Discussion: Connecting Your Learning

Discussion: Connecting Your Learning

 

Photosynthesis is a biological process that occurs in plants, some bacteria, and some protists. This process relies on pigments, most importantly chlorophyll, to capture light energy and drive the chemical reaction of photosynthesis. This lab involves the extraction of several plant pigments that convert light energy into glucose.

Resources and Assignments

Multimedia Resources

None

Required Assignments

Lesson 5 Lab 5

Required Materials

From the Lab Kit:

· 1test tube

· 4 strips of chromatography paper

· 10 mL syringe

· 2 Phenol red tablets

· Test tube stopper

· 2 Micropipettes

· 2 paper clips

· Rubber band

· 100 mL graduated cylinder

· Mortar and pestle

· Bag of sand (about 2 teaspoons)

· Goggles

· Metric ruler

· Hole punch

· Straw

· Forceps

Student Supplied:

· Acetone (either nail polish remover or acetone from paint section of a hardware or home improvement store)(Acetone is flammable so be certain to keep it away from open flames.)

· Light source (lamp with 75W or greater light bulb)

· Scissors

· 1/4 teaspoon baking soda

· 2 cups plus 50 mL distilled water

· Liquid dish soap

· 10 Spinach leaves (large, standard-sized leaves; do not use small, baby spinach leaves)

· 1 large glass jar with lid

· Paper towel

· 2 Glass bowls

· 2 Cups or Glasses (4 oz.)

· Hairdryer

· Pencil

· Stopwatch or clock

· Tape

· Marker or Pen

Focusing Your Learning

Lab Objectives

By the end of this lab, you should be able to:

1. Study the nature of light and its effect on biological systems.

2. Provide the chemical reaction for photosynthesis.

3. State the importance of pigments to photosynthesis.

4. List variables which affect the rate of photosynthesis.

5. Describe the separation of photosynthetic pigments by chromatography.

6. State the purpose of determining Rf values.

7. Calculate Rf values.

Background Information

Energy from the sun travels in waves, similar to the way that waves move across the ocean. All waves share characteristics, such as a crest, the highest part of a wave, and a trough, the lowest part of a wave. The distance measured between either the crests or troughs of two successive waves is knows as wavelength.

Waves of energy from the sun are comprised of photons. Low energy photons travel in longer wavelengths while high energy photons travel in shorter wavelengths. Wavelength is measured in nanometers (1 nm = 1 billionth of a meter). Photosynthetic organisms absorb light of wavelengths between approximately 380 and 750 nanometers.

Waves of radiant energy are organized according to the electromagnetic spectrum. The portion of the electromagnetic spectrum that is visible to the human eye is called the visible spectrum and ranges in wavelength from 400 nm to 700 nm. Different wavelengths of light are viewed as different colors. Wavelengths of 700 nm are seen as red light, while wavelengths of 400 nm are seen as violet light. From highest wavelengths to lowest, the visible colors are red, orange, yellow, green, blue, indigo, and violet. This order is often abbreviated as ROYGBIV, from the first letter of each color. Beyond the visible spectrum are other forms of energy, such as infrared and gamma rays, which are undetectable without specialized equipment.

Pigments are molecules that absorb only photons of specific wavelengths of light. Photons that are not absorbed are reflected back as a color.Chlorophyll a and chlorophyll b reflect wavelengths of approximately 510 nm, which appear green in color. Carotenoids reflect wavelengths of 650 nm (red), 590 nm (orange), and 570 nm (yellow). Xanthophylls reflect wavelengths of 650 nm (red) and 570 nm (yellow).

Click on image to enlarge.

Plants absorb sunlight and convert the solar energy into chemical energy in the form of glucose as a food source through a process called photosynthesis.  Photosynthesis  is the process of combining water and carbon dioxide in the presence of light energy to produce glucose and oxygen. The chemical equation for photosynthesis is:

Click on image to enlarge.

The process of photosynthesis is carried out in the  chloroplasts  of plant cells, which are concentrated in the interior of the leaves. Gas exchange of carbon dioxide and oxygen occurs through small openings in the leaf called  stomata . The figure below details the location of the stomata in comparison to the chloroplasts in a leaf.

Click on image to enlarge.

All life on earth is either directly or indirectly dependent on photosynthesis. Each chloroplast consists of a double membrane. The inner membrane houses a chamber filled with a fluid called  stroma . Suspended in the stroma are membranous sacs called  thylakoids , which are stacked into structures called  grana . Below is an image that details the structure of a chloroplast.

Click on image to enlarge.

Chloroplasts appear green in color due to the presence of a light-absorbing pigment called  chlorophyll , which is found in the thylakoid membranes. The process of photosynthesis mainly employs two types of chlorophyll ( chlorophyll a  and  chlorophyll b ), each activated by a different wavelength of light. In order for the process to proceed, the plant cell must absorb (take in) the particular color (wavelength) of light needed to activate chlorophyll. Chlorophyll a and b absorb blue and red light while they reflect green light; giving leaves their green appearance.

While chlorophyll is required for photosynthesis, it is not the only pigment that plants contain. Many plants contain accessory pigments, including carotene  (orange) and  xanthophyll  (yellow). Even though these pigments do not directly participate in photosynthetic reactions, they do transfer the energy they receive to chlorophyll, which, in turn, uses the energy for photosynthesis. Because these pigments absorb light at different wavelengths, the presence of accessory pigments allows a plant to maximize the amount of sunlight captured. Another pigment is  anthocyanin  (red or purple), which helps protect the plant from ultraviolet damage. These accessory pigments contribute to the colors found in fall foliage. As chlorophyll levels decline in the fall, the accessory pigments are able to be seen, producing the vivid orange, yellow, and red colors seen in leaves.

The first part of this lab will involve a demonstration of CO2 use in photosynthesis. To test if plants require CO2 for photosynthesis, an experiment will be conducted using phenol red. Phenol red is an indicator that turns yellow in the presence of CO2. If the CO2 is removed, phenol red returns to its original color (red).

In the second experiment in this lab, four pigments found in plant leaves will be identified through the process of  chromatography . The four pigments to be observed include carotene (yellow-orange color), xanthophyll (yellow color), chlorophyll a (blue-green color), and chlorophyll b(yellow-green color). The pigments will be extracted using one-way paper chromatography. To conduct this type of chromatography, an extract of the compound must first be obtained. The chromatography paper must be marked with a pencil, as ink will dissolve in the solvent used in this experiment and will also travel up the paper. At the conclusion of the experiment, the Rf factor will be calculated for each pigment.

In chromatography, chemicals can be compared to one another based on their Rf values. Rf stands for “ratio of fronts” and is characteristic for any given chemical. Rf values are calculated using the following equation:

Rf =

Distance the pigment traveled

Distance the solvent traveled    (solvent front will be near the paper clip)

For example, if the solvent travels 10 cm, and the pigment travels 3 cm, the Rf value for that pigment would be:

3 cm/10 cm = 0.3000

Note that the Rf value should be calculated to four decimal places. For example, if the result is 0.345678, the Rf value should be documented as 0.3457.

The higher the Rf factor, the more soluble that pigment is in the particular solvent.

As will be demonstrated in this experiment, pigments have different Rf values. This occurs because pigments travel at different rates depending on their solubility in the solvent, molecular mass, and affinity for bonding with the paper (or their chemical charge). In general, the less chemically charged and lighter pigments will travel further up the paper. Those that are heavier (have a higher molecular weight) and are chemically charged travel a shorter distance. If the pigment does not travel, it is NOT soluble in the particular solvent.

The final experiment will determine the influence of light intensity and carbon dioxide concentration on the rate of photosynthesis in plant leaves. Plant leaves contain extracellular spaces that are filled with gases that enter and exit the leaves. Recall that the overall reaction for photosynthesis includes an input of carbon dioxide gas and an output of oxygen gas. As described earlier in the lab, these gases are exchanged through small openings in plant leaves called stomata. Air that is found in the extracellular spaces of leaves gives them buoyancy, causing them to float on water. In the experiment, a solution of baking soda and water will be used to supply a source of carbon for photosynthesis. Circular disks will be cut from spinach leaves for the experiment. The air will be extracted from the extracellular space of the spinach leaves and replaced with water, which will cause the leaves to sink in a sodium bicarbonate solution. If cells in the leaves are performing photosynthesis, oxygen gas will be generated as a by-product, causing the leaves to float in the solution.

The amount of time it takes for the disk to float will be used as a measure of photosynthetic activity. In order to account for variability in photosynthetic rates between the disks, the time required for 50% of the disks to float (five disks) will…