Chromatography Forum

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:

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.

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.

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?

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.

Thank you for your response.

Yes, that's correct, we don't have temperature control on the semi-prep HPLC, but there is a column oven on LC-MS.

Unfortunately we only have C18 columns, not HILIC.

Gerhard Kratz wrote:
Thanks for the details.
So you have no temperature control? When you did the methode development did you tried a HILIC column?

Thank you for your response.

Yes, that's correct, we don't have temperature control on the semi-prep HPLC, but there is a column oven on LC-MS.

Unfortunately we only have C18 columns, not HILIC.

Gerhard Kratz wrote:
Thanks for the details.
So you have no temperature control? When you did the methode development did you tried a HILIC column?

Good Morning,

My recommendation is to use your MP also with 30°C. 5ml flow rate with a temperature difference 30°C to room temperature (21°C) is in my opinion too high. I would insulate the capillaries between injection system and the column. And please take into account what Vlad has posted.
Take care and stay healthy! Have a nice and sunny day!

Phytochem wrote:
Is it because the baseline in my semiprep HPLC curved? Is there a way to further straighten the baseline and improve separation?

Could be. Your baseline is quite elevated. So you're collecting also what's forming this baseline.
So, what about tannins/catechins?
As polymeric compounds, they may form such unspecific humps.
How does the peak/hump UV-spectra looks like?

Depending on your compound of interest, removal by filtration/adsorption on polyamide or Gelatin may help but may also adsorb your desired compounds.