ANALYTICAL cHEMISTRY - II

Experiment 6

Principle

• In chemistry, Column chromatography is a technique that is used to separate a single chemical compound from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow them to get separated fraction-wise. This technique can be used on a small as well as large scale to purify the materials that can be used in future experiments. This method is a type of adsorption chromatography technique.

• When the mixture, that needs to be separated, is introduced from the top of the column along with the mobile phase, the individual components of the mixture move at different rates. The components with lower adsorption and affinity to the stationary phase travel faster, compared to the components with greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last.

• The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as the retention factor, or R_f, which is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

𝐑𝐟 = D𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐬𝐨𝐥𝐮𝐭𝐞 / 𝐃𝐢𝐬𝐭𝐚𝐧𝐜𝐞 𝐭𝐫𝐚𝐯𝐞𝐥𝐞𝐝 𝐛𝐲 𝐭𝐡𝐞 𝐬𝐨𝐥𝐯𝐞𝐧𝐭

Materials required

Glass column equipped with stopcock, Cotton plug, Solvents (mobile phase), Silica gel / Alumina, Glass rod, Mixture to be separated, etc.

Procedure

Part-1: Preparation of the silica gel bed (Loading of the column)

1. Take an empty, clean, and dried column with a Teflon stopcock.

2. Put a cotton plug at the bottom of the column near the Teflon stopcock.

3. Pour the prepared slurry of silica gel, in an appropriate solvent, into the column using the funnel. Ensure that the cotton plug should not be disturbed and stay at its position while pouring the silica gel slurry.

4. Open the stopcock and collect the solvent in a suitable container.

5. Tap the column gently and constantly to ensure proper settling of the silica gel bed.

6. Avoid formation of cracks and voids in the silica gel bed.

7. After pouring the entire slurry into the column while allowing the solvent to elute from the column via stopcock, add fresh solvent on the top of the column using the funnel. The silica gel bed should be disturbed while pouring the fresh solvent.

8. Once the bed has been settled completely (ensured by constant tapping) stop collecting solvent from the bottom of the column.

Part-2: Sample loading

1. In a clean dry beaker take the given mixture of analytes.

2. Add 20 mL of CH₂Cl₂ into it and swirl it well (ensure dissolution of analyte).

3. Add small portion of silica gel into the clear solution of analytes in CH₂Cl₂.

4. Evaporate the excess amount of solvent by putting it in the water bath kept at 50 ^oC.

5. Constantly stir the mix. of silica gel and analyte using glass rod in order to ensure uniform coating of the mix. on silica gel particles.

6. After complete evaporation of the solvent, the silica gel + analyte mixture should be completely free flowing and should not contain any lumps.

7. Introduce this free-flowing silica gel + analyte mixture from the top of the column, on the top of the prepared silica gel bed without disturbing the bed using funnel.

8. To avoid formation of voids and cracks, constantly tap the column.

9. After it, put a cotton plug on the top of the prepared bed.

Part-3: Elution of the components of the mixture from the column

1. Elute the mixture of the analytes by adding freshly distilled solvent.

2. Increase gradually the polarity of the mobile phase by adding small portions of ethyl acetate into the freshly distilled petroleum ether (i.e., 5% v/v, 10% v/v, 15% v/v, etc.).

3. Collect mobile phase fractions (50 mL) into the conical flask/Nessler’s tube.

4. Check the presence of analyte into each fraction by repetitive TLC analysis.

5. The Fractions containing pure compound [Aniline/Acetanilide] should be kept separate from the fractions containing the mixtures of compounds.

6. Collect all the fractions having same compound [Aniline/Acetanilide] into 250 mL round bottom flask and evaporate the mobile phase by either simple distillation or vacuum distillation.

7. Collect the pure compound and weigh it. Find out the isolated yield.

8. Record the isolated yield and efficiency of the separation by column chromatography in the result.

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. Total weight of pure aniline in the given mixture = _______ gm

2. Total weight of pure acetanilide in the given mixture = _______ gm

3. Total weight of pure aniline collected from the column = _______ gm

4. Total weight of pure acetanilide collected from the column = _______ gm

5. % loss of aniline during the column chromatography = _______

6. % loss of acetanilide during the column chromatography = _______

Result

Weight of aniline collected = __________ gm.

Weight of acetanilide collected = __________ gm.

Efficiency of separation = __________ gm.

Reference Material

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