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- Posts: 6
- Joined: Mon Oct 12, 2020 5:41 am
I am new to separation science and still learning, and would be lovely to hear some thoughtful advice.
I am fractionating crude polar plant extract using a semiprep C18 column (250 mm) at 0-40 B% gradient over 50 min:
https://drive.google.com/file/d/1JRcKSciER61wRo7IxNf8QMnHGnNRMGOz/view?usp=sharing
When I collect what looks a well separated peak and run on an analytical C18 column (150mm) 0-40 B% over 17 min to check purity I get this "hump" in my chromatography, which is more prominent at 210 nm than 254 nm.
https://drive.google.com/file/d/1HrhdJRm3vOiz7Uh0TJX_0L97lfd1Hfdp/view?usp=sharing
If I do another round of semiprep HPLC on this hump and compound mixture, I am able to purify the compound, but I am hoping there is a way to maximize the yield and fractionation efficiency.
So where is this hump coming from? Is this a protein? Is it because the baseline in my semiprep HPLC curved? Is there a way to further straighten the baseline and improve separation?
Thank you so much in advance for any help.