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I prepared anisaldehyde charring solution, and refrigerated it. Its color was yellow. However when I am using it for staining tlc plate, it is giving dark pink color on tlc plate surface and unclear blue stains... Also not all the stains are visible. If I am not wrong it should have white background with dark brown spots appearing clearly.

What mistake I am doing here possibly?

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p-Anisaldehyde (PAA) stain

This posting from www.ochemonline.com (which is available under a CC-By-SA-3.0 licence) provides the following information about the stain and its behavior. The information below is consistent with my personal experience using PAA stains.

  • Color: Range of colors on a light pink background (my experience was mostly blue spots)
  • Medium shelf life

The post on stains goes on to say:

This stain is light and oxidation sensitive and will gradually turn pink/orange. The stain should be kept in an aluminum-covered jar while in use, and the excess should be kept cold and in the dark. Once the stain turns dark red, it should be discarded and made fresh again.

Now you have your answer - the stain should turn pink over time, it should produce (mainly) blue spots on a pink background, and you have a visual indicator of its shelf-life. Once it is dark red, it is done. To keep the stain around longer, wrap the jar in foil.

Brown spots on white background

The iodine stain is the only stain I know that does this for most compounds.

Not all the spots are visible

PAA is principally an electrophile and stains nucleophilic compounds more strongly than other types of compounds.

Personal recommendation

I grew frustrated with the PAA stain because it degraded quickly and switched to the vanillin stain. The recipe is in the link posted, and reproduced below scaled down. Make small batches to avoid waste. You want to use the stain faster than it decomposes.

Dissolve 3 g of vanillin in 50 mL of ethanol. Slowly add 0.5 mL of concentrated sulfuric acid.

This stain is very sensitive to functional groups and produces a wider range of functional group specific colors than PAA. Vanillin (the structure of vanillin is simiular to PAA, but with an additional OH on the ring) is both an electrophile and a nucleophile and so reacts with a wider range of compounds.

Ochemonline indicates that it has the same "medium shelf-life" as PAA, but in my experience, PAA decomposed more quickly. I left my vanillin stain in an amber glass jar without foil wrapping in my hood at room temperature, and since I was doing lots of TLC every day, I used it faster than it degraded. PAA stain degraded much more quickly for me under these conditions, and I was throwing half of it out despite the number of TLC plates I was doing. If you are not doing TLC every day, then wrap in foil and refrigerate.

Best reason to use vanillin: The awesome smell!

Poor spot quality

This main not be a problem with your stain.

  • Big streaky spots are indicative of too high concentration in your solutions.
  • Alternatively, you may need a more optimal solvent. For example, when doing amines on silica, you may want to add 1% triethylamine to your TLC solvent since silica is acidic. Amines and other basic compounds will streak less in these conditions.
  • Big streaky spots may also mean poorly resolved ocmpounds
  • Faint spots may mean poor compatibility with stain or that the concentration was too low.
  • If spots run together, perhaps your lanes are too close, the bottom of the TLC plate is not level, or your plate touched the side of the developing chamber.
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