1/10/2013

Lecture 2

Thin Layer Chromatography (TLC)

Reading: Lab manual: Expt. 1

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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Theory of Chromatography: To Adsorb or Not to Adsorb

A polar molecule (nitrobenzoic acid) will adsorb (stick) to a polar surface (silica). A non-polar solvent (hexane) will not be able to rinse it off. A more polar solvent (acetone, acetic acid) will compete with the surface and move the solute with the solvent flow.

Solute molecules adsorb to surface or flow with solvent

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

Note: in your experiment, you will spot solutions, not ink!!

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2. A mixture of organic solvents

More polar solvents compete better with the polar silica/alumina, and so compounds travel faster with the mobile phase

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.

Solvent Front (Finish Line)

Spotting Line (Starting Line) Mobile Phase

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Spot the TLC plate

Place the TLC plate in the elution container

The solvent will rise up the TLC plate by capillary action.

Remove the plate from the beaker.

View the plate under a UV lamp (or place in an iodine chamber, or both)

Mark the spot(s) with a pencil.

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4. Calculation of Rf value

Rf =

distance traveled by spot distance traveled by solvent

= 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

Which one is correct?

(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.

(3) Many random spots are seen on the plate.

Likely, you accidentally dropped or splashed an organic compound on the plate as you leave it on the workbench

(4) No spots are seen on the plate.

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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)?

Experiment of the Week

(starting Tuesday, January 22) You will use TLC to determine the composition of various over-the-counter analgesics

Acetaminophen (Ac)

Caffeine (Cf)

Acetylsalicyclic acid (Asp)

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

1. Anacin 2. Excedrin 3. Tylenol

Aspirin frequently contains a small amount of salicyclic acid impurity

Aspirin

Salicyclic acid

Why does it contains this impurity?

Why does salicyclic acid have a lower Rf value than Aspirin?