Chemical Forums
Chemistry Forums for Students => Organic Chemistry Forum => Organic Chemistry Forum for Graduate Students and Professionals => Topic started by: alexia on August 13, 2009, 03:47:49 PM
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Hello. I made a compound that I can't figure out how to isolate, I was wondering if anyone could help. This compound contains an amine and pyridine group. I took a TLC on silica plates in 100% methanol, and the compound is very polar, it hardly moves off the baseline. Still, I tried a small test column, not surprisingly nothing came off. I tried to do prep TLC, but the compound could not be extracted off of the silica gel. Then, tried doing prep HPLC, but that didn't work out either. I'm wondering if the silica gel is the problem, I really don't know... Thanks if anyone can offer any help or advice...
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Is the compound you've drawn the one you want to isolate, or is it the starting material for which you are deprotecting the t-Boc amine?
If the former, and maybe the latter as well have you tried an acid base wash with something like NaHCO3 and e.g. NaOH?
I'm suprised it's not moving at all in 100% MeOH, though this can dissolve silica, so be careful there. Can you try alumina based systems, or reverse phase plates/silica?
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try to ppt w/ ether (from min. amount of MeOH, or whatever its soluble in)
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What are your impurities? You might be able to use ion exchange.
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The picture I drew is my product which I'm trying to isolate. The other impurities present appear to be some boc lysine, 3-pyridinepropionic acid, and N-hydroxysuccinimide. Thanks for the advice, this is helping a lot.
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Peptides (which are similar in structure to the amine you have) are generally purified on an HPLC using a reverse phase column using water/acetonitrile as the solvent. You can find a protocol in the Current Protocols in Molecular Biology (doi:10.1002/0471142727.mb1014s54 (http://dx.doi.org/10.1002/0471142727.mb1014s54)).
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Hello Alexia
(please forgive the repetition - I emailed this message to your Inbox, but I decided to post it anyway because others may also find it useful).
I can think of 3 ways to help you with this purification:
1) Are you familiar with cyano-functionalised silica gel? Silica gel that has been functionalised with cyano (CN, otherwise known as “carbonitrile” groups) has the exceptional property of being both a “normal” AND a “reverse” phase. The polarity of this sorbent is exactly half way between the polar and non-polar phases, therefore it can behave as a normal sorbent or a reverse phase sorbent depending on the solvent system that you employ. The beauty of CN-functionalised silica gel is that it is the least retentive sorbent in either situation; in other words, your amine would move down the column much faster and more readily than with regular silica gel. The only downside of using cyano-functionalised silica gel is that it is potentially quite expensive – slightly more expensive than C18 reverse phase silica, in fact.
2) The second choice, which happens to be much more affordable, is to use Florisil®. The Florisil® sorbent is a magnesia-silica gel that has been activated at high temperature. It is great for lipids, fatty acids, cholesterol esters, amines, basic compounds, natural products and alkaloids, nitrogen-containing heterocycles etc. It is slightly basic in its nature and it would allow an amine to flow fast and efficiently down the column. Since no real fictionalisation is involved with Florisil® the cost of the sorbent is low; however, it is not as “flexible” in its use as CN-silica gel, which can be used as both a normal and a reverse phase sorbent.
3) Last but not least, you have the option of doing some “catch and release” purification. To do this, you will need a functionalised silica gel that is a “strong cation exchanger”: for example propylsulfonic acid-functionalised silica gel, or tosic acid-functionalised silica gel. If you pass your solution through a column containing the cation exchanger, your product will be “caught” by the silica gel and you can wash off the non-basic impurities. Then, you can pass ammonia-methanol through the silica gel, and your compound will be released.
Best wishes
Athan
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Determine the pKa of your amine. Adjust the pH of your solution to 2 pH units below the pKa. Load on a cation exchange SPE column at 1 drop per second. Dry sorbent under vacuum pressure for 5 minutes. Elute with a buffer that is 2 pH units above your pKa. You have isolated your amine.
Contact me with questions.
Don dshelly@unitedchem.com