Why do different pigments travel at different rates during plant pigment chromatography?

Different pigments have varying solubility in the chromatography solvent, causing them to travel at different rates up the paper. Pigments that dissolve more readily in the solvent move faster and travel farther, while less soluble pigments move slower. This separation allows you to identify individual pigments by calculating their retention factor (Rf) values and comparing them to literature standards.

What does the retention factor tell you about a pigment in chromatography?

The retention factor (Rf) is calculated by dividing the distance a pigment traveled by the distance the solvent traveled. This ratio is consistent for each pigment under the same conditions, allowing you to identify unknown pigments by comparing your calculated Rf values to reference values in literature tables. Higher Rf values indicate pigments that are more soluble in the solvent.

How does vacuum infiltration help measure photosynthesis in leaf discs?

Vacuum infiltration removes air from leaf tissue spaces, allowing the discs to sink initially. During photosynthesis, oxygen produced by the leaves fills these air spaces, causing the discs to float. By counting floating discs over time, you indirectly measure the rate of photosynthesis. This method also allows observation of cellular respiration quantification using microrespirometers procedure when comparing light and dark conditions.

Why do leaf discs sink in the dark during the floating disc experiment?

In darkness, photosynthesis stops and leaf cells perform cellular respiration, consuming oxygen from the air spaces in leaf tissue. As oxygen is depleted, the discs become denser and sink. This demonstrates that photosynthesis, not respiration alone, produces the oxygen that causes discs to float in light conditions.

What is the purpose of using bicarbonate solution versus water in the leaf disc experiment?

Bicarbonate solution provides additional carbon dioxide to fuel photosynthesis, increasing the rate at which leaf discs produce oxygen and float. The water control lacks this extra CO2 source, allowing you to compare photosynthetic rates between the two treatments. This comparison demonstrates how substrate availability affects the rate of photosynthesis.

What does ET50 represent in the leaf disc floating experiment?

ET50 is the effective time at which 50% of leaf discs are floating. By determining when this threshold occurs in both light and dark conditions, you can quantify the rate of photosynthesis and respiration. Comparing ET50 values between bicarbonate and water treatments reveals how substrate availability influences photosynthetic rate.

How do you prepare chromatography paper to separate spinach leaf pigments?

Draw a pencil line two centimeters from the paper's bottom, then roll a fresh spinach leaf over this line using steady pressure with a coin. This transfers pigment onto the paper as a concentrated band. The paper is then suspended in chromatography solvent, which travels up the paper and carries pigments at different rates, creating separated bands of distinct colors.

Photosynthesis

Concepts Instructor Prep Student Protocol

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Have you ever wondered why every autumn leaves on trees change from green to shades of yellow, orange, and red? What gives leaves their bright colors in the first place? The answer lies in the plant organelles called chloroplasts which contain pigments that absorb certain wavelengths in sunlight and reflect others. One particular pigment, chlorophyll, is the most abundant in summer. It absorbs high-energy, purple, blue, and red wavelengths from sunlight, and reflects green wavelengths giving leaves their green appearance. There are other pigments in leaves, such as carotenoids, which reflect red and yellow light. In autumn the leaves stop replenishing their pigments. Since chlorophyll degrades faster than the other pigments, the colors of these carotenoids are unmasked.

The presence of different pigments in a green leaf can be demonstrated with chromatography paper, a hydrophilic polymer that separates molecules based on their solubility in a particular solvent. First, leaf extract is loaded onto the paper. When the paper is dipped into an organic hydrophobic solvent, the solvent travels along the paper due to capillary action and along the way it separates different pigments in the leaf extract. The pigments that are the most hydrophobic are carried further up the paper. Whereas hydrophilic pigments bind to cellulose which hinders their movement. After all of the pigments are sorted by their hydrophobicity, we can calculate the retention factor or Rf values. The Rf value is the ratio of the distance traveled by a pigment to the distance traveled by the solvent. Each pigment has a unique Rf value and we can easily identify pigments by comparing calculated values to standards. Photosynthesis, the process by which plants convert carbon dioxide, water and light energy into chemical energy and oxygen, is carried out primarily in the leaves of a plant, and chlorophyll plays a critical role in this process. Chloroplasts contain dozens of chlorophyll molecules, each performing a specific task and interacting in complex ways. Ultimately, light energy causes chlorophyll molecules to give up electrons which are utilized in other metabolic processes. Therefore, chlorophyll needs a continuous supply of electrons to replace the ones it loses. These replacement electrons come from splitting water molecules into protons, electrons and oxygen molecules. At high rates of photosynthesis water is split faster to replenish electrons and oxygen is generated rapidly.

This phenomenon helps us to assess the rate of photosynthesis in the lab by simply suspending leaf discs in a bicarbonate solution where bicarbonate acts as a rich source of carbon. At the beginning of the leaf disk experiment the gases are forced out of the leaf discs by applying negative pressure with a vacuum in a syringe. The leaf discs with their gases expelled become heavier and sink to the bottom of the bicarbonate solution when transferred to a beaker. When photosynthesis takes place water in the environment is split in order to replenish chlorophyll electrons. The resulting oxygen makes the discs lighter causing them to float to the surface over time. Environments that allow for higher rates of photosynthesis have discs that float faster.

In this lab you will first separate and identify pigments in spinach leaves using chromatography paper. Then you will assess the rate of photosynthesis in water and in bicarbonate solution with a leaf disk experiment.

Print Procedure Steps

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Plant Pigment Chromatography
- NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments. HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
- Use a pencil to make a line two centimeters from one end of the chromatography paper.
- Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
- Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
- Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
- When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
- Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
- Next, place 8 mL of chromatography solvent in the beaker.
- Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
- Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
- When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
- Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
- Next, record the number of visible bands and describe their color and relative size.
- Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1. Click Here to download Table 1
- Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
Floating Leaf Discs in a Vacuum
- NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO₂ to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
- To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe “bicarbonate”, and label the other syringe “water”.
- Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
- Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
- Push as much air out as possible without expelling the solution or damaging the leaf discs.
- Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
- To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
- Then, release the vacuum. NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don't sink, you can repeat the vacuum creation up to three times.
- Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
- For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
- Place both cups under a light source.
- Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed. Click Here to download Table 3
- Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
- Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
- To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
Results
- NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
- Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
- Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
- Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. Click Here to download Table 2
- Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
- Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
- Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
- Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50. NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
- Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
- Finally, compare your ET50 values and graphs with the rest of the class.