Tuesday, March 25, 2008

Let's talk about TLCs Part 3 - Phosphomolybdic Acid (PMA) Stain

PMA Stain is a highly versatile general stain for developing TLC plates. The stain is easily prepared by dissolving 10 wt. % phosphomolybdic acid in ethanol. More PMA can be used however as stains go PMA is relatively expensive.
TLCs developed with PMA stain upon heating give green/dark green/dark blue spots on a yellow/green background (see picture).
Phosphomolydic acid (PMA) Stain Recipe
100 ml container
100 ml Ethanol
10 g Phosphomolybdic acid, H3[P(Mo3O10)4] · xH2O
Alternatively, you can go for the more expensive solution and buy ready made PMA stain from Aldrich (see picture). Where I've worked in the past we simply poured some of the ready made stuff into a jar and use it for our TLC plates. At the end of the day we poured it back into the Aldrich flask and rinsed with some ethanol. Using this method the stain lasts for a very long time. I never had any problems storing the stain at room temperature despite Aldrich's recommendation to refrigerate.
See under Vanilin Stain for general staining guidelines. D!


Anonymous said...

PMA solution is light sensitive - it goes green fast if stored in light bottle. I wrapped my bottle in Al foil. I would use 30% solution in EtOH and sprayer - the problem is it leaves an goddamned mess so I had special trap box in the hood where I would spray.

Later I completely converted to CAM, cerium ammonium molybdate dip solution - it has a better sensitivity and is less messy and it is cheaper also/

Anonymous said...

I've learned to love PMA stain at my current job. It's extremlely versatile. My previous lab was a fan of p-anisaldehyde. I didn't find it as versatile, but at least it smelled nice!

Unknown said...

Both PMA & CAM are excellent stains; the colour contrast is much better for CAM though (blue on white) than PMA (dark green on light green).

I'm told that PMA is meant to be good for amines, but didn't seem any better than good ol' iodine. haven't found the light sensitivity of PMA to be a problem; even if the solution changes colour it still seems to stain well.

Of course the best way is to have at least a couple of different stains; my preferred set up is UV-Iodine-PMA: you can blow the iodine off (air/heat gun) and get three visualisations from one plate!

Irene said...

Hi, I am doing a TLC job with PMA stain. I read your blog, and wondering the specfic meaning when you said the stain appears while heating. Do you mind talk more details about the procedure, such as what the temperature following the spray and how long for the heating? I used to heat at 100 C for 5 min. Does this sound correct to you? Thank you. You can also email me at elitelc@hotmail.com if you don't mind.

Daniel Sejer said...

Irene, you have to hit it hard and fast. High temp (highest setting on your heat gun) and blast away until spots appear on the plate. On my heat gun they claim it goes to 700 degrees C so it's pretty hot. D!

ChemGrad said...

I have another recipe:

3.0g PMA
0.5g Cerium sulphate
5.0ml Sulphuric acid
95.0ml water

So far, I get sudden pink spots without heat and blue or brown for others, with yellow background upon heating. If over-heat, blue background while the pink/blue/yellow spot fade away.

How is this different than PMA/ethanol recipe? What Cerium sulphate does?

Daniel Sejer said...

@ Curious Chemistry Grad: Check out my post on Hanessian Stain: http://curlyarrow.blogspot.com/2006/11/lets-talk-about-tlcs-part-2-hanessians.html

ChemGrad said...

thank you Curly Arrow

Unknown said...

This blog just saved my life! I'm working with fatty acids and posphoglycerolipids and CAM works only when they are highly concentrated. Result: try to make a FLASH chromatography, but blind! PMA is much more sensitive to these compounds and is a must for anyone working with fatty acids. Thanks!

JJ said...

I see one red/violet spot on my PMA-sprayed TLC plates between all the dark gray ones - does anyone have an idea what that could be?
I'd appreciate any comment!

COT said...

Can anyone tell me the meaning of the different colours on p-anisaldehyde and PMA stained plate? I would appreciate it if a literature reference is given.

Unknown said...

This is a really useful series of posts on stains! One important thing to remember, which I discovered the hard way, is that silica apparently plays a role in some of the staining chemistry. Many stains, especially PMA, behave completely differently on C18 reverse-phase silica.

DJ Korey said...

At my lab we have a bunch of molybdic anhydride but no PMA, anyone ever made it from molybdenum trioxide and phosphoric acid?

EmeraldBiology said...

I'm no chemist but I would like to know about the chemical reaction that takes place between PMA and TLC spot chemical groups. Even general chemistry pointers would be helpful. Tak skal du have!

Danta CC said...

KMnO4 solution could be an alternative general TLC staining reagent.