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Duke University - Organic Chemistry Laboratory

Lab

COLUMN CHROMATOGRAPHY SEPARATION OF LEAF PIGMENTS1

Background
Reading Assignments

Photosynthesis and Plant Pigments

Spinach, like other green plants, contains pigments that make the process of photosynthesis possible. The structures of several spinach pigments are shown in the figure
below (Fig. 1). As you will learn in Chem 202, molecules with a large number of consecutive (conjugated) double bonds tend to absorb light in the visible region of the

Techniques:
Extraction (review): Fessenden2, p. 49-61;
Blackboard (Bb) links
Column Chromatography: Fessenden2, p. 119129, particularly section.11.3; Sapling links

electromagnetic spectrum. The light absorbed by the pigments is converted into

chemical energy. Our eyes see the light that is transmitted. As a result, highly conjugated molecules like the pigments of spinach, are colored (See 15.2C in Loudon for a complete description.)
New Techniques
Column Chromatography. In the previous experiment we
used the analytical method of thin-layer chromatography to determine the purity and identity of the isolated samples of caffeine and aspirin. Column chromatography is another example of a separation technique
used often to isolate and purify compounds from natural product mixtures (as in todays lab) or from a reaction mixture. Chances are great that if you looked in
the workspace of any organic graduate student more
than 50% would have a column running at this very
moment!
Like thin-layer chromatography, column chromatography requires both a mobile and stationary phase.
The polar stationary phase (most commonly silica gel,
SiO2, or alumina, Al2O3) is packed into a tall glass cylinder along with the solvent (the mobile phase). The
sample is dissolved in a minimum amount of solvent
and loaded (carefully) on the top of the column. The
solvent is allowed to flow through the column separating the compounds in the mixture based on their affinity for the solvent versus the adsorbent. When milliFigure 1. The structures of several pigments in spinach: chlorophyll a (blue-

gram amounts of material are being used, the column

green), chlorophyll b (green), pheophytin a and b (grey), carotene (yellow-

can be as small as a Pasteur pipette. This method is

orange) and xanthophyll (yellow).

especially effective when the fractions are colored because the different fractions are easily identified based

solely on visual inspection. More commonly the compounds to be separated are colorless, in which case small fractions are collected and analyzed by TLC.

A m a n d a K a s p e r, P h . D . O r g a n i c L a b o r a t o r y M a n a g e r 1 2 2 5 F r e n c h F a m i l y S c i e n c e C e n t e r

See links on the Sapling site for this course under Lab 7 and Fessenden2 for additional info.
Safety Issues
Standard issues apply for the use of gloves, safety glasses and pipettes (see Lab 1). Take care not to inhale the alumina
(aluminum oxide) as it can cause irritation to the respiratory tract. All of the eluent solvents are highly flammable.

Procedure
1.

In your notebook predict the relative polarity of -carotene versus the chlorophylls. Which one do you expect to
travel through the column the fastest or up the TLC plate the farthest? Why?

2.

Extract the pigments from the spinach leaves. Spinach leaves were pureed in 95% ethanol prior to lab and this
mixture is available in the laboratory. Measure out 5 mL of the spinach mixture with a graduated cylinder and
add 5 mL of cyclohexane. Stir the contents of the graduated cylinder well. The pigments are highly soluble in
cyclohexane. (What does this tell you about their general polarity?) Using a pipette, remove the upper cyclohexane layer and place it in a separatory funnel. Add another 5 mL of cyclohexane to the spinach mash and mix
well. Remove the upper layer of cyclohexane and add it to the first fraction. Discard the remaining spinach mash
in a waste container.

3.

Remove ethanol. Add an equal volume of water to the organic layer and rock the sep funnel gently. If an emulsion forms add 2 mL of saturated NaCl solution (brine) to the mixture and rock the sep funnel gently. Separate
the layers and wash the organic layer with a second portion of water. (Which layer is on top? Bottom?)

4.

Remove water and solvent. Transfer the organic layer into a dry Erlenmeyer flask and add anhydrous Na2SO4.
Vacuum filter the solution into a round bottom flask, then remove the solvent on the rotary evaporator. (Reminder: why did you add Na2SO4?)

!Hint:

While one partner is watching and waiting for the rotovap, the other can begin preparing the

plate for the TLC (Step 5) or getting the column ready (Step 6).
5.

Analyze the spinach extract using thin-layer chromatography. Materials available: Alumina TLC plates, glass
spotters and the eluent system: 60% petroleum (pet.) ether: 16% cyclohexane: 10% ethyl acetate: 10% acetone: 4%
methanol. Draw a picture of the TLC plate in your notebook (be sure to note colors!). Calculate Rf values for each
of the spots. Used TLC plates can go in the standard trash, glass spotters should go in the broken glass bin.
Note: we expect that you may observe the following pigments in the crude extract: Carotenes (1 spot, yellow
orange), phenophytin a (grey, can be as intense as chlorophyll b; might not see this band), phenophytin b (grey;
might not see this band), chlorophyll a (blue-green, more intense than chlorophyll b), chlorophyll b (green) and
the xanthophylls (possibly 3 spots; yellow). You may observe other spots/colors. This is a result of the pigments
undergoing chemical reactions such as air oxidation, hydrolysis, etc.

6.

Prepare the column. A Pasteur pipette is used for the column in this experiment. Add a small piece of cotton to
the pipette and push it into the tip using a small rod. Add a small amount of sand. This cotton and sand keeps
the solid phase from running out of the column. Using a piece of filter paper like a funnel, pour the stationary
phase (alumina) into the column in small portions. Tap the pipet gently to pack the alumina tightly. If there are

D u k e U n i v e r s i t y!

Lab 7: Column Chromatography - Separation of Leaf Pigments

cracks or spaces in the alumina the cyclohexane and pigments will take the path of least resistance and slide
freely down the cracks resulting in no separation. Fill the column to approximately height then add a thin
layer of sand. The sand will prevent the top of the solid phase from being disturbed when the sample and cyclohexane is placed on the column. Carefully clamp the column in place, being careful not to tip it. It should be at a
height just above the base of the stand so that a test tube can rest against the tip and also be easily inserted and
removed.
Slowly add cyclohexane to the column. You may use the house air to push the solvent through the column so
that the alumina is packed as tightly as possible, but be careful not to force the level of the cyclohexane below
the level of packed alumina. This causes cracking, etc. Collect the cyclohexane as it exits the column and add it
to the top until you are satisfied that the packing is sufficient.
7.

Redissolve your spinach extract in a MINIMUM amount (a few drops) of cyclohexane. Allow the cyclohexane
level to drop just to the level of the sand and carefully add ~2 drops of your sample via pipette. Use care not to
disrupt the solid phase for optimum separation the adsorbent should be completely flat! Allow the sample to
drop to the level of the sand then fill the column with cyclohexane.
Note: be sure that your sample has completely diffused into the sand before adding more cyclohexane. If the
cyclohexane is turning green as you add it, it means you are diluting the sample you took such efforts to concentrate.

8.

Continue to add cyclohexane to the column, being careful not to disturb the alumina. Allow a total of ~1-2 mL of
cyclohexane through the column. If necessary, use house air to gently push the cyclohexane through the column.

H
! int: At least one partner should maintain the column at all times to ensure that it doesnt run dry. In
the meantime, the other partner should start Step 9.
9.

Prepare 20 mL total volume of a new mixture of solvents: 75% TLC eluent (60% pet. ether, 16% cyclohexane,
10% ethyl acetate, 10% acetone, 4% methanol) : 25% cyclohexane (volume %:volume %). Add this new solvent
mixture to the top of the column (if necessary, your TA may instruct you on using the house air to carefully push
solvent through the column. This technique is called flash chromatography, and has advantages and disadvantages
over standard gravity chromatography). Again, take care not to force the solvent level below the level of the
alumina. You should see different colored bands moving through. Collect each individual colored band in a clean
test tube. The solvent in between bands should be collected in a separate tube and set aside.

10. Analyze individual fractions (bands) by TLC to determine the success of the column separation.
11. If you did not achieve satisfactory separation of the pigments on the column (at least two visible bands, four
even better) prepare a new column and re-run the experiment 1-2 more times to find the solvent system that effects the best separation.
12. Pay careful attention to washing your glassware at the conclusion of the experiment (Buchner funnel, sep
funnel, etc.). The green chlorophylls can persist throughout the term if the glassware is not washed well today. Use soap and water, and other solvents if necessary (What other solvents might be effective? What have

D u k e U n i v e r s i t y!

Lab 7: Column Chromatography - Separation of Leaf Pigments

you used already to dissolve these compounds?). You can dispose of used columns and alumina (no need to
empty the columns) in the glass waste disposal containers.

Prelab Assignment
Prereading - Be sure to read the sections pertaining to new techniques and chemistry in this lab (see the first page).
Prelab Quiz - You will need to complete Prelab Quiz 7 on Sapling prior to your lab meeting.
Prelab Notebook:
Objective Include main goals of the experiment. Structures not necessary. Refer to this handout or other
source.
Table of Reagents - not required this week.
Separation Scheme - begin with spinach and finish with pure pigments.

In-Lab Notebook
Procedure/Observations: Write directly in your lab notebook what you do, as you do it. Remember the goal is for
someone else to be able to follow the procedure exactly based only on your lab notes. Include specifics for column
and TLC (kind/amount of stationary phase, volumes of each eluent, etc.) and pictures to represent observations
made. Also note any changes made to written procedure. This should be written directly in your notebook as you
complete the lab.
All information above should be written directly into your lab notebook. The duplicate pages will be turned in to your
TA after they have signed them before you leave lab.
All information below will be included on the Post-Lab Report Worksheet or typed Discussion and does NOT have to
be written separately in the notebook.

Post-Laboratory Report
Complete the report as directed on the next pages.

References
a) Roy, C.P., Sebahar, H.L. Organic Chemistry Laboratory Handout, Duke University, 2005, 2008. a) Bell, C.E., Clark, A.K., Taber, D.F., Rodig, O.R., Organic Chemistry Laboratory, 2nd ed., Saunders College Publishing, New York, 1997. b) Quach, Stepper, Griffin J. Chem. Ed. 2004, 81, 385-387. (eluent system for TLC) c) Still, W.C. et al, Journal of
Organic Chemistry, 43, 2923-2925 (1978). This is an excellent reference for flash column chromatography a must read for those of you who are considering doing research in an
organic laboratory. You may access this journal article at the following url: http://pubs.acs.org/cgi-bin/archive.cgi/joceah/1978/43/i14/pdf/jo00408a041.pdf
1

Fessenden, Fessenden, Feist. Organic Laboratory Techniques, 3rd Edition. Brooks/Cole, Pacific Grove, CA, 2001.

Loudon, GM. Organic Chemistry. 5th ed. Roberts & Company Publishers; 2008

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Lab 7: Column Chromatography - Separation of Leaf Pigments

Chemistry 201L - Post Lab Report for Column Chromatography (Lab 7)

Name:__________________________________

TA name/Section number:______________________________

Write out your revised separation scheme for this experiment (begin with spinach and end with pure pigments)

D u k e U n i v e r s i t y!

Lab 7: Column Chromatography - Separation of Leaf Pigments

Table of Results

TLC Analysis of the Crude Spinach Extract

TLC Analysis of Each Band from the Column

Indicate the approximate location, shape and

Indicate the approximate location, shape and

size of each of the spots on the TLC plate.

size of each of the spots on the TLC plate.

In

In

the columns to the right indicate the color and

the columns to the right indicate the color and

Rf value of each of the spots (calculations not

Rf value of each of the spots (calculations not

required).

required).
Color of spots

Rf value

Color of spots

Rf value

1 = band 1
2 = band 2
3 = band 3

!
Column chromatography
Draw a picture of your column and
indicate the number and color of bands
collected from column and the order of
elution:

D u k e U n i v e r s i t y!

Lab 7: Column Chromatography - Separation of Leaf Pigments

Discussion:
Please attach a discussion (2 pages double spaced, 12 point font, 1 inch margins maximum!) addressing the questions
listed below. Consider your results from 1) the TLC analysis of the crude spinach extract, 2) separation of the pigments via column chromatography, and 3) how the results of the column relate to your initial TLC analysis.
Discuss the results of the original TLC experiment on the crude plant extract.
How many compounds were present in the original extract? Make predictions as to which spots
corresponded to the possible pigments (refer back to Figure 1). Do your best to predict which spots
correspond specifically to -carotene and the chlorophylls. Compare their Rf values (observed polarity) to what you predicted. Considering only -carotene and the chlorophylls, how do their differences in polarity relate back to their differences in structure ?
Discuss the results of the column chromatography experiment.
How well were you able to separate the desired compounds? Discuss your visual inspection of the
pigment bands traveling down the column and your TLC results of the individual fractions. Compare these results to your initial TLC analysis of the crude extract. Were you able to isolate all of the
spots you initially visualized? Did you observe mixed fractions? Were the results what you anticipated based on the TLC analysis of the crude extract? Include both the visual inspection of the
bands traveling down the column and the TLC results of the individual fractions. Include a thorough error analysis and changes you would make if you were to repeat the experiment again.

D u k e U n i v e r s i t y!

Lab 7: Column Chromatography - Separation of Leaf Pigments