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Fluorescence of Chlorophyll

Abstract: Fluorescence occurs when a compound, in this case chlorophyll, absorbs a specific wavelength of light and subsequently emits light at another wavelength. This experiment shows the fluorescent character of chlorophyll when exposed to either UV light or a He-Ne laser. In both cases, the emitted light is in the red region of the visible spectrum. In addition, spectral analysis can be performed using a spectrophotometer to determine the wavelengths of light in the visible spectrum that are absorbed by chlorophyll, revealing the cause of its observed green color. The chlorophyll used in this experiment can be extracted easily from fresh spinach leaves.

Under visible light

Under UV light


mortar and pestle centrifuge test tubes laboratory balance Pasteur pipettes 3-5 spinach leaves He-Ne laser (optional)


ethyl acetate (or ethanol) methanol anhydrous sodium sulfate (optional) alumina (optional)

Safety Hazards:

Ethyl acetate, methanol, and ethanol are all flammable and should be kept away from open flame. They are volatile, release vapor, and should be stored in tightly sealed containers. Avoid prolonged contact and/or inhalation when using any chemical. Refer to the MSDS for these chemicals before proceeding.

Extraction Procedures:

The two options for extracting chlorophyll from spinach leaves are described in Procedure A and Procedure B. Procedure A for dry spinach leaves: 1. Dry 3-5 spinach leaves over night in a dry location at room temperature or in an oven at 45-50 oC for 2-3 hours. Be sure the leaves are completely dry and brittle before continuing. 2. Grind leaves with mortar and pestle in approximately 5-10 mL solvent (enough to cover the leaves). Either ethyl acetate or ethanol can be used as a solvent. 3. Add the leaf mixture to a centrifuge tube. Use additional solvent to rinse the contents from the mortar and pestle into the tube. If more than one tube is needed, be sure to divide the leaf mixture evenly to balance the centrifuge. If only one tube is used, prepare a blank of the same weight to balance the centrifuge. 4. Centrifuge about 5 minutes. The mixture is sufficiently centrifuged when the solid material is a pellet at the bottom and the remaining liquid fraction is green. Discard any solids 5. Using a pasteur pipette, extract the green liquid fraction containing chlorophyll dissolved in ethyl acetate and transfer to a clean, dry test tube. Cap the test tube to prevent evaporation.

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Procedure B for fresh spinach leaves: 1. Use 3-5 fresh spinach leaves. (Do not dry.) 2. Grind the leaves with a mortar and pestle in 5-10 mL of ethyl acetate. (Ethanol is not an appropriate solvent for the extraction of pure chlorophyll, because water is soluble in ethanol.) 3. Add the leaf mixture to a centrifuge tube. Use additional solvent to rinse the contents from the mortar and pestle into the tube. If more than one tube is needed, be sure to divide the leaf mixture evenly to balance the centrifuge. If only one tube is used, prepare a blank of the same weight to balance the centrifuge. 4. Centrifuge about 5 min. 5. Using a pipette, remove the green liquid fraction containing the ethyl acetate and chlorophyll and transfer to a clean, dry centrifuge tube. Discard any solids and the aqueous layer. 6. Add 2-3 mL of water to the tube containing the chlorophyll solution, shake, and re-centrifuge. 7. Remove the lower aqueous layer by pipette and discard. 8. Cap the remaining green liquid, which contains the chlorophyll.

Fluorescence under ultraviolet light:

To observe fluorescence in the ultraviolet (UV) range, use a black light that emits in the 365 nm range. a common black light will suffice. All background lights should be turned off. Bring the test tube containing chlorophyll to within a few centimeters of the black light, and note the color of the chlorophyll extract.

Purification procedure for laser or spectral analysis:

1. Use chlorophyll extracted by either procedure A or B. 2. Evaporate the solvent in a water bath at 40-45 oC in a fume hood until the chlorophyll solution is reduced to about 2 mL. 3. To purify and dry the product, set up a Pasteur pipette column with alumina to extract the carotenes. Place a small piece of glass wool in the tip of the pipette and add the alumina until the test tube is 3/4 full. Tap lightly to pack it. Assemble the apparatus by attaching the upright column to a ring stand and placing a dry, clean test tube below the column for collection. Two to four test tubes will be necessary for a complete collection. 4. First rinse the column with pure ethyl acetate. (Do not allow the solvent level to fall below the top of the alumina during the elution process.) Now add the 2 mL sample. Add ethyl acetate to the column in order to elute the carotenes. The carotenes will appear yellowish green in color. (The carotenes can be saved for use in laser analysis). 5. Once all carotenes are eluted, the green chlorophylls will elute next. These may be easier to elute using a different solvent such as methanol or ethanol. 6. Next, prepare a column with anhydrous sodium sulfate to remove the small amounts of dissolved water. The column is prepared by placing a small amount of cotton in a Pasteur pipette as a plug. Add about 0.5g anhydrous sodium sulfate to the column in a vertical position and tap it lightly to pack the sodium sulfate. 7. Pre-weigh a clean, dry test tube for the collection process. 8. Clamp the column vertically to a ring stand. Place the pre-weighed test tube under the column for collection. 9. Using a Pasteur pipette, transfer the green extract to the column. Rinse the column once with ethyl acetate. 10. After collecting a dried sample, the solvent can be evaporated using the process described in Step 2. Chlorophyll will be a dried residue at the bottom of the test tube. 11. Find the mass of chlorophyll by subtracting the mass of the original clean, dry test tube from the total mass of the test tube containing the chlorophyll. Dilute the dried chlorophyll to a desired concentration, cap, label, and refrigerate. This solution can be used in other experiments.

Laser Analysis (adapted from Zare et al.):

A He-Ne laser or a comparable laser of approximately 633 nm, and a 675 nm cut-off filter are needed for this experiment. Place the laser in line with a cuvette or test tube holder and the filter at a 90 degree angle from the test tube holder. Set up a test tube with carotenes from the extraction described above, and place it in a test tube holder. Observe the test tube through the cut-off filter. No red should be seen. Now replace the carotenes with chlorophyll. If the extract is pure, fluorescence should occur and appear as a red beam within the test tube when viewed through the filter.

Spectral Analysis:For the procedure for spectral analysis click here . Use ethyl acetate or methanol as a blank. Discussion: Chlorophyll is a natural pigment found in green plants.

It is the primary pigment that absorbs light energy from the sun for photosynthesis. This energy is then used by the plant to synthesize glucose from carbon dioxide and water. The structure of the chlorophyll molecule consists of several conjugated nitrogen-containing rings surrounding a magnesium ion by coordinate covalent bonds. Molecules such as this with a metal ion coordinated to an organic compound are called coordination compounds. Coordination compounds are found elsewhere in nature, and generally have distinct spectral characteristics that account for their energy-transfer function in metabolic reactions. This chemical behavior is also evident in the photoresponse of these compounds. For example, hemoglobin changes from a deep purplish red to a bright red upon the binding of oxygen. In the case of chlorophyll, a spectral analysis shows the wavelengths of sunlight absorbed, which is actually the combined absorption of two different chlorophylls, a and b. The maximum absorbance of chlorophyll a is at 420 and 660 nm and the maximum absorbance of chlorophyll b is at 435 and 643 nm. In leaves, chlorophyll is bound to thylakoid membranes in the chloroplasts, and absorbed wavelengths of light are converted to chemical energy. When chlorophyll is extracted from leaves, light energy cannot be transferred to the chloroplasts. Instead, the light is re-emitted and/or absorbed as heat. The emission of light is

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known as fluorescence and occurs between 675 and 685 nm (in the red region of visible light). In the laser analysis, the fluorescence is distinguished from scattered red light by the use of a cut-off filter that allows only this range of wavelengths to pass through. This longer wavelength light has a lower energy, as predicted by the expression: E = hc/, where h is Planck's constant (6.63 x 10-34 J s), c is the speed of light (3 x 108 m/s) and is the wavelength. The actual energy absorbed as heat can be calculated by subtracting the energy emitted from the energy absorbed as ultraviolet light: E400 - E675 = E heat.

Calculations: 1. Energy at 4.00 x 10 -9 m __________J 2. Energy at 6.75 x 10 -9 m __________J 3. Heat absorbed (1-2) __________J

References: 1. Hall, D. O. & Rao, K. K. (1994). Photosynthesis. Cambridge: Cambridge University Press. 2. Pavia, D., Lampman, G, & Kriz, G. (1982). Introduction to Organic Laboratory Techniques . (pp. 368-375). Philadelphia: Saunders College Publishing. 3. Zare, R. N., Spencer, B. H., Springer, D. S. & Jacobson, M. P. (1995). Laser Experiments for Beginners . (pp. 165-68). Sausalito, CA: University Science Books.


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