Chlorophyll Lab - eweb.furman.edu
Chlorophyll Lab Photosynthesis consists of the light and dark reactions (Calvin Cycle). It is the Calvin Cycle that sequesters CO2 but it is the light reaction which produces the chemical energy to drive the Calvin Cycle. The light reaction also produces the oxygen we breath. The light reaction functions by capturing photons of light and transferring that energy to NADP+ and by producing ATP through a process called chemio-osmosis. Capture of photons involves a series of pigments (proteins complexes) which capture the energy of photons and transfer it from pigment to pigment through Photosystems II then I. Figure 1.The absorption curve for photosynthetic pigments. CLa = chlorophyll a, CLb = chlorophyll b, B-CT = beta carotene, PE= phycoerythrin (red algae), and PC = phycocyanin (bluegreen algae). Bacteriochlorophyll (purple and green sulfur bacteria) has peak absorptions between 300-400 nm and 700-800 nm. Most plants appear green because they reflect green portions of the visible spectrum and absorb the red and blue portions of the spectrum. A plot of absorption as function of wavelength for different pigments is illustrated in Fig. 1. Photosynthetic bacteria absorb wavelengths lower and higher than blue-green algae, algae and higher plants. The bluegreen algae or “cyanophyta” and red algae (mostly marine) utilize pigments in the phycobiliprotein family of pigments which includes allophycocyanin, phycocyanin and phycoerythrin. Phycocyanin is used mostly by bluegreen algae (PC in curve Fig. 1) and has a peak absorption around 600 nm. Phycocyanin’s peak should clearly be identifiable from the peaks of chlorophylls a and b. Chlorophyll a and b are pigments utilized by green algae and higher plants (eukaryotes). Generally speaking, nuisance blooms or outbreaks of noxious algae are blooms of bluegreen algae. Some bluegreen algae outbreaks render water supplies unfit for water filtration plants for domestic water. The purpose of this exercise it to determine the absorption spectrum for the algaes that were grown in the Eutrophication lab. Samples of algae have already been filtered and the pigments extracted in acetone. You will use a cuvette with about 3 ml of acetone as your "blank" (i.e., calibration for 0% Absorbance). Read and record the absorbances at 10-nm (an 5 nm) intervals from 330 nm to 800 nm. How many peaks were there? _____ What were the wavelengths corresponding to the peak(s)? Did the right-most peak indicate a green algae or pigments of a cyanophyta (peak around 600nm)? Fill out the data sheet or linked spreadsheet and then graph the results. Measurement of Absorption Curve of Algal Chlorophyll using Spectronic 20 Genesys Spectrophotometer™ A spectrophotometer measures the percentage of light of particular wavelength passed through a sample (transmittance). Absorbance is the percentage absorbed. When you turn on the Spectronic 20 Genesys™ instrument, it performs its automatic power-on sequence (check to be sure the cell holder is empty and its cover closed before turning on the instrument). This includes a self-check of the software, and initializing the wavelength filter mechanism. The sequence takes about 2 minutes to complete; do not interrupt during this sequence. Allow the instrument to warm up for about 15-20 minutes before you are ready to use it. When your samples and blanks (or calibration tubes) are ready, follow the steps below to operate the instrument. 1. Press 'A/T/C' to select the absorbance mode. The selected mode will appear on the display. (Use A) 2. Press nmˆ(up) or 'nm?(down) to select the initial wavelength of 330 nm specified in your exercise. Note: holding either key will cause the wavelength to change more rapidly than pressing many times. 3. Insert your blank (containing just acetone; a "control') into the cell holder and close the sample door. Be sure to position the cuvette so that the light passes through the clear walls from front to back. 4. Press '0 ABS/100%T' to set the blank to 0% absorbance since the Absorbance Mode was selected in #1 above. 5. Remove your blank and insert your chlorophyll-acetone sample into the cell holder. The sample measurement appears on the LCD display. Record the Absorbance and Wavelength. 6. Raise the wavelength by 10 nm to 340. Insert the black acetone, Press '0 ABS/100%T', insert the chlorophyll sample and record the Absorption and Wavelength. 7. Repeat steo 6, increasing wavelength by 10 nm (or 5 nm) until you have reached 800, recording the absorbance at each wavelength. Remember the sequence of steps: Raise wavelength → insert acetone blank→ Press '0 ABS/100%T' → insert chlorophyll sample, record Absorption. 8. Plot Absorbance of the algal chlorophyll extract as a function of wavelength. It should appear something like this if the sample was derived from a green algae.: Figure 2. Typical Absorbance vs Wavelength for green algae and higher plant pigments. Table 1. Data sheet for recording wavelength and absorbance. Notice that in the shaded areas wavelength increases by 5 nm. Wavelength (nm) 330 340 350 360 380 390 400 405 410 415 420 425 430 435 440 445 450 460 470 480 490 500 510 520 530 540 550 Absorbance 560 570 580 590 600 610 620 630 635 640 645 650 655 660 665 670 675 680 690 700 710 720 730 740 750 760 770 780 790 800
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