Methanol + 0.1% TFA pH

[kpld]

Dear All,

Lately I have been having problems with retention times for a RP gradient run. At a suggestion, I measured the pH for my methanol + 0.1%TFA bottle and I noticed interesting results.

When I prepare a new bottle of MeOH of 1L with 1ml of 99.9% TFA, I noticed the pH is around 0.98. When I let the bottle sit for a few days, the pH becomes around 1.68. When I let the bottle sit for a few weeks, the pH is around 3.14. When I look at the datasheet for pre-mixed MeOH + 0.1% TFA from vendors, they list the pH as 2.9.

Can anyone explain this phenomenon? Is it TFA esterization (someone from another post mentioned that) or could it be evaporation? Also can anyone tell me what the correct pH I should be seeing? I have tried with different vendor's MeOH and TFA, and they all produce similar pH. Since I am using a Waters T3 prep column which has a pH range of 2-9, I am very afraid I might be continuously damaging my column. If so, how much TFA should I actually add to 1mL bottle of MeOH to create a pH above 2?

Thanks in advance.

[Vlad Orlovsky]

lets start with the statement that measuring pH in 100% organic does not make a lot of sense. Your phenomena can be explained by first class of organic chemistry: your TFA is reacting with MeOH to form ester, so you are depleting amount of TFA. I would suggest that you add TFA to your aqueous portion of RP chromatography...unless you are running normal phase and you don't have water in your system.

[kpld]

Okay so we currently use a gradient run for our method where the MeOH percent in our flow increases over time. We we worried that if we only add TFA in water, then the TFA concentration over time will decrease as MeOH percent increases. That is why we add 0.1% TFA into both our water and MeOH. We have noted that others have done so in previous publications and MeOH distributors also sell MeOH premixed with 0.1% TFA (http://www.sigmaaldrich.com/catalog/pro ... &region=US). My organic chemistry is a bit rusty, so will TFA react with MeOH to form ester readily? Or after maybe a few hours. We are trying to make fresh MeOH + 0.1% TFA and used it all up within a few hours and not let it sit as a solution.

[Vlad Orlovsky]

You will have reaction between TFA and MeOH. I don't know kinetics of this reaction but at the end you will have no TFA in MeOH. What is your gradient? may be I will advice how to modify your run. Unless you are going to 100% MeOH you can try to create gradient from two channels. If you have 4 channels you would not have problem creating any gradients.

[kpld]

So we are using a 4channel pump. Our method is as follows (with MeOH in 1 channel and water in another):

At equilibration and starting point: 30% MeOH + 0.1% TFA, 70% Water + 0.1% TFA
0-10 min: 30% MeOH + 0.1% TFA, 70% Water + 0.1% TFA
10-40 min: 30% MeOH + 0.1% TFA, 70% Water + 0.1% TFA to 80% MeOH +0.1 TFA, 20% Water + 0.1% TFA
40-60 min: 80% MeOH +0.1 TFA, 20% Water + 0.1% TFA
60+ min: 100% MeOH

Any help would be appreciated.

[Vlad Orlovsky]

If you have quaternary pump you don't need to mixt TFA with MeOH.
the way you do it is set up three channels:
A: 100% water
B:100% MeOH
C: 1% TFA in water

In order to create your gradient:
0-10 min: 30% MeOH + 0.1% TFA, 70% Water + 0.1% TFA
10-40 min: 30% MeOH + 0.1% TFA, 70% Water + 0.1% TFA to 80% MeOH +0.1 TFA, 20% Water + 0.1% TFA
40-60 min: 80% MeOH +0.1 TFA, 20% Water + 0.1% TFA
60+ min: 100% MeOH

You do:
0-10 min A 60%, B- 30%, C-10%
10-40 min from A 60%, B- 30%, C-10% to A-10%, B-80%, C-10%
40-60 min A-10%, B-80%, C-10%
60+ B-100%
Don't forget that you need to equilibrate column back to your initial point of gradient.
In this case you will never have case when you are depleting amount of TFA due to reaction with MeOH

[Victor]

Why not just use acetonitrile instead of methanol?

[kpld]

Why not just use acetonitrile instead of methanol?

MeOH is cheaper for us and our previous methods all have used MeOH + 0.1% TFA. I am just following what others have done before me at my workplace. If I change the method, then downstream there will have to be a lot of retesting.

[Robert Classon]

There was an excellent poster on this subject given by the folks at Burdick & Jackson (Honeywell) at the ACS meeting in August 2007. They showed that methanol when relatively dry reacts with just about any acid to form an ester. They proved this by looking for the corresponding ester by GC. The kinetics were approximately the following:

For formic acid in methanol:
25% conversion in 25 hours
60% conversion in 100 hours
80% conversion in 350 hours

Results were comparable for TFA and acetic acid as well. Methanol reacts with those also.

This helps to explain the 'it worked on Friday phenonenom, but isn't working on Monday' situation. Just letting the methanol plus acid sit for a weekend changes the acid content quite a bit and the pH change is enough to change the HPLC results.

The easiest fix is to just eliminate the acid from the methanol. Just add it to the aqueous solvent for your gradient. You will obtain more stable results and will have less trouble with pH shifting as a function of the clock.