Let's Talk About TLCs Part 5 - Iodine Stain

The iodine stain is by far the easiest stain to prepare and use. The classic way is to simply have a chamber with some iodine crystals that you put your TLC plate into. The iodine vapours will react with many functional groups but is particularly good with unsaturated compounds (e.g. alkenes and alkynes) to give reddish brown spots (see TLC plate below). Another way to prepare the iodine stain is to mix silica gel with iodine crystals (see pic to the right). Stick the TLC plate into the red silica and wait a few minutes to develop your plate. This approach is a bit gentler and in my experience gives a better result. If you want to keep your TLC result for later remember to take a picture of your plate or circle the spots as they fade relatively fast. D!

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.

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Ninhydrin Stain Recipe

100 ml container

0.2 g Ninhydrin

0.5 ml Acetic acid

100 ml n-Butanol

4.5 ml Water

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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!

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).

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Phosphomolydic acid (PMA) Stain Recipe

100 ml container

100 ml Ethanol

10 g Phosphomolybdic acid, H3[P(Mo3O10)4] · xH2O

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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!

Let's talk about TLCs Part 2 - Hanessian's Stain

Don't you just love that feeling when you are checking your reaction mixture by TLC and no matter which stain you use nothing appears on the plate. If your compound also isn't UV active you've got a real problem. Well the good news is that there is one stain that will do the job for you - Hanessian's Stain! Hanessian's stain is an excellent multi-purpose stain that when used the right way usually gives blue spots (TLC plate A). Since it is a water based stain it requires vigorous heating for development. However, if you overdo it the entire plate goes dark blue (TLC plate B).

Hanessian's Stain Recipe 1

100 ml container
90 ml Water
5 g Ammonium molybdate, (NH4)6Mo7O24-4H20
1 g Cerium sulfate, Ce(SO4)2
10 ml Concentrated sulfuric acid

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Hanessian's Stain Recipe 2

100 ml container

90 ml Water

2.5 g Ammonium molybdate, (NH4)6Mo7O24-4H20

1 g Cerium ammonium sulfate, Ce(NH4)4(SO4)4-2H2O

10 ml Concentrated sulfuric acid

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Dissolve ammonium molybdate and cerium sulfate in water (with heating if required) followed by careful addition of concentrated sulfuric acid. Sometimes an insoluble residue is observed. If that happens remove it by filtration. Cerium sulfate can be replaced with Cerium ammonium sulfate which is significantly cheaper (Recipe 2). Hanessian's stain is used just as previously described for the Vanillin Stain although it requires more vigorous heating. Because rather harsh heating is required this stain may prove inefficient with volatile compounds. Also keep in mind that this a very sensitive stain so even trace impurities can appear as significant spots on your TLC plate. D!

Let's talk about TLCs Part 1 - Vanillin Stain

I've lost count of the number of times I've searched the web for TLC stain information long ago. I have a feeling that this is a reoccurring issue for many synthetic organic chemists so we've decided to start a "Let's talk about TLCs"-series on Curly Arrow. With time this site will hopefully be the only place you'll ever have to visit for information on TLC stains and related stuff. Not only will we present the recipe for any particular stain but there will also be some comments regarding what it's good for detecting and some pretty pictures so you can see what it looks like after the stain treatment. Let's start the series with a classic: The Vanillin Stain. This is my favourite all purpose stain and after a quick look under the UV lamp this is my next stop. It virtually always works well to give spots in a wide variety of colours which can be handy for spotting what your after when you have multiple spots.

Vanillin Stain Recipe:
100 ml container
6 g Vanillin

1.5 ml Conc. sulfuric acid
95 ml 96% Ethanol

Add the vanillin to your container followed by ethanol to give a clear solution. Carefully add the sulfuric acid. The final product is a clear colourless solution. However, after some dipping of plates it will quickly become a clear yellow solution (see picture above).

How to use the vanillin stain: Run your TLC and let it dry. If you want to check for UV activity you should do so now and not later. Dip the TLC plate in your vanillin stain and heat it using a heat gun (gently at first so that you don't spray the stuff all over your hood) until coloured spots appear. Remember to put the lid back on tightly otherwise it'll dry out.

To the left there's a picture of a TLC plate that I developed today using the vanillin stain.

Alternatively, you can use an atomiser and spray the TLC plate with the stain. This prevents the stain from turning yellow and makes it last a lot longer since you are using much less per treatment. D!