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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) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 1: Gather the materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 2: Grind the Silica Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 3: Weigh out and mix the silica gel and plaster of paris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 4: Suspend the powder in water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 5: Coat glass slides with suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 6: Air dry followed by activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 7: Some final notes on preparing the plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 8: Construct the developing chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . step 9: Extracting the plant pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3 4 4 5 5 6 7 8 8
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
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 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).
Image Notes 1. The Eluent, the solvent being used to push the mixture, moves up the plate from bottom to top by capillary action 2. The original spot, placed on the plate by way of an eye-dropper above the solvent-line 3. Three distinct species are separated as the solvent pushes up. The blue is more strongly held by the silica and thus lags further behind whereas the red is less 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 the distance 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 then the R.F.s of unknown species simply looked up.
Image Notes 1. As the Analytes (stars) are pushed past the silica particle, they interact (gold arrows). These interactions slow them down. The strength of the interactions governs how much they are slowed down.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder 2. 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 the edge)
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
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.
Image Notes 1. The contents of one packet. Large beads that are too large and round to grind properly.
Image Notes 1. After much beating, complaining, and cheating by using a ball-grinder at work.
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.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. these plates have just been coated with their respective suspensions
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. the suspension, loaded into the syringe (with air that will be pushed out later)
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder 2. 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 the edge)
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. cut the filter paper so that it will fit in the jar
Image Notes 1. note how the filter paper fits along the wall of the jar.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. Acetone 2. Hexane 3. The graduate cylinder for making up the mixed solvents.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. This is the final liquid that was drawn off
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. the pigment moving up the plate (I spotted a line to make it easier to see with the camera) 2. beaker with a watch glass for a lid simply so I could take pictures through the wall. 3. the eluent, a 6:4 mixture of hexane and acetone
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
Image Notes 1. there's another line down here you can't see very well 2. before developing 3. after developing, you can see where I pressed too hard with the pipette one time 4. chlorophyll and xanthopyll close to togethor 5. there's a faint green line up here that didn't show up in the picture very well
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Comments
12 comments Add Comment
bioman says:
Aug 13, 2007. 8:37 PM REPLY 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.
allanf0 says:
Aug 15, 2007. 2:11 PM REPLY 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).
bioman says:
Aug 16, 2007. 1:21 AM REPLY 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!
allanf0 says:
Aug 15, 2007. 2:16 PM REPLY 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.
lemonie says:
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
allanf0 says:
Aug 1, 2007. 1:03 PM REPLY 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.
lemonie says:
Thanks, it would be good to see more of this. L
allanf0 says:
Aug 2, 2007. 7:55 PM REPLY 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.
lemonie says:
Great, hope it wins a prize. L
ewilhelm says:
This technique looks like it could be used as part of a great entry in our Science Fair.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/
allanf0 says:
Jul 31, 2007. 12:48 PM REPLY 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.
CameronSS says:
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.
http://www.instructables.com/id/Preparing-your-own-thin-layer-chromatography-plate/