Objectives:
1. To determine the pigments responsible for leaf color.
2. To investigate differences in leaf color within and between trees.
I. BACKGROUND MATERIAL
During autumn in Vermont, tree leaves "paint the landscape" with awe-inspiring colors. While some plants exhibit a single shade of color in the fall, such as birch and aspen that have yellow leaves and sumacs that have deep red leaves, other species can have multiple color signatures, such as maples that dazzle with red, orange, and gold, and ashes that show maroon and yellow. The colors of maples and ash, among others, can vary considerably from one locality to another, or even from one leaf to another, depending on the combination of pigments present in the fall leaves.
In this lab you will separate out the pigments in leaves by paper chromatography, then measure the quantity present by spectrophotometry.
Through chromatography, individual pigments are isolated from the many other substances found in living tissues. Once separated, the amount of pigments present can be determined with spectrophotometry, which measures the light absorbed by a given substance.
A. PHOTOSYNTHESIS
Photosynthesis n: A process by which green plants and other organisms produce simple carbohydrates from carbon dioxide and hydrogen, using energy that chlorophyll or other organic cellular pigments absorb from radiant sources.
Photosynthesis is the most important series of chemical reactions on earth. Without photosynthesis, life as we know it would not exist. It is a complex chemical process that converts radiant energy (light) to chemical energy (sugar):
Light + 6 CO2 + 12 H2O à C6H12O6 (sugar) + 6 H2O + 6 O2
B. PIGMENTS
Pigment n: A natural substance in plant or animal tissue that absorbs light and gives the tissue its color)
The chloroplasts (those photosynthesizing organelles) of mature leaves contain several groups of pigments:
Chlorophylls
chlorophyll a – grass green
chlorophyll b – yellow-green
Carotenoids
carotenes – orange/yellow
xanthophylls – pale yellow
Each of these pigments plays a role in photosynthesis. Research suggests that the large amounts of chlorophylls and their intense green color usually hide the presence of the carotenes and xanthophylls.
Most plants regularly destroy and re-synthesize their chlorophyll during their growing season, but as the fall progresses, the rate of chlorophyll synthesis lags behind that of its breakdown. The decreasing amounts of chlorophyll no longer mask the other pigments, and the fall color change begins. (Additional color may also occur due to an increase in production of two other common pigments, anthocyanin or betacyanin. These water-soluble pigments are non-photosynthetic and are not present in the chloroplasts, but instead are localized in vacuoles, especially in epidermal cells.)
Eventually the leaf cells break down and die, and the leaves eventually turn a shade of brown or tan. This browning is due to a reaction between leaf proteins and tannins stored in the cell vacuoles. (This is akin to the tanning of animal hides that produces leather, in which tannins react with proteins.)
C. PAPER CHROMATOGRAPHY
Chromatography n: A method for determining which chemical components a gaseous or liquid mixture contains. It involves passing it through or over a medium that absorbs the different components at different rates.
Separation of a compound from others present in a given tissue can be done with a simple piece of paper!
Pigments have an affinity for paper, and are also easily dissolved in solvents. This technique takes advantage of these two facts. Paper is made up of cellulose, which has many -OH groups present. These groups hydrogen bond to other hydrophilic groups, and as a result many substances (like chlorophyll) hydrogen bond to cellulose. Yet, pigments can be dislodged from their cozy hydrogen bonds if a solvent is present. If we apply a tissue extract of pigments to paper and then wet the paper gradually with a solvent, a pigment molecule can be displaced slightly from its original position. The pigment will migrate over the paper as the solvent flows over it. The other pigments present in the tissue extract also bind to the cellulose, but with different affinities since different types of pigment molecules have different chemical structures, sizes, polarities and solubilities. Therefore, the substances in the mixture separate: some are slightly soluble in solvent and don't migrate very far on the paper, while others are more soluble and migrate farther, separating from each other.
D. Spectrophotometry – Review the workings of the spectrophotometer.
How did you utilize it for the Cellular membrane experiments? Also, remember there is the Spectrophotometry animation.
Use of the spectrophotometer - summary:
Allow spec to warm up for 30 minutes before use.
1. Press A (from A/T/C) for “Absorbance”. Mode appears on display.
2. Press nm D or nm Ñ to select wavelength.
3. Insert cuvette filled with the blank solution into cell holder and close sample door.
4. Press 0 ABS/100% T to set blank to 0A.
5. Remove blank and insert cuvette sample into cell holder. Measurement appears on LCD displayII. Context for the Exercise
Recall the components of the Scientific Process:
The Observations behind this exercise include: Plant leaves are various shades of green while they are photosynthetic but often change to other colors as the autumn season progresses.
This leads to two basic questions: Why are tree leaves green? What changes occur when leaves change color?
You should now try to rephrase these questions as testable Hypotheses, which include an aspect of the mechanism of color production, using the background material discussed above. This might have the form; Plant leaves are green because they contain ….. and Fall leaf color changes are caused by changes in …...
You will use the experimental techniques of paper chromatography and spectrophotometry to test these hypotheses as they relate to specific trees on campus. See methods, section III.
Record your results in self-explanatory, legible tables and graphs. What inferences can you make from your own results or from compiled results?
What you will do in this lab:
Week #1: Selection of research tree, sampling and ESIQ.
You and your partner will select a tree as your test specimen. Using the basic observation, question and hypothesis outlined above, plan a set of experiments to test your hypothesis and predictions concerning why your tree is green.
In the first week you will sample leaves and learn the basics of ESIQ: Extraction, Separation, Identification and Quantification of the pigments responsible for most leaf color.
Week #2: Measuring differences in leaf color.
Plan a set of experiments that address the observation, question and hypothesis concerning why leaf color differs within your tree.
In the second week you must take two separate samples of leaves from your tree.
III. METHODS
1. PIGMENTS
Routine laboratory procedures including paper chromatography and spectrophotometry can be used to (E) extract, (S) separate, (I) identify, and (Q) quantify leaf pigments - ESIQ.
Procedure for ESIQ:
A. Chromatography
1. Each group of students will collect leaves to analyze for pigments.
2. Weigh approximately 5 grams of fresh leaves. Chop the leaves into small pieces and place some in a chilled mortar, add 10 mL of cold acetone, (a *small* pinch of sand may help the grinding process) then grind the leaves with a pestle. The goal is to grind until all color is in the liquid phase, and the pulp is white or colorless. (Why is this important?) Add the remaining chopped leaf , 10 ml more acetone, and grind until all the leaf has been extracted. Caution: acetone is flammable!
3. Pour off the liquid extract from the mortar into a 50 ml graduated cylinder being careful to get as little pulp in the cylinder as possible. Rinse the pulp in the mortar with a small amount of acetone, and add this to the graduated cylinder. Place the extract in an ice bath (Why?) allowing any solids to settle for 5 minutes. Be sure to record the total volume of liquid extract.
4. Draw a light pencil line across the chromatography paper 2 cm from the bottom. Apply the extract carefully and as evenly as possible along the pencil line. Make sure you don’t tear the paper by dragging the pipette tip aggressively. Allow the paper to air-dry before applying each successive sample. The tighter and neater the line of pigment you can make on the paper, the better the chromatography will work!
You should apply 500 to 1000 uL (0.5 to 1.0 ml). RECORD VOLUME APPLIED. The line of green tissue extract is called the origin.
5. Roll the paper (lengthwise) into a tube, hold edges 1/8" apart, and staple. Place the tube in the chromatography tank. The tank contains 50 mL of 10% acetone/90% petroleum ether solvent, which is 1 cm deep. (Solvent must not cover the origin.) Cover tightly.
6. The solvent will travel upward, wetting the paper. This process is called irrigation. Allow the solvent to irrigate until the solvent front is 1-2 cm from the top of the paper (this should take 15 minutes). Remove the chromatogram from the tank, mark the solvent front and let the paper dry.
7. Locate the chlorophyll a and b bands and the xanthophyll and carotene bands. (Note: In this procedure, you have extracted only non-polar photosynthetic pigments. For example, the water-soluble anthocyanin pigments were not extracted and cannot be measured. Fortunately, these usually represent a small percentage of most leaf pigments.)
8. Mark the bottom of each pigment. Measure the distance each pigment migrated from the bottom of the pigment origin to the bottom of the separated pigment band. Record the distance that each front, including the solvent front, moved.
The relationship of the distance moved by a pigment to the distance moved by the solvent is a constant called the Rf value. It can be calculated for each of the pigments using the formula:
Rf = distance pigment migrated (mm)/distance solvent front migrated (mm)
Calculate an Rf value for each pigment extracted.
9. Cut out each band separately and place each band in a vial with the correct labeled top to which 10 mL of acetone has been added. The acetone will dissolve the pigment in about 5 minutes. Remove the strip with forceps. Remember: acetone is flammable!
Note: Some species may contain more than four pigment bands.
B. Spectrophotometer
1. Read and reread the procedure for using the spectrophotometer. IT IS A VERY DELICATE INSTRUMENT,
2. Carefully swirl each of the four vials of acetone with dissolved pigment (after removing the paper strip).
3. Set the wavelength of the spectrophotometer to 400 nm and blank (set the absorbance to zero) with a cuvette of pure acetone.
4. Transfer an aliquot of one of the four well-mixed acetone and pigment solutions to a cuvette and measure the absorbance of each sample at 25 nm intervals from 400 nm to 775 nm. Blank with acetone each time the wavelength is changed. You can measure all four (pigment) samples at each wavelength.
5. Record your data in a table and plot it to produce an absorption spectrum for each pigment. What is the peak absorbance for each pigment? Once you know the peak absorbance for each pigment, you need only measure the absorbance at this wavelength for successive trials.
6. For each pigment, calculate the number of grams that were in 1 gram of leaf material:
g of pigment / g fresh weight of leaf material. Record your results in a table.
C. Calculations
For the calculations you will need the following information:
Pigment Molecular weight Molar extinction coefficient
chlorophyll a = 894 g/mol 89 / mM cm (milimole/L)(cm)
chlorophyll b = 907 g/mol 56 / mM cm
carotene = 536 g/mol 2500 / mM cm
xanthophyll = 568 g/mol 2500 / mM cm
Absorbance, A, = peak absorbance measured. Path length, l, = 1 cm.
1. Start by solving the Beer-Lambert equation for concentration, C, which is expressed as molarity (M), or moles/L.
2. Calculate a value of C (concentration) for each pigment.
3. Multiply this value (C) by the molecular weight for that pigment to convert the units of concentration from molarity, to grams/liter (g/L). Be sure to pay attention to units!
4. This value is the concentration (g/L) of pigment in your cuvette, and thus, the vial you extracted the strip of chromatography paper in. You need to calculate the amount (g) of pigment in that vial. Multiply the concentration by the total volume in the vial (g/L x L = g). This represents the amount of pigment in your vial, which is only a fraction of the total amount of pigment extracted from the leaf. Continue to work backwards to determine the total amount of pigment extracted. For instance, where did the grams of pigment you just calculated come from? It came from the volume of leaf extract you placed on the chromatogram… what can you calculate with values for amount and volume?
5. Eventually you should get the number of grams of pigment in the sample of leaves used. This value needs to be adjusted to express your final value as grams of pigment/ gram fresh weight of leaf material.
IV. THOUGHT QUESTIONS
These questions can be used to gain perspective on this lab.
1. Do your individual results support or fail to support your hypotheses? Do the compiled results support or fail to support the hypotheses?
2. How many grams of cholorphyll a are there per gram of the leaf material you analyzed? How many grams of chlorophyll b? Xanthophyll/ carotene?
3. The leaves of some trees or plants, such as maples and ashes, change to different colors in the fall – sometimes exhibiting different colors on even the same leaf. What do you think controls this apparently haphazard distribution of pigments?
4. Why do different pigments have different Rf values? Would a change in solvent change the Rf value for each pigment? Explain.