Harristotle
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Posts: 138
Registered: 30-10-2011
Location: Tinkerville
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Mood: I tink therefore I am
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Preparation of TLC Plates and quantitative analysis of paracetamol
Greetings to all.
The following is a summary of a technique that I have used to prepare silica-based TLC plates (Mg Silicate or talc)
Talc plates can be prepared in one of two ways:
Using Methyl Cellulose as a binder. This method produces plates which are highly stable in aqueous solution, but which have high background staining
with stains such as vanillin.
Methyl cellulose version (makes 12-15)
Advantages: strong, reliable
Disadvantages: Not compatible with vanillin stain
NOTE, WEAR GLOVES IN ALL STEPS – you will contaminate the plates otherwise
1. On the day before making the plates, dissolve 1g of methyl cellulose in 100ml of distilled water using a magnetic stirrer and bar on low stir rate.
It will take 8h to fully dissolve so leave it to run over night.
2. Take 20g of talcum powder, and add 15ml of 0.25% methyl cellulose solution, and 15ml of ethanol. (Good quality methylated spirits 96% ethanol will
work)
3. Stir until an even and consistent slip, with no lumps forms.
4. Use a Pasteur pipette to place very approximately 1.5-2ml of the mixture onto the slide.
5. Allow to air dry overnight.
6. Activate the slides by placing in an oven or frying pan at 120 oC for 10 minutes.
7. Lightly smooth the irregularities out of the surface by rubbing the whole slide very gently with a gloved finger.
Using gypsum as binder. These plates have low background staining and are suitable for use with acidic ethanolic vanillin stain, but are somewhat
delicate in polar/aqueous solvents.
Note my inovation here was to prepare a slip out of ethanol/gypsum/talc. Gypsum is very slow to pull out the 5% or so of water in the ethanol, if it
is even capable of doing so at all. This ensures that you can get a good homogenous slurry. Furthermore, when you do add water to start "setting the
mix", the concentration of water is lower, and hence the rate of setting is slowed. This gives you much more time than if you just add water, and you
can get a very even plate.
Gypsum version (makes 10-12)
Advantages: can be used with vanillin stain
Disadvantages: Very fragile in water
1. Mix 20g Talc powder with 2g of gypsum in a ziplock back. Roll, mix and agitate the two powders for at least a couple of minutes.
2. Add approximately 20ml of ethanol (clean methylated spirits), and then quickly and thoroughly mix it up in the ziplock bag.
3. When thoroughly mixed, add 6ml of water and mix again.
4. Using a plastic pasteur pipette with the tip cut off, transfer the talc in an even manner to a glass slide. Microscope slides work, or for a smine
and 50 cents at your local Op shop, you can get an old picture frame and cut that to larger sized backings.
5. Tap the desk that the slides are on a couple of times and then leave to air dry.
6. Leave to dry over night.
7. When dry, lightly rub the surface with a gloved finger to make it even.
To separate out paracetamol (rf ~ 0.45).
I use a 20% acetone, 20% diethyl ether, and 60% heptane solvent system. Into a 5ml measuring cyllinder transfer 1ml of acetone. Spray 4ml of "Start Ya
Bastard" 25% ether starting fluid. Note, transfer the acetone first so that the Start Ya Bastard doesn't boil and change its solvent ratio.
Spot out 10 - 50 µg of paracetamol from a standard of paracetamol (3x crystalised from Coles paracetamol tablets, $1 per packet) in ethanol (meths).
Run in a sealed chamber ($1 glass with silicone seal from the Reject Shop) for approximately 10 minutes.
Remove the slide, and allow to thoroughly dry (note you can help with gentle warming from hair dryer).
Staining and visualising
When thoroughly dry, spray with a 1% H2SO4 /Ethanol/Vanillin solution. Vanillin can be bought as a food flavouring from ebay. It should cost around
$10 per 100g. Don't get one with sugar in it.
To make the acidified ethanol, approximately 300ml of ethanol was added to an old coke bottle into which had been added 30g of dehydrated Epsom salts
(MgSO4). 10ml of 30% non-fuming pool acid (H2SO4) was added, with shaking, and stored over night. 100ml of this dehydrated acidified ethanol was added
to 5g of vanillin, and the solution was stored in a spray bottle in the dark in the fridge between use.
To visualise the paracetamol, the slide was lightly sprayed with the vanillin solution, then heated for approximately a minute until the yellow bands
that formed from the reaction between vanillin and paracetamol were visible.
Densitometry
Photograph the slide in even lighting.
To quantify the paracetamol (or other stain) please see the attached document below. In general terms, use ImageJ to perform densitometry, after you
have decomposed the photo into its channels. This allows you to subtract a channel that the stain does not appear in (or minimally appears in) from
the channel that is the stain shows up in. This allows you to compensate for non-specific variation in slide background and lighting. Don't worry if
this is too much - just follow the recipe - it is very robust.
For your reading pleasure, I also have attached some polarity data on solvents, and highlighted common safe solvents. Together these will allow you to
produce safe chromatography solvent systems to replace some of the older benzene-containing nasties which might be problematic or hard to obtain.
I thank Loptr for his encouragement to write this up, and my students for their hard work on aspects of this project.
Attachment: Performing densitometry on Thin Layer Chromatography plates.docx (3.7MB)
This file has been downloaded 599 times
Attachment: Relative polarity of solvents.docx (26kB)
This file has been downloaded 620 times
Attachment: Recipe for Vanillin stain.docx (15kB)
This file has been downloaded 665 times
References:
http://www.sciencemadness.org/talk/viewthread.php?tid=78671#...
http://www.sciencemadness.org%2Ftalk%2Ffiles.php%3Fpid%3D297...
http://home.kpn.nl%2Fvheeswijk%2FTalc.pdf&usg=AOvVaw1EzF...
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CharlieA
National Hazard
Posts: 646
Registered: 11-8-2015
Location: Missouri, USA
Member Is Offline
Mood: No Mood
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Thanks for posting this, especially with the links to so many references. Maybe now I'll get off of my seat and do some TLC; but probably not this
year.
CharlieA
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