HPLC Column Care

[Varta]

Do you know why some manufacturer's of silica C18 columns do not recommend using mobile phase combinations of methanol/water/TFA (trifluoroacetic acid)?

[Rafael Chust]

Eventually because those are old technology columns that does not allow low pH.

"Old"silicas can not withstand pH < 2 and > 7. "New" columns easily operate from 1 < pH < 10, so TFA should not be a problem.

[Victor]

Golnar-the answer to your question is very complex, because it depends on the type of column that you are working with and how long you want your column to last.

At low pH, RP columns are susceptible to hydrolysis of the bonded ligand. C18 ligands are generally more stable than C1 ligands used in endcapping. At a pH value less than about 3, there is a danger of removing the endcapping from the column. The rate of loss will depend on the particular column you use. TFA used at the common concentration (0.1%) has a pH well below 3.0. The consequences of the loss of endcapping may not be serious, but you could see a loss in performance for some active compounds. Of course some columns are not endcapped at all, and in addition have stabilising groups which hinder the hydrolysis of the C18 ligands. I believe the Zorbax SB columns are of this kind.

I would note the recommendations of the column manufacturer, although I would guess they may not themselves have investigated in great detail the stability of their phases. Really you will have to investigate this yourself -but I might start by selecting a column which is recommended by the manufacturer for use with TFA!

Your question was not about high pH, but the silica on many RP columns may start to dissolve gradually above pH 7. Some columns are designed for use above this pH. Again the loss in performance may be very gradual, but it will certainly depend on the particular column.

[Varta]

Thank you Victor. That was my initial thought too. Then I started to wonder maybe there is something curious about that certain combination (methanol/water/TFA (trifluoroacetic acid), as against Acetonitrile/water/TFA that might be harmful to the column.

[Victor]

Golnar,

That is a thoughtful comment. I did not know that the recommendation was against particularly the use of methanol-TFA. I cannot particularly see a reason for this, especially because in most applications the concentration of methanol or ACN used in combination with TFA is quite low, and therefore I would not expect much difference between these solutions. I still think the pH of the solution is the important factor.

I know that some manufacturers recommend storage of columns in pure ACN. Pure methanol or methanol-water can cause some hydrolysis or methanolysis of bonded ligands. However, I don't think this is very relevant to the TFA issue.

[tom jupille]

Will TFA esterify MeOH?

[Victor]

Tom-that's a great suggestion and one we have all missed so far- I guess that 's the answer!

I would guess that TFA does esterify methanol but whether this occurs at an appreciable rate under the conditions of HPLC (usually low temperature, low concentrations and the absence of a catalyst) I don't know, or what dire consequences it might have if this happened to a small degree-but the reproducibility might be an issue.

[Peter Apps]

Hi Tom

Does TFA react with methanol in HPLC mobile phases, my initial response was no - mainly becuase there is a high concentration of water - but then I started wondering about the effect of high pressures on reactions that do not happen when chemicals are mixed on the bench.

Are there are organic synthesis experts out there who could shine some light ?

Peter

[yangz00g]

No, TFA won't react with methanol under normal condition. As we know, COOH + ROH=CO-OR needs catalyts highly affilated with H2O such as H2SO4.

[Rafael Chust]

I have used TFA to precipitate proteins before plasma analysis, but I never used as mobile phase... but, in theory, I would say it does not react with MeOH...

[HW Mueller]

A simple ester like the one from TFA and MeOH should be slightly more stable than the precursors, Thus, since TFA should be able to catalyze the reaction (probably slowly at ~ pH of 2) one may expect the ester to have, very roughly, a concentration ratio (TFA to CF3CO2ME at equilibrium) that corresponds to that of MeOH to Water. Now, if these mobile phases stand around for some time...Pure TFA in pure MeOH should give almost 100% ester, but in what time??
On the pressure factor: Difficult to asses for liquids. What takes more volume? The TFA + MeOH or the ester + H2O? The ester is less soluble in H2O.... might be more volume "intense"? Thus literally squeezed toward acid and alcohol?

[tom jupille]

I didn't mean to open a "can of worms"; I only asked because TFA is commonly used with acetonitrile, much less commonly used with MeOH and I wondered if a low level of the methyl trifluoroacetate building up might cause problems. I don't see how that could hurt the column, however.

[DR]

pH MeTFA>>pH TFA?

[tom jupille]

I don't know, DR. I was speculating that MeTFA would be non-polar (using ethyl acetate as the analogy), and so might affect retention and selectivity at fairly low levels.