Problem with cleaning column??

[monic]

Hi everyone!!
I have a question if someone's had the same problem. I determinate amines and use water:methanol phase and C18 column. I did couple injections and noticed that i have some ghost peak from phase in PDA. I wash out the column, the HPLC system and wash that by methanol again. I've checked methanol and it was ok. But I checked my water and there was a signal in PDA at about 200nm. I wasn't sure if water is clear so i did the different injection from different type of water so i appeared again. Is there any chance that I,ve got something on column that is not washed properly by methanol and it could be washed by water?? Amines can do so?? It is strange because the sample is transparent so there was no doubt to used methanol. For more information I use isocratic separation, the amines and another components are not seen in UV properly that i needed to use RI detector. I cleaned detector with 6M nitric acid at first, so it is not from detector. It is still there.
Thanks in advise for Your replay
Monika

[Gerhard Kratz]

Hi Monika,
What is your mobile phase composition? If you have 50:50 Methanol:Water and you inject pure Water you will get a peak, and that for sure at 200nm. It is due to refractive index in your PDA flow cell. That's all. Please use your mobile phase composition also for your sample, and your ghost peak will dissapear. Good luck

[monic]

Hi Gerhard,
Thank you for Your replay
I used methanol:water 25:75 mobile phase, but I have used mobile phase composition to dissolve my sample from the beginning, so it is not that probably. All I have done was washed the system by 6M nitric acid, because I started to think it could be an injector and a needle. Hope to have problem resolve tomorrow.

[monic]

Hi Gerhard
Unfortunatly i still got is at my system somewhere. Do you know how to wash amine or phenols out of the system?? I suspect that may be one of those.
I've checked different columns and different solvent and it still appears. In water it has max at 201.5nm and at around 265. Even if I inject just pure water to pure water it's the same. If I made 0 ul injection it appears
How longe shoulde I wash my injecton port??
Do you have any suggestions??

[Gerhard Kratz]

Hi Monic,
first, disconnect your column. Than rinse your system first with 10% sulfuric acid, than with Water, than with Acetonitril. Check your injector, the rotor seal respectively - replace it. If you have stainless steel tubing in your system, replace it and make NEW dead volumn free connections. Check your solvent delivery parts. Than rinse the column first with Water, than with Acetonitril, than take the certificate of analysis what should be provided to you with the column, and do the QC check, under the same conditions as discribed in the Certificate of analysis, to check if the column is still ok. What is the matrix of your sample? Is it a little bit viscose? It seams that you have a very triggy problem. Good luck.

[monic]

Hi Gherard
Thank you for advice
I will try what you suggest tomorrow.
My matrix is phosphorous ester additionally with amines part. I suspected also phenols as my contamination because I've done couple injections recently. The problem with system and ghost peaks starts two weeks ago. I forgot to add that sometimes it appears not just as one peak depends on condition run and solvent. Really strange.

[HW Mueller]

Sounds to me like a simple carryover (not ghosting) due to phenols getting stuck between the rotor and stator or even material that is sort of dissolved in the teflon (or similar) rotor. This has been discussed before. I think I even related the story that this sort of carryover led a group here in Giessen and another in the USA to possibly wrongly suggest that digitalis or analogs are a new human hormone.