Separate a mixture of Methylene blue and Fluorescein (sodium salt) by column chromatography using aluminium oxide as adsorbent.

 

Separate a mixture of Methylene blue and Fluorescein by column chromatography using aluminum oxide as an adsorbent.

Theory:

Separation of the mixture can be done by dissolving the mixture in ethyl alcohol or rectified spirits. When the mixed solution is added from the top of the column and ethyl alcohol is used as the solvent, fluorescein is strongly adsorbed on the top and methylene blue passes through the column. Thus methylene blue can be extracted with ethyl alcohol. When the blue color is removed from the column, the fluorescein is extracted with water. The two components are recovered by evaporation of the solvent.

Requirements:

Chromatography tube, adsorbent Alumina, Sodium salt of Fluorescein, Methylene blue, Ethyl alcohol or rectified spirit.

 Procedure:

1. Prepare an alumina slurry by mixing about 25.0 g of alumina in about 40-50 ml. of ethyl alcohol. 

2 Pour this slurry into the chromatography tube and prepare a column about 25 cm long. Place the filter paper disc at the top of the column. 

3. Take about 10.0 mg Methylene blue and 15.0 mg Fluorescein sodium salt Mix the two and dissolve in 15-20 ml ethyl alcohol or rectified spirits in a boiling tube.

4. Open the tap of the chromatography column to drain the excess solvent. When the column surface is almost dry, add the mixed dye solution to the top of the column. 

5 Attach a tap funnel to the chromatography tube at the top and fill it with alcohol.

6. When the last drop of the dye solution has gone into the column, start adding alcohol from the tap funnel.

7. The blue color of the Methylene blue dye will start moving downwards in the column while Flourescein will be adsorbed on the top.

8 Continue the solvent flow until all the methylene blue has entered the receiving container and the filtrate turns colorless. 

9 Now replace the alcohol in the tap funnel with water and let it fall drop by drop on the top of the column. The Flourescein ribbon will start moving downwards in the column. Continue elution until all fluorescein passes to the receiver. 

10. Evaporating the receiving solvent and collecting pure components.

Precautions : 

1. There should be no air bubbles trapped in the alumina column.

2. Only chromatography grade aluminia should be used for filling the column. 

3. For effective separation, the rate of the flow down the column should be about 10 drops per minute.

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