Introduction
The purpose of this lab was to see separate and identify the pigments of spinach cells through the use of paper chromatography. Through this lab, one can understand the rate of photosynthesis and absorbency rate of chloroplasts in different light intensities.
Background:
Paper chromatography is used to separate and to identify the mixtures of pigments and other molecules of a substance. In chromatography the solvent moves up a piece of paper by capillary action, which occurs because of the attraction of the solvent molecules to each other and the thin paper. As the solvents moves up the paper it separates the pigments into its components. The substances that are seen on the paper are plant pigments. Plant pigments are chemical compounds that absorb certain wavelengths of light while reflecting others. Some of the light absorbed will be used for chemical reactions while the other reflected wavelengths will determine the color the pigment will be appear to the eye.
Chlorophylls are greenish pigments that have a special structure to provide energized electrons to other molecules. There are several types of chlorophyll but the most important one if chlorophyll a. Chlorophyll a passes its energized electrons on to molecules that will manufacture sugars and other carbohydrates. All plants that photosynthesize contains chlorophyll a, while, chlorophyll b is a second kind of chlorophyll that occurs only in green algae plants. Accessory pigments absorb light energy and transfers energy to chlorophyll a during photosynthesis. Two accessory pigments are carotenes and xanthophylls. Carotene is the most soluble pigment because it makes no hydrogen bonds with cellulose, a major component of cell walls, so this pigment will be “carried†furthest by the solvent. Xanthophyll isn’t carried as far as carotene because it is less soluble and is slowed down by the hydrogen bonding to cellulose. Using the formula, R(f)= distance pigment migrate (mm) / distance solvent front migrated (mm), one can determine the distance moved by the pigment to the distance moved by the solvent. Pigments capture the light energy needed for photosynthesis.
Photosynthesis is a process that uses the energy from sunlight and converts the carbon dioxide into organic compounds, like sugars. The equation for photosynthesis is 6CO2 + 6H2O (with light energy) –> C6H12O6 + 6O2 where we see that carbon dioxide with water and light energy makes carbohydrates and oxygen. There are two parts in photosynthesis the light dependent reactions and the dark reactions also known as the Calvin cycle. Light reaction occurs in the thykaloid membranes of chloroplasts and converts light energy into chemical energy. Chlorophyll and beta- carotene are all in the thylakoid membranes and involved in the light reaction. Each of the pigment passes the energy they absorb from light to the chlorophyll molecule to do photosynthesis. In the light dependent process, light energy is absorbed and the electrons are boosted to a higher energy level. After a series of reactions, the energy is then concerted along an electron transport process and ATP and NADPH is produced. In this process water serves as a by-product of the reactions. The ATP and NADPH are used in the dark reactions as energy for carbon fixation, a process where carbon dioxide is converted into carbohydrates and sugars. This process occurs in the stroma of chloroplasts. RuBP captures six molecules of carbon dioxide and eventually produces one molecule of glucose. ATP and NADPH are made in light reactions and are used in the dark reactions, after use it returns to light reactions as NADP+ and ADP which doesn’t contain much energy.
In this lab, a spectrophotometer was used to measure the level of light transmitted in the chloroplasts of spinach leaves. Chloroplasts from spinach leaves were taken and put into tubes so one can understand the rate of photosynthesis in spinach cells at different light intensities. In this lab, a dye reduction technique was used. A solution called DPIP (2, 6-dichlorophenol-indophenol) was used in place of the NADP. Since DPIP has a dark blue color, DPIP was used during the lab to show that photosynthesis was occurring in the cuvettes. A cuvette is like a small test tube that fits perfectly into the colorimeter. The colorimeter is a lab material that measures the amount of light absorbed by the solution. When the light shines on the chloroplasts, the light energy will boost the electrons to higher energy levels and will reduce DPIP. A reduction of DPIP will change the color in the tubes from blue to colorless so one can judge the color change of the chloroplast solutions
Procedure:
First we turned on the flood lamp already prepared for us, and placed a 600mL beaker of water in front of it to protect the chloroplasts from being warmed up by the flood lamp. This was done to prepare for the second part of the lab. Then we prepared for the first part of the lab. First we obtained a piece of filter paper, spinach leaf, and a penny. Then we obtained a vial with 1 cm of solvent in the bottom. We made sure the filter paper fit into the vial and cut a point at one end of the paper. We used pencil to draw a line 1.5 cm from the point just cut. Then we placed the spinach leaf on top of the filter paper and started to crush the cell pigments onto the filter paper just above the pencil line using that penny. We did this step several times using different spots of the leaf each time. Then we placed the chromatography paper in the cylinder and made sure the point of it is barely in the solvent. Then we sealed the lid of the cylinder and waited for the solvent to reach about 1 cm from the top of the filter paper. We removed the paper and quickly marked the location of the solvent front. Then we measured the distance each pigment migrated front the point of the pigment origin to the bottom of the separated pigment band. Then we recorded the distance each front moved and recorded our data. After we finished this part of the lab, we prepared for the second part of the lab.
First we connected the Colorimeter with the computer interface. Then we obtained 4 cuvettes and their lids. We marked one (BL) for blank, one (U) for unboiled, one (D) for dark and one (B) for boiled. The blank cuvette was used as the control and no DPIP was added. The cuvette marked (U) was to see the absorbance of light with unboiled chloroplasts. The (B) was to see the absorbance of light with boiled chloroplasts. The (D) is for unboiled chloroplasts in the absence of light. We covered all four sides and bottoms of the (D) cuvette with aluminum foil. Then we added the phosphate buffer, distilled H2O, and DPIP to each cuvette. In the blank cuvette we dropped 1 mL of phosphate buffer, 4 mL of distilled water, and 3 drops of unboiled chloroplasts. Then in the (U) cuvette we added 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of unboiled chloroplasts. In the dark cuvette, we added 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of unboiled chloroplasts. Finally in the (B) cuvette, we added 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP and 3 drops of boiled chloroplasts. After this we calibrated the colorimeter. We made sure the outside of the cuvettes were clean and free of fingerprints. Then we opened the lid of the colorimeter and placed the blank cuvette inside. (The calibrate part was done by the teacher). After we calibrated the colorimeter, we placed all three of the cuvettes in front of the beaker and light. After waiting for 5 minutes we removed the cuvettes from the light and put the (U) cuvette in the colorimeter. After we closed the lid, we waited for 10 seconds and recorded the absorbance value. When it was done calculating we placed the cuvette in front of the light and beaker. Then we did the same thing with the (D) cuvette and (B) cuvette right after. Then we waited for another 5 minutes, we did the same thing and put each cuvette in the colorimeter. After recording the data we placed them in front of the light again. After another 5 minutes removed the cuvettes from the light and took turns placing them in the colorimeter. When the data was recorded we placed them in front of the light for the final trial. When time was over we placed them into the colorimeter and recorded the results. When we finished the lab, we cleaned up and poured the cuvettes containing DPIP into a special beaker and poured the ones without down the drain. We turned off the computer interface and colorimeter and wiped the tables.
Hypothesis:
I predict that the dark cuvette (D) and the cuvette with the boiled chloroplasts in the light (B) will have slight changes in the reduction of DPIP; while, the cuvette with unboiled chloroplasts in the light (U) will have the highest reduction of DPIP.
Conclusion and Error Analysis:
My results supported my hypothesis. I predicted that the dark and the cuvette with the boiled chloroplasts in the light will have slightly changes in DPIP and that the cuvette with the unboiled chloroplasts will reduce more DPIP.
Everything that occurs during photosynthesis needs the energy from light, even the dark reactions because the dark reactions need the products of the light reactions to properly function. In the dark cuvette, though the chloroplasts are unboiled, the cuvette was completely covered in aluminum foil. The foil serves as a barrier between the light and chloroplasts. So, the chloroplasts could not properly absorb the light. Therefore, the DPIP was only slightly changed. The boiled chloroplasts did not reduce much DPIP because when the chloroplasts were boiled, it denatures the chloroplasts along with the enzymes within them so they could no longer properly function. DPIP is reduced when light reaches the chloroplasts and the enzymes are boosted to a higher level of energy. But the chloroplasts and the enzymes are already non-functional so even if light were to reach it, it could no longer perform photosynthesis. The cuvette with the unboiled chloroplasts in the light had the highest reduction of DPIP because this cuvette has the proper conditions for photosynthesis to occur. Unlike the other cuvettes, this one has light and the chloroplasts were not denatured from boiling. When the light reached inside the chloroplasts the enzymes inside was also boosted to a higher level of energy. DPIP is normally blue but when it is reduced, or when more electrons are gained it turns colorless. So as more and more electrons were boosted and more electrons were gained, the DPIP was slowly reduced along with its color. Many sources of errors could have occurred but one of them was that the cuvettes were probably not handled well before they were inserted into the colorimeter. We did not handle the cuvettes only by the top edge of the ribbed sides but we touched the 4 sides of the cuvette as well. This probably left a lot of fingerprints that could have blocked the light to enter the chloroplasts at its potential and cause the colorimeter to miscalculate the amount of DPIP reduced. When placing the 3 cuvettes in front of the light we did not remember the positions were placed them in. After each absorbance reading for the cuvettes, the cuvettes were placed in a different spot in front of the light. This could have caused more light to enter one part and less to enter another causing our data and results to not be a sufficient as they could have been. A source of error that occurred during the first part of the lab was we did not rub enough spinach pigments onto the filter paper so we did not have that many pigment bands. I feel like we did carry enough trials for our experiment to have accurate results. But maybe next time have more time to see if more DPIP will be reduced with more time under the light. To make the experiment more scientifically sound, one could avoid careless errors that we did like leaving fingerprints and placing them in different spots of the light with wearing gloves and marking where the cuvettes were placed each time. Some further experiments could be done to expand my knowledge could be to see how photosynthesis occurs in photoautotroph or algae. This would be interesting because instead of seeing how photosynthesis works in chloroplasts we could see how photosynthesis works in animals that can provide its own food.
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