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 has since 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 might see 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 many forms 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 the stationary 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 move slowly 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 be separated 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 it from 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 for a 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 phase is 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 and the 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 up the 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 the degree 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 phase instead of silica. Paper chromatography has its limitations, however, usually making themselves visible as smearing or poor separation. This is why thin layer chromatography 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).

Step 1: Gather the Materials

First off you need some basic supplies:
  • an oven, generally comes with houses
  • a weigh scale, nothing too fancy should be accurate to one tenth of a gram, e.g This digital scale from Amazon
  • an 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 ones
  • a syringe, 10cc minimum, plastic works fine, I got mine from Home Depot
  • glass 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 friend
  • Water, from the tap, or distilled if impurities are an issue
  • Silica 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 easily
  • filter paper, you can also use sturdy sketch paper, I use 10mm filter paper
  • eye dropper or pasteur pipette
  • acetone 50mL
  • hexane 50mL
  • a pencil
  • a graduate cylinder
  • some leaves, from which to extract the chlorophylls and xanthophylls
  • clean 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 what constitutes appropriate safety you shouldn't be messing with organic solvents.

Step 2: Grind the Silica Gel

I 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 break up 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 and mineral 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 it done by hand. Also had I decided not to grind up all those packets and limited myself to smaller volumes it would have gone faster.

Step 3: Weigh Out and Mix the Silica Gel and Plaster of Paris

The 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 mix works well.

For this project, weigh out:
  • 1.0g plaster of paris
  • 4.0g 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.

Step 4: Suspend the Powder in Water

Transfer 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 as lazy 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.

Step 5: Coat Glass Slides With Suspension

Draw 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 to the 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 back and 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.

Step 6: Air Dry Followed by Activation

You 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 -OH groups 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 be done 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.

Step 7: Some Final Notes on Preparing the Plates

These 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, and wield 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 no advice 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 from the 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 not absorb 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 hear that 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 literature out 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 against the white backdrop of the plate do not need to be further developed by UV or Iodine to be seen.

Step 8: Construct the Developing Chamber

The 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.

Step 9: Extracting the Plant Pigments

You 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 and gets 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 plates directly)

Step 10: Spotting and Developing

Carefully 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 the filter 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 plate than 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 above separation was adapted from Adams' Laboratory Experiments in Organic Chemistry 6th Ed., in which petroleum ether was used instead of hexane.
<p>This is an amazing project that I will test out really soon.</p><p>I only wished to know one thing, is hexane available to the general public? I'm a chemist, of course I could get some, but I'm interested in making projects that anyone could do.</p><p>Thanks for the instructable!</p>
<p>Another question just popped in my head, how do you discard your organic solvents?</p><p>Thanks</p>
This is a nice introduction to TLC. I would suggest that the tedious procedure of pulverising silica gel can be avoided by using Plaster of Paris itself as the stationary phase. ( See Amoozadeh.A, Asian Journal of Chemistry Vol. 20, No. 8 (2008), 5873-5877 &amp; JSUT 33(2), 2007, p. 23-24 ). Commercial Plaster of Paris is calcined gypsum and does not require any grinding in a mortar before use. The slurry for coating the plates is prepared from 10 grams of Plaster of Paris and 20 mL water. <br>Also, it is not necessary to use filter paper for the lining of the developing chamber, a paper towel is quite satisfactory and is much cheaper. Lighter fluid ( sold in some countries as Shellite ) can be substituted for hexane.
This 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 project has, 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 to look like. I had the same problem and now I buy the foils and cut them in small stripes. That way it is pretty cheap.
The 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 otherwise they all bleed together. That and my eluent mix was more towards separating the different xanthopylls than chlorophylls (which somehow I thought would look 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 used glass microscope slides as they are convenient (and about the same size as TLC plates I used in organic chemistry).
I don't neccesarly think so. The mixture of solvents is correct. You could also use benzine. The bad distinction between the different components is due 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 this with a mortar. I would rather suggest to use a different material. For example you can buy aluminum oxide (Al2O3) as a polishing agent for minerals or glas. There are finely ground types of this around. Or why not table chalk? Also small particle size!
Oh 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 TLC plates 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 lot of patience.
These 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
I would hesitate to use these for anything requiring more precision than simple testing. Simply because the thickness and uniformity of the layer is difficult to 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, but next 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 have no idea what you would use either.
Thanks, it would be good to see more of this. L
I expanded this instructable to include developing plates. I originally took pictures at home but you couldn't see much of anything through the mason 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.
Great, hope it wins a prize. L
This technique looks like it could be used as part of a great entry in our <a rel="nofollow" href="https://www.instructables.com/id/ESGO9GBF3R9YIXH/">Science Fair</a>.<br/>
I never thought of that. I guess I should get to work making "infographics" and fleshing it out some more, when I get the chance.
Eric, 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 more sciency Instructables from you to lessen the K'nex gun inundation.

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