Plant Pigments and Photosynthesis Lab

Introduction: This lab explores the process of photosynthesis, purpose of plant pigments, and relationship between light and photosynthetic rates.

4A - Plant Pigment Chromatography

Purpose:

This exercise will separate and identify pigments within spinach. This allows us to understand the roles of different pigments.

Procedure & Methods:

1. Obtain a 50 mL graduated cylinder, and fill it 1 cm high with solvent. Keep the cylinder slightly stoppered because the solvent is volatile. Keep the stopper on as much as possible.

2. Cut a piece of filter paper long enough to reach the solvent. Cut one end to a point and draw a pencil line 1.5 cm above the point.

3. Using a coin, extract pigments from spinach leaf cells. Place a small section of the leaf over the pencil line, and use the ribbed edge of the coin to crush the cells onto the line. This should leave a line of pigment on the pencil line. Repeat this procedure 8-10 times, using a new section of the leaf every time.

4. Place the chromatography paper in the cylinder so that the pointed end is barely immersed in the solvent. Do not allow the solvent to touch the pigment line.

5. Stopper cylinder. When solvent is 1 cm from the top of the paper, remove the paper and quickly mark the location of the solvent before it evaporates.

6. Mark the bottom of each pigment band. In Table 4.1 record the distance each pigment and the solvent moved. Depending on the plant species, you may see 4 or 5 distinct pigment bands.

Data:

Analysis and Conclusion:

Chlorophyll a is the primary pigment utilized in photosynthetic reactions. This is the most abundant, along with Chlorophyll b. These two pigments are utilized by plants to synthesize glucose from harnessing light energy. Carotene and Xanthophyll (also known as Carotenoids) are pigments that primarily protect photosystems from light damage and bring captured light energy to the Chlorophyll a. Because Chlorophyll a and b do more of the ‘heavy lifting’ work in photosystems, they are found further down on the chromatography paper as they form greater hydrogen bonds with the paper. Chromatography paper pulls solvent and material in the solvent up through capillary action and the formation of hydrogen bonds. Chlorophyll a and b are able to form more hydrogen bonds with the paper, causing it to move more slowly.

As calculated in the data on Table 4.2, Rf helped us determine which pigments are found in spinach. The higher the Rf value is, the less likely the spinach would be able to absorb that color as demonstrated from Chlorophyll a and Chlorophyll b at Table 4.2. Their Rf values are much higher compared to Carotene and Xanthophyll. Think of it this way: the farther the distance between the solvent and the pigment, the more that they “repel”. In this case, this means that spinach cannot absorb green wavelengths and therefore they reflect it. This is the reason why most plants are green.

4B - Photosynthesis/ The Light Reaction

Purpose:

The purpose of this dye reduction experiment is to gain an understanding of light energy and how different amounts of energy applied to differently manipulated photosystems impacts the rate of photosynthesis.

Procedure & Methods:

1. Turn on the spectrophotometer to warm up the instrument and set the wavelength to 605 nm by adjusting the wavelength control knob.
2. While the spectrophotometer is warming up, your teacher may demonstrate how to prepare a chloroplast suspension from spinach leaves.
3. Set up an incubation area that includes a light, water flask, and test tube rack (see Figure 4.2). The water in the flask acts as a heat sink by absorbing most of the light’s infrared radiation while having little effect on the light’s visible radiation.

4. Your teacher will provide you with two beakers, one containing a solution of boiled chloroplasts and the other containing unboiled chloroplasts. Be sure to keep both beakers on ice at all times.
5. At the top rim, label the cuvettes 1, 2, 3, 4, and 5, respectively. Be sure to follow your teacher's directions on how to label cuvettes. Using lens tissue, wipe the outside walls of each cuvette. Cover the walls and bottom of cuvette 2 with foil to make a foil cap to cover the top. Light should not be permitted inside cuvette 2 because it is a control for this experiment.

6. Refer to table 4.3 to prepare each cuvette. To each cuvette, add 1 mL of phosphate buffer. To cuvette 1, add 4 mL of distilled H2O. To cuvettes 2, 3, and 4, add 3 mL of distilled H2O and 1 mL of DPIP. To cuvette 5, add 3 mL and 3 drops of distilled water, and 1 mL of DPIP.

7. Bring the spectrophotometer to zero by adjusting the amplifier control knob until the meter reads 0% transmittance. Add 3 drops of unboiled chloroplasts to cuvette 1. Cover the top with Parafilm® and invert to mix. Insert cuvette 1 into the sample holder and adjust the instrument to 100% transmittance by adjusting the light-control knob. In other words, you will measure the light transmitted through each of the other tubes as a percentage of the light transmitted through this tube. For each reading, make sure that the cuvettes are inserted into the sample holder so that they face the same way as in the previous reading.
8. Obtain the unboiled chloroplast suspension stir to mix, and transfer 3 drops to cuvette 2. Immediately cover and mix cuvette 2. Then remove it from the foil sleeve and insert into the spectrophotometer’s sample holder, read the % transmittance, and record it as the time 0 reading in Table 4.4. Replace cuvette 2 in the foil sleeve and place it in the incubation test tube rack. Turn on the flood light. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette’s contents just prior to each reading. Remember to use cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.
9. Obtain the unboiled chloroplast suspension stir to mix, and transfer 3 drops to cuvette 3. Immediately cover and mix cuvette 3. Insert into the spectrophotometer’s sample holder, read the % transmittance, and record it in Table 4.4. Place cuvette 3 in the incubation test tube rack next to cuvette 2. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette’s contents just prior to each reading. Remember to use cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.
10. Obtain the unboiled chloroplast suspension stir to mix, and transfer 3 drops to cuvette 4. Immediately cover and mix cuvette 4. Insert into the spectrophotometer’s sample holder, read the % transmittance, and record it in Table 4.4. Place cuvette 4 in the incubation test tube rack next to cuvette 2 and 3. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette’s contents just prior to each reading. Remember to use cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.
11. Cover and mix the contents of cuvette 5. Insert into the spectrophotometer’s sample holder, read the % transmittance, and record it in Table 4.4. Place cuvette 5 in the incubation test tube rack next to cuvette 2, 3, and 4. Take and record additional readings at 5, 10, and 15 minutes. Mix the cuvette’s contents just prior to each reading. Remember to use cuvette 1 occasionally to check and adjust the spectrophotometer to 100% transmittance.

Data:

**The “real” Cuvette 1 was not included in these graphs since it was only supposed to be a baseline for our comparisons. Therefore, Cuvette 2 from the experiment is Cuvette 1 in these graphs. (Cuvette 3 is Cuvette 2 in these graphs, etc.)

Analysis and Conclusion:

Cuvette 1: A Blank Cuvette that has no DPIP to demonstrate that unboiled chloroplasts do not react on their own without an electron acceptor (DPIP). This Cuvette does not contribute to our data, because there is no reaction therefore no significant data to record. There was an initial reading of 80% transmittance and 0.097 absorbance, which serves as a baseline.

Cuvette 2: Contains unboiled chloroplasts which were kept in the dark to demonstrate how chloroplasts function differently in no light. This cuvette represents “Cuvette 1” on the graph, which is, by far, the most flawed item of our data. The graph shows that for cuvette 2, as more time passed, the transmittance rate increased by a drastic amount compared to our other data. On the absorbance graph, it can be seen that as more time passes, the absorbance rate decreases dramatically, as well. The line for both Absorbance and Transmittance should have shown very little change, seeing as chloroplasts should not be able to work effectively with no light. This could be due to the possible accidental exposure to light during transportation from its tin foil cover to the device used to measure our data, resulting in this odd line formation compared to all of our other data.

Cuvette 3: Contains Unboiled chloroplasts which were placed in the light. This was meant to show how a fully functional chloroplast operates in the light, measured by it’s use of the DPIP. When the chloroplasts are exposed to light, the DPIP is reduced by the excited electrons in the chlorophyll, which changes its color from blue to colorless as it accepts electrons. On the absorbance and transmittance graphs, there seems to be a relatively constant rate throughout the whole 15 minutes. The percent transmittance stays at around 67%, with very slight decrease over time. The absorbance stays at around 170, with a very small increase over time.

Cuvette 4: Contains Boiled chloroplasts which are exposed to light. However, it makes no difference if these chloroplasts are in the light or not, they will not work regardless. This is because they have been denatured by a dramatic change in temperature, disabling them entirely. By doing this, they have been rendered unable to do anything your average chloroplast can be seen doing on a Friday night. These chloroplasts have failed to thrive. It appears that, according to the graph, that once again absorbance and transmittance rates stay constant throughout the whole time it was exposed to light. The transmittance rate was lower than it was for cuvette 3, at around 60 % with very small increases throughout the entire 15 minutes the cuvette was in the light. The absorbance is at around 0.219 with very slight decreases as the time went on.

Cuvette 5: This last cuvette contains all “ingredients” except for any chloroplasts. This cuvette is designed to show us how DPIP does not react and change colors on its own. It needs (living) chloroplasts to reduce it so it is able to switch colors, indicating that there was an acceptance of electrons. Once again, the transmittance rate and absorbance rate stayed relatively constant. The transmittance stayed at around 58% with a very small increase throughout the time the cuvette spent under the light. The absorbance rate was at around 0.23 with very small decreases as the 15 minutes went by.