In the synthesis of peptides by SPPS, at the end of the cleavage it is precipitated in cold ether and the ether is allowed to evaporate and the solid is obtained. This is not happening, the peptide disappears along with the ether. Something slimy remains in the tube. Somebody could help me? In the image you can see what remains in the tube.
Deisy Yurley Rodríguez Sarmiento
After the cleavage of the peptide with TFA (resin and protecting group cleavage), you need to evaporate the TFA. The residue is treated with cold ether and you should observe a yellowish-white precipitate. You can rub the flask and let the flash in a freezer for a few hours to enhance the precipitation. The precipitate is the peptide and the ether contains the dissolved protecting groups. The next step is filtration with a funnel and washes with cold ether. You keep the white solid and you discard the filtrate (ether). The final step usually involves dissolving the white precipitate with aq. CH3COOH, water and MeCN and lyophilization, to afford the crude peptide.
Hello Deisy
I am also currently doing chemical synthesis and cleavage of peptides. Sometimes I am also encountering colloidal-like or slimy-like precipitates in my tube that I precipitated and washed in cold ether, and dried with nitrogen in contrast to what I should ideally get which is solid and powder-like ppt.
What I normally do is that I store the peptide precipitates in cold ether in the fridge for around 10-20 min just to make sure that I see visible precipitates before washing and drying.
If it doesn't work, I improve my cleavage cocktail conditions, and increase my cleavage time (~4hr for R-rich peptides), and do a Kaiser test on the resin before and after cleavage to see if the peptides were completely removed in the resin.
Finally, if my previous methods still give me colloidal-like or slimy-like precipitates. I improve my synthesis conditions preferably on the activating and coupling reagents based on the recommendation on the technical bulletin and literature search.
Hope my inputs would help you.
Good luck with your work!
Your peptide, if not volatile enough and which will not be even if it's a dipeptide, is not vaporizing out in ether at RT, higher temperature (and under reduced pressure) is a different story. The other impurities, parts of the protecting groups, small molecules, etc. all volatiles at the (given) temperatureare are vaporized. Better would be to precipitate first after some scratching the flask wall and keeping it in fridge-cold condition. Washings with cold ether (since you precipitated in cold ether) will provide the product with little loss in repeated washings.
Hi Deisy,
Peptides commonly precipitate when you add ether to your cleavage cocktail after cleavage (unless they are too hydrophobic). My usual approach is to centrifuge 3 times discarding the supernatant, this way you remove from your reaction mixture all the protecting groups, the TFA and the scavengers. Then I dry the pellet and redissolve it in ACN/H2O for HPLC analysis and purification.
Alternatively, if your peptide does not precipitate upon ether addition, you can just evaporate the cleavage cocktail in the rotavap.
Hope this helps!
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