Report On Lab 6: Photosynthesis: Capturing Energy

Abstract:

The main purpose of conduction the photosynthesis experiment was to carry out paper chromatography in order to determine the pigments involved in photosynthesis. Secondly, the absorption spectrum for chlorophyll was determined in order to determine the wavelengths useful in photosynthesis. Third, the experiment aimed at examining chloroplasts utilizing the Hill reaction. Lastly, the relationship between light, photosynthesis, and chloroplasts was to be investigated. This would help in providing insight into the electron transport and carbon fixation phases of photosynthesis. Paper chromatography was used to separate pigments in plants. Xanthophyll was found to travel the fastest. The absorption spectrum of chloroplasts was plotted and it is evident that the maximum wavelengths are 490 nm and 680 nm. DCIP was used in the experiment to replace the action of NADP+. From the experiment, it is evident that photosynthesis relies heavily on light. Furthermore, there are two stages in the entire process. The data and results obtained from some of the experiments did not match up to what was expected.

Introduction

Photosynthesis is the process by which green plants and bacteria manufacture their own food (Nobel, 34). For photosynthesis to take place, there are a number of conditions that have to be met. There must be carbon dioxide and sunlight. Photosynthesis takes place in the chloroplasts in the plants. These contain a green coloring matter that gives leaves their characteristic color (Alberts, 10). The process of photosynthesis allows a plant to make sugars from carbon dioxide in the presence of sunlight. The process of photosynthesis takes place in two distinctive stages. The first stage, which is light dependent, involves the use of energy of the sun to break down carbon dioxide into sugar and water. This stage is usually known as carbon fixation. During this stage, the ATP and NADPH are produced. The second stage does not need light and results in the production of water. Both ATP and NADPH are utilized in the second stage (Starr, 84)
The aim of the experiment is to measure the pigments in chloroplasts where photosynthesis takes place. In order to identify the pigments that allow photosynthesis to take place, we are going to use a process called chromatography. Chromatography aims at separating the various pigments in a color and analyzing it. Chromatography is generally any technique that can be used to separate mixtures. Colors usually contain a mixture of various colors (Heldt, and Fiona, 166). Chloroplasts, which is the green coloring matter found in leaves is made up of a number of different pigments. Some are more suited to photosynthesis than others are (Blankenship, 30). The aim of this experiment is to separate the various pigments and determine which of these pigments are best suited for photosynthesis. This is achieved since pigments move at different speeds due to their composition. The retention factor can be used as a measure for describing pigments. It is evaluated as the ratio of the distance moved by each pigment to the distance moved by the solvent front. A spectrometer can also be utilized in determining the absorption spectrum of a molecule.

Materials and Methods:

Experiment 1
The aim of the first experiment was analyzing the Chromatogram. This was aimed at utilizing paper chromatography to identify the pigments involved in photosynthesis. The chromatogram from experiment 6.1, a solvent, a chromatography jar, and cover were used for the experiment. A pencil was used to mark the origin on the chromatography paper. The experiment was set up as instructed with the standard on the left side and the sample on the right side. The set up was left to sit and react. Once the experiment was over each visible spot on the chromatogram was outlined using a pencil and its color was noted. A dot was made using a pencil at the center of each observed spot. The distance from the origin to the solvent front was measured and recorded. In addition, the distance from the origin to the pencil dot created earlier was also measured and recorded. The Rf for each identified spot was calculated and its appearance under visible light

Experiment 2

The second experiment was aimed at analyzing the absorption spectrum. For the experiment a spectrophotometer, 80% acetone chlorophyll and extract graph paper were utilized. The spectrophotometer was turned on in order to allow it to warm up. The spectrophotometer cuvette was then filled halfway with the 80% acetone. This was used as the blank for the experiment. A second tube was filled halfway with the diluted chlorophyll extract. The wavelength on the spectrophotometer was set to 400 nm and the first tube was inserted. The machine was then blanked, the tube containing the chlorophyll was inserted, and the absorbance reading was taken. The wavelength was changed to 405 nm and the second absorbance reading was taken. The wavelength was increased by 5 nm and once the reading became low the wavelength was increased by 10 nm or 20 nm. This was done until the wavelength reached 700 nm.

Experiment 3

This experiment was aimed at investigating the hill reaction. For this experiment, the spectrometer and three tubes were used. The three tubes were labeled appropriately. The first tube was labeled as the blank since it contained 0.5mL chloroplast, 3.0 mL cold buffer, 1.5 mL distilled water and 0 mL DCIP. The second tube was labelled as the reaction since it contained 0.5ml chloroplast, 3.0ml cold buffer, 0.5ml distilled water and 1 mL DCIP. The third tube was the control which contained 0 ml chloroplast, 3.0ml cold buffer, 1 ml distilled water and 1 mL DCIP. The contents of the tubes were covered using Parafilm and the contents mixed. An aluminum foil was used to cover the tubes at all times except when they were being measured. Tube 1 was inserted into the spectrophotometer and absorbance was set to 0. The second tube was inserted into the machine and the absorbance was recorded. The tube was covered for 3 minutes and the readings taken again. This was repeated until 12 readings were obtained. The same was done for the control tube and the readings were recorded.

Experiment 4

This experiment is aimed at observing the light dependence of photosynthesis by using DPIP as an indicator. Five accurately labelled test tubes were used in the experiment. The fourth test tube was wrapped in foil. All the five test tubes contained 2.5 ml of chloroplast dilution buffer. The first test tube (the blank) contained 1.3 ml of water and 0.2 ml chroloplast. The second test tube received 0.5ml distlled water, and 1 ml DPIP. The third test tube received 0.3 ml distlled water, 0.2 ml chloroplasts and 1 ml DPIP. The fourth tube received 0.3 ml distilled water, 0.2 ml chloroplasts and 1 ml DPIP. The fifth tube received 0.2 ml chloroplasts, 1 ml DPIP and 0.3 ml sarkosyl. The mixtures in the tube were mixed thoroughly. The tubes were inserted into the spectrophotometer and was set ast 605 nm. The transmittance value for tube 2, 3 and 5 wwas determined and recorded. The foil on tube 4 was removed and it was quickly palced into the machine and the measurements taken. Tube 4 was then wrapped quickly after the readings were taken. Tube 2 to 5 were transferred to a beaker and placed eight inches from the photosynthesis incubator’s light source. The transmittance for each of the tubes was determined after every 5 minutes and recorded.

Results:

Figure 1: Image showing the separated pigments by chromatography

Discussion:

The results for the chromatography experiment indicate that the molecule that travelled the fastest is xanthophyll. From the results, the retention factor for this molecule is equal to 0.68 cm. Its visible color is yellow with a wavelength of between 570 mm and 590mm. On the other hand, the molecule that travelled the slowest is chlorophyll b. The results above indicate that this molecule has a retention factor of 0.3 cm and in visible light; it is green with a wavelength of between 500 nm – 550nm. The results also indicate that polarity affects the rate of diffusion of molecules down a gradient.
Results from the hill experiment indicate that the percentage of DCIP reduced increased with an increase in time the mixture is exposed to light. This is evident since at 0 seconds the amount of DCIP reduced was 0%, however, this value continued to increase with time and at 360 seconds the amount of DCIP reduced was 10.04%. It was also noted that the value of absorbance decreased with time. For the control, it is evident that the % DCIP varied slightly with changes in light.

Works Cited

Alberts, Bruce. Molecular Biology of the Cell. New York: Garland Pub, 1989. Print.
Blankenship, Robert E. Molecular Mechanisms of Photosynthesis. , 2014. Print.
Heldt, Hans-Walter, and Fiona Heldt. Plant Biochemistry. Amsterdam: Elsevier Academic Press, 2005. Print.
Nobel, Park S. Physicochemical and Environmental Plant Physiology. Burlington: Elsevier Science, 1991. Print.
Starr, Cecie, Christine A. Evers, and Lisa Starr. Biology: Today and Tomorrow : with Physiology. Australia: Brooks/Cole Cengage Learning, 2010. Print.