Chromatography Forum

I am working on the OIL spill. I am currenlty looking toindentify some fluorescing compounds found in BP crude for use as indicators.
I did a normal phase column and collected a fraction. Through MS, this sample is still very complex. I decided to use RP HPLC to further separate it. This fraction was in Hexane. I forgot to evap the hexane and replace it with Methanol.
I was just doing some initial exploratory runs with a linear gradient from 0-100% MeOH (B) and Water (A).
Needless to say Hexane as my sample solvent was a bad idea. I forgot.

The first run looked good. saw a decent band. But towards the end, the signal began to rise as the run ended.
Now, when I start a run, even wen injecting plain water, the signal of the fluorescence Spectrometer is maxed out in the beginning. Somewhere between 30-70% MeOH, the sgnal drops to zero, and then towards the end goes back to max signal.

This seems like there is something stuck in the C18 guard.

My question is: Do you feel this is the correct conclusion, or is it that these compounds could be that fluorescent?
Since all of the compounds are fluorescent in Hexane, I was thinking of flushing the system with it, then to Isopropranol, then back to MeOH. I think that should pretty much get rid of the problem.

Is this a good idea?
The spectrometer zeros, every run, so, technically I can still see my peaks, but I would like to clean the guard column, since it is not mine. The column is, but I wonder if it is worth cleaning. I could unhook it and just hook the guard up to the Fluorescence Detector. i also have a PDA... but dont use it as much

Sorry, this is a long post. Thanks for your help! great forum guys.

Following the cleaning procedure of the manufacturer of you C18 column, and additionally do the QC test for the C18 column, you will spend minimum 1 day in your lab. More cost effective would be to purchase a new C18 column, and a guard column for your colleague. Otherwise you must get rid of the Hexane on your column. Rinse your column with MeOH and Inject 50°C hot water several times. Good luck.

I spent all night doing the cleaning procedure.

20 CV water
20 CV ACN
5 CV Isopropranol
20 CV Hexane
5 CV Isopropranol
20 CV ACN

The signal subsides for a while then comes back. And just stays for ever...
If the detector has signal, this means to me that my compound is peeling off. I only injected a total of 20 uL onto the column. It has to go down sometime!?!

Any further advice, besides buy a new Guard column? I just want to learn how to fix it. With the range of solvents I passed through, I should of cleaned out everything. Why would the detector continue to go up, level off, and sit endlessly?


Thanks!

Maybe some compounds stick to the frit of your column. Take out the column and guard column and flush your system with your mobile phase with low flow rate to see if you still have the problems with your detector.
If your detector shows no signals your system is free of contaminations. Open your columns and replace the frits.Than install your guard column first with reversed flow direction - no detector connected. I hope that will help. BR