Separating the Pigments in Spinach Leaves by Thin-Layer Chromatography

Thin Layer Chromatography Separating the Pigments in Spinach Leaves

Nabona, Kathy. "Thin Layer Chromatography Separating the Pigments in Spinach Leaves." Handout to Organic Chemistry I students. Austin Community College. Austin, TX. n.d. Print.

I.  Introduction

For the purpose of this experiment we are to extract the colored pigments within spinach leaves. Once extracted we are to separate the pigment from from remainder of solvent used in extraction and then identify those pigments using thin layer chromatography.

II.  Experimental Procedure

SAFETY NOTES

- Petroleum ether, Ethanol, and Acetone are flammable, no open flames

- If solvents make skin contact they will irritate and cause a burning sensation despite not really physically burning.

PROCEEDURE

* Developing Chamber Prep *

1. Select a convex bottom, glass jar

2. Pour enough [70:30 petroleum ether : acetone] to cover base entirely

3. Close jar & allow solvent to evenly saturate

* Pigment Extraction *

4. Select 2 spinach leaves and break apart into pestle dish.

5. Pour 4mL of petroleum ether & 2mL ethanol together into a10mL-graduated cylinder

6. Add solution to spinach & mix with mortar until pulpy, solvent will evaporate.

7. Add another Pour 4mL of petroleum ether & 2mL ethanol together to the pulp.

8. Swirl contents together and let sit to dissolve until deep green in color.

9. Transfer liquid ONLY (strain pulp) to the separatory funnel using disposable pipet.

10. Note the volume obtained and add an equivalent amount of water (Note 2 separate layers) and swirl gently to remove ethanol.

11. Drain bottom layer from funnel and dispose into NON-HALOGENATED ORGANIC WASTE container.

12. [Steps 9 & 10 are repeating steps 7 & 8 twice more, this is to remove all remaining ethanol. (Note remaining layer decreases with each rinse)]

13. Again, note the volume obtained, add an equivalent amount of water and swirl gently before again, draining and disposing the bottom layer.

14. Drain top-layer from separatory funnel into a 50mL Erlenmeyer flask.

15. Add some anhydrous sodium sulfate (Na2SO4) and swirl gently to remove excess traces of water.

16. Wait 5 min (Note the solution will become clear while white sodium sulfate remains)