Increasing retention time

[carnie]

I'm running a method using a water/MeOH/CH3CN/Formic Acid (700:2005) mobile phase. The retention time of the sample continues to increase daily. I get it back to the required time by reducing the amount of formic acid in the mobile phase. This operates well for a period but then the next day I have to reduce the amount of formic acid again. I'm now running at approximately 1/4 of the initial volume of formic acid. Can anyone help explain why this is happening. I can't give any information on the compound as I don't know, however based on the above I'm assuming it's an acid. The sample solvent is MeOH/H2O/Phosphate buffer(pH3.0)/saline (1:1:1:1). The column is an ODS-2.

[Consumer Products Guy]

Are you pre-mixing the mobile phase, then putting into a single reservoir, and using that for a few days? Maybe some organic is being lost through evaporation, making your mobilephase more aqueous in nature, causing longer retention times.

[carnie]

The solvents are pre-mixed in a single resservoir, however even if I prepare fresh mobile phase the change is still apparent. Also, ther is an internal standard which is unaffected.

[HW Mueller]

Sounds like you are killing your column (this assumes that washing the column doesn´t reverse things).

[carnie]

If I were killing the column, wouldn't the Internal standard peak show a similar effect.

[HW Mueller]

Only if the internal standard was behaving chromatographically similar.
Also, is your analyte a single entity (molecule) and stable?

[SMC]

Just a few thoughts:
Please excuse my ignorance but why do you assume it is an acid?

I have seen this with a tricyclic antidepressant (e.g. Nortriptyline). It seemed that I had to saturate the column with this compound prior to getting stable retention times (after something like 20h of continuous injection).

Are you always injecting the same sample size of your unknown compound? Especially for ionic compounds column overload could be an issue in respect to retention; e.g. had you injected a larger sample size first (under overload conditions) and then you reduced the sample size retention could probably increase.

Is your ODS2 column temperature controlled? Changes in temperature can change the pKa of the compound thus leading to shifts in retention at given pH, if the pKa of the compound is close to mobile phase pH. The pH of FA probably is not markedly affected by T-change.

Still this would not explain your observation with respect to the reduction of FA…mmh…I will think about it a bit more...

[carnie]

Just to give a little more background. This method had been operating over a number of weeks without any problems. A total of about 150 injections. The first 100 injections were at 20 microliters and then changed to 50 microlitres. The change in retention time occured between one days analysis and the next with no changes to the system.
I'm assuming that the analyte is stable as I 've established stability in the autosampler for 24 hours and that it is a single molecule. I made the assumption that it was an acid because if I reduce the amount of formic acid in the mobile phase the retention time increases which would be the expected behaviour of an acidic component. The column is temperature controlled.
I'm thinking that the column may have become contaminated and the contaminant has an affinity to the analyte but not the internal standard. I'll try a thorough washing sequence and see where that takes me.

[HW Mueller]

carnie, on this formic acid concentration effect: Are you thinking of a solvent effect of formic? If you decrease the formic acid you are increasing the pH. This could have the tendency to increase the ionization of your acidic analyte, and thus decrease its retention (if the final pH is still ~2 units below the analytes pKa it wouldn´t change much).

[pawan ratra]

I will suggest to incorporate some buffer (keeping the same pH) in the mobile phase and then check whether u get the same observation or not.