Retention time change
Posted: Thu Jan 17, 2013 9:08 pm
by jeffchem
I am using HPLC to determin monosaccharides. I am derivatizing with anthranilic acid and using a Phenomenex kinetex column/ 4.3pH acetate buffer/acetonitrile with a gradient. I had to change the guard column and my retention times shifted about 2 min (slower) but I didn't change anything but the guard column. Could the guard column cause a shift like that?
Re: Retention time change
Posted: Fri Jan 18, 2013 10:17 am
by Gerhard Kratz
Guard column and analytical column are the separation unit! Both should be brand new when you do validation of a new developed method. When changing guard and/or analytical column give time for equilibration.
What is the dimension of your guard and analytical column?
Re: Retention time change
Posted: Fri Jan 18, 2013 4:05 pm
by lmh
That's worrying, because it's got slower (which implies the new guard column is holding up analyte for 2 minutes more than the old, and no guard column should have such a significant retention time chromatographically, relative to the analytical column, which is the same stuff but a lot longer!).
The most likely explanation is that you have a slight leak somewhere in fitting your guard (so less solvent is passing through the column, which means it takes longer to elute things) or, if you can't trace any leak, it could be this: the old guard may have become blocked, meaning the pump was having to operate at a much higher pressure. And depending on the state of your pump, it may have been pumping a different solvent mix at the higher pressure (for example, binary pump with slightly leaky "B" channel will deliver less than the specified flow, and higher in "A" at high pressure. If "A" is organic, this means retention times in reverse phase can be shortened at high pressure...
If you can, check the pressure traces before and after the change. If either trace saw-tooths up and down, expect its retention times to be very late because it probably means there was an air-bubble in the pump (means less solvent delivered), or a wildly leaky seal or valve somewhere. If the pressure changes smoothly in runs (no saw-tooth) but wildly different before and after changing guard, consider the possiblities in previous paragraph!