Online SPE with HPLC

[Christine]

I have developped a method which I am doing a SPE online with HPLC.

Samples are injected on a Oasis SPE cartridge and washed with 5% MeOH to remove salts (we have to remove salts because molecules are detected by light scattering). After, molecules are eluted from the SPE cartridge (2.1 by 2 0mm) with 85% MeOH, 10% water and 5% ACN. This solution is used also to separate molecules (isocratic conditions) on a Zorbax C18 column (4.6 by 150 mm). While a sample is washed by SPE cartridge, an other one is eluted from a second SPE cartridge and molecules are separated on the Zorbax column and vice versa.

This method has been working perfectly for a year and now I am starting to observe peak splitting only for the first component eluted on the Zorbax column.

I have replaced everything, switching valve, peak tubing, fittings, buffers, column, SPE cartridges, SPE cartrige holders.

Any other suggestions or explanations

Thanks a lot

[Uwe Neue]

I have seen events like this being caused by residual strong solvent somewhere in the lines, causing the elution of the analytes from the pre-column to happen in two slugs. Check if 1. there is a line that is not flushed completely, and 2. if something in the set-up has changed just before you saw this peak splitting for the first time.