Name: spinach pigments and leaf disc photosynthesis
Uploaded: 2019-01-29T11:04:45.000Z
Duration: 8 min 20 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

North Carolina K-12 Science Standard Course of Study

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms- Structures and Processes

LS.Bio.3.2

Photosynthesis

<ul><li><a href="#" data-time="8">Plant Pigment Chromatography
</a></li><li><a href="#" data-time="165">Floating Leaf Discs in a Vacuum
</a></li><li><a href="#" data-time="381">Results
</a></li></ul>

photosynthesis

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Plant Pigment Chromatography
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Use a pencil to make a line two centimeters from one end of the chromatography paper.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-8"><div class="lab-step">Next, place 8 mL of chromatography solvent in the beaker.
</div></li><li data-sub-note-item="1-9"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-10"><div class="lab-step">Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
</div></li><li data-sub-note-item="1-11"><div class="lab-step">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.
</div></li><li data-sub-note-item="1-12"><div class="lab-step">Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
</div></li><li data-sub-note-item="1-13"><div class="lab-step">Next, record the number of visible bands and describe their color and relative size.
</div></li><li data-sub-note-item="1-14"><div class="lab-step">Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1.  <a href="https://www.jove.com/CDNSource/lm/labs/7/Table_1_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 1</a>
</div></li><li data-sub-note-item="1-15"><div class="lab-step">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.
</div></li></ul></li><li data-note-item="2" data-parent-collapse="">Floating Leaf Discs in a Vacuum
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="2-1"><div class="lab-step">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 CO<sub>2</sub> 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.
</div></li><li data-sub-note-item="2-2"><div class="lab-step">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 &#8220;bicarbonate&#8221;, and label the other syringe &#8220;water&#8221;.
</div></li><li data-sub-note-item="2-3"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-4"><div class="lab-step">Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
</div></li><li data-sub-note-item="2-5"><div class="lab-step">Push as much air out as possible without expelling the solution or damaging the leaf discs.
</div></li><li data-sub-note-item="2-6"><div class="lab-step">Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
</div></li><li data-sub-note-item="2-7"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-8"><div class="lab-step">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&#39;t sink, you can repeat the vacuum creation up to three times.
</div></li><li data-sub-note-item="2-9"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-10"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-11"><div class="lab-step">Place both cups under a light source.
</div></li><li data-sub-note-item="2-12"><div class="lab-step">Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed.  <a href="https://www.jove.com/CDNSource/lm/labs/7/Table_3_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 3</a>
</div></li><li data-sub-note-item="2-13"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-14"><div class="lab-step">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.
</div></li><li data-sub-note-item="2-15"><div class="lab-step">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.
</div></li></ul></li><li data-note-item="3" data-parent-collapse="">Results
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="3-1"><div class="lab-step">NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
</div></li><li data-sub-note-item="3-2"><div class="lab-step">Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
</div></li><li data-sub-note-item="3-3"><div class="lab-step">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.
</div></li><li data-sub-note-item="3-4"><div class="lab-step">Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. <a href="https://www.jove.com/CDNSource/lm/labs/7/Table_2_Photosynthesis.xlsx" style="display:block;margin:20px;" target="__blank">Click Here to download Table 2</a>
</div></li><li data-sub-note-item="3-5"><div class="lab-step">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.
</div></li><li data-sub-note-item="3-6"><div class="lab-step">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.
</div></li><li data-sub-note-item="3-7"><div class="lab-step">Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
</div></li><li data-sub-note-item="3-8"><div class="lab-step">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.
</div></li><li data-sub-note-item="3-9"><div class="lab-step">Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
</div></li><li data-sub-note-item="3-10"><div class="lab-step">Finally, compare your ET50 values and graphs with the rest of the class.
</div></li></ul></li></ol>

Plant Pigment Chromatography
ExpandNOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments ...

Education

JoVE Lab Manual

Cellular Processes

Lab Manual Sub Articles

spinach pigments and leaf disc photosynthesis

<p class="jove_content">Spinach leaves contain pigments that can be separated by chromatography. In this lab, chromatography paper is used to move the pigments at different rates so their colors and positions can be compared. By measuring how far each pigment travels and calculating its retention factor, or Rf value, students can identify the pigments by matching them with literature values.</p>

<p class="jove_content">The chromatography setup starts with a pencil line drawn 2 centimeters from one end of the paper. A spinach leaf is pressed along that line with a coin until a green band appears. The strip is then placed in a beaker with chromatography solvent, but the green line must stay above the liquid. As the solvent rises, the pigments separate into clear bands, and the paper is removed after about 10 minutes, when the solvent has moved about halfway up the strip.</p>

<p class="jove_content">The second part of the activity shows photosynthesis and cellular respiration with floating leaf discs. Leaf discs are placed in syringes with either bicarbonate solution or water, and a vacuum is created so the solution enters the air spaces in the tissue. The discs sink after this step. The bicarbonate solution provides added carbon dioxide for photosynthesis, so the experimental hypothesis is that more discs will float in that treatment. The null hypothesis is that there will be no difference between bicarbonate and water, or between light and dark conditions.</p>

<p class="jove_content">After the vacuum step, the discs are placed in cups under a light source. Every five minutes, students count how many discs float on the surface for up to 20 minutes. Then the cups are moved to a dark place, and the counts are repeated while the cups are swirled before each reading. In the dark, the discs are expected to sink as cellular respiration continues. The results are graphed with time on the x-axis and number of floating discs on the y-axis, and the effective time, or ET50, is the point when 50% of the discs are floating.</p>

Spinach Pigments and Leaf Disc Photosynthesis

spinach-pigments-and-leaf-disc-photosynthesis

Spinach leaves contain pigments that can be separated by chromatography. In this lab, chromatography paper is used to move the pigments at different rates so their colors and positions can be compared. By measuring how far each pigment travels and calculating its retention factor, or Rf value, students can identify the pigments by matching them with literature values.<br /><br />The chromatography setup starts with a pencil line drawn 2 centimeters from one end of the paper. A spinach leaf is pressed...

Leaf Pigment Prep for Chromatography Labs

Leaf pigment chromatography and floating leaf disc preparation are part of a photosynthesis lab. The work begins the day before the experiment. Spinach leaves are placed in a container of water and kept fully submerged overnight at room temperature.<br /><br />The setup also includes chromatography paper for the pigment test. Wearing gloves, prepare and measure 18 cm x 304 cm sections of the paper. For the leaf disc activity, make a bicarbonate solution by mixing 0.25 g NaHCO3 with 300 mL water. Add one...

lab manual sub articles

Separate spinach pigments by chromatography, calculate Rf values, and use floating leaf discs to compare photosynthesis in light, dark, bicarbonate, and water.

Spinach Pigments and Leaf Disc Photosynthesis | JoVE K12

Pennsylvania State Academic Standards

Life Science

matter-and-energy-in-organisms-and-ecosystems

Matter and Energy in Organisms and Ecosystems

3.1.9-12.E

3.1.9-12.J

3.1.9-12.K

Louisiana Student Standards for Science

High School

Environmental Science

ecosystems-interactions-energy-and-dynamics

Ecosystems: Interactions, Energy And Dynamics

HS-LS2-4

ohios-learning-standards-and-model-curriculum

Ohio’s Learning Standards and Model Curriculum

Cells

Cellular processes

Virginia  Standards of Learning

HIgh School

BIO.2

BIO.2.e

Next Generation Science Standards

From Molecules to Organisms: Structures and Processes

HS-LS1-5

new-york-state-science-learning-standards

New York State Science Learning Standards

HS-LS1-5.

<p>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.</p><p>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.</p><p>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.</p><p>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.</p>

photosynthesis: light, sugars, and carbon

<p class="jove_content">Photosynthesis turns sunlight into chemical energy in the form of glucose. This process powers most life on Earth by helping autotrophs, or primary producers, make food. The glucose they make can provide quick energy or be stored as starch for later use. Consumers depend on these producers for energy.</p>

<p class="jove_content">Photosynthesis happens in many kinds of organisms. Cyanobacteria, some protists such as algae, and plants can all carry out this process. In eukaryotic cells, it takes place in the chloroplast. Chloroplasts look green because they contain chlorophyll, a pigment that absorbs light at specific wavelengths.</p>

<p class="jove_content">Plants use several pigments to capture light. Chlorophyll absorbs red and blue light and reflects green light. Other pigments, including carotenoids, anthocyanins, and betalains, absorb or reflect different wavelengths and often appear yellow to red. In autumn, when chlorophyll levels drop in temperate regions, these pigments become visible and leaves change color.</p>

<p class="jove_content">Photosynthesis has two main stages: light-dependent reactions and light-independent reactions. These stages happen in different parts of the chloroplast. The chloroplast has an outer membrane, an inner membrane, and an inner thylakoid membrane that forms disc-like folds. The space between the inner membrane and thylakoids is the stroma.</p>

<p class="jove_content">The light-dependent reactions begin in the thylakoid membrane when sunlight excites electrons in chlorophyll. Those electrons move through an electron transport chain, dropping to lower energy levels and releasing energy. The cell uses that energy to make ATP and NADPH. Water splitting, also called hydrolysis, replaces the lost electrons in chlorophyll. This process releases oxygen into the atmosphere and helps keep the flow of electrons going.</p>

<p class="jove_content">The light-independent reactions take place in the stroma. They use ATP and NADPH from the first stage to build a three-carbon sugar from carbon dioxide. That sugar can then be turned into glucose or other biomolecules. This is also called carbon fixation or carbon sequestration because carbon is pulled from the atmosphere and stored in living molecules.</p>

<p class="jove_content">Photosynthesis plays a major role in the carbon cycle. Carbon dioxide moves between reservoirs at a steady pace, but changes in that flow can upset the balance. Because carbon dioxide is a greenhouse gas, more of it in the atmosphere can raise temperatures. Much of the extra carbon dioxide comes from burning fossil fuels, which returns ancient carbon to the air very quickly.</p>

<p class="jove_content">Forests and ocean algae help lower carbon dioxide levels, so deforestation is a serious concern in a changing climate. Scientists are also studying artificial photosynthesis as a way to make carbon-neutral biofuels. Like solar panels, these methods take energy from the sun and store it as chemical energy instead of electricity. Researchers have also used photosynthetic bacteria to make simple sugars, lactic acid, biodegradable plastics, and even medically useful compounds, including substances studied for cancer diagnosis and treatment.</p>

Photosynthesis: Light, Sugars, and Carbon

Autotrophs

Almost all living organisms on Earth depend on photosynthesis, which is the process that converts sunlight energy into a simple sugar called ...

Test your understanding of Photosynthesis with this Biology assessment quiz on JoVE Lab Manual. Covers key concepts through video-based questions designed to evaluate student learning.

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Prepare spinach leaves, chromatography paper, bicarbonate solution, and leaf discs for a high school photosynthesis lab on pigments and floating discs.

Leaf Pigment Prep for Chromatography Labs | JoVE K12

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Cytoplasm

The cytoplasm consists of organelles and a framework of protein scaffolds called the cytoskeleton suspended in an aqueous solution, the cytosol. The ...

The Nucleus

The nucleus is a membrane-bound organelle that acts as a control center in a eukaryotic cell. It contains chromosomal DNA, which controls gene expression ...

Endoplasmic Reticulum

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Ribosomes

Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that ...

Golgi Apparatus

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Lysosomes

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Peroxisomes

Peroxisomes and mitochondria are two important oxygen-utilizing organelles in eukaryotic cells. Mitochondria carry out cellular respiration&#x2014;the ...

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Introduction to Actin

Actin is a highly conserved cytoskeletal protein found abundantly in eukaryotic cells. It constitutes 10% weight of the total cellular protein in muscle ...

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Types of Intermediate Filaments

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The Extracellular Matrix

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LS.Bio.1.3

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the-tree-of-life-bacteria-archaea-and-eukaryotes

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LS.Bio.1.4

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RNA Structure

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Transcription Elongation Factors

Alternative RNA Splicing

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Transfer RNA Synthesis

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The Nucleolus

Construct an explanation to summarize how DNA and RNA direct the synthesis of proteins.

LS.Bio.1.5

Analyze how the relationship between structure and function supports life processes within organisms.

Spinach Pigments and Leaf Disc Photosynthesis

Procedure

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