Plant Pigments & Absorption Lab
SBI4U Metabolic Processes Lab 1

Kevin Salwach
P.1 Rm. 208 De Cat
November 5 2013
Lab 1 Pigments & Absorption
Introduction
Autotrophs, a group of organisms to which plants belong, obtain their own food and energy through ways other than hunting and ingestion. Almost all plants use photosynthesis to obtain their energy. Using the CO2 in the air that surrounds them, plants can create the energy needed to survive. The process is split into two parts; the “light” reactions occur by using light from the sun to activate electrons in the photosystem to produce adenosine triphosphate (ATP) the main requirement for metabolism. After this has occurred, the “dark” reactions occur where ATP is reduced to coenzymes in several complex cycles, and with the help of CO2, glucose is produced. This is a simplified explanation of course, as the entire photosynthetic process contains many different reactions and cycles. Overall, this is a very effective way of obtaining energy, and allows a whole manner of organisms to thrive almost anywhere on Earth.
Purpose
The purpose of this lab is conduct three experiments that answer the questions:
- Where to plants conduct photosynthesis?
- What photosynthetic pigments do they use?
- What wavelength of light works best for photosynthesis?
Materials
-Microscope -Elodea
-Microscope slide / cover slip -Spinach leaves
-Mortar / pestle -Distilled water
-80% acetone -Sand
-Filter paper -Coleus / Germanium
-Funnel
-Spectrophotometer
-Chromatography paper
-Spec tubes
-Kimwipes
-Test tube rack
-Chromatography solvent
-Coloured pencils
Procedure
Refer to “Metabolic Processes Lab 1: Plant Pigments and Absorption” in the SBI4U Metabolic Processes Learning Guide under “Activities”

Observations & Analysis
Part A
Observation (sketch) of the magnified spinach leaf is attached to the back of the report.
What might the purpose of cytoplasmic streaming be?
Cytoplasmic streaming is the movement of fluid within the cytoplasm around the cell. The main liquid constituent of cytoplasm is cytosol or cytoplasmic matrix. Although it is not fully understood, it is believed that cytosol flows around the cell with the help of special proteins acting as motors. The purpose of this cytoplasmic streaming is to deliver nutrients, proteins, organelles, genetic material and other molecules to parts of the cell where they are needed.
Part B
How many pigments are present on your chromatogram? Why do plants have more than one?
There 5 observed pigments present on our chromatogram. The reason that a plant has more than one type of pigment is the broad spectrum of solar light. Although it comprises only a small sliver of the electromagnetic spectrum, sunlight still contains a fair amount of different light. Stretching from violet to red, the spectra of sunlight covers about 370 nm. Because it is a wide spectrum it is exceedingly difficult for a single pigment to cover and absorb all of the light. As a result, a plant contains many different pigments, so it can maximize its absorption of light and collect it across varying wavelengths, as opposed to being confined to a single one. Pigments such as chlorophyll absorb red light and reflect green, while other pigments such as carotenoids do the opposite.
Can You Explain, based on chemical structure and what you know about chromatographic separation, the relative positions (distances migrated) if your pigments?
The different positions of pigments on the paper indicated the different concentrations (or abundance) of that pigment. For example, the chlorophyll remained near the bottom of the paper and showed a dark green band. Being the most abundant pigment, it showed up the clearest. The carotenoids were less abundant, and showed up fainter and farther from the bottom. Finally the anthocyanins and xanthophylls were the least abundant, and faintly showed up near the top of the paper.
Do different leaves have the same pigments?
If the leaves are of the same plant then yes. Different leaves of the same plant contain the same pigments. Leaves of different plants however, do not always share the same pigment. Different plants of different environments, shapes and sizes have all evolved differently, < and therefore may require different wavelengths of light and may have different methods of absorption for photosynthesis. This is why some plants are dark green, while others are light green, red or even beige and brown.
Why do leaves of many plants change colour in the autumn? What happens to the pigments?
To begin with, most leaves are of a green shade during the summer months. This is due to an abundance of chlorophyll A and B in the leaf, both of which reflect green light, giving the leaf its colour. The growth, production and abundance of chlorophyll are all a result of light. Chlorophyll can only be produced in the presence of light (i.e. leaves are only green in light; they become white and pale in darkness). During autumn, when the days become shorter and the amount of sunlight hours decreases, the leaf does not receive as much light as in the summer months. As a result, less chlorophyll is produced. With less of the strong chlorophyll being produced, other pigments such as carotenoids and anthocyanins are able to be seen as they are o long being overpowered. These pigments reflect yellow and red light, respectively, and as a result leaves appear yellow and red during autumn.
Part C
The following table shows the absorption of spinach at different wavelengths. Absorption Data For Spinach Leaves | Wavelength (nm) | 350 | 375 | 400 | 425 | 450 | 475 | 500 | 525 | 550 | 575 | 600 | 625 | 650 | 675 | 700 | Reading | 0.75 | 0.49 | 0.32 | 0.35 | 0.31 | 0.205 | 0.07 | 0.03 | 0.03 | 0.04 | 0.05 | 0.07 | 0.11 | 0.39 | 0.5 |

What “blank” did you use to calibrate the spectrometer?
The blank that we used was the pigment solvent, which was 80% acetone. We took the readings off the of the spectrometer using only the solvent, so when the spinach and pigments were added, the solvent would be cancelled out, revealing only pigment.
The graph to the left shows an absorption spectrum using the data we obtained from the spectrometer. Is shows the different absorption of spinach at different wavelengths.
Do different leaves (and pigments) have different absorption spectra? Explain.
Different leaves will have different absorption spectra. The spectrum depends on which pigments are present in the leaf, and at what concentrations. One leaf could have a slightly lower concentration of chlorophyll or a slightly higher concentration on anthocyanin, making the leaf a different colour, and thereby giving it a different absorption spectra, as the pigment reflects a different wavelength of light.
Using your graph, discuss which light wavelengths are best absorbed by plant pigments. What does your absorption spectrum tell you? Can you relate this to photosynthesis?
The wavelength that was best absorbed by the pigments was 350 nm. Wavelengths between 500 and 650 nm are those least absorbed. The area least absorbed, the green light, is the wavelength of the sun’s most powerful and energetic light. As a result, absorbing the most intense and energetic part of the spectra could easily destroy the chlorophyll pigments, so they are simply reflected back to avoid damage.

References
Encyclopedia Britannica. “Cytoplasmic Streaming” (2013). [Web] Retrieved November 4 2013 from http://www.britannica.com/EBchecked/topic/148976/cytoplasmic-streaming
“Light absorption for photosynthesis” (2013). [Web] Retrieved November 4 2013 from http://hyperphysics.phy-astr.gsu.edu/hbase/biology/ligabs.html