Hi! Using Chiral HPLC column (OB-H), I am trying to analyze final enzymatic reaction products of NADPH dependent aldo ketoreductase for possible formation of asymmetrical alcohols of pharmaceutical potential.
I have been notified by the Chiracel company ( chiral column manufacturer) about non compatibility of this super FRAGILE column with any traces of salts (phosphate or Tris) or ethyl acetate in the final sample for column injection!
So, I used SPE strata column to remove all salts before final HPLC injection.
The chiral separation pattern is not matching with retention times of standard chiral forms of target compounds.
The reference articles have mentioned use of ethyl acetate extraction method for the buffered enzymatic reaction . Citing a few for reference here. Also sharing method snapshots from the articles
doi.org/10.1186/s40643-018-0220-x
DOI 10.1007/s10529-016-2167-3
DOI 10.1007/s12257-010-0214-9
So, can someone with relevant research expertise speculate
1) whether it can destroy the chiral column if I follow the cited method of ethyl acetate extraction ? We do not have purchased guard column for this fragile column.
2) Can ethyl acetate extracted sample be considered completely salt free, NADPH free?
I can use vacuum assisted ultracentrifuge to evaporate ethyl acetate and resuspend final extracted sample into IPA or n-hexane for final column application.
Expert opinion would greatly help me troubleshoot further
Thanks a lot,
Aniket
The chiral columns are quite expensive but generally robust if handled as per instructions of manufacturer. The mobile phase is normally hexane-isopropanol mixture. If you inject sample with salts, the salts will precipitate and block the column. Since the manufacturer specifically mention that ethyl acetate is bad fo your column, it is best to remove it by evaporation, redissolve your product in mobile phase and then inject. The retention times do not always match with reported values due to several factors like pump pressure, age of column, whether TFA or DEA were added to the mobile phase etc. Don't worry If your retention times are not matching but they should follow the same trend; e.g. R eleuting before S etc You should anyway always confirm the structure by NMR and Mass; and Configuration by Specific rotation value.
Dr.Nitin Fadnavis ,thanks a lot for sharing your insight in detail.
Just to get more clarity before implementation, can you please verify my understanding :
If I use ethyl acetate for extraction of my enzymatic reaction done in 25 mM Tris -Cl with pH 8 and NADPH as cofactor, I can be safely extracting my target chiral alcohols without any Tris salt or NADPH.
Does this make sense?
Thanks a lot,
Aniket
No. Ethyl acetate has ability to dissolve some water and some salts along with it. For preparative purpose it is OK to extract with EtOAc and dry the organic extracts over anhydrous magnesium sulfate but for chiral HPLC you need to remove this solvent and redissolve in mobile phase
Dr. Nitin Fadnavis
Our institute has vacuum enabled centrifuge to evaporate organic solvents.
So, if I extract my enzyme reaction in Tris buffer by using ethyl acetate and then evaporate it to complete dryness in the vacuum centrifuge; Should I worry about some residual salts that may have got extracted in ethyl acetate ? Or This vacuum centrifuge evaporation is good enough for chiral column ? Also, I understand before final sample application, the dried samples must be redissolved in mobile phase and filtered with 0.2 micron PTFE membrane
You should worry about the salts. After evaporation they're not going anywhere. You have to dissolve the residue in the mobile phase, filter through the membrane filter and then inject.
Thank you very much.
So, the membrane filtration will eventually block the salts from entering into chiral column otherwise. This seems to be the key step in salt removal from the chiral sample, Dr Nitin Fadnavis .
Dear Aniket,
many thanks for sharing this very interesting technical problem with the RG community. As you already know, we are synthetic inorganic chemists, so I'm absolutely not an expert in this field of research. Thus I'm not really qualified to provide you with an expert answer. However, to my knowledge compounds like NADPH and Tris-hydrochloride are soluble in highly polar solvents like water, ethanol, or DMSO. I might be proven wrong, but I assume that both compounds are more or less insoluble in ethyl acetate. To try it out in a quick experiment, just take a few crystals of Tris-hydrochloride (I assume that's what you mean by Tris salt) and add a few ml of ethyl acetate to see if it dissolves or not. In any case I'm pretty sure that both NADPH and Tris-salt will remain in the water phase and can thus be separated by extraction with thy acetate.
On a more general note I would like to mention that RG is much more than just Q&A. As you know, all RG members are asked to post their "Skills and expertise" on their profile page. Thus you can use the "Researchers" section of RG to find and contact researchers who are proven experts in your field. For example, if you are looking for colleagues who also use chiral HPLC, just search RG for the term "chiral HPLC" and then click on "Researchers". This will provide you with a list of RG members who also use this method and who will certainly be glad to help you. Just go to one of the selected profile pages and click on "Experience". Then a button "Send message" will appear on the right hand side. This way you can easily get in directly contact with expert RG members.
In a similar manner, you can also use the search function of RG to find useful literature references and closely related technical questions. Once again, search for e.g. "chiral HPLC" and then click on "Publications" or "Questions". The first link will provide you with a long list of potentially useful articles about this topic, and the other one lets you find closely related questions and answers which have been posted earlier on RG.
Good luck with your work and best wishes, Frank Edelmann
I think Nitin gave fairly good advice on this issue. I can only add that filtration through special membrane filter - which should be always used for all HPLC samples in general - will only remove salts if they are insoluble and present in your solution as a mechanical impurity.
Also, if you use gradient elution, dissolve your sample in the mobile phase containing the lowest amount of the polar component (which is isopropanol for normal phase column); in this case no salt precipitation would occur when the sample is injected into the column.
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