1. business and industrial
2. chemicals industry
3. dyes and pigments

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