Basic Column Chromotagraphy

1. Introduction: Hello my name is James from HSA-SA and my demo is basic column chromatography. In this experiment i will be testing spinach powder. Spinach is a green vegetable rich in iron, but there are additional pigments that are not visible to the naked eye that. The presence and identity of the additional pigments in the spinach can be found by using Basic column Chromatography

2. Materials: Acetone 10-ml, Aluminum oxide, Hexane 10-ml, Hexane-acetone mixture 50/50 10-ml, sand .5-g, spinach powder .5-g, Chromatography column, Balance .1-g precision, Beaker 250-ml, Beakers or Erlenmeyer flasks 50-ml, Clamp, Graduated cylinder 10-ml, Marker, Pipets thin-stem, Spatula, Stoppers, Support stand Test tubes 13x100 mm, Test tube rack, Weighing dish

3. Procedure: 1.Attach the clamp to the support stand.

2.Place the tip on the bottom of the chromatography column. Pour about 2.5 mL of hexane into the column so that the liquid fills the narrow portion of the column.

3. Slowly pour 2g of the aluminum oxide into the column. Fill the narrow portion of the column until it is about 3/4 full. Tap the tip of the column on the lab bench while pouring the aluminum oxide-tapping the column sufficiently will eliminate holes or spaces. Having holes or spaces in the packing of the solid will leave channels for the substances to travel through and lead to poor separation.

4.Place the column in the clamp and tighten until the wide portion of the column is held firmly by the clamp.

5.Place an empty 250mL beaker underneath the column.

6.Carefully pour a small amount of sand into the column so it forms a 2-mm layer on top of the aluminum oxide

7.Remove the column from the clamp and tap the column on the lab bench again to ensure the sand forms an even layer.

8.Place the column back in the clamp so that it is positioned above the beaker.

9.Obtain 10mL each of the following solventers-hexane, 50/50 hexane-acetone, and acetone. Place each solvent in a clean 50mL beaker or Flask.

10.Weigh 0.5 g of spinach powder and place it in a 13 X 100 mm test tube. Add 1 mL of the 50/50 hexane-acetone mixture.

11. insert a stopper into the test tube and shake vigorously. Continue shaking until the liquid (extract) begins to turn a dark green. Place the test tube in a test tube rack.

12.Remove the tip from the chromatography column and allow the hexane in the column to drain into the beaker below. Allow it to drain until the liquid level is just above the sand layer. Replace the tip on the column.

13.Shake the test tube containing the spinach extract and allow the solid to settle. Place a clean pipet into the test tube and fill the pipet with only the liquid portion of the extract. Avoid obtaining solid in the pipet.

14.Remove the tip from the column and add 5 drops of the spinach extract to the top of the column by running it down the inside of the column in a circular fashion. Do not simply squirt in into the center of the column as this will disturb thee sand layer.

15.As soon as the extract is absorbed into the sand layer, carefully add a pipet-full of hexane to the column by running it down the inside of the column in circular fashion. Continue adding hexane in this manner until the entire column is full.

16.As solvent moves through the column, it will begin to carry one of the pigments with it. This will take several minutes as the pigments interact with the adsorbent phase. Colored bands should become visible in the column.

17. Label three clean test tubes pigments 1, pigment 2, and pigment 3.

18.As the first pigments band begins to exit the tip of the column, place the appropriate test tube underneath the column to collect the slovent containing this pigment. As soon as the band has completely exited the column, remove the test tube, stopper it and see it aside. Record observations in the data table. The same general procedure will be used to collect additional pigments as they pass through the column. Different solvents, first 50/50 hexane-acetone and then acetone, will be needed to collect pigments 2 and 3, respectively. In each case the same general procedure should be followed. Never allow the top of the column to run completely dry. Add each solvent carefully by running it down the inside of the column in a circular fashion so as not to disturb the sand layer. Collect each pigment band solution in a different test tube. Replace the test tube with a beaker to collect eluent that does not contain a pigment.

19.Collect remaining hexane in the 250mL beaker. Do not allow column to go dry.

20.Using pipet, add the 50/50 hexane-acetone mixture to fill the entire column.

21.As the hexane-acetone mixture begins to flow down the column, it will carry another pigment band with it. Collect the eluent in the 250mL beaker until the colored band is at the bottom of the column.

22.Collect the solvent containing the second band in the appropriate test tube. Record observations in the data table.

23.Allow any remaining hexane-acetone to drain into the 250ml solvent beaker. Leave a thin layer so the column remains wet.

24.Using a clean pipet, add acetone to fill the column.

25.As the acetone begins to flow down the column, it will carry the last spinach pigment with it. Add more acetone as needed to make sure the column does not dry out until the third band has almost reached the bottom.

26. Collect acetone eluent in the the 250ml solvent beaker until the third band approaches the bottom of the column. Replace the beaker with the third test tube to collect the last pigment band. As soon as the band has completely exited the column, remove the test tube, stopper it and see it aside. Record observation in the data table.

4. Scientific Principle: The purpose of the experiment is to learn column chromatography by separation the spinach pigments. the spinach pigments will be separated and collected by the use of the multiple eluents that vary in polarity so you can see the changes in the pigments and how they vary.

5. Safety Regulations: Acetone and hexanes are flammable liquids and dangerous fire risks. Acetone is also slightly toxic by ingestion and inhalation. Hexanes are a respiratory irritant. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory.