Preparation of the Chromatography Chamber

The solvent mixture is extremely flammable; the chromatography should be carried out in a fume hood. Before handling the paper, hands should be washed and thoroughly dried. The paper should be handled as little as possible and only on the edges.

1. Obtain a piece of chromatography paper (18 cm X 18 cm).
2. Add the solvent mixture to the chromatography chamber at least 30 min. before adding the streaked chromatography paper so that the atmosphere in the chamber will become saturated with solvent vapor.
3. Add freshly prepared solvent (9 vols. petroleum ether *:1 vol. acetone, mixed well) to a height of approximately 2 cm and cover the chamber with a lid that is sealed at the rim with Vaseline. Be careful not to get any solvent on the Vaseline.

* CAUTION: Petroleum ether and acetone are extremely flammable and should be kept away from heat, sparks, or an open flame.

Preparation of the leaf extract for the whole class

1. Weigh out 15 g of fresh spinach leaves after removing major veins. Cut into small pieces.
2. Place into blender; add 60 ml 100% acetone and homogenize.
3. Filter first through cheese cloth and then through Whatman filter paper in a Buchner filter under vacuum.
4. Place filtrate in a sealed container and store on ice until ready to use.

Preparative Paper Chromatography

1. With a pencil and ruler, draw a light line across the width of the chromatography paper, about 3 cm from the bottom. This will insure that the extract, which will be streaked on this line, is not immersed in the solvent. Make a light mark on each end of the line about 1 cm from the edge of the paper.
2. Using a capillary tube, make 10 streak applications of the pigment extract along the line between the two marks. Do not let the pigment touch the edge of the paper. The capillary tube is filled by immersing the tip in the extract. Hold the tube at a 45^o angle and draw it along the pencil line. Move your arm quickly so the pigment does not form a large spot at the beginning of each streak.
3. Streak each successive application in the direction opposite of the preceding one to insure an even line at the end of the ten applications. Allow each application to air dry before making the next. The final thickness of the streak should be no more than 6-7 mm.
4. Use two paper clips and thread to place the streaked chromatography paper in the equilibrated chromatography chamber, being careful not to get any Vaseline on the paper. Allow the chromatogram to develop in the dark or in very dim light for 45-60 min. or until there is clean separation of the 5 bands. Stop the development before the solvent front reaches the end of the paper.
5. Remove the chromatogram from the chamber and put a pencil mark at the leading edge of the solvent front before it dries. Hang it in the hood to dry. Record the distance traveled by the solvent front and by the leading edge of each band. Also record the color of each band and what pigment it contains. This must be done before elution.

Elution and Spectrophotmetry

1. Allow the spectrophotometer to warm up for at least 5 min. Set the wavelength at 400 nm. (See the Spectrophotometry Guide for information on how to use the Spectrophotometer.)
2. Label 5 cuvettes as follows: chl b, chl a, carotenols, b-carotene, and blank.
3. Cut out each of the 5 bands on the chromatogram; cut them into thin strips and place them in the appropriate cuvette (cut them long enough so you can remove them from the cuvettes but not so long that they extend out of the acetone); place the two carotenol bands, violaxanthin and lutein, in the same cuvette. Thus, you will be eluting 4 pigments or pigment groups: chl b, chl a, carotenols, and b-carotene.
4. Add 5.0 ml acetone to each cuvette, cover each tube with parafilm, and allow the pigments to elute for 5 min., occasionally swirling each tube. Invert each cuvette twice to mix the contents, then remove the paper strips with forceps, draining the paper against the side of the tube.
5. Measure the absorbance of each sample at 20 nm intervals starting at 400 nm and proceeding to 700 nm. The spectrophotometer must be adjusted with the reference blank prior to the absorbance readings at each wavelength. All 4 cuvettes can be read with the one adjustment for the blank at each wavelength. The filter in the spectrophotometers must be changed for the 600 nm reading.
6. To determine the concentration of chl a and chl b, dilute the solvent to 80% acetone as follows:

   Transfer 4 ml of the chl a eluate and 4 ml of the chl b eluate to two clean cuvettes. To each add 1 ml distilled water, cover, and invert each tube twice to mix the contents. Also prepare a reference blank with 80% acetone (4 ml acetone and 1 ml distilled water).

7. Measure the absorbance of the chl a solution (in 80% acetone) at 663 nm and the absorbance of the chl b solution (in 80% acetone) at 645 nm. Be sure to adjust with the reference blank at each wavelength.

RESULTS AND DISCUSSION

The following should be included in the results and/or discussion of the lab report. Incorporate them in the appropriate section. Do not write the results and discussion as a list of answers to the questions.

1. Calculate the R_f value for each pigment.
2. What do the R_f values indicate about the relative solubilities of the pigments in the solvent? What would be the order of solubility if the pigments were separated in water instead of the nonpolar solvent mixture (petroleum ether and acetone).
3. Explain the relative solubilities of chl b and chl a in the solvent on the basis of their molecular structures.
4. Explain the relative solubilities of the three carotenoids in the solvent on the basis of their molecular structures.
5. Using a spreadsheet program, plot wavelength (ordinate, x axis) versus absorbance (abscissa, y axis ) for chl b, chl a, carotenols, b-carotene, and the diluted extract. Include a title, axes labels, and legends. Place all pigment values on the same graph. When printing the graph choose the landscape choice for paper orientation and make the graph fill 75% of the page.
6. What is the wavelength of the absorption maximum (or maxima) for each pigment or pigment group? Compare your results to references.
7. Why does a plant use several pigments instead of one or two? Why are plant leaves green?
8. From the absorbance readings at 663 nm and 645 nm and the Beer-Lambert equation, determine the concentration of chl a and chl b in the diluted eluates.
9. What is the ratio (chl a)/(chl b) in spinach?
10. Do the results support the hypothesis in the lab introduction? Why or why not?