Introduction:

Why are so many plants green?  Could there possibly be a natural predilection for green pigmentation? To answer this question, consider the part of the plant that is typically green and the function of this part of the plant.  What molecular machinery has nature used throughout the plant kingdom to carry out this role?  This experiment should help you shed some light on this subject!  The purpose of this experiment is to isolate and identify plant pigments. This will be accomplished in two parts. The first step is the separation and isolation of a plant’s pigments using thin layer chromatography. These pigments can then be identified using the chromatographic data in combination with spectroscopic (what is spectroscopy?) data collected on each pigment of interest.

Experimental Details:

Cut a rectangular strip of chromatography paper so that it will stand by leaning against the  inside of the development flask provided. (consider height and width). Ideally it should only touch the side of the flask near the very top while the lid is in place.  Record your measurements since you will likely need to cut many of these strips. Near one of the short edges, make a mark with a pencil (not a pen!) about 2 cm up the side length (not the middle) of the strip to indicate the starting level of the plant pigments on the paper. 

Prepare several micropipets that will be used to spot the plant pigments (in solution) onto the chromatography plate.  Obtain several thin-walled capillary tubes (often used for melting point determinations).  Heat the center of a tube while rotating it lengthwise in a microburner flame.  When the tube is soft, it is pulled apart by holding onto both ends of the tube and drawing them away from each other until a constricted portion is formed measuring about 5 cm in length.  Carefully score the center of the constricted region with a file and break into two micropipets.  One of these two will have a closed end that you will need to open (score and break close to the end).

You are now ready to choose a development solvent.  This will be made up of any combination of the solvents listed in Table 1.  You may use pure solvents or mixtures.  Ideally, you need to choose a solvent system that will allow you to separate all the pigments cleanly from each other based on their rate of travel up the chromatographic plate.  To begin, notice that Table 1 lists solvents based on their relative polarities.  Depending on the polarity of the pigments, they will travel at different rates in the presence of different polarity solvent systems.  Choose at least three different solvent systems to use in your experiment.  If at least one of these does not give a satisfactory result, repeat with more trials until you obtain a plate with some separation of the pigments.  Note that the development process can take as much as an hour a plate so time is of the essence.  Do simultaneous trials.

Obtain a piece of  filter paper for each development chamber that will be used.  If necessary, cut the paper into a large rectangle that can be curled inside the flask without lining the entire circumference of the jar.  You'll need to leave a large enough opening to see the chromatographic plate that will be standing inside the flask.  Record the measurements.  Fill the bottom of the flask with the chosen solvent system so that the filter paper is completely moistened with solvent and the remaining solvent stands about 1 cm above the bottom of the flask.  Cover the flask with the lid.

You will be provided with 4 mL of spinach juice that was obtained using a juice extractor.  The extracted juice was filtered once to remove bulk pulp.  Divide your spinach juice into two equal volumes and place them into two 10-12 ml capacity centrifuge tubes that have stoppers.  Add about 0.5 mL of methanol, 3.5 mL of pentane, and 0.5 mL of deionized water to each tube.  Stopper and shake the resulting solutions vigorously for 1 minute.  Immediately spin the samples in a centrifuge for two minutes at medium speed.  Be certain to balance out the samples in the centrifuge!  The resulting transparent green top layer can now be transferred to a clean test tube using a Pasteur pipet .  This top layer contains the pigments which are now isolated in pentane solvent.   Concentrate the pigments (evaporate most of the solvent) to about a half a milliter using a hot water bath in the hood.  Remember to avoid ambient light and overheating as much as possible.  If the sample happens to dry out completely, just add some more pentane to redissolve. 

Spot the green pigment solution onto the chromatographic plate in as small a spot as possible at the level previously marked with a pencil.  Dip an empty micropipet into the green solution and notice how the solution rises inside the tip by capillary action.  Now, try to barely touch the tip of the micropipet to the chromatographic plate briefly in the marked location.   Make as small a spot radius as possible for the best results.  Allow the spot to dry; blowing on it should speed the drying process.  Reapply more solution to the exact spot in the same manner and dry the spot again.  Repeat this process about 3-5 times until a reasonably large quantity (intensity of color) of material is spotted in a small dot on the plate.

Once you have an adequate amount of dried sample applied to the chromatographic plate, insert the plate into the development chamber being careful not to let the solvent slosh around or the front of the plate stick to any of the walls of the flask or filter paper.   You should stand the plate inside the flask so that you can see the spot on the plate easily.  It is crucial that the spotted  plant pigments do not touch the solvent in the bottom of the flask directly or this plate will be ruined and unusable.

The liquid should now begin to rise up onto the plate. The solvent will continue to creep up towards the top over time. Record observations as this movement progresses. What happens to the pigments? Once the solvent fails to rise any further up the plate or if it comes within a centimeter of the top, remove the plate from the flask. Immediately mark the highest point the solvent has risen on the paper with a pencil. Make a scaled drawing of the results including distances traveled  and spot shapes for each different pigment observed and for the solvent front.  If you have time, you may wish to repeat the "successful" trials.

Green plants typically contain pigments called chlorophylls, which gives them their green color. Normally, you would want to include standard samples of pure chlorophylls to compare with your sample.  It is a good idea to run the standard and the unknown sample side by side on the same plate to make a direct comparison.  Unfortunately, you will not have standards available, but there is another way to identify your pigments.

Further identification of the pigments can be obtained by visible spectroscopy since the pigments are colored.  The spectra that you obtain can be compared to literature data to make an identification.  Prepare solutions of the isolated pigments, particularly the green pigments which are likely chlorophylls, from your chromatography experiments by the following method.  Cut out the pigment (spot) from your chromatography experiment using scissors and soak it in 3 mL of methanol solvent to extract the pigment into solution from the plate.  A capped sample vial works great for this purpose.  (Note:  depending on the type of pigment isolated, you may need to choose a different solvent to extract the pigment into solution.)  Remember that the pigments may degrade once you remove them from the development chamber so you will have to carry out this process quickly.  Obtain copies of the visible spectra of these pigments using an appropriate spectrometer (select here to find out how to operate this instrument) and report your findings.  Be sure to collect data on the green pigments first, and then on any other pigments you would like to explore as time permits.

Food for thought:  From your chromatographic and spectroscopic data, can you identify the pigments in your plant’s leaves?  What else besides chlorophyll might you expect to find? What is the chemical structure of chlorophyll?  Is it possible that there is more than one type of chlorophyll?  How does the chemical structure relate to its function in green plants?  What happens to the color of the pigments over time as they are exposed to the surroundings?  Why?

Something else to explore:    What happens if you expose the chromatographic plate to long-wave ultraviolet light in a darkened room?  Try shining a flashlight on it and see what happens.   Can you explain it?   How might this be useful?    How does this relate to the function of this compound in green plants?

Created by S. Phillips on 9/10/98