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Lecture 2
What is Chromatography?
Simple way of separating a mixture of chemicals based on polarity Molecules partition between a stationary phase (e.g. silica) and a mobile phase (solvent) Every chemical possesses an intrinsic polarity p-nitrobenzoic acid: red (electron rich d-), blue (electron poor d+)
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TLC Basics
1. TLC Plate silica gel (SiO2) or alumina (Al2O3), usually impregnated with a fluorescent dye
Alumina is more polar than silica Plate glows green at 254 nm under a UV lamp
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Eluting power of organic solvents Alkanes (hexanes, petroleum ether) toluene Increasing polarity (more powerful eluent) Halogenated hydrocarbons (dichloromethane) Diethyl ether Ethyl acetate Acetone Alcohols Acetic acid If the compound is colored, they are easy to see by eyes. If not, a UV lamp or iodine chamber is used.
When the compound has the fluorophore, UV lamp is used. If no fluorophore, the iodine stain is used.
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3. Typical Procedure
Line part of the inside of the beaker with filter paper. Cover the beaker with a watch glass
Pour solvent into the beaker to a depth of just less than 0.5 cm.
Using a pencil, draw a line across the plate at the 1 cm mark. This is the origin: the line on which you will "spot" the plate. Measure 1 cm from the bottom of the plate.
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View the plate under a UV lamp (or place in an iodine chamber, or both)
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4. Calculation of Rf value
Rf =
= 0.25
5. Key Hints
If the eluent is too polar everything will just elute together If the eluent is too non-polar nothing will eluteneed to find the perfect combo. If you dont let your mobile phase travel far enough up the plate you will not give your chemicals enough time to separate Be sure to not put your spotting line (start line) directly into your solvent or youll just wash away your chemicals (sharks will eat them!) Always be sure to cap your chamberWHY?
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6. Troubleshooting TLC
(1) Overloading
(2) Plate solvent front runs crookedly, harder to measure Rf values accurately.
Two possibilities: The adsorbent has flaked off the sides of the plate The sides of the plate are touching the sides of the container as the plate develops.
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Troubleshooting TLC
The left: has a large yellow smear; this smear contains two compounds. The middle: nicely resolved. The right: A UV visualization of the same plate on the left What is (are) the problem(s)?
Acetaminophen (Ac)
Caffeine (Cf)
Ibuprophen (Ibu)
Salicylamide (Sal)
No physical constants are needed in this TLC pre-lab, but they are required for the rest of the labs.
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On one plate you will spot 5 On the other plate you will spot various common drug components over-the-counter meds and one unknown.
Silica gel TLC plate Eluent: 0.5% glacial acetic acid: ethyl acetate
Ref: Ac+Asp+Cf+Ibu+Sal
Salicyclic acid