HPLC peak tailing

Hi all,
I recently encountered a problem for one of my tests on HPLC. The pic shows a peak with shoulder which should never happen. It's a well established method in my lab and it's the first time I had this problem. This API is being detected at above 300 nm. Along with this API, there are several other APIs in the solution. All of them are detected below 300nm and they all showed normal peak shape. Can someone have some input about what caused it and how to fix it? Thanks.

I have done several trouble shooting steps:
1. washed entire system with large amount of hot water

2. cleaned inline filter

3. switched mobile phases to different lines/prepared new mobile phase/used new column

4. switched inlet and outlet connection order on flow cell

5. switched test to another instrument with the same column and mobile phases

Step 1-3 didn't work at all. Step 4 seems had a little improvement on the shape, however, the shoulder still shows just a bit smaller. Step 5 assured me this is not a problem due to column or mobile phase deteriorating because the peak looked completely normal on the other instrument.


  • Try sonicating the check valves or changing the solvent filter frits.
  • Check the lines on each end of the column. Are they cut cleanly and square? If not, fix that. Also make sure they are fully seated when the column in installed. If that isn't the issue, you may have to more fully clean the flow cell and/or the rest of the system. Search the Waters website for their passivation procedure.
  • Check the system pressure.. I think seals are blocking just check the service engineer