[Babcock_Hall]

We deprotect our amino acids using TFA, and we purify them over Dowex-50 using water first, then HCl.  We have not seen loss of the double bond.

[rolnor]

I guess you see coupling pattern on the double bond, if this is intact you just have to run the deprotection for shorter time as you suggest. Whats happening with the trityl is hard to say but it seems like a dead end.

[kriggy]

We deprotect our amino acids using TFA, and we purify them over Dowex-50 using water first, then HCl.  We have not seen loss of the double bond.

Hm I dont think we have dowex but im going to look around if I can get some. Also, how do you know how much water / HCl to use? I suppose you cant realy TLC it to see how it moves on dowex column?

I guess you see coupling pattern on the double bond, if this is intact you just have to run the deprotection for shorter time as you suggest. Whats happening with the trityl is hard to say but it seems like a dead end.

I guess. Im worried that the double bond moved around but thats not something I can figure out now without having the purified amino acid.

Thanks for help guys

[rolnor]

TFA will not make a double bond move, if there is a double bond I think its OK.

[Babcock_Hall]

For Dowex-50 we use step gradients, such as water then 4 M HCl.  We make large pools, comprising several column volumes.  We often load the amino acid in 0.5 column volumes of water, then use 1.5 column volumes of water as a wash.  Then we use 2-3 column volumes of HCl.  If an amino acid elutes slowly (and when there is a hydrophobic side-chain, it has in our hands), then the product still make show up in more than one pool.  We take several pools, rotovap/vacuum, and check masses.

Dowex-50 is an old-school technique for purifying amino acids.  Often people used water followed by 2 M ammonia , and I have used this many times myself in the past.  Although I have seen people back the ammonia down to 0.5 M once or twice (presumably for sensitive side chains), I decided to avoid any possibility of its attacking the amino acid, and that is why I chose to elute with HCl.  4 M HCl is probably overkill, but people have used it before, so I just decided to try it.  We only occasionally use TLC on amino acids, but Stahl's venerable TLC book gives five common solvents.  70:30 ethanol/water is one of them.  There are both acidic and basic conditions.

We occasionally use Dowex-1 in the acetate form, but it does not always work in our hands for reasons that are not known to me.  The idea is for the carboxylate group of the amino acid to displace an acetate ion on the resin.  Acetate is less strongly bound to the resin than chloride; the conversion of the resin from the chloride form to the acetate form is lengthy but not difficult.  If you need any references or protocols, just let me know.

This brings me to a question.  Someone once asked me if I knew of any papers on the purification of amino acids by ion-exchange.  I said that I did not, but that I could almost write one.  Since that time my students and I worked out one additional method that is applicable when the side chain carries a charge.  Would any journal or serial publication (by which I mean something like Methods in Enzymology) be interested in such a work?
EDT
Just FYI, there are some reviews (I have not read them in a while) of unsaturated amino acids that deals a little with isotope labeling:
Kaiser J et al., Org. Biomol. Chem. 2005 3:3435-3467. (a review which covers 2000-2005)
DOI: 10.1039/b001538p (a review which covers 1990-2000 IIRC)

[rolnor]

You could use some MeOH in the eluent to push out the product from the resin if you have lipophilic groups in the product. 4M sounds really strong, I would try 0,5M for sensitive compounds. But if it works good, there is no reason to change anything offcourse.

[kriggy]

@Babcock_Hall:

Thank you very much for your reply and the references, it was very helpful.

I have few question towards the work with dowex. At first, I need to wash with HCl to make sure its in the H+ cycle, then with water untill the elution is neutral. Then add my aminoacid in water (free base not salt)  and elute with water and then with HCl. ?

If my reaction mixture contains various salts (NaIO4) I suppose they are converted into HIO4 and still elute while the Na+ is retained on the ion exchange?

Thank you.

[Babcock_Hall]

When converting from a more tightly bound ion to a less tightly bound ion, one has to use a greater volume of the new ion.  "As a general rule, use 1 bed volume of 1 M counterion solution for each unit difference in relative selectivity. For example, converting AG 50W-X8 resin from the K+ form (relative selectivity 2.5) to the H+ form (relative selectivity 1.0) would require 2-3 bed volumes of 1 M HCl. The conversion is complete when all the K⁺ ions are displaced by the H⁺ ions."  This information is useful when one reuses Dowex-50 that is in the ammonium form (see below).

Regarding the rinse after the conversion (for example) from H⁺ to Na⁺, BioRad recommends 4 bed volumes of water and checking the pH.  The protons on Dowex-50 should protonate your amino acid.  The sodium ions will also stick.  Therefore when you calculate how much resin you need, you will need to sum the moles of amino acid and the moles of NaIO₄.  I typically use 20-fold capacity of resin, relative to this sum, but a lower ratio may also work.  You can elute with acid or with ammonia.  In the presence of a sensitive group, I have seen a few authors elute with a lower concentration of ammonia than is typical (0.5 M vs. 2.0 M), but if HCl is compatible with your functional group, then it should work fine.  We have used HCl in the presence of either an aldehyde or a Michael acceptor.

[Babcock_Hall]

http://www.jbc.org/content/213/1/39.citation
Black and Wright, J. Biological Chemistry (1955) 213:39-50 converted allylglycine into aspartate beta-semialdehyde and then purified over Dowex, using 4 M HCl.  There are probably other examples of acid elution in the literature, but my paper copies of articles on this subject are in arrears (I would start by looking up papers by Stein and Moore, who did a great deal of analytics biochemistry on amino acids and proteins many years ago).  I think that 4 M is overkill and 1 M would probably work fine, but we used 4 M twice, both in the presence of a Michael acceptor.  The alternative to HCl elution is elution with ammonia, which deprotonates the bound amino acid. 

[rolnor]

Yes, if it works its fine. I guess you have to rotavape a lot of acid, othervise it does nor matter.