ANALYTICAL cHEMISTRY - II

Experiment 5

Principle

• TLC is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are there in a mixture. TLC is also used to support the identity of a compound in a mixture when the R_f of a compound is compared with the R_f of a known compound (preferably both run on the same TLC plate).

• A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action.

• As the solvent moves past the spot that was applied, an equilibrium is established for each component of the mixture, between the molecules of that component which are adsorbed on the solid, and the molecules which are in the solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others.

• When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually, the compounds are not colored, so a UV lamp is used to visualize the plates. (The plate itself contains a fluorescent dye that glows everywhere except where an organic compound is on the plate.)

Interactions between the compound and the adsorbent

• The strength with which an organic compound binds to an adsorbent depends on the strength of the following types of interactions; ion–dipole, dipole–dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. With silica gel, the dominant interactive forces between the adsorbent and the materials to be separated are of the dipole-dipole type.

• Highly polar molecules interact fairly strongly with the polar Si-O-H groups at the surface of these adsorbents, and will tend to stick or adsorb onto the fine particles of the adsorbent while weakly polar molecules are held less tightly. Weakly polar molecules generally tend to move through the adsorbent more rapidly than the polar species. Roughly, the compounds follow the elution order given above.

The R_f value

• The retention factor, or R_f, is defined as the distance travelled by the compound divided by the distance travelled by the solvent.

• For example, if a compound travels 2.1 cm and the solvent front travels 3.5 cm, the R_f is 0.60.

• The larger the R_f value of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger R_f is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger R_f value than a polar compound run on the same plate.

Materials required

Ethyl acetate, Petroleum ether, Unknown mixture, and their corresponding standard compounds, Filter paper, TLC sheets, TLC chamber, Capillary tubes, Iodine chamber, UV-visible chamber for the visualization of developed TLC, etc.

Procedure

Part-1: Preparation of the TLC plate

1. TLC Plates used in the laboratories are purchased as 20 cm x 20 cm sheets.

2. Each large sheet is cut horizontally into plates which are usually 4 – 5 cm tall having width as per requirement; the more samples you plan to run on a plate, the wider it needs to be.

Part-2: Spotting of the sample on the TLC plate

1. Mark a reference line about 1 – 2 cm from the bottom edge of the plate with the pencil gently.

2. The standards provided to you must be applied as tiny dots on the starting line and well-spaced from each other.

3. You need to put individual spots on the starting line corresponding to the standard, unknown mixture along with a co-spot of the standard as well as the unknown mixture.

4. To put a spot, draw a small amount of solution in a capillary, say 1 – 2 cm and lightly touch the capillary to silica surface so that sample is soaked up by silica and forms a tiny spot.

5. Do not use the capillary for another sample to avoid contamination.

Part-3: Solvent run on the prepared TLC plate

1. After spotting, the TLC plate is transferred to a glass chamber containing the solvent to be used as a mobile phase (ethyl acetate & petroleum ether).

2. The solvent level in the chamber must be a few mm below the starting line.

3. Allow the solvent to rise along the Silica surface and as soon as the solvent front reaches the finish line, remove the plate from the solvent chamber and allow the solvent to dry out.

4. Repeat this procedure using mobile phases of different polarity, until the mixture is separated properly on the TLC plate.

Part-4: Visualization of the developed TLC

1. Transfer the plate to Iodine / UV chamber and watch the spots appear on the plate.

2. In the Iodine chamber, the sample spots absorb iodine and develop a yellow-brown color, the density of which depends on the solubility of I₂ in the given sample.

3. After withdrawing the plate from the iodine chamber, circle the spots with a pencil and measure the distance traveled by each spot.

4. Draw the chromatogram in your journal sheet and calculate the Rf value for each spot and use it to identify the constituent in the mixture.

5. The components can also be identified by comparison of the color of spots with those given by standards.

Calculations

1. Distance traveled by the solvent (X) = _________ cm.

2. Distance travelled by compound – 01 (Y) = _________ cm.

3. Distance travelled by compound – 02 (Z) = _________ cm.

4. R_f of compound – 01 = Y / X = _________.

5. R_f of compound – 02 = Z / X = _________.

Result

1. The R_f of compound–01 is _________.

2. The R_f of compound–02 is _________.

Reference Material

1. G H Jeffery, J Bassett, J Mendham and R C Denney, Vogel's Textbook of Quantitative Chemical Analysis, 5th Edition