To ALL Chromatographers:
I am about to run the method, see condition listed below, there is no TFA in ACN. Is it a common practice? Any suggestions or comments?

Detection = 254nm
mpA = 0.1% TFA in water
mpB = acetontrile
Gradient
0min = 10%B
10min = 50%B
15min = 50%B
25min = 85%B
29min = 85%B
Equilibration


Thanks

Dear Anonymus;

It is common to maintain the % TFA constant during separations of peptides and similar molecules. However, is not unheard of to have the situation you described.

TFA is normally used to supress ionization of acids by lowering the pH of the mobile phase, and also to ion-pair some compounds (amines) and enhance retention.

TFA is adsorbed on most reversed-phase columns and in your case you are running a sort of double gradient changing the TFA and ACN concentrations simultaneously. In both changes you are likely to reduce retention of most molecules.

I hope this helps. Good Luck.

Benjamin

I recommend that you also adjust the composition:

mpA = 90% water/10% ACN, 0.1%TFA
mpB = 15% water/85% ACN, 0.1%TFA

You will avoid some mixing problems compared to the use of "pure" solvents.

I beleive it is common pratice, isn't it?

The primary reason for using TFA at a constant concentration is to maintain a flat baseline at low UV. Since you are working at 254, you'll get away with an unbalanced composition. However, the other reason for adding TFA was most likely pH control. You'll get better pH control with a balanced TFA composition.

Thanks for the comments that all of you have given to me. Have a great day

re Einar's Q - I'm assuming that your tag question refers to the common practice of not pumping 100% ACN. In many LC pumps, particularly those that use sapphire & ruby check valve components, pure ACN can be problematic, hence the practice of cutting it with at least a little bit of the aqueous phase. If you really want or need to pump pure ACN, ceramic check valve cartridges are suggested.

Mr. Einar's suggestion of premixing the solvents has a couple of advantages. One is that you can run steeper gradients which are more accurate (and precise) on older solvent delivery systems. You may also cut down on bubble formation when the aqueous and organic fractions mix. Lastly, to DR's point, a few percent of either water or MeOH in ACN will help any pump.

I've found 1-5% MeOH in ACN is just enough to allow any pump to work flawlessly without a noticeable effect on RTs.