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Duke University - Organic Chemistry Laboratory

Lab

COLUMN CHROMATOGRAPHY SEPARATION OF LEAF PIGMENTS1

Background
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 on the next page (Fig. 1). As you will learn in Chem 152, molecules with a large number of consecutive (conjugated) double bonds tend to absorb light in the visible region of the 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 the organic graduate students upstairs more than 50% would have a column going 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, dissolved in a minimum amount of solvent, is loaded (carefully) to the top of the column. The solvent is allowed to ow through the column separating the compounds in the mixture based on their afnity for the solvent versus the adsorbent. When milligram
Figure 1. The structures of several pigments in spinach: chlorophyll a (bluegreen), chlorophyll b (green), pheophytin a and b (grey), carotene (yelloworange) and xanthophyll (yellow).

Reading Assignments Techniques:

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

amounts of material are being used, the column can be as small as a Pasteur pipette. This method is especially effective when the fractions are colored because the different fractions are easily identied 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.

James A. Parise, Ph.D. Organic Laboratory Manager 1225 French Family Science Center

See links on the Blackboard (Bb) site for this course under Laboratory > 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. The eluent solvents are each highly ammable.

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. Place 5 mL (measure with graduated cylinder) of the spinach mixture in a test tube and add 5 mL of cyclohexane. Stopper and shake the test tube 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 shake well. Remove the upper layer of cyclohexane and add it to the rst 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 to the mixture and rock the sep funnel gently. Separate the layers and wash the organic layer with a second portion of water. 4. Remove water and solvent. Transfer the organic layer into a dry Erlenmeyer ask and add anhydrous Na2SO4. Vacuum lter the solution into a round bottom ask, then remove the solvent on the rotary evaporator.

! int: H
5.

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). Analyze the spinach extract using thin-layer chromatography. Materials available: Alumina TLC plates, glass spotters and the eluent system: 60% petroleum 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. 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 weigh boat or piece of lter 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 cracks or spaces in the alumina the cyclohexane and pigments will take the path of least
D u k e U n i v e r s i t y! Lab 7: Column Chromatography - Separation of Leaf Pigments

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 satised that the packing is sufcient. 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 at! Allow the sample to drop to the level of the sand then ll 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.

Hint: 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, 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 ash 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 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 nd 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 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.

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

Lab 7: Column Chromatography - Separation of Leaf Pigments

Prelab Assignment
Prereading - Be sure to read the sections pertaining to new techniques and chemistry in this lab (see the rst 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 nish 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 specics 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, Grifn 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 ash 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 2 3

Fessenden, Fessenden, Feist. Organic Laboratory Techniques, 3rd Edition. Brooks/Cole, Pacic Grove, CA, 2001. Loudon, GM. Organic Chemistry. 5th ed. Roberts & Company Publishers; 2008

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

Lab 7: Column Chromatography - Separation of Leaf Pigments

Chemistry 151L - 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 Indicate the approximate location, shape and size of each of the spots on the TLC plate. In the columns to the right indicate the color and Rf value of each of the spots (calculations not required). Color of spots Rf value TLC Analysis of Each Band from the Column Indicate the approximate location, shape and size of each of the spots on the TLC plate. In the columns to the right indicate the color and Rf value of each of the spots (calculations not required). 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!) answering the following questions. Label your discussion in two parts: Part 1: Results of TLC Experiment on Crude Sample: ! Discuss the results of the original TLC experiment on the crude sample. What did you learn from the TLC results? How many compounds were present in the original extract? Do your best to predict which spots correspond to -carotene and the chlorophylls. Compare the observed polarity of these pigments (based on your calculated Rf values) to your predictions based on the structures. Discuss the differences in the structures and how these differences may affect their relative polarities. Part 2: Results of Column Chromatography and Pure Pigments: ! Discuss the results of the column chromatography experiment. To what extent were the pigments separated? How do you know? Include both the visual inspection of the bands traveling down the column and the TLC results of the individual fractions. Discuss the different solvent systems used and which effected the best separation (if applicable). 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