1. A Green Approach to Separate
Spinach Pigments by Column
Chromatography

2. Today's Lab
• Principles of Green Chemistry
• Two types of chromatography
– Column Chromatography
– Thin Layer Chromatography (TLC)
• First, column chromatography will be used to
separate pigments from a spinach extract
• Second, TLC will be used to identify the
pigments

3. Principles of Green Chemistry
• Renewable feedstock
– Spinach is a safe, cost effective, and easily obtainable
starting material
• Safer solvents
– Traditional plant extraction solvents: CH2Cl2, CHCl3,
CCl4, benzene, etc.
– Greener alternatives: alcohols, acetone, hexanes
• Recycling
– Acetone and hexanes may be re-purified by distillation
– Alumina may be washed, filtered, and reused

4. Flow of Lab
• Demonstrations (Column chromatography & TLC)
• Videos (Column chromatography & TLC)
• Conduct lab
• Turn in lab notebook pages, TLC plate, and product
report form for collected pigments

5. Video 1: Column Chromatography
• Column chromatography basics
• Williamson microscale chromatography column
• Video link: http://youtu.be/vHcWWIU-7Og

6. Demo 1: Column Chromatography
• Column chromatography setup and operation
– Williamson microscale column
– Alumina/hexanes column packing
– Acetone (Recycle/reuse: safer solvent)
– Load ferrocene/acetylferrocene
– Demonstrate use of flash chromatography
– Collection of separated fractions

7. Column Construction
• Adsorbent, guard
sand, and sample
• Vertical orientation
of column
• Fractions can be
seen as they elute

8. Video 2: TLC
• Thin Layer Chromatography basics
• Video link: http://youtu.be/D1MoWtOr5f4

9. Demo 2: TLC
• TLC
– Plate = silica, aluminum backed,
UV active
– Developing chamber = 60:40
hexanes:acetone
– Standards = xanthophylls (X),
beta-carotene (B), co-spot
– Setup, develop, calculate Rf
• Rf values of standards for
comparison to experimentally
obtained pigments
– Spot distance Solvent distance

10. Additional Notes
• There is a report form to turn in at the end of
the lab (product report form for pigments)
• Record the appearance of the pigments as
they migrate on your column
• Draw a picture of your finished TLC plate in
your lab book
• TAPE your TLC plate to your notebook pages
when you turn them in

11. Visual Aids (to be left up during lab)
The “dark green” of
your initial sample
The three fractions to
collect from the column: -
carotene, xanthophylls,
and chlorophyll a.