Peak Splitting on exsiting method - not the column/system

[kevinkol]

Hello,

I have a problem with peak splitting and I'm hoping I can get some ideas.

The method is a 0.1% formic acid / ACN gradient from 5-95%B on a PackPro C18 column. Sample concentration is 0.2 mcg/mL with 20 mcL injection volume. Sample solvent is 50/50 ACN/Water.

This method has been in place and running well for ~18 months when suddenly, I observe peak splitting of multiple components.

I switched columns (three different) and systems (N=2) and observe the same problem, though it does vary in intensity between columns (from shoulders to nearly resolved split peaks).

All mobile phases, diluents, and samples have been reprepared with no significant changes.

Preparing samples in weaker solvent or decreasing the injection volume does seem to improve peak shape. So the obvious solution is to do just this.

However, this is a validated method and it has been working well for all this time. What can be causing this sudden change?

Thanks for taking the time to consider this problem and I appreciate any ideas.

Kevin

[Uwe Neue]

It appears that you are aware of the problem of injecting a large sample volume in a solvent which is a strong eluent. When you do that, it is common, even normal, to get distorted peaks, double peaks or fronting peaks. To prove that this is the problem, inject sample made up in a much more polar solvent, preferentially in the mobile phase composition at the beginning of the gradient. If the double peaks disappear, your issue is related to changes in the precolumn mixing volume. Maybe you have shortened the tubing, or selected a tubing with a smaller i.d., or made changes in the injector etc.

If the problem does not go away completely with injecting sample made up in the initial mobile phase, then your columns may have died. It is rare that three different columns die at the same time, so this is why I would look at the sample solvent first. However, it is not impossible that three column die at the same time, if changes have been made recently to the column storage solvents or related things.

Assuming that you can prove that it is an issue related to the strong sample solvent, you may be able to save your method in all details by prescribing some additional mixing tubing between injector and column.

[Bruce Hamilton]

Uwe has probably given you the solution, however with regard to your question of why it changed from working previously, you may discover the cause during the inevitable process of review before you modify the method.

The most obvious reason is that the sample has changed slightly, assuming nothing else has changed, as you state. Other possible reasons could be an uncontrolled parameters, such as water quality, needle wash composition ( if used ), or sample temperature, were maginal.

As it is a validated method, I'd ask why was the method subjected to validation with such a high risk sample solvent?. I'm bemused by the expensive effort to validate methods that use high risk sample solvents, without investigating first whether good practice ( eg sample dissolved in initial mobile phase, or similar ) is being followed.

Bruce Hamilton

[kevinkol]

Uwe and Bruce,

Thanks for the quick response. As would be expected, Uwe had it exactly correct. I added an extra length of tubing between injector and column, and peak shape is back to normal.

I misstated the injection volume of the "validated" method - it was only 10 mcL not 20. I know that it is hard to generalize, but I'm somewhat surprised to have this solvent strength problem with such a small injection volume.

Thanks again for your help. Uwe, as a debt of gratitude for solving my problem, I promptly went and purchased a copy of your book.

Thanks

Kevin