Idea Transcript
Thin Layer Chromatography Chromatography Overview Chromatography is the general name for a variety of techniques used in the physical separation of mixtures. Chromatography separates compounds based on their differing affinities for a medium known as the stationary phase. The stationary phase is a solid material that organic compounds will adhere to in varying degrees depending mostly on their polarity and molar mass. Chromatography techniques also have a gas or liquid mobile phase which pushes the mixture through the stationary phase. Substances with a low affinity for the stationary phase will move through the medium quickly and those with a strong affinity for the stationary phase will move slowly. Common chromatography techniques include Gas Chromatography (GC), Column Chromatography, High Performance Liquid Chromatography (HPLC), and Thin Layer Chromatography (TLC). Thin Layer Chromatography Thin Layer Chromatography or TLC is one of the most quick and useful analytical techniques in organic chemistry. A TLC chamber can be found on the bench of virtually every organic chemist and the technique is used several times a day by most of them. TLC can be used to confirm the presence of a compound in a mixture, to check for the presence of impurities, to monitor to progress of a reaction and in some cases to separate a mixture on a preparative scale. TLC uses silica gel (or less frequently alumina) spread on a plastic or glass backing as the stationary phase - this is known as a TLC plate. Silica gel is fine particles of porous, amorphous, solid silica (SiO2). Silica is a polar substance, therefore the more polar the compound, the more it will to stick to the silica. Since water is polar and silica attracts it, you will often find small packets of silica gel in new leather goods where is it used as a desiccant. The mobile phase in TLC is an organic solvent or mixture of solvents known as the eluent. A pencil line drawn 1 cm from the bottom of the TLC plate indicates the starting point of the material to be chromatographed. Pen should not be used on TLC plates because ink pigments are soluble in organic solvents and will be chromatographed along with your material. The mixture gets deposited with a capillary tube as a small spot on the pencil line. To spot a TLC plate, dip a capillary tube in a dilute solution of your material so that a small amount in small amount of liquid is picked up by capillary action. Quickly touch then lift the filled capillary tube on the line where you are spotting and allow the solvent to evaporate. This creates a small spot of your material. If you let the entire capillary tube drain onto the plate you will create a large spot which does not separate as well. If you need to put more of the compound on the plate, it is better to spot several times in the same location, allowing the solvent to evaporate each time, rather than making a large, spread-out spot. To the left is a TLC plate ready for the developing chamber. The spot on the right is too big – the separation will not be good and it may bleed over into the other spots. The middle spot is a bit large but acceptable. The spot on the left is perfect. Once ready, the TLC plate is placed in what is known as developing chamber. Developing chambers are covered containers which have a few millimeters of eluent at the bottom. The liquid level should be below the level of the pencil line because we want the compound to stay on the plate, not get dissolved into the solvent. The eluent rises up the plate through capillary action and will eventually saturate the entire plate, just as a paper towel with one edge dipped in water will eventually become entirely wet. The edge of where the eluent has climbed is known as the solvent front. The plate is removed from the chamber when the solvent front is about 1 cm from the top of the plate. As the eluent rises, the compounds in the mixture start moving up the plate at different rates thus separating them. The rate at which a compound moves up a plate depends on both its attraction to the silica and its solubility in the eluent. The more polar a compound is, the more attracted it is to silica and the slower it will move up the plate. The more soluble a compound is in the eluent, the faster is moves up the plate.
The illustration on the left shows a TLC plate immediately after it has been placed in the developing chamber. The middle illustration represents a half-developed plate. When the solvent front is within 1 cm of the top of the plate (illustration on the right) it should be removed from the chamber. In this example, the spot separates into two compounds. The red compound is the less polar and the blue compound is the more polar. Visualizing Spots Most organic compounds are colorless therefore a visualization aid is needed once a mixture has been separated by TLC. Most TLC plates have a fluorescent compound mixed with the silica gel so that the plate glows green under a UV lamp. Compounds that absorb UV light (usually those with bonds) will appear as a dark spot on the green plate. Compounds may also be visualized by staining the TLC plate. Different stains are can be used depending on the functional groups of the molecules that need to be visualized. Calculating Rf The retention factor (Rf) is used to compare how far different spots travel up the plate. The Rf is calculated as follows: Rf =
distance from pencil line to center of spot distance from pencil line to solvent front
Note that all measurements taken from the spot’s starting point – the pencil line. We divide the distance a spot traveled from its starting point, by the maximum distance it could have traveled, from the starting point to the solvent front. All Rf values will be between 0 and 1. If a spot did not move from the pencil line, it will have an R f, of zero, such as spot A in figure (2) – this spot is very attracted to the silica. If a spot moves with the solvent front, it will have an Rf of 1, such as spot B which is not very attracted to the silica. For spot C, the distance from the pencil line to the spot is 1.0 cm, from pencil line to the solvent front is 4.0 cm, making the Rf = 0.25.
Comparing Different Samples The number of spots that separate indicates the number of compounds in the sample. In the figure to the right, three samples (D-F) were developed with TLC. Samples D was a pure substance –one spot on the plate indicates that it contained only one compound. Sample E contained two compounds and sample F contained three compounds. Spots that have the same Rf are probably the same compound. One way to confirm that an unknown sample is the same as a standard is to use a technique called co-spotting. This makes it easy to tell if you really have the same compound, or two different compounds with similar Rfs. On the developed plate to the right, a standard “A” was spotted on the left side and in the middle. An unknown was spotted on the right and directly on top of the A spot in the middle. If the unknown is compound A, we would expect the middle spot to not separate, as it would be a pure compound. However, we see that the co-spotted sample separated into two spots, so the unknown is not compound A.
Procedure In this lab you will extract the organic analgesics from an unknown painkiller tablet and compare it to standard solutions to determine the identity of the pill. Below is a table of some common over-the-counter analgesic pills and the drugs that they contain. Acetaminophen Commercial Painkillers Anacin Bufferin Excedrin Tylenol Motrin
H N
Aspirin O
N
O
O
HO
O
X X
Caffeine
Ibuprofen
OH
X X X
N
O
O
N
N
OH
O
X X X
You will be given a tablet by your instructor. Crush the tablet well with a mortar and pestle. Mix the powder with 3 mL of 1:1 ethyl acetate: ethanol and stir with a glass rod for at least one minute. Allow the solid to settle and decant the mixture into a glass vial. Keep the vial capped when not in use.
x
x
x
x x
Obtain two TLC plates; hold them only by their edges (oils on your fingertips can ruin the plate). Gently draw a line in pencil 1 cm from the bottom of each plate. For the first plate, mark 5 x's along the line and label them with the names of the 4 standards and your unknown as shown on the left. Using a micropipette, spot your unknown solution and the 4 standard solutions on the appropriate x on your plate. The diameter of the spots should be as small as possible (practice on a paper towel first!). Before developing, check the plate under a UV light – if the spots are not dark enough to be seen before developing, they will not be dark enough to see afterwards. Re-spot samples until they can all be seen with a UV light.
Prepare a developing chamber by putting a small amount of ethyl acetate in a clean 1000 mL beaker then covering the beaker with aluminum foil. Use just enough liquid to cover the bottom of the beaker (~5 mL). Place the first TLC plates in the chamber and re-cover with foil. Make sure the side edges of the plate do not touch the beaker as this causes the solvent front to be uneven. When the solvent front is one centimeter from the top of the plate, remove the plate from the chamber and mark the solvent front with a pencil. Put the developed plates under UV light. Circle the outline of the spots with a pencil. You will make a preliminary guess as to the identity of your compound and choose two standards to run on a co-spotting plate. You should choose whichever two standards would make it easiest to confirm your guess. Set up your co-spotting plate as to the right, where “A” and “B” are the two standards you chose. Develop the second plate and circle spots under UV light as before. Chemicals: ethyl acetate, ethanol, aspirin*, acetaminophen*, caffeine*, ibuprofen* *Safety data is not needed for starred compounds Waste - Used TLC plates should go in the specified box. - Developing chamber liquid and unknown spotting solution can go in Disposal D. - Used glass micropipettes go in the melting point waste jar.