Change in the standard chromatogram

[Chamila]

I got a question with the HPLC method I am working now and need immediate solution.....

Please help if anyone has an idea for this....
I am running several standards with C30 column in a RP-HPLC. I have 5 standard compounds and when I develop the method I got a good baseline separation for all the compounds. And I used the same method for quantification of samples too...
And yesterday when I tried to inject the standard mixture again, I did not get a baseline separation for two of them. they were half overlapped...and today i tried again and I only got frour peaks!!!! those two compounds are completely overlapped and appeared as one compound!!

I have no idea what is happening inside the column....Has anyone experienced this before????? anybody knows a solution.....Its urgent, please help.....

[Gerhard Kratz]

Could be a column problem, but it could be also a problem with degradation of your standards in solution. Did you prepare fresh standard solution yesterday and today? What are the chromatographic conditions, mobile phase, buffer, organic modifier, flow rate, temperature etc.? Please prepare mobile phase and standard solution fresh and try it again.

[LC_labrat]

If the fresh solutions aren't seperating the peaks enough I would try a new column.

[cody84]

I would wash the column really well and inject same vial again. Are both peaks getting bigger each injection?