Chromatography Forum

Good morning all,

I have just started working on the whey proteins ( alfa-lactalbumin, beta-lactoglobulin, bovine serum albumin and inmunoglobulins) analysis by HPLC using a C18 column and the following solvents:

Solvent A: 100% ultrapure water + 0.1% TFA
Solvent B: 20% ultrapure water + 80% ACN + 0.085% TFA

I achieved to develop a method for the compounds separation and I was quite happy, but...one day I opened a new bottle of TFA and every retention time changed in the chromatogram and the compounds separation is not possible anymore using the previous gradient.

I think it is because the TFA is more concentrated because it was recently opened ( TFA is a very volatile compound).

Now I feel quite desperated because my previous work is not valid ( including calibration curve).

PLEASE, COULD ANYONE HELP ME? anyone was in the same situation as me?
I will have the same problem everytime I open a new bottle?how can I reproduce the previous results without modifying the gradient and then the calibration curve?

THANK YOU very much for your help and have a nice day!

V.

I'm not sure that the TFA is your problem. Do you have some of the original TFA left so that you could go back and reproduce your original results. Please post as much information as possible about your system, gradient, etc. so that we can help you solve your problem. Did the retention times increase or decrease? Post some before and after chromatograms if possible.

Why haven't you run less concentrated TFA mobile phase with TFA from your new source to confirm your suspicions? If it turns out you're right, you don't have to invalidate your old runs.

You can buy TFA in 1 mL ampules. Not sure if that is really the problem you had, but I feel more comfortable using an unopened ampule than a bottle that someone else may have left open...