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Preparing your own thin layer chromatography plates (and then using them)by allanf0 on July 30, 2007

Table of Contents

intro:   Preparing your own thin layer chromatography plates (and then using them) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

step 1:   Gather the materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

step 2:   Grind the Silica Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

step 3:   Weigh out and mix the silica gel and plaster of paris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

step 4:   Suspend the powder in water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

step 5:   Coat glass slides with suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

step 6:   Air dry followed by activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

step 7:   Some final notes on preparing the plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

step 8:   Construct the developing chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

step 9:   Extracting the plant pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

step 10:   Spotting and Developing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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intro:  Preparing your own thin layer chromatography plates (and then using them)Chromatography is a widely used technique in both analytical and preparative chemistry. It was first made practical by Tsvet for the separation of chlorophylls but hassince been expanded into a huge field of study with a wide range of instruments and techniques. Fundamentally, however, the principle underlying the GC-MS you mightsee on CSI and that simple column of calcium carbonate Tsvet used to separate plant pigments remains the same.

An unknown mixture is pushed passed what is called the stationary phase in a continuous stream, it can be a solid in the case of TLC, or a liquid as is the case in manyforms of GC. The exact nature of the stationary phase is not so important, what is important is that the individual components of the unknown mixture interact with thestationary phase in some way. Ones that interact more strongly and prefer to stay associated with the stationary phase versus in solution (or in the gas phase) will moveslowly and be held back in the stream. Species that interact weakly move more swiftly through the stream. In this manner the different components of the mixture can beseparated out by the amount of time it takes for them to elute : move through a column of stationary phase.

A measure of how strongly or weakly a compound is retained is the Retention Factor (RF) or Retention Time. In TLC the RF of a compound can be used to identify itfrom tabulated values. The retention factor is the ratio of the distance traveled by the compound up the plate versus the distance traveled by the solvent front. The RF fora given compound is (relatively) unique as it depends upon the structure and chemistry of that compound.

In this instructable I will describe how one can prepare their own silica gel TLC (Thin-Layer Chromatograph) plates. In this form of chromatography the stationary phaseis a thin layer of silica, a type of finely divided silicon dioxide, deposited on a glass slide. The analyte is "spotted" on the plate using an eye-dropper or micropipette andthe whole plate is placed in a beaker with a small amount of solvent in the bottom such that the solvent level is just above the bottom of the plate. The solvent moves upthe plate by capillary action, pulling the pigment along with it. The different chemical species in the pigment interact with the silica in differing ways and this affects thedegree to which they are pulled up the plate, which is how the separation is effected.

A more basic version of this substitutes a strip of sturdy paper in place of the TLC plate, in which case the cellulose of the paper fills the role of the stationary phaseinstead of silica. Paper chromatography has its limitations, however, usually making themselves visible as smearing or poor separation. This is why thin layerchromatography is usually employed. It operates in much the same way as paper chromatography in so far as development, however peak separation is generally better(amongst other things).

Image Notes1. The Eluent, the solvent being used to push the mixture, moves up the platefrom bottom to top by capillary action2. The original spot, placed on the plate by way of an eye-dropper above thesolvent-line3. Three distinct species are separated as the solvent pushes up. The blue ismore strongly held by the silica and thus lags further behind whereas the red isless attracted to the silica and spends more time in the solvent.4. Species can be identified by their Retention Factors. This is the ratio of thedistance traveled by the spot to the maximum distance traveled by the solvent.The retention factor given a certain set of variables can be tabulated and thenthe R.F.s of unknown species simply looked up.

Image Notes1. As the Analytes (stars) are pushed past the silica particle, they interact (goldarrows). These interactions slow them down. The strength of the interactionsgoverns how much they are slowed down.

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Image Notes1. These were made with lab grade silica gel, on glass slides, with plaster ofparis as the binder2. These were made with silica gel from dessicator packets, prepared in thesame way as above but with less suspension to work with (hence the gaps nearthe edge)

step 1: Gather the materialsFirst off you need some basic supplies:

an oven, generally comes with housesa weigh scale, nothing too fancy should be accurate to one tenth of a gram, e.g This digital scale from Amazonan old plastic bottle you don't care about, not too large (I used a 150mL one)a pan, for resting the plates on and for putting in the oven.a mortar and pestle, larger ones are easier to work with than smaller onesa syringe, 10cc minimum, plastic works fine, I got mine from Home Depotglass slides, you can also use sheets of tin or plastic, basically anything stiff that won't interact with water

Then you need your "chemicals" for preparing the slides:

Anhydrous Calcium Sulfate, a.k.a. Plaster of Paris, I liberated mine from an artsy friendWater, from the tap, or distilled if impurities are an issueSilica Gel - This is the desiccant in those little packets you find in medicine bottles and assorted what-nots.

Note: Silica Gel is hygroscopic, and its fine particles can be harmful if inhaled. It is not a bad idea to wear gloves and a mask while grinding this stuff.

The final materials are needed for constructing a developing chamber and developing slides of plant pigments:

a mason jar with lid. it should be just taller than the glass slides such that you could prop a slide up in it easilyfilter paper, you can also use sturdy sketch paper, I use 10mm filter papereye dropper or pasteur pipetteacetone 50mL

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hexane 50mLa pencila graduate cylindersome leaves, from which to extract the chlorophylls and xanthophyllsclean sand for grinding with, mine is from the beach.

Note: When using organic solvents ensure appropriate safety measures are in place. Such as proper ventilation, safety glasses, &c. If you are unsure as to whatconstitutes appropriate safety you shouldn't be messing with organic solvents.

step 2: Grind the Silica GelI cannot stress enough how much of a pain it is to grind silica gel. It is glass, and the packets tend to contain it in bead form. I found it is relatively pain-free to breakup the beads first by putting them between two pieces of stiff cloth and beating it with a hammer. Transfer this to the mortar, pick out the lint, and grind away.

For disclosure: I cheated after grinding for a while in a mortar and pestle. I took mine in to the lab I work at and used the ball grinder there. Its set up for grinding soil andmineral samples and did a fine job of pulverizing this. That said I was making head-way with the mortar and pestle, were I not impatient I imagine I could have gotten itdone by hand. Also had I decided not to grind up all those packets and limited myself to smaller volumes it would have gone faster.

Image Notes1. Remember: Do not eat. Image Notes

1. The contents of one packet. Large beads that are too large and round to grindproperly.

Image Notes1. After much beating, complaining, and cheating by using a ball-grinder atwork.

step 3: Weigh out and mix the silica gel and plaster of parisThe plaster of paris is the binder and is present only to keep the silica from sloughing off the glass slides. I have found 10-20% (by weight) plaster of paris in the final mixworks well.

For this project, weigh out:

1.0g plaster of paris4.0g silica gel (ground)

Combine these in the mortar and pestle and grind together very well. The mixture should be very homogeneous and the finer the particles the better the separation.

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step 4: Suspend the powder in waterTransfer the powder into an old plastic bottle that you will never want to use for anything else again (especially if you let the plaster set before cleaning it out and are aslazy as I).

Add 10mL water (or a water to powder ratio of 2:1), the syringe is handy for this.

Cap the bottle and shake violently for one minute, the bottle that is. The goal is to form a slurry of all the solid in water.

Image Notes1. no longer in here2. powder transfered to here

Image Notes1. into the bottle goes 10mL of water

step 5: Coat glass slides with suspensionDraw up the newly formed suspension into the syringe.

With the slides cleaned and dried (and free of fingerprints or oils), move the tip of the syringe back and forth width-wise across the slide while applying gentle pressure tothe plunger. The motion is sort of like tiling a field. You don't want to pour or dispense it all at once as the layer will form a hill instead, and will be too thick. By going backand forth slowly you can dispense a reasonably even, thin, layer of suspension across the plate.

The thickness of the layer is important, less than 1mm when dry is preferable so be careful not to overdo it.

Image Notes1. these plates have just been coated with their respective suspensions

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Image Notes1. the suspension, loaded into the syringe (with air that will be pushed out later)

step 6: Air dry followed by activationYou want to air dry the slides to allow the plaster to set. Just leave the slides in a calm place for about an hour, or until they are white and smooth.

Prior to using, the tlc plates need to be activated. This entails driving off any remaining water that would still be held by the silica gel. This, apparently, frees-up the -OHgroups of the silica gel to do your bidding as a stationary phase does.

Activation is done by heating the plates in an oven at 120C for 30-45 minutes. At this stage there really is no harmful chemical residues to worry about and this can bedone in a regular household oven (unless the oven caretaker objects of course).

After they have cooled the plates are ready for use so that you may elute to your heart's content.

The advantage of using glass slides is that when you're done with the plate you can always scrape off the stationary phase, clean it, and re-layer it.

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Image Notes1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder2. These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with (hence the gaps near theedge)

step 7: Some final notes on preparing the platesThese plates can be used like any other, though generally home-made plates have a more brittle stationary phase so be gentle. Tweezers are a good investment, andwield the "science tongs" carefully for with great power comes great responsibility.

I've heard that some silica gel desiccant packets contain fungicides and other chemical dopants, they may interfere with the operation of your plates. I have absolutely noadvice on how to deal with this besides, perhaps, cleaning the silica powder before hand with a non-polar solvent. I did not have this problem and the plates I made fromthe desiccant packets worked the same as ones I made from lab grade silica gel (for chromatography, oooh).

Other stationary phases can be used as well, alumina for example. In this case slightly less water can be used, about 1.5:1 ratio instead of 2:1 as alumina does notabsorb as much water as silica (and you want to maintain the consistency of the suspension). Cellulose can also be used, though I haven't experimented with it, I hearthat you don't need to use a binder as cellulose is sticky enough on its own. You can, of course, experiment with your own stationary phases. There is a lot of literatureout there, ripe for the googling.

The following steps describe a simple experiment to show how TLC works by way of separating plant pigments. Pigments are chosen as they are clearly visible againstthe white backdrop of the plate do not need to be further developed by UV or Iodine to be seen.

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step 8: Construct the developing chamberThe developing chamber is just a fancy name for a jar with a piece of filter paper in it. A 1cm layer of solvent (eluent) will be put in the bottom, the plate propped up inside,and the lid placed over. The point of the filter paper and lid is to ensure the atmosphere inside the chamber is well saturated with eluent.

First take a piece of filter paper and cut it such that there is one straight edge. When rolled and put inside the mason jar it should fit snugly along the wall of the jar.

That is it, the developing chamber is complete.

Image Notes1. cut the filter paper so that it will fit in the jar

Image Notes1. note how the filter paper fits along the wall of the jar.

step 9: Extracting the plant pigmentsYou will need a large mortar and pestle, some sand, the plant leaves, and the organic solvents for this step.

Cut up the plant leaves (say with scissors) and grind with the sand in the mortar, add 20mL 1:1 acetone:hexane and continue to grind until the juice is very green andgets no greener. You can experiment with how many leaves you need to get the juice its greenest. Draw off with an eye-dropper and put in a vial (or spot onto the platesdirectly)

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Image Notes1. Acetone2. Hexane3. The graduate cylinder for making up the mixed solvents.

Image Notes1. an office plant and some sand ready to be ground

Image Notes1. Mashing away.

Image Notes1. The end result

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Image Notes1. This is the final liquid that was drawn off

step 10: Spotting and DevelopingCarefully mark a line 1.5cm from the base of one plate.

Place as small a drop as possible on this line, centered as much as possible. Blow on it until it dries and repeat two or three more times.

Place a 1cm layer of 6:4 hexane:acetone in the bottom of the mason jar/developing chamber, put the lid on and set aside for a few minutes to allow the eluent to wet thefilter paper. In the pictures I went in to work and used a beaker since the jar is too hard to see through.

Using tweezers gently place the plate in the jar such that the spot is just above the level of the hexane/acetone mixture, propped up against the filter paper.

Sit back and watch as the solvent line climbs up the plate and pulls the pigments along with it. The green is the chlorophylls and they should move further up the platethan the yellow lines which are the xanthophylls.

When the solvent is about 0.5cm from the top of the plate quickly remove it and mark the solvent line with a pencil.

To obtain the RF for any spot (or band) on the plate, divide the distance from the bottom pencil line to the spot by the distance between the two pencil lines. The aboveseparation was adapted from Adams' Laboratory Experiments in Organic Chemistry 6th Ed., in which petroleum ether was used instead of hexane.

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Image Notes1. this is a very big spot

Image Notes1. the pigment moving up the plate (I spotted a line to make it easier to see withthe camera)2. beaker with a watch glass for a lid simply so I could take pictures through thewall.3. the eluent, a 6:4 mixture of hexane and acetone

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Image Notes1. there's another line down here you can't see very well2. before developing3. after developing, you can see where I pressed too hard with the pipette onetime4. chlorophyll and xanthopyll close to togethor5. there's a faint green line up here that didn't show up in the picture very well

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Comments12 comments Add Comment

 bioman says:  Aug 13, 2007. 8:37 PM  REPLYThis is a neat little project. I used to prepare TLC slides to when I was younger. I made the stuff out of chalk and some hydrolysed cellulosis to make it stick.Worked pretty well, but there is just no way around buying at least a chromatographic static phase, if not the complete foils/ slides. The problem this projecthas, is that the surface is to small. The chlorophyll is not well seperated. Just look up Google Images with chlorophyll tlc. There you will see how it has tolook like. I had the same problem and now I buy the foils and cut them in small stripes. That way it is pretty cheap.

 allanf0 says:  Aug 15, 2007. 2:11 PM  REPLYThe poor separation is mainly due to my terrible spotting technique. With plates this short you have to spot very very thin lines or small spots otherwisethey all bleed together. That and my eluent mix was more towards separating the different xanthopylls than chlorophylls (which somehow I thought wouldlook cooler except the yellow basically didn't show up at all in the picture).

That said, there's nothing stopping you from pre-cutting strips of tin or plastic to a much longer length and coating them in the same manner. I just usedglass microscope slides as they are convenient (and about the same size as TLC plates I used in organic chemistry).

 bioman says:  Aug 16, 2007. 1:21 AM  REPLYI don't neccesarly think so. The mixture of solvents is correct. You could also use benzine. The bad distinction between the different components isdue to the size of silica gel particles, I bet you! Comercially available tlc slides have extremly fine ground powder on them, you can never reach thiswith a mortar. I would rather suggest to use a different material. For example you can buy aluminum oxide (Al2O3) as a polishing agent for mineralsor glas. There are finely ground types of this around. Or why not table chalk? Also small particle size!

 allanf0 says:  Aug 15, 2007. 2:16 PM  REPLYOh and regards to spotting, which I didn't really mention: the stationary phase here is thicker than you would probably be used to with bought TLCplates and so spotting is a little more tricky. Basically spots spread out more in my experience and getting them to stay nice and small requires a lotof patience.

 lemonie says:  Jul 31, 2007. 7:19 PM  REPLYThese look thick enough for preparative TLC(?)Could you add-in (or create another) for your tank, and mention Rf?Oh and what is it that you add to make these fluoresce under UV?

L

 allanf0 says:  Aug 1, 2007. 1:03 PM  REPLYI would hesitate to use these for anything requiring more precision than simple testing. Simply because the thickness and uniformity of the layer is difficultto control precisely without the use of those fancy plate coaters. But yes they tend to come out thick enough for preparative.

My developing tank is just a mason jar in which the slides are propped up, and I also have another jar with iodine crystals in it for resolving the spots, butnext time I get the chance I'll take some pictures. Since I don't have a UV lamp to make anything fluoresce under I didn't add anything else, I really haveno idea what you would use either.

 lemonie says:  Aug 1, 2007. 2:17 PM  REPLYThanks, it would be good to see more of this.

L

 allanf0 says:  Aug 2, 2007. 7:55 PM  REPLYI expanded this instructable to include developing plates. I originally took pictures at home but you couldn't see much of anything through themason jar I have. It has fruits and berries embellished on it in glass and it distorts everything.

So I went into work and used a beaker and the fume-hood there.

Hopefully this gives a better description of how TLC works.

 lemonie says:  Aug 4, 2007. 4:35 AM  REPLYGreat, hope it wins a prize.

L

 ewilhelm says:  Jul 30, 2007. 10:32 PM  REPLYThis technique looks like it could be used as part of a great entry in our Science Fair.

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 allanf0 says:  Jul 31, 2007. 12:48 PM  REPLYI never thought of that. I guess I should get to work making "infographics" and fleshing it out some more, when I get the chance.

 CameronSS says:  Jul 30, 2007. 11:20 PM  REPLYEric, you stole my comment!

This is very well done, and if you enter this is the science fair contest you'll put me out of a grand prize. Oh well, I'll vote for you anyway. I hope to see moresciency Instructables from you to lessen the K'nex gun inundation.