For many centuries, people believed that the increase in the size of a plant was caused by the intake of material from the soil. It was not until a Belgian physician, Jan Baptista van Helmont (circa 1577-1644), performed an experiment that demonstrated conclusively what we accept today: the increase in the size of a plant is not due simply to the plant obtaining a mystery substance from the soil; plants gain what they require through the process of photosynthesis.
Photosynthesis uses energy from light captured by photosynthetic pigments, and splits water molecules in the process. The plants fix carbon from carbon dioxide into glucose and fructose chains and oxygen is released as a byproduct. In many plants the sugars then combine to form long chains known as starches. Many plants store their photosynthetic products this way.
Most plants produce their own organic molecules through photosynthesis and do not need to take them from another organism; these plants are called autotrophs. However, not all plants are able to carry out photosynthesis. Many parasitic plants that don’t have photosynthetic pigments rely entirely on other host species for nourishment; these plants are called heterotrophs.
Photosynthetic pigments include chlorophyll a, chlorophyll b, and the carotenes. Green plants usually have high chlorophyll content; in a typical plant, approximately three-quarters of the chlorophyll is chlorophyll a and the rest is chlorophyll b. In some plants, the presence of other pigments becomes apparent in the fall when the chlorophyll no longer masks their presence. Other plants are high in pigments that
mask the chlorophylls during the whole growing season (e.g., red cabbage remains red because of the presence of anthocyanin). Each pigment absorbs a specific range of wavelengths. Chlorophyll a, the primary pigment used in photosynthesis, absorbs blue and red light. Chlorophyll b absorbs light in the blue-green and orange-red portions of the spectrum. Carotenoids absorb light in the blue and blue-green regions.
The first step in the conversion of light to chemical energy is the absorption of light by a pigment system. In all photosynthetic cells, except photosynthetic bacteria, the pigment system includes chlorophyll a. In vascular plants, bryophytes, green algae, and euglenoid algae, chlorophyll b is also found and functions as an accessory pigment. In the leaves of green plants, chlorophyll b generally constitutes about one-fourth the total chlorophyll content. It extends the range of light that can be used for photosynthesis because it absorbs wavelengths different from chlorophyll a. At the same time, it shares the ability to absorb light energy and produce an excited state in the molecule with chlorophyll a. The excited molecule of chlorophyll b
transfers its energy to a molecule of chlorophyll a, which then transforms it into chemical energy. Chlorophyll c or chlorophyll d takes the place of chlorophyll b in other groups of plants.
Certain carotenoids are also accessory pigments involved in the capture of light energy in photosynthesis. Carotenoids are red, orange, or yellow fat-soluble pigments found in all chloroplasts and also, in association with chlorophyll a, in the prokaryotic blue-green algae. Carotenoids are not found free in the cytoplasm, but like the chlorophylls are bound to proteins within the plastids. There are two classes of carotenoids: those that do not contain oxygen are called carotenes, and those that do contain oxygen are called xanthophylls. In green leaves, the color of the carotenoids is masked by the much more abundant chlorophylls; in some tissues, such as those of a ripe tomato or the petals of an orange flower, the carotenoids predominate. During autumn, chlorophyll begins to break down as the leaf begins to senesce, allowing the carotenes and xanthophylls to display the brilliant colors we associate with fall.
One technique that can be used to separate extracted plant pigments is called chromatography. It separates liquid components into individual components based on their specific affinity, or attraction, for a solid surface, known as the stationary phase, and a specific solubility in a chromatography solvent, known as the mobile phase. Initially, an extract of the plant is placed on the bottom of a strip of chromatography paper, which is then placed in a vial with solvent covering the bottom. The chromatography strip and solvent are then placed in a vial. The paper acts as a wick, drawing the solvent upward by capillary action and dissolving the mixture as it passes over it. Different components of the mixture interact differently with the two phases. Some components will be more strongly attracted to the stationary phase and adsorb to the filter paper while others will be more attracted to the mobile phase and will migrate with the solvent. As the mobile phase passes over the stationary phase, the components more strongly adsorbed to the stationary phase will travel more slowly than those soluble in the mobile phase. The final result is that different pigments in the mixture show up as colored streaks or bands on the strip. The pattern formed on the paper is called a chromatogram.
To establish the relative rate of flow for each pigment, the Rf value of each pigment is calculated. The Rf value represents the ratio of the distance a pigment moved on the chromatogram relative to the distance the solvent front moved. It is calculated using the following formula:
Rf= Distance pigment traveled / Distance solvent traveled
For example, the following sample chromatogram displays four different pigments: A,B,C, and D as well as the solvent front.
In order to determine the Rf for pigment B, for instance, the distances that pigment B and the solvent front traveled would be placed into the Rf formula:
Rf = 42 mm / 80 mm
Rf = 0.525
Any molecule in a given solvent matrix has a uniquely consistent Rf, so the Rf value can be used by scientists to identify molecules.
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See more plant lab activities at wardsci.com.
Chromatography of Spinach Lab Activity
The procedure presented in this activity quickly yields large amounts of plant pigments, making it easier to complete than traditional solvent extractions. Students will learn the basics of paper chromatography, including the use of petroleum ether/acetone solvent, and calculating the rate of flow (Rƒ) for each pigment. Once students have completed the experiment they will be rewarded with a vivid chromatogram displaying chlorophyll a and b, xanthophyll, and carotene pigments, as well as an understanding of the different affinities for the mobile and stationary phases.- Photosynthesis Demonstration Model
Easily demonstrate quantitative examples of photosynthesis and various environmental effects on plants with WARD’S photosynthesis apparatus. The variety of suggested experiments work with either aquatic plants or terrestrial plants. You can change the variables affecting photosynthesis and measure the rate of oxygen emitted as a byproduct, or test the influence of light intensity and wavelength, carbon dioxide and oxygen concentration in water, and other environmental factors. - Separation of Plant Pigments by Gel Filtration Lab Activity
Using less-volatile ethanol rather than petroleum ether or acetone, students can achieve results superior to paper chromatography in less than 30 minutes. Four major plant pigments separate based on molecular charge and structure differences, creating differently colored bands. 
Photosynthesis Lab Activity
Choose just how in-depth you’d like to go with photosynthesis studies, with a two-part activity that explores different aspects of the complex process.The first part of the activity focuses on the Hill Reaction, as students carry out partial photosynthesis in the lab by splitting water. In the second part of the activity, students obtain the enzyme phosphorylase and use it to catalyze the synthesis of starch.
