Peak splitting in HPLC

[meadowlane]

Hi,

I recently worked on a group of compounds, the peaks split at 10-uL injection size at different concetrations, but look all fine at 5uL injection size. For ease of method transfer, I would like to keep 10uL injection size.

My mobile phase is Water/ACN/TFA, diluent is 50/50 water/ACN. Can anyone suggest some way to reduce the peak splitting?

Thanks,

[tom jupille]

Use more water in the diluent.

[HW Mueller]

Maybe also TFA in the sample solvent, the pH incompatibility might play a role also.

[Consumer Products Guy]

all fine at 5uL injection size. For ease of method transfer, I would like to keep 10uL injection size.

Why would 10 uL provide "ease of method transfer", I don't understand. Is a location still using manual injection and would need to physically swap over an injector loop (isn't it the 1990s now?) ?

[tom jupille]

Why would 10 uL provide "ease of method transfer"

At the risk of misinterpreting the OP's intentions, a method that touchy about injection volume has *serious* robustness problems.

The reason I suggested more water in the diluent (i.e. make it weaker) is that I have run into a couple of situations where the "strong diluent effect" was on the ragged edge such that the method would work on one system but not on another (presumably due to differences in mixing characteristics).

[DR]

And, if it turns out that the diluent composition does not matter, it's probably time to check for poorly seated fittings or a void in the column as these can cause splitting also.

[meadowlane]

thank you all, incerase water in the dluent helped to eliminate the pek splitting.

[Maheswaera Reddy]

What is the intial cmpoistion of your mobile phase in the gradient or if it is isocrotic mode use the same ratio as diluent.