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- Posts: 3
- Joined: Wed Jun 10, 2020 7:26 pm
- Location: Slovakia
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