Hi everyone,
I normally use sep pak C18 cartridges to separate my molecule of interests from copper. I successfully used cartridges with 360mg sorbent weight and I recently bought the cartridges from same supplier with 5g of sorbent to scale up the purification. I applied the same protocol with same elution gradients and I couldn't obtain my product, it wasn't eluted in any of the gradients that I used. Do you have any suggestions on why this happened? How does the increase in sorbent weight affect separation?
Thank you in advance!
I'm reading your question as "I used the same volume of solvent for the 5 g cartridge as I used for the 0.26 g cartridge" since I saw this: "I applied the same protocol with same elution gradients and I couldn't obtain my product, it wasn't eluted in any of the gradients that I used"
If I interpreted that statement as you intended, the compound is likely still stuck on the column. You need to scale up the solvent too in each gradient step.
5 g/ 0.360 g = 13.9, so you will need to apply that multiplier to all your gradient steps. Assuming you used 1.5 mL per gradient step for the small sep pak, you'll need 20.8 mL per step for the larger cartridge (13.9*1.5)
Jack Silver thank you for your reply. Actually I used 10 ml for the small cartridge, and I don't think is feasible to use 139 ml of solvent for each gradient. How can I solve this issue? Also, how would you increase the flow, since also that must be scaled up? with a pump? For the small cartridge I used to use a syringe to just slowly push the solvent to obtain a flow of circa 1 ml/min.
I can't answer the first part of your question because I don't know details of the solvents used, nor details of the steps used. you may be able to optimize that.
As for the question about flow rate, you don't need to scale it up precisely. Sep pak cartridges are conceptually similar to flash chromatography (or any other chromatography, including HPLC) and the compound elutes in the same number of column volumes (CV) when using the same media and solvent composition. One CV is approximately the void volume. The elution time in CV is mostly independent of flow rate. In fact, that's how I calculated your scale-up- the CV is proportional to the amount of material packed. Ideally, the flow rate is proportional to the square of the cartridge radius when scaling up.
You may find flash chromatography to work better for this application, too (please note I work for Teledyne ISCO, a manufacturer of flash chromatography systems).
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