Chromatography Forum

I have always separately pumped my TFA in from a different line (as opposed to adding TFA directly to acetonitrile) to avoid production of acetamide.

How facile is the conversion of CH3CN to acetamide in presence of acid acid (TFA)?

Since this rxn proceeds through the addition of water, would it not be avoided if neat TFA were added to pure acetonitrile?

To say the truth I have never heard of anybody introducing 100% TFA through a separate HPLC line. All problems about not premixing your mobile phase will be singificantly magnified and TFA is quite corrosive so introducing it like that it must not be very healthy for your HPLC.

About the conversion of CH3CN to acetamide, I have heard about it but never really experienced it even with mobile phase that have been sitting around for a while (i.e. a couple of months). I was told that you can see it as a layer in the bottom of your bottle when it happens and that you can get rid of it by washing (the bottle or HPLC) with warm water... but again never experienced it... Most of the time we add TFA in 100% acetonitrile.

I don't introduce 100% TFA. It is 0.25% aqueous TFA, and is pumped in at a constant 40% of the total mobile phase to get a final concentration of 0.1%

And the rest 60% is only water or only ACN which you use to do perform gradients from 0% to 60% ACN?

And the rest 60% is only water or only ACN which you use to do perform gradients from 0% to 60% ACN?

Yes

It's an equilibrium reaction so you need a lot of water around to favor that transformation.
We usually just add a whole 1 mL ampule of TFA to the acetonitrile and have never had a problem.

It's an equilibrium reaction so you need a lot of water around to favor that transformation.
We usually just add a whole 1 mL ampule of TFA to the acetonitrile and have never had a problem.

Just to clarify, you add an ampule of neat TFA to pure CH3CN, and experience no increase in background absorbance at 220 nm?

We typically run this for research purposes and have Solvent A as Water with 0.1% TFA and Solvent B as Acetonitrile with 0.08% TFA and typically monitor at 210 nm for peptides.
At the beginning where TFA is retained (10-15% acetonitrile), the baseline is noisy, but once the gradient is past this point the baseline is smooth and acceptable to us and we've had adequate sensitivity for our methods. I've never tried another way of mixing. Maybe it is not the best way of preparing, but it's easy enough and suits our needs.
I suppose as long as you have a pump capable of mixing 3 solvents then your method may be better, but we often use a binary high-pressure gradient pump.

I doubt if there would be production of acetamide. I always add TFA directly to acetonitrile to analyze chiral amino acids and other chiral compounds , and have never had a problem. (Instrument: Angilent 1200 HPLC & DAD detector)

I don't introduce 100% TFA. It is 0.25% aqueous TFA, and is pumped in at a constant 40% of the total mobile phase to get a final concentration of 0.1%

We can't but add TFA directly to acetonitrile if use NP-HPLC, because water will be avoid in this system .

Noser222, is there a reason why you use 1ml TFA ampules rather than pipetting 1 ml from a larger volume, eg. a 100ml TFA by Sigma Aldrich?

I realised that old TFA can become discolored (from clear to brown) but the run seemed to look fine - is this one of the reason why you prefer using 1ml ampules?

We use the ampules for simplicity. Since we are a small lab we might use 10-20 mL of TFA in a year. There is no need to keep a larger volume around and risk the discoloration or contamination from pipetting.