Experiment 16 Extraction of pigments from spinach

Prepare a chromatography developing chamber with a wick and about 0.5 cm of 70/30 hexane/acetone. Seal with saran-wrap and a rubber band and leave in the hood.
1. Tear 0.5g spinach leaves (no stems or veins) into small pieces.
2. Place in mortar with 1 ml acetone; grind into paste. Add more acetone if it evaporates.
3. Put mixture into centrifuge tube without a cap; add 1 ml more acetone to mortar and pestle and rinse into centrifuge tube; centrifuge.
4. Pipet liquid to centrifuge tube with tight lid – you should have about 2 ml.
5. Add 2 ml hexane, cap and shake (vent).
6. Add 2 ml water, cap and shake (vent).
7. Centrifuge.
8. Remove bottom water layer with Pasteur pipet-dump.
9. Filter organic layer thru 0.5 g of anhydrous sodium sulfate in a Pasteur pipet column – clamped to a ringstand. Collect the green solution in a small beaker labeled E for extract. See diagram on p. 138
10. Rinse column with 0.5 ml hexane into beaker E.
11. Evaporate solvent off solution E in sand bath on low. When solution is concentrated spot a TLC plate several times with your dark green solution, 1 cm from the bottom of the plate, keeping the spot as small as possible. Cover TLC plate with plastic wrap and keep in the dark in your lab drawer. You will spot the same plate with the individual bands that are collected from the column chromatography (2 or 3 more spots.)
Preparing for column chromatography (while solvent is evaporating.)
1. Use 5 3/4 inch Pasteur pipet for your column and clamp it to a ringstand.
2. Pack the column as described on p.765, Dry Pack Method 2. Place a small wad of glass wool, loosely packed, into the constriction and then add a 1/8 inch layer of sand.
3. Weigh 1.25 g alumina on a piece of paper and add it slowly to the column while tapping the column with your finger to settle the alumina without air pockets.
4. Add another 1/8 inch layer of sand to the top of the column.
5. Label 3 small beakers: #1, #2, #3.
6. Put about 5 ml of hexane in a small, labeled flask. Do the same for 70/30 hexane/acetone, pure acetone, and 80/20 acetone/methanol.
Doing the column chromatography of spinach pigments.

Never let the liquid go below the sand level.
1. Add 0.5 ml hexane to the dried pigments in beaker E.
2. Add 3 ml hexane to the column carefully and collect in a clean flask or beaker.
3. As soon as the hexane level just reaches the sand on the top of the column add all of solution E to the column.

4. When E just penetrates the sand layer, add hexane again to the column. Continue adding hexane to the column, noting when a faint yellow-orange band appears to be coming down the column. The green band should be staying at the top of the column.
5. To separate the yellow-orange band from the green band , start adding 70/30 hexane/acetone to the column.
6. Collect the orange band in beaker #1.
7. Place this beaker in the sand bath to evaporate off the solvent.

8. Elute the green band and collect it in beaker #2 by adding pure acetone to the column. Evaporate the solvent off on the sand bath.
9. Finally, try to elute any material remaining on the column, using methanol/acetone, and collect in beaker #3. Evaporate the solvent off.
Thin-layer Chromatography
1. Add 2 drops of 70/30 hexane/acetone to the dried pigments in #1,#2,#3.
2. Spot your three solutions on the TLC plate, making the spots as small and dark as possible but spotting several times, especially the yellow band.
3. Develop the plates in a 70/30 hexane/acetone solvent.
4. When the plate is removed from the chamber, circle all individual spots and immediately note the color of each spot - these colors do fade. You will see yellows, various shades of green, and gray.
5. Sketch the TLC plate in your notebook and Identify as closely as possible each of the spots in the four samples. The TLC plate will be your “product” for this lab. You will be graded on the quality of the TLC plate.