Monday, August 25, 2008

Let's talk about TLCs Part 4 - Ninhydrin Stain

2008 was the year where I became an amino acid chemist and as a consequence the Ninhydrin Stain has become my new friend. Many of the amino acids I handle these days are not UV active but fortunately the Ninhydrin Stain is great for visualising free amines and primary amines. Most of my amines are BOC-protected but they still develop nicely with ninhydrin because the BOC group is cleaved upon treatment with the stain.
Ninhydrin Stain Recipe
100 ml container
0.2 g Ninhydrin
0.5 ml Acetic acid
100 ml n-Butanol
4.5 ml Water
After treatment with a heat gun Ninhydrin Stain tends to give brightly coloured pink to purple spots as shown above. The spots can fade rather fast so record the result immediately. In the past I have been using a Ninhydrin Stain that didn't contain water, however adding a bit of water seems to improve the result a fair bit. D!


milkshake said...

the Boc gets cleaved by simple pyrolisis at around 200C, but AcOH probably helps a bit too.

You can spray your TLC with a solution of a copper or cadmium salt after developing it with ninhydrin - the colors will get a bit stronger and more stable. Cadmium is unhealthy though.

The reason for colored background is that anhydrous ninhydrin (the triketone) is pink-maroon but it hydrates back to the colorless hydrate.

For a very sensitive detection of all NH bonds (promary secondary amines,amides and carbamates, the TLC can be sprayed with a diluted solution of tBuOCl in CCl4, dried with a hair drier without heating, until you can no longer sniff the tBu hypochlorite, and the produced N-chlorinated spots are then developed by a tolidine spray at room temp, as black spots on white. Tolidine is a methylated analog of benzidine, hence it is very unhealthy also. But the detection works like a charm.

Anonymous said...

After many years of tedious recording of TLCs by drawing them in my notebook with all the loss of information inherent in that process, I wised up. An HP scanner can be obtained for $100. I put one in the lab and scan every TLC I want to save to a jpeg. I circle UV active spots and write on the plate any reference notes needed to understand it. This process captures all the nuanced colors of the plate (ninhydrin stains included).

Daniel Sejer said...

I have to agree. I never quite got the redrawing your TLC plate stunt. A scanner is a great idea. Personally I take photocopies of my TLC plates. Remember not to put the TLC plate directly on the photocopier. I'm sure that the secretary won’t appreciate some PMA stain on her hands. I attach the TLC plates to an A4 sheet and slip it in a plastic pocket to protect my colleagues.
I know of a lab that has a digital camera attached directly to the UV viewing cabinet making recording UV activity simple. D!

John Fetzer said...

I have a special fondness for the ninhydrin reaction, not only for its widespread utility, but because it was A. J. P. Martin's undergraduate research project to develop it. He went on to win a Nobel in chemistry, but just having something like this reaction as an accomplishment would highlight most careers.

Anonymous said...

What do you mean when you say that it will react with "free amines"? By the way, your blog is starting to get known around here (UW-Milwaukee :)


Daniel Sejer said...

An actual amine is required to give a reaction with ninhydrin. If you for example acetylated the amine to give an amide nothing would be seen without extensive heating and a fair bit of strong acid. Boc protected amines do show up as the Boc group comes off when heated. Great to hear I have a following in Milwaukee. Thanks for taking an interest. D!

kemisten said...

Hi! As an organometallic chemist who finds herself needing to use more and more organic chemistry, I'm just delighted to have found you're blog. Excellent initiative! Now I'm off to make a ninhydrin stain!

nina said...


I have tried dipping my peptide which is protected with BOc into ninhydrin solution..Ninhydrin solution (ninhydrin/ethanol/acetic acid/water)..The mobile phase that I used is 2-butanol/acetic acid/pyridine/water..I found that some colour changes appeared but not in good just spread something everywhere not in circle shape.., but it turned to purple..I dont know why..anybody can help me to produce good resolution for the spots..thanks..

Daniel Sejer said...

Nina, Isn't that a strange TLC mobile phase to employ? For starters two of the solvents will actually react to form pyridinium acetate! Maybe there is something here I don't know about. Is the phenomenon you are experiencing streaking? Is it like the spot is dragging a tail, comet style? Also actually dipping the plates can sometimes result in blurring of spots. This can be fixed by spraying the plate with an atomiser. D!

nina said...

Yeah..the spot was dragging like a tail..but performed purple colour.. for your info, i dissolved my peptide in DMSO..Is DMSO caused a problem during staining? or do you have any suggestion? many thanks :)

Anonymous said...


That is a common solvent system for ion exchange columns. Acetic acid and pyridine can be used to elute amino acids from a Dowex-50 resin.

@ Nina
Try applying a finer mist to the plate if you are using an atomizer. If you are dipping the plate in the stain (applying it with a pipet), it maybe useful to switch to butanol instead of ethanol in your ninhydrin solution. The lower polarity will reduce the bleeding of your spots.

CAY said...

Hi, do you know if the spot is not in purple color but orange color. What would the compound possible be? because i am dealing with unknown simple molecule that stained orange in the test (control of any amino acids stained pink-purple).

freezedream said...

I know this is an old post now, but I recently discovered that phosphines also show up well with ninhydrin. Typically as orange spots. This may be well known but I thought I'd share anyway as it may be useful to someone.