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gradient problems_!!!!!
Posted: Fri Oct 02, 2009 5:20 pm
by Pao
Hello, my name is Paola,
I am having some problems tryng to implement EP method for Related subtances.
EP method has a gradient, FM A is (NH4)2CO3 solution + THF and FM B is ACN. wavelength is 220. Column: inertsil ODS 3
For some reason in the place where it should appears my interest peak it also appears a huge mountain of "ghost" peak. So I can not define LOQ or LOD properly and I also can not define correctly my peak. I am using HPLC Waters 2695.
May it be the cut off of THF (225nm)?? I also have a terrible base line!!!
What can i do??
Posted: Fri Oct 02, 2009 5:52 pm
by DR
220nm is low enough that you could be having peroxide issues with the THF (I'm assuming it is not stabilized THF - if it is stabilized, you need some unstabilized THF). The other possibility is that your water is not quite up to the task at hand. Search the forums for "Empore Extraction Disks" - you will find several threads discussing their use to prepare water for use in your A phase.
Posted: Fri Oct 02, 2009 5:53 pm
by grzesiek
" I am having some problems tryng to implement EP method for Related subtances." - that usually means you are normal human
"What can i do??" - forget about the metod, use another
Posted: Tue Oct 13, 2009 3:51 pm
by seamoro
Clean your system out 6M nitric acid..that will get rid of any built up contamination
Posted: Tue Oct 13, 2009 5:09 pm
by lmh
if you follow seamoro's advice, DON'T, DON'T under any circumstances let your 6M nitric acid get in contact with organic solvents. It explodes. Really. It did in a building close to where I sit, and it hurt someone badly.
Posted: Fri Oct 16, 2009 9:17 pm
by Chromatographer2010
To isolate where the ghost peak is coming from you should do a couple of isolation experiments.
First do a diluent blank by running an injection of your normal sample diluent. If the peak goes away then the contaminant is in your sample.
Next try an injection of your A mobile phase. If the peak goes away then the contaminant is in your diluent.
Then run a gradient blank by doing a zero volume injection or an inject immediate if you are using Empower. If the peak goes away then the contaminant is coming from your off-line portion of your injector. Wash solvents or seal wash frits typically.
Next bypass the injector and plumb your pump directly to your detector. Run another inject immediate. If the peak goes away then the contaminant is in your on-line portion of your injector.
Next try two gradient blanks with a different reequilibration times between injections with 100% of the A solvent (more aqueous). If the ghost peaks are different heights then the contamination is in you A solvent or A solvent lines.
Next try two gradient blanks with 1-5% of the B solvent with different reequilibration times between injections. If the ghost peaks are different heights then your contamination is in your B solvent or B solvent lines.
Next try changing the solvent that has the contamination to a different source. Another brand, different water system or even bottled water. This will tell you if its the solvent lines or the solvent themselves.