Has anyone got a working method for methylene
blue. I've been trying diode PDA detection
at 660nm with no luck.

Thanks.

You may be able to use an alkyl sulfonate ion-pairing agent in conjuncton with a reversed phase column and PDA detection at 652 nm. Methylene blue is a commonly used reagent for quantiative detection of anionic detergents. The assay involves the ion pairing of methylene blue with the detergent under acidic conditions followed by extraction of the methylene blue-detergent complex with CHCl3.

While I've never chromatographed methylene blue, I'd bet that methylene blue complexed with a pure short chain (5 or so carbons) alkyl sulfonate at low pH would be amenable to separation on a C8 or C18. I'd try the C8 first.

Alternately, a cation exchange column might do it, too.

let me know how you make out, I'm curious

Chris

There is a literature regarding analysis of methylene blue on reverse phase column. Anal. Chem. 1989, 61, 728-732.

Please let me know your fax number if you want the literature.

Sha

Thank you both for the reply.

It's actually detergent analysis that I'm trying to do. I have been using the method which Chris has described binding MB to anionic surfactants and quantifying at 660nm. I was curious as to whether it could be performed by LC using DAD.
I tried some initial injections on a C18 using a 50% MeOH pH5 phosphate buffered mobile and scanned at various wavelengths 660, 650 etc.
When no peak appeared I tried some injections of MB on its own. Tried several injections with several different mobiles ranging up to 70% ACN and still no peak.
My query is;
If MB is very polar and clearly should give a response at 660 or 650nm why am I getting no peak
even with a very strong mobile (70% ACN)??
Where is the MB going??

I would be very interested in that reference Sha
Thank you very much for your offer.
FAX: Ireland (353) 0902 24492

Adrian Redmond.

Hmmm...This is interesting! I'd bet that unless you're using a base de-activated column AND your pH is acidic that the MB may be binding to free silanols on the column.

There are a number of chromatographic assays for the analysis of anionic surfactants, though DAD is not the best detection method for this.

Let me think on this one for a while, do some homework, and I'll get back to you. If you like, you can e-mail me at juddman@peconic.net (home) in the meantime.

Chris

I believe the cause of your problem (failure to elute methylene blue from a reversed phase column) is due to be chemical nature of methylene blue. Since this dye contains two quaternary groups, it undoubtedly sticks quite tenaciously to any residual silanols. It's probably better to do this sort of compound by cation exchange. We have successfully separated methylene blue by cation exchange on a Dionex OmniPac PCX500 column with a perchlorate acid, sodium perchlorate and acetonitrile eluent system. If you are interested in the specifics let me know.