Instructions for Lab

Before you begin your lab read the objectives and the introduction.

Biology Lab Photosynthesis

Complete the report sheet as you do your lab. Turn your completed report sheet in to your instructor. This is a dry lab. You will not have to gather materials and can begin your lab immediately.

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Objectives
In this section you will Understand the nature of visible light as a part of the electromagnetic spectrum Describe pigments extracted from leaves and separated using paper chromatography Explain the importance of photosynthetic pigments in absorbing light energy Understand the structure and function of the chloroplast Understand the importance of starch as an energy storage molecule within the plant

Introduction
The sun is the ultimate source of energy. Plants utilize that energy in a process called photosynthesis. The chloroplasts in the plant leaves harness that light and convert it into chemical energy. Plants are called autotrophs because they use sunlight energy directly and combine inorganic compounds to form organic molecules. Heterotrophs, on the other hand, obtain their energy by consuming other life forms. Humans are one example of heterotrophs and break down the complex molecules they consume to obtain energy. Balanced equation for photosynthesis 6 CO2 + 12 H2O + light energy carbon dioxide water C6H12O6 + 6 O2 + 6 H2O glucose oxygen water

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Introduction
Photosynthesis occurs in the chloroplasts and consists of two linked processes called the light reactions and the Calvin cycle (dark reactions). The light reactions occurs when light splits water into oxygen and hydrogen. The hydrogen is then further split into a hydrogen ion and a free electron. The reaction is driven forward by the presence of a coenzyme, NADP+, which accepts the electron and the hydrogen ion to become NADPH, preventing the reformation of water. the light reactions also form ATP by photophosphorylation. Light reactions: 6 CO2 + 12 H2O + light energy carbon dioxide water C6H12O6 + 6 O2 + 6 H2O glucose oxygen water

Introduction
The Calvin cycle (dark reactions) is where the sugar is formed. Carbon dioxide from the air is taken into the chloroplast and is fixed into an organic molecule. The chloroplast uses products from the light reactions (ATP + NADPH + hydrogen ions) to convert CO2 into a carbohydrate. Calvin cycle: 6 CO2 + 12 H2O + light energy carbon dioxide water C6H12O6 + 6 O2 + 6 H2O glucose oxygen water

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Introduction
The light that comes from the sun looks white or colorless to us, but in fact is made of all wavelengths (colors) or light. When it rains you can sometimes see a rainbow, which is the result of light hitting raindrops and being split into its different wavelengths, similar to what happens when light hits a prism. The colors we are able to see are in the visible range, but there are other wavelengths of light we cannot see. We see objects in color because when light hits the object, most of the wavelengths are absorbed, but some are reflected. It is the reflected wavelengths we see as the color of the object. Why are plants green in color? The leaf contains pigments, which absorb the wavelengths of light required to split water. Leaves contain different levels of these pigments depending on the time of year. The main pigments found in the chloroplasts are chlorophylls a and b (which reflect green) and carotenoids (which reflect red-orange-yellow). Continue

Visible light consists of electromagnetic energy of different wavelengths ranging from about 380 to 750 nm. Our eyes perceive these different wavelengths as different colors. This colored spectrum can be absorbed or reflected by an opaque surface or transmitted by a translucent substance. We can use a prism to break white light into the colors of visible light: red, orange, yellow, green, blue and violet. The shorter the wavelength of the light, the stronger the energy with which it travels. Thus, the blue-violet end of the spectrum is more energetic than the red end. A spectroscope and a spectrophotometer will be used to analyze the wavelengths that are absorbed by a pigment extract from spinach leaves and from carrots.

I. Using the Spectroscope Experiment (dry lab)

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In lab we have two types of spectroscopes, handheld (about 15 cm long) and tabletop (about 30 cm in height).

I. Using the Spectroscope Experiment (dry lab)

A spectroscope is a device that shows the wavelengths of light that enters it. Allowing white light through the prism of a spectroscope will display all the colors of the visible light spectrum. Using a colored filter or test tube of pigment extract to block the entrance slit of the spectroscope will display only some of the colors because the others, not displayed, are absorbed by the filter. An absorption spectrum is the range of light wavelengths absorbed by a particular colored filter or pigment.
Colored filters: red, green, blue

I. Using the Spectroscope Experiment (dry lab)

Spinach leaf extract

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Allowing white light through the prism of a spectroscope will display all the colors of the visible light spectrum. The image you see will appear something like the diagram below.
Visible light spectrum

I. Using the Spectroscope Experiment (dry lab)

This image was taken by placing a red filter over the entrance slit of the spectroscope.

I. Using the Spectroscope Experiment (dry lab)

The color not absorbed is the color seen. What color(s) are not absorbed by the use of a red filter? Record your answer on your report sheet.

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This image was taken by placing a blue filter over the entrance slit of the spectroscope. What color(s) are not absorbed by a blue filter? Record your answer on your report sheet

I. Using the Spectroscope Experiment (dry lab)

This image was taken by placing a green filter over the entrance slit of the spectroscope.

I. Using the Spectroscope Experiment (dry lab)

What color(s) are not absorbed by a green filter? What is the relationship between the color of the filter and that filters absorption of light? Record your answers on your report sheet.

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This image was taken by placing a test tube of spinach leaf pigment extract over the entrance slit of the spectroscope. What color(s) are not absorbed by the extract? How does the spinach extract compare to the absorption spectrum of the green filter? Record your answers on your report sheet

I. Using the Spectroscope Experiment (dry lab)

A spectrophotometer can be used to measure absorption and transmittance of light through solutions.

II. Absorption Spectrum Experiment (dry lab)

Spectrophotometer

The solutions that will be tested are solutions of carrot pigment extract (#1) and spinach leaf pigment extract (#3). A blank (#2) will be prepared to calibrate the spectrophotometer. Refer back to Spectrophotometry unit to understand the importance of a blank tube.

#1

#2 #3

Spinach leaf extract

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The spectrophotometer is calibrated before the start of the exercise and every time the wavelength is readjusted (review Spectrophotometry unit). You will determine the absorption spectrum of the pigment extract (both carrot extract and spinach extract) by recording the %transmittance of light at 25 nm intervals starting at 400 nm and continuing to 700 nm. Record your data in the table of your report sheet.
Carrot extract

II. Absorption Spectrum Experiment (dry lab)

With the carrot extract test tube in the sample holder, record the %transmittance of light at 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm and 550 nm.

II. Absorption Spectrum Experiment (dry lab)

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II. Absorption Spectrum Experiment (dry lab)

Carrot extract

Continuing with the carrot extract test tube in the sample holder, record the %transmittance of light at 575 nm, 600 nm, 625 nm, 650 nm, 675 nm and 700 nm. Plot your data on the absorption spectrum graph. What wavelengths (colors) of light are not absorbed well by the carrot extract? Record your answers on your report sheet.

Spinach leaf extract

With the spinach leaf extract test tube in the sample holder, record the % transmittance of light at 400 nm, 425 nm, 450 nm, 475 nm, 500 nm, 525 nm and 550 nm.

II. Absorption Spectrum Experiment (dry lab)

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Spinach leaf extract

Continuing with the spinach leaf extract test tube in the sample holder, record the % transmittance of light at 575 nm, 600 nm, 625 nm, 650 nm, 675 nm and 700 nm. Plot your data on the absorption spectrum graph. What wavelengths (colors) of light are not absorbed well by the spinach extract? Record your answer on your report sheet.

II. Absorption Spectrum Experiment (dry lab)

II. Absorption Spectrum Experiment (dry lab)

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Paper chromatography is used to separate substances from one another based on their characteristics. This exercise will separate pigments of a spinach leaf. The separation is due to the solubility of the pigment in the chromatography solvent (10% acetone in petroleum ether) and the affinity of the pigment to the paper surface. The finished product is called a chromatogram.

III. Paper Chromatography Experiment (dry lab)

Adding pigment to the chromatography paper

III. Paper Chromatography Experiment (dry lab)

Chromatography paper strip

On a 3 cm x 20 cm strip of chromatography paper, a baseline is drawn with pencil (graphite) about 2 cm from the bottom. On this pencil line the spinach leaf pigment is deposited by rolling a coin over the leaf, onto the paper.

Chromatography solvent

Roll of chromatography paper

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Placing the strip into the chromatography chamber

III. Paper Chromatography Experiment (dry lab)

The chromatogram

III. Paper Chromatography Experiment (dry lab)

The paper with pigment is placed into a test tube containing 1 cm of chromatography solvent. Left undisturbed for 15 minutes the solvent moves up the paper and carries with it the pigments of the spinach leaf. The pigments move at their own characteristic rate.

There are four pigments that are seen on the chromatogram. - Carotene (orange) - Xanthophyll (yellow) - Chlorophyll a (blue-green) - Chlorophyll b (yellow-green) Which pigment has the greatest affinity for the solvent (is most soluble in the solvent)? Which pigment has the greatest affinity for the paper? Record your answers on your report sheet.
Baseline

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Determine the Rf (ratio factor) value of each pigment. To determine the Rf value of a pigment you must measure the distance traveled by the pigment/distance traveled by the solvent. -To determine distance traveled by the pigment you must measure from the middle of the pigment band to the baseline. -To determine the distance traveled by the solvent you must measure from the solvent line to the baseline. Record the Rf values on your report sheet.

III. Paper Chromatography Experiment (dry lab)

Solvent line

In the spring the new leaves are bright green in color. What pigment do you think is present in the highest concentration during the spring season? In the fall, leaves turn red, orange, yellow, and eventually brown. Where did the green color go? Record your answer on your report sheet.

III. Paper Chromatography Experiment (dry lab)

Baseline

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When plants photosynthesize, they produce organic molecules which can be used to make glucose, a carbohydrate. Glucose can be broken down to provide energy for metabolic processes, or it can be converted to another carbohydrate for storage. The most common from of carbohydrate stored by plants is starch, a polymer of glucose. The formula of photosynthesis indicates a carbohydrate as an end product. 6 CO2 + 12 H2O + light energy C6H12O6 + carbohydrate 6 O2 + 6 H2O

IV. Starch Localization Experiment (dry lab)

The leaf will be tested to determine which area contains starch. Part of the leaf contains green pigment while some parts do not (see image on right). QUESTION: What would be your hypothesis on the localization of starch? To test where starch will be seen in the leaf, the leaf pigments must first be removed by placing it in boiling 95% ethanol. (Ethanol cannot be boiled directly on the hotplate. A beaker containing ethanol is placed into a larger beaker of boiling water.)

IV. Starch Localization Experiment (dry lab)

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Boiling 95% ethanol

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Iodine is then added to the leaf. If starch is present the area will become blue-black (review macromolecules unit). Which part of the leaf stores starch? Record your answer on your answer sheet.
Before boiling After staining

IV. Starch Localization Experiment (dry lab)

Photosynthesis occurs in the chloroplasts of a cell. The stages of photosynthesis are the light reactions and the Calvin cycle (also known as the dark reactions because they are light-independent). The light reactions occur at the thylakoid membrane. The Calvin cycle occurs in the stroma. Plant cells contain chloroplasts. In this cell model, chloroplasts are represented by green structures. Below you will find enlargements of chloroplasts to show the cross section and surface view.

V. Chloroplast Structure Experiment (dry lab)

Plant cell

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Cross section

Surface

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Regions of the chloroplast are 1-outer membrane 2-inner membrane 3-intermembrane space 4-stroma 5-thylakoid disk granum one stack grana multiple stacks

V. Chloroplast Structure Experiment (dry lab)

3 4 1 2

Match the descriptions to the labels below a. Site for light reactions, electron transport chain and ATP-synthesizing enzymes b. Site for Calvin cycle c. Surrounds the organelle
Thylakoid Stroma Outer membrane Starch grain

V. Chloroplast Structure Experiment (dry lab)

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End of Lab