Compound isolation

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

5 posts Page 1 of 1
Hi everyone,

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:

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.

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.
More chromatography details would be helpful. Maybe it is a temperature problem. Temperature control not only the semiprep column, also the injection system, short capillaries, same temperature for the solvent reservat, what is your pH? And give my regards to Prof. Hans Rausch.

Happy new year to all of the community.
Gerhard Kratz,
Thank you.

The buffers for semiprep HPLC are water with 0.05% TFA and 90% ACN with 0.05% TFA - column and buffers at room temperature.

The buffers for analytical HPLC are water with 0.1% FA and 90% ACN with 0.1% FA - buffers at room temperature, column at 30C.

The loading method for semiprep is 5 ml line loading (flow rate 5ml/min), column 21.2 mm ID - fractions collected automatically based on the peak up/down slope.

Loading for analytical is automated injector, 5 uL, flow rate 0.8 ml/min, column 4.6 mm ID.
Thanks for the details.
So you have no temperature control? When you did the methode development did you tried a HILIC column?
Gerhard Kratz,
Mixed-mode columns will provide you with 5-20 times loadability (and retention) and additional mechanism of interaction (ion-exchange), which will help you achieve desired result. We have customers who used 4.6x250 mm columns for isolation of impurities instead of using a much bigger column. Contact me through our website if you would like to discuss your separation.
Vlad Orlovsky
HELIX Chromatography
My opinions might be bias, but I have about 1000 examples to support them. Check our website for new science and applications
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