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Effects Of Wavelength And Light Intensity On Photosynthetic Activity

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Effects of Wavelength and Light Intensity on Photosynthetic Activity


The photosynthetic process of eukaryotes revolves around chlorophyll, the substance that give plants their green color. Plants convert light energy into chemical energy by means of photosynthesis. This experiment tests the reaction rates of a chloroplast suspension against variables of wavelengths and light intensity. Both a control and an experimental cuvette were exposed to a range of 450 to 750nm of light and varying intensities to test for reaction rates. These effects on rate were obtained by measuring the absorbance of DCPIP on a spectrophotometer after 16 minutes.

We hypothesize that the least effective wavelength will be that which is reflected back at us in broad day light, this being green light of about 545 nm in wavelength. In regards to intensity, we hypothesize that the most intense source of light will result in the greatest photosynthetic activity based on the resource availability principle. The results clearly demonstrate the ideal conditions in each set of given variables for an optimum reaction rate and also reflect upon the chemical structure of chlorophyll itself.


The goal of this experiment is to test the effects of wavelength and intensity on a solution of photosynthetic chloroplasts.

We tested photosynthetic activity through the absorption of light on 2,6-dichlorophenolindophenol (DCPIP). DCPIP is an artificial electron acceptor; DCPIP serves as the electron acceptor to the light reactions of photosynthesis and will substitute for NADP+. Absorbance can be measured using a spectrophotometer because prior to being reduced by the electrons from the light reactions, DCPIP is blue in color, and as it accepts electrons it becomes colorless. (Vliet, 2006)

The complex photosynthetic process utilized by eukaryotes is one that converts gaseous CO2 into glucose through the use of light energy, water, and adenosine triphosphate (ATP). Photosynthesis takes place inside the chloroplasts of plants and algae, chloroplasts are found in disk-like sacs called thylakoids. Thylakoids are stacked onto one another to form structural columns called grana; the cytoplasm surrounding the grana is called the stroma. The light reactions of photosynthesis take place inside the thylakoids and the dark reactions, or Calvin cycle, take place in the stroma. (Vliet, 2006)

Water absorbed by the plant is hydrolyzed into gaseous O2, H+, and electrons using light energy. These electrons then go on to an electron acceptor that donates electrons to NADP+. The electrons then drive NADP+ and hydrogen protons to form NADPH, the NADPH and ATP generated in the light reactions go on to the Calvin cycle to make carbohydrates. (Vliet, 2006)

On the surface of chlorophyll exist two types of pigments: the chlorophylls, which include chlorophyll a and b, and the accessory pigments, which include the carotenoids and phycobilins.

The chlorophylls are arranged to maximize light absorption, and accessory pigments act like antennas and channel electrons to energy sink molecules. These energy sink molecules have absorbance maxima of 700 and 680 nm. (Larrys, 2005)

Because different photosynthetic pigments specialize in the absorption of certain wavelengths, we hypothesized that the greatest activity will result from the exposure of high intensity blue light for the given constant of time. The purpose of this experiment was to test the effects of wavelength and intensity on reaction rates for photosynthetic activity.


Procedure: Two variables were tested in this experiment, wavelengths ranging from 450 to 750 nm and light intensities at varying distances from a source against absorbance of the DCPIP. Absorbance was measured via a spectrophotometer which was calibrated before each reading using a blank cuvette containing 2.0 ml PO4 buffer, 4.5 ml of de-ionized water, and the 0.2 ml suspended chloroplast solution for a total volume of 6.7 ml.

The control consisted of 2.0 ml of PO4 buffer, 2.0 ml of de-ionized water, 0.2 ml of the chloroplast suspension, and finally 2.5 ml of DCPIP also for a total volume of 6.7 ml. The experimental cuvette contained the same contents as the control cuvette with the same volume of 6.7 ml, all three cuvettes were kept on ice throughout the experiment to prevent damage to the heat-sensitive chloroplasts, the only time the cuvettes weren’t on ice was when they were being measured by the spectrophotometer.

Prior to the addition of the 0.2 ml of chloroplast suspension, all the lights in the room were turned off so that the chloroplasts would not begin to react with other light sources. This meant that every experiment was run at the same time for each variable to prevent excess light from other sources.

Variables: The variable of wavelength was measured at four different values. A source of white light was passed through a colored water medium that allowed only the passing of one part of the light spectrum. The four values were blue light with a wavelength of approximately 450 nm, green light at approximately 545 nm, red light at approximately 650 nm, and lastly far red light at approximately 750 nm.

For the variable of light intensity, beakers were placed at different distances from a white light source to measure the effects of intensity on photosynthetic rate. Intensity of light is given by the following formula IпЂ»1/d2. To maximize light absorption, the cuvettes were placed at the given intensity in a wooden tunnel that trapped the light.

Intensity was measured in пЃ­Eisnteins/m /sec with values ranging from 3 to 35. In both of the experiments, the control cuvette was wrapped in aluminum foil to prevent exposure to any light. Once the lights were all turned off, the foil was taken off for the cuvette to be measure by the spectrophotometer. After the measurements the control was wrapped in foil before the lights came back on. At all times with the exception of when the cuvettes were in the spectrophotometer, all of the cuvettes were kept on ice in a glass beaker to prevent damage to the heat sensitive chlorophyll.

Prior to each reading the spectrophotometer was set to 600 nm and zeroed using the blank cuvette. Readings were taken every two minutes for a total of 16 minutes.

Calculations: The data obtained from both experiments was tabulated and graphs were made comparing the experimental variables in absorbance versus time in minutes. A best fit line was made for all variables



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