I am a graduate student trying to learn some chemistry for some analysis I'm doing on HPLC and need help! I want to synthesize and purify retinyl laurate to use as a standard on HPLC. I've been told I need to TLC-purify the stuff I make. I need a primer in TLC and how it is used to purify. What is TLC? How is it used to purify compounds? I know how to purify on HPLC -- I simply collect my peak of interest. How do I purify using TLC?

Don't bother with purifying by TLC if you know how to do it by HPLC. You can purify MUCH more with HPLC than with TLC.

Anon 2: Well, if you have prep HPLC that's fine.
But I have and I still choose to prep TLC many
small samples with much success.

Anon 1: TLC stands for thin layer
chromatography. We usually use glass or
aluminium backed plates coated in a thin
layer (hence the name!) of silica. The mixture
is applied as a small spot in a volatile solvent
at the bottom of the plate, allowed to dry, and
the plate is stood in a tank. The tank should
contain solvent to just below the level of the
spot. The solvent in the tank depends on the
mixture you have to separate. You will have to
experiment with several mixtures of common
solvents to fing a system that separates your
compounds best.

My favourite systems are ethyl acetate and
hexane in various ratios. You allow the solvant
to travel up the plate to almost at the top (but
not all the way to the top) and then remove the
plate from the tank. You should mark where
the solvent came to so that Rf values can be
calculated (Baseline to spot distance divided
by baseline to solvent front distance).

To visualise the spots, UV lamps are often
used, but in cases where there is no
chromophore, dips can be used to develop
the plate (such as KMnO4, CAM etc etc).

This briefly covers analytical TLC. Prep TLC is
basically the same. The plates are larger, with
a thicker silica layer. The mixture is applied in
the same way, but as a line along the botom
of the plate instead of a single spot. The plate
is run in a tank in the same way as before.
Then you see where your componants are on
the plate by UV, mark it in pencil, and just
scrape off the silica containing your
compound, wash the compound off with
solvent and revover pure material.

remember not to use stains with prep TLC, as
you will not recover any compound from the
silica after it has reacted with the staining
mixture!

Any good practical organic chemistry text book
will have a better description of the techniques
.
If things are still not clear, or if I've just made
you more confused, post a message to say
so, and I'll endevour to explain better.

Good luck,

Will

Hi, I'm the graduate student with the original question about TLC. Thanks SO MUCH to those who commented, i.e., Will and Anonymous. I had no clue that you actually scrape off the spots to recover the purified substance. Thanks again.

When I was in school, we used to isolate the various components from our reaction mixtures by prep TLC. Much easier than prep LC in my opinion. We used to use prep TLC plates manufactured my Alltech if I remember correctly. There are other vendors. Again, if I remember correctly, these were about 10 x 10 or 8 x 8 inch glass plates with up to 2 mm silica thickness. We would make a "paint brush" out of a pasture pipette (tip snipped off a bit) and cotton. Load the pipette with the sample dissolved in solvent and streak a fine line across the plate. This took some practice, but once you get it, it's like riding a bike. We would typically load 50 to 200 mg of material. Please refer to Will's discussion above for TLC conditions… nice job.

Once the plate was developed, we would visualize, scrape off the silica (lot's of powder at this point), mix with strong solvent such as ethyl acetate, filter and evaporate. If we could not visualize by UV, we would put a piece of tape across the plate at one side from top to bottom. When you pull off the tape some silica adheres to it, and you can then develop the tape with something like Iodine. Then, match the tape to the plate to get the position of your band.

Anon 3, Nice trick with the tape!! I will bear it in
mind.

Someone has an idea to develop allantoin in TLC