Chromatography Forum

Before I developed reversed phase HPLC methods for small molecules. pH, organic modifier type/percentage and column temperature were usually one of the most important parameters for development of the method.
Now I work at a biopharmaceutical company. For their methods they often use a gradient with:

A: 0.1% TFA in 100% H2O
B: 0.1% TFA in 100% Acetonitrile

For some methods we have a critical separation. We want to develop newer methods. What would be the best parameters to vary?

- Will pH have a significant effect on the separation of peptides on a RP column? Or is there a reason that only TFA is used (they don't seem to know)
- Will Methanol give a different selectivity for the peptides or only changes the retention time?
- Is column temperature an important factor?

- Will pH have a significant effect on the separation of peptides on a RP column? Or is there a reason that only TFA is used (they don't seem to know)

It can. TFA controls pH, acts as a weak ion-pairing reagent, and is chaotropic enough to prevent folding of peptides. Changing the TFA concentration can affect selectivity

- Will Methanol give a different selectivity for the peptides or only changes the retention time?

It can do either. It's not often used because it tends to give broader peaks (arguably due to its greater viscosity, which slows down diffusion and results in a larger van Deemter C-term).

- Is column temperature an important factor?

Yes

If you can change only one parameter at a time, my suggestion would be:
1. gradient steepness
2. temperature
3. TFA concentration

If you have appropriate modeling software (e.g., DryLab, ChromSword, etc.), then you can look at parameters 2 at a time (using 4 experimental runs). The recommendation there would be:
1. gradient steepness x temperature
2. gradient steepness x TFA concentration

Hi Tom Jupille,

Thanks for you explanation!
Do you think the use of a buffer instead of TFA is a good option?

Do you think the use of a buffer instead of TFA is a good option?

You can always try, but I'd say "probably not". Remember that the TFA does more than simply control pH. In any case, TFA *is* effectively a buffer, in that it will prevent the pH from going up when perturbed. It won't prevent the pH from going down, but then how often will you inject a sample with pH < 2 ?

in some cases formic or acetic are used and this is generally for MS purposes because TFA is a problem there
but you need to use a lot more of them to get good results

if you go for more TFA check the pH you get
most columns will not hold lower the 2.0 and with 0.1% you are already very close there

you can also play on the column characteristics
less or more carbon loading can make a difference
try a 3u column
generally in contrast to small molecules proteins love those silanols that we try to get rid of in the columns

If the amino acids of your peptides have charged side groups (e.g. aspartic acid, glutamic acid), then pH will play a big role for your separation.

For the peptides I work with pH is was more important than column chemisty, gradient steepness or organic modifier.

If the amino acids of your peptides have charged side groups (e.g. aspartic acid, glutamic acid), then pH will play a big role for your separation.

For the peptides I work with pH is was more important than column chemisty, gradient steepness or organic modifier.

For my small molecule separations the pH was also one of the most important parameters. Usually we started our method development by varying the pH and modifier percentage. For some reason they only use TFA at my 'new' company. And for some reason I still don't know why.