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.
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="http://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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