar mobile phase that only hydrophobic interactions can occur and the glycerol will be highly retained.

Hmmm, some confusion I think.

In reverse phase chromatography, the column is non-polar.
Chemicals (analytes) interact with the column by hydrophobic interactions.
Chemicals are retained when the mobile phase (solvent) is hydrophilic.
Chemicals are less retained when the mobile phase is less hydrophilic and more hydrophobic, more like the column.
(i.e. they partition; if a chemical is capable of making hydrophobic interactions, it will do so with the column, but as the solvent becomes more hydrophobic, the chemical has less reason to interact with the column rather than the solvent, so it will partition more into the solvent, and move faster).

To get retention, lower the organic content in the solvent. To get less retention, increase the organic content.

(To others: yes, I'm aware that there are other interactions going on in reverse phase chromatography, maybe with silanols etc., but generally I'd hope that the simple hydrophobic interactions ought to dominate everything when looking for rule-of-thumb explanations about how retention time will vary with choice of solvent strength).

The general explaination of reverse phase mechanism is o.k. , but the question is for a very polar compound .

Actually , normal phase may be more suitable , but if reverse phase is required then using high organic mobile phase seems more reasonable for higher retention , since increasing water in the mobile phase will move a polar compound faster.

Actually , normal phase may be more suitable , but if reverse phase is required then using high organic mobile phase seems more reasonable for higher retention , since increasing water in the mobile phase will move a polar compound faster.

At which point, you are arguably doing HILIC rather than reversed-phase. It's actually not unheard of to have the retention mechanism change "mid-stream", with retention first decreasing and then increasing as the ACN concentration increases.

Yes, you're either doing Hilic relying on the highly dubious hydrophilicity of a reverse phase column for the aqueous partitioning bit (or relying on the analyte making hydrophilic interactions to a column whose designer was probably doing their best to prevent any possibility of hydrophilic interaction), or on some polar analytes, you're testing the concentration at which they redissolve having been precipitated by the running buffer.

If you need normal phase or hilic, I personally would suggest using normal phase, or a column that is intended for hilic applications.

Yes, Tom, I've had awful problems with a very amphiphilic compound, sulphonate groups on one end and a long fatty chain on the other; in gradient elutions it initially came out 1 or two runs after the injection, because it was really only mobile during a narrow period in the middle of the gradient. I was completely flummoxed until a nice person here gave me the explanation. Solution in my case was to increase flow-rate, shorten the column, and slow the gradient a bit, so it had a chance to elute properly before the concentration of organic increased too much.