Intro to Molecular, Cellular, and Developmental Biology Labs: Spring 2010

Announcements

Download a PDF version of this page and print it out prior to attending lab.

Overview

The photosynthetic pigments are responsible for absorbing and trapping light energy in the early steps of photosynthesis. Before coming to lab develop a hypothesis relating to pigments and light (i.e. why are there several pigments in green leaves?). What benefits do the plants get by having several pigments? Also, consider what happens to leaves on deciduous trees in the Northern Hemisphere in the fall and pose a hypothesis to explain this phenomenon. Which of the hypotheses developed can be tested using this experiment? Why? Be sure to include these hypotheses in the introduction of your lab report.

The major pigments of photosynthesis are the chlorophylls. The two chlorophylls found in green plants are chlorophyll a (chl a) and chlorophyll b (chl b). Certain other chlorophylls (chlorophyll c and bacteriochlorophylls) are found in non green algae, protistans, and photosynthetic bacteria. Other pigments include carotenoids and phycobilins, sometimes referred to as the accessory pigments. Carotenoids occur in all photosynthetic organisms, while phycobilins occur in the red algae and cyanobacteria.

In this experiment, the photosynthetic pigments from spinach leaves will be extracted and separated using the technique of paper chromatography. After separating the pigments, their absorption spectra will be obtained using a spectrophotometer.

Chlorophylls

The chlorophylls have a similar molecular structure. Each has a porphyrin ring and a long phytol side chain. Although the porphyrin ring resembles the prosthetic group of hemoglobin and cytochrome, it has a central magnesium atom instead of iron. The alternating double and single bonds of the porphyrin ring make chlorophyll an efficient light absorbing molecule and determine the general shape of the absorption spectrum. The phytol chain, which is almost devoid of double bonds, contributes little to the absorption spectrum (view chemical structures). As is the case for other compounds, the specific absorption maxima of any chlorophyll depends on the solvent in which it is dissolved.

Carotenoids

There are two classes of carotenoids, the carotenes and the carotenols. All carotenoids have long isoprenoid chains, with alternating double and single bonds. Structurally, the carotenes are composed entirely of carbon and hydrogen, whereas the carotenols also contain oxygen in the form of hydroxyl or keto groups (view chemical structures).

Paper Chromatography

Chromatography is a technique used to separate the components of a mixture. There are various types of chromatography (column, paper, thin layer, gas), but in all cases the separation is achieved by distribution of components between a fixed or stationary phase and a moving or mobile phase. In paper chromatography, the components of a mixture are separable into discrete zones on a sheet of filter paper.

The mixture is initially spotted or streaked near one end of the paper. If the separated substances are to be extracted later for further analyses, the procedure is called preparative paper chromatography. With a capillary tube, the mixture is streaked on the chromatography paper: enough sample is applied so that there will be an adequate amount for subsequent extraction and spectrophotometric analysis.

For ascending paper chromatography, the appropriate solvent is added to the bottom of a chromatography jar. The atmosphere in the jar should be saturated with solvent vapor prior to adding the paper. The paper is placed in the jar so that the streak is above the level of the solvent. Then, the solvent moves up the paper by capillary action, past the sample, toward the end of the paper.

During this process, termed development, the solutes separate and form a trail of discrete bands on the chromatogram. Separation of the components is usually measured by the R_f value. The R_f value is given by the equation:

	Distance traveled by the solute
Rf =
	Distance traveled by the solvent from the origin

For the numerator, the distance is measured from the origin either to the center or to the leading edge of each spot or band. The denominator is the distance from the origin to the solvent front. The Rf values can be used to identify the various solutes when the experimental conditions are very carefully controlled.

Analysis of Spinach Pigments

The photosynthetic pigments are extracted from spinach by grinding the leaves in acetone. The paper is then streaked with the spinach extract and suspended in a chromatography jar previously equilibrated with vapors of the solvent, a 9:1 mixture of petroleum ether and acetone.

When separation is completed, identify the pigment bands by their colors and relative positions on the chromatogram. The major pigments appear in 5 bands: in order, from the origin to the solvent front, they are chl b(olive green), chl a (blue green), violaxanthin (yellow), lutein (yellow), and β carotene (yellow orange). (Sample chromotagraphic images)

p>Each pigment or pigment group will be eluted from the chromatogram by cutting out each band and soaking the strips of paper in acetone. Violaxanthin and lutein, the carotenols, will be combined and treated as a single group. The absorption spectrum of each pigment or pigment group will then be determined.

You will also perform a quantitative analysis for the two major pigments, chl a and chl b. The absorption coefficients (α) for chl a and chl b in 80% acetone are: for chl a, α₆₆₃ = 82.04, and for chl b, α₆₄₅ = 45.60. Using the Beer-Lambert equation you will determine the concentration (mg/ml) of each chlorophyll in a dilution of the eluted sample. Finally, you will calculate the ratio (chl a)/ (chl b), a value that is characteristic for each plant species.

Beer-Lambert equation

A = αcl

A = absorbance α = absorption coefficient c = concentration l = length of light path (l=1)

Procedure

Preparation of the Chromatography Chamber

The solvent mixture is extremely flammable so that 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. Cut it to 19X19 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. 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 foil covered 150 ml flask, cap, and put on ice.

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 be 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 45o 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.
   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.
3. 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.
4. 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.
2. Label 4 cuvettes as follows: chl b, chl a, carotenols, β carotene.
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 β 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. Prepare a fifth cuvette with a dilute solution of the original extract (0.2 ml of extract + 4.3 ml acetone). Invert twice to mix the solution. Also prepare a reference blank with 100% acetone.
6. 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 5 cuvettes can be read with the one adjustment for the blank at each wavelength. Move the filter lever to the right before you do the 600 nm reading.
7. 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.
8. 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 Excel, plot wavelength (ordinate, x axis) versus absorbance (abscissa, y axis ) for chl b, chl a, carotenols, β 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.
   (chl a) = _____ mg/ml;
   (chl b) = _____ mg/ml.
   Show the calculations in the results.
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?

Lab Report

A typed, complete lab report (introduction, methods, results, and discussion sections) will be submitted no later two weeks after the lab to earn credit for this exercise. Submit this report on paper.

The Introduction section includes sufficient background information to justify your hypotheses addressing absorbance values of various photosynthetic pigments. Use background information from your reading (e.g., textbook) and your everyday experiences to derive a specific hypothesis about photosynthetic pigments. Reading the introduction to the lab should help you focus your thoughts.

The Materials and Methods section should be a very brief description of the extraction of the pigments from spinach, the separation of pigments by chromatography, and the spectrophotometric analysis of the pigments. This section must be written in past tense and should be limited to one paragraph with between six and ten sentences.

The Results section begins with a brief paragraph describing the observed trends in the graph of the spectrophotometric analysis and a report of the relative concentrations of the different chlorophylls. A graph (produced in a spreadsheet program) of the absorbance values (dependent variable) vs. wavelength (independent variable) is presented as part of the results section and the graph is referred to in the text.

The Discussion section is the place where you discuss the reasons why your results support or do not support your hypotheses. If your results support your hypotheses, then explain why. If your results do not support your hypotheses, explain why they don't. You should also compare your results with previously recorded results (from the web or from your textbook). In your comparison with previous results you should attempt to explain any differences observed.

References used to write the report should be listed on a Literature Cited page at the end of the report. Each reference should also be noted in the text where it is used.

Requirements:

1. Were hypotheses presented and derived from sufficient information in the introduction? If so, 20 points.
2. Were all the methods briefly described? If yes, 5 points.
3. Were all the results presented and described? If yes, 25 point.
4. Was one graph of the absorbance values vs. wavelength for all pigments presented? If yes, 20 points.
5. Did the discussion section include the information described above and general answers to the questions presented in the lab handout? If yes, 25 points.
6. Was there at least one reference used other than the lab handout? If yes, 5 points

Note: You might find the guidelines presented in the Westfield State College Writer's Guide helpful as you write your lab report.