Zic-HILIC retention time shift

[zoltan]

Hi,
firstly, I want to tell I am PhD student (biochemistry) and I trying to isolate and identify glucosinolates from various plant sources. We successfully optimised the isolation steps and found out that Zic-HILIC analysis is the best way of basic analysis (according to Wade, et al. 2007, doi: 10.1016/j.chroma.2007.04.034).
After few months I have some trouble with the analysis. We are limited only for isocratic run of the LC system. I recognised that the retention time of the most analytes in the mix of glucosinolates are shifted, e.g. retention time for glucoraphanin increased from 9,000 to 9,520 min and sinalbin increased from 9,943 to 10,140 min. I tried to regenerate the column, but the retention times did not change back to original (e.g. retention time of glucoraphanin is now 9,333 min and for sinalbin is 10,033 min). I run the regeneration protocol according to the manufacturer (we have Zic-HILIC 150 x 4.6 mm, 5 μm, 200 Å PEEK HPLC Column SeQuantTM), but at flow rate 0,5 mL/min as we use for analysis. Before the regeneration I washed the system (without column) in 80% (v/v) methanol to wash the flow cell in detector (approx. 2 mL/min). Can anyone give some suggestion what can be the problem or any solution for the problem?
Thank you for help.
Zoltan

[Ice9]

What type of chromatic system. Regardless, with HILIC chromatography wash solvent composition if it enters flow path (waters acquity classic weak wash) is extremely important that it be identical to in water.organic concentration to initial conditions. I usually just put the wash (weak wash for acquit classic) in the mobile phase itself as long as there is not a good reason not to (extremely high salt concentrations, corrosive MP etc. Typically we would need more info to give a good answer.

[zoltan]

Hi,
thanks for your answer. We use an older system, which include:
pump: Shimadzu LC-10ATvp
detector: Shimadzu SPD-10Ai (UV-VIS detector)
The recommended regeneration (according to manufacturer) is:
30 column volumes (CV) of water
30 CV of 0,5 M NaCl
30 CV of water
and after that the column is equilibrated in the 5mM NH4Ac in 80%(v/v) acetonitrile (pH = 6,8)
I run this regeneration step, but later I read, that the equilibration shoul be done at lower flow rate of mobile phase, but I don't know what they mean by "lower flow rate" (the exact flow rate).
Thanks for help.
Zoltan

[Ice9]

I think the lower flow rate is not in relation to your flow rate for a run, just that when regenerating a column you don't want the pressure to be too high, so use a low flow rate and do it for a long time. Well depends on the column too, I've regenerated c18 colums at pretty hgih pressure flowing backwards through it, so results may vary.

[Vlad Orlovsky]

Are you keeping your temperature above "ambient"? If you are not controlling the temperature of the column, you might be seeing effect of temperature or you have problem with your HPLC system. If you are using HILIC mode, the only reason for increased retention might be increase in polarity or basicity of your column (since your compounds are acidic). It is hard to believe that this is happening. Another reason for shifted retention time might be equilibration. You might want to consider to switch to RP/IE mixed-mode column (or HLIC/anion-exclusion). My guess is that this should be a 5 minutes isocratic method on 50 mm column. Contact me if you are interested.