0.1 TFA is the mobole phase. I am using symmetry column.Temperature is maintained at 40 degrees. RT shifts in both directions. I tried longer equilibriation, but that didnt help. Along with this some time my peaks are tailing.
The responders above offered general advice and asked for more detailed information, and the above information is insufficient for further detailed suggestions.....
TFA is 0.1% of the mobile phase. Would you please look at the method you are following and share with us the detail of the preparation and composition of the remaining 99.9%?.
Knowing whether you are using a gradient ( with details of the programme ) or isocratic method would also help. Details of the flow rate, and column pressure at the initial flow, would be helpful, as would whther the pressure changes with the number of sample injections.
As a very first step, you should measure the flow coming from the detector cell and ensure the flow is constant throughout several sample runs, and that there is no correlation between pressure and retention time variations. If there are variations, you should look for leaks or air bubbles in the pump, and also ensure sample solution is completely miscible with the mobile phase.
Can you look on the column ( or the Waters column box ) and provide more details like whether it is RP8, RP18, along with the particle size, column internal diameter and length. An indication of whether you are using a guard column, and how frequently you change it would be nice.
Could you look at the detector and tell us what sort ( UV ) and what the detection wavelength is. An idea of the sample sequence used and confirmation that any standard is stable would also help.
Could you also add the identity and nature of the sample, and how you prepare it ( what solvent, any filtration or centrifugation).
Can you provide some historical details - eg were you using the method previously for a long time, or is it a new method?. How long has the column been used, and whether the pressure has changed.
Bruce Hamilton
