Materials Included In Kit

Additional Materials Required

Prelab Preparation

Experiment I. Separation of Pigments in Inks

Prepare individual chromatography strips from the chromatography paper sheets provided.

1. From a chromatography sheet, cut six strips, each 13 cm long x 2 cm wide.
2. Using a pencil, lightly draw a line across the width of each strip, 2 cm from one end (see Figure 7a).
   {12602_Preparation_Figure_7}
3. Cut off the bottom corners of each strip to create a point, as shown in Figure 7b. Staple or tape the strip to a wooden splint, as shown in Figure 7c. Repeat for all six strips.

Experiment II. Separation of Plant Pigments

Prepare one chromatography strip as described in the Preparation for Experiment 1. Also, shake the bottle containing chromatography solvent to ensure that the two components are mixed well.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The water in the Erlenmeyer flasks may be poured down the drain. The chromatography strip and spinach leaf can be placed in the trash. The chromatography solvent should be returned to the instructor and disposed of according to Flinn Suggested Disposal Method #18a.

Lab Hints

• Proper technique is required for students to see well-separated compounds on the chromatography splint. Poor separations or results are usually caused by too much starting material placed on the initial spot or band, initial spot or band too large or the initial spot or band is below the solvent level in the developing chamber.
• Allowing enough time for the development of the strip is critical. The strip must be left in the chromatography chamber long enough for the solvent to be drawn up near the top of the strip. Do not stop the development until the solvent front nears the top of the strip. Underdevelopment will lead to incomplete separation.
• Solvent used for development can be recycled. Do not discard leftover chromatography solvent. Save it for use by another class or other chromatography development. Do not leave the chromatography solvent uncovered for long periods of time—one component may evaporate faster than the other, changing the polarity of the solvent.

Teacher Tips

• Experiment I. Separation of Pigments in Inks
  
  1. If you do not have enough Erlenmeyer flasks for Experiment 1, you can do one of three things
     1. Have each student or group only use one pen and strip, or
     2. Use an alternate chromatography chamber set-up, as shown in Figure 8, using a large test tube. (A graduated cylinder may also be used.)
        {12602_Tips_Figure_8}
     3. You may try to place two strips in each flask. However, the strips cannot touch each other.
  2. The apparent pigment colors we found in our six pens, using water as a solvent were: (If the pigment fluoresced under UV light, it is marked with a ‡.)
     1. Black: dark blue, light blue, orange, yellow
     2. Blue: dark blue, light blue
     3. Brown: dark purple, yellow,‡ pink‡
     4. Green: light blue, yellow
     5. Red: yellow,‡ pink,‡ (under UV light, a fluorescent orange also appeared to be present)
     6. Yellow: yellow‡

        Note: The particular pens we used may or may not be the ones shipped with this kit. Manufacturers may change ink formulations without notice, or pens may be substituted depending on availability.

  3. A terrific extension of this activity is to adjust the polarity of the solvent to try to achieve better separations. In fact, when we tried a 50/50 mixture of water and acetone, we got some very interesting results. The yellow ink, which we previously thought contained only one pigment, separated into two yellow pigments, only one of which was fluorescent. The yellow and pink pigments in the red and brown inks actually reversed their order on the strips.

• Experiment II. Separation of Plant Pigments
  Each strip will reveal four to five different pigments. The pigments can be identified by their colors and their relative positions on the chromatogram. The major pigments seen in spinach are (in order from the initial spot; see Figure 9).
  {12602_Tips_Figure_9}
  1. chlorophyll b (olive-green) 
  2. chlorophyll a (blue-green) 
  3. violaxanthin (yellow)
  4. lutein (gray-yellow)—(very difficult to see) 
  5. carotenes (yellow-orange), near the solvent front
  Other pigments may be visible but these are more difficult to identify. Sometimes neoxanthin is also present but is usually very close to the chlorophyll b; and therefore not visible. Neoxanthin, violaxanthin and lutein are all xanthophylls.
  
  As an extension, have students run chromatograms prepared from other plants, including red- and yellow-leaved plants, and compare results.
  
  The chromatography solvent contains 90% petroleum ether and 10% acetone.

References

Bregman, A. A. Laboratory Investigations in Cell Biology, 2nd ed.; John Wiley & Sons: New York, 1987; pp 119–123.

Green, N. P. O.; Stout, G. W.; Taylor, D. J. Biological Science: Organisms, Energy, and Environment: 2nd ed.; Saper, R., Ed.; Cambridge University: Cambridge, MA, 1990; pp 255–257.

Russo, T.; Meszaros, M. W. Vial Organic; Flinn Scientific: Batavia, IL, 1996; pp 25–33.

Wilkins, M. B., Ed. Advanced Plant Physiology; Pitman: Marshfield, MA, 1984; pp 221–224.

Introduction

Chromatography is a popular method used to separate organic compounds for identification or purification. Discover the multitude of pigments present in ink and spinach by analyzing a chromatograph.

Concepts

• Chromatography
• Plant vs. ink pigments
• Photosynthesis

Background

Chromatography is one of the most useful method of separating organic compounds for identification or purification. There are many different types of chromatography but most work on the concept of absorbance. The two important components of chromatography are the absorbent and the eluent. A good absorbent is usually a solid material that will attract and absorb the materials to be separated. Paper, silica gel or alumina are all very good absorbents. The eluent is the solvent which carries the materials to be separated through the absorbent.

Chromatography works on the concept that the compounds to be separated are slightly soluble in the eluent and will spend some of the time in the eluent (or solvent) and some of the time on the absorbent. When the components of a mixture have varying solubilities in the eluent, they can then be separated from one another. The polarity of the molecules to be separated and the polarity of the eluent are very important. Changing the polarity of the eluent will only slightly change the solubility of the molecules but will greatly change the degree to which they are held by the absorbent. This affinity for the eluent versus the absorbent is what separates the molecules.

Paper chromatography is often used as a simple separation technique. In paper chromatography, the absorbent is the paper itself, while the eluent can be any number of solvents. The polarity of the eluent is very important in paper chromatography since a small change in polarity can dramatically increase or decrease the solubility of some organic molecules. Many times, a mixture of a nonpolar solvent and a polar solvent is used to achieve an optimum polarity. When placed in a chromatography chamber as shown in Figure 1, the eluent moves up the strip, being drawn by capillary action. The organic molecules, which were “spotted” onto the paper chromatography strip, separate as they are carried with the eluent up the strip at different rates. Those molecules that have a polarity closest to the polarity of the eluent will be the most soluble, and will move up the strip the fastest.

The choice of the eluent or solvent is the most difficult task. Choosing the right polarity is critical because this determines the level of separation that will be achieved. Common solvents used in chromatography, in order of increasing polarity, are: petroleum ether or hexanes, cyclohexane, toluene, chloroform, ethyl ether, acetone, ethanol, methanol, and water. Sometimes mixtures of solvents are used to achieve the desired degree of polarity. A general rule of thumb is if the substances to be separated are polar, the developing solvent should be slightly less polar. Likewise, non-polar substances would require slightly polar solvents.

Experiment I. Separation of Pigments in Inks 

Many inks are actually mixtures made up of several basic pigments. Each of these pigments has a different molecular structure and, usually, a different polarity. Many of these pigments can be easily separated using paper chromatography. 

Experiment II. Separation of Plant Pigments

I. Photosynthetic Pigments
Photosynthesis is the process by which plants use the energy in sunlight to convert carbon dioxide and water to glucose. Almost all living organisms directly or indirectly rely on photosynthesis to provide the basic building blocks for cells and tissues.

The first step of the photosynthetic process involves the absorption of sunlight by various pigment molecules in the plant. These pigment molecules absorb certain wavelengths of visible light very strongly, giving them characteristic colors. Structurally, it is the multiple conjugated (alternating) double bonds in these pigment molecules which allow them to absorb light energy (see Figures 2–5). The major pigments of photosynthetic organisms are the chlorophylls. Chlorophylls are responsible for the green coloring of most plants, as these pigments absorb light strongly in the red and blue-violet regions of the visible spectrum and transmit or reflect most light in the green region (see Figure 6). There are two types of chlorophyll found in higher plants, chlorophyll a and chlorophyll b. Other chlorophylls occur in some types of single-celled organisms and algae (see Table 1). Besides chlorophylls, plants also contain other pigments used to collect light energy. Sometimes known as accessory pigments, these molecules include carotenes, xanthophylls and phycobilins. Normally, the abundant chlorophylls mask the colors of these other relatively scarce pigments. However, in autumn, as chlorophylls begin to break down and lose their color, it is these accessory pigments (which are still active) that give autumn leaves their brilliant red, yellow and orange colors.

II. Structure and Function of Photosynthetic Pigments
Figure 2 (see Part I) shows the structural formula for the chlorophylls. In chlorophyll a, the “R” group is a methyl group (–CH₃). In chlorophyll b, the “R” group is an aldehyde (–CHO). Essentially, it is the conjugated double bonds of the porphyrin ring (shown in the figure) which determine the shape of the absorption spectrum (see Figure 6 in Part I). While only chlorophyll a participates directly in the conversion of light energy to chemical energy, chlorophyll b assists the process by broadening the range of wavelengths absorbed and then transferring energy directly to chlorophyll a.

Figures 3–5 (see Part I) show the structures of three common carotenoids, which include the carotenes and xanthophylls. β−carotene is a carotene; and lutein and violaxanthin are common xanthophylls. Structurally, the carotenes are composed entirely of carbon and hydrogen while the xanthophylls also contain oxygen. Note the multiple conjugated double bonds in these molecules. It is these conjugated double bonds which are responsible for the carotenoids’ absorption of blue light and reflection, or transmission, of yellow, orange and red light.

Like chlorophyll b, the accessory pigments serve to further broaden the range of wavelengths which can be utilized by the plant for photosynthesis, also transferring energy to chlorophyll a. Carotenoids may also protect the chlorophylls from excess light and from oxidation by oxygen produced in photosynthesis.

In this experiment, separate and identify five pigments found in spinach leaves using paper chromatography.

Materials

Procedure

Experiment I. Separation of Pigments in Inks

1. Add 50 mL of water to each Erlenmeyer flask.
2. Using a chromatography pen, place a small dot on the center of the drawn line on one chromatography strip. Repeat for each pen on a separate chromatography strip. Using a pencil, write the color of the pen on the top of the strip or on the wooden splint.
3. Slowly lower one chromatography strip into an Erlenmeyer flask. The sample spot should remain above the solvent (the water). If it is not, your sample will dilute into the solvent.
4. Repeat step 3 for each chromatography strip.
5. The solvent will be drawn up the chromatography strips by capillary action. As it is drawn up, it will carry the pigments in the samples up the strips at different rates depending on the characteristics of the individual compounds.
6. When the solvent front is within 0.5–1.0 cm of the top of the chromatography strip, the run is stopped by removing the strip from the flask.
7. It is a good idea to carefully mark the location of each of the pigment spots on the strips and the final solvent fronts, again using a pencil. This is done because some of the color and brightness of each of the spots may be lost over time. During this time, the residual water on the strips may continue to be drawn up the strip slightly by continued capillary action. If you have marked the location of the pigment spots in pencil, this is not a concern.

Experiment II. Separation of Plant Pigments

1. Add 50 mL of chromatography solvent (containing about 90% petroleum ether and about 10% acetone) to the 250-mL Erlenmeyer flask. Cover with Parafilm or aluminum foil.
2. Place the spinach leaf on top of the chromatography strip. Slowly roll the penny over the leaf along the pencil line drawn on the strip. Repeat this action 3–4 times. It is important that your chromatography strip contains a single, relatively narrow, horizontal green line. Note: This step may take some practice!
3. Remove the Parafilm or aluminum foil (Do not discard!) and slowly lower the chromatography strip into the Erlenmeyer flask. The green line containing the plant pigments should remain above the solvent. If it is not, the pigments will dilute into the solvent.
4. Carefully replace the Parafilm or aluminum foil to cover the mouth of the flask.
5. The solvent will be drawn up the chromatography strip by capillary action. As it is drawn up, it will carry the pigments in the sample up the strip at different rates depending on the characteristics of the individual compounds.
6. When the solvent front is within 0.5–1.0 cm of the top of the chromatography strip, the run is stopped by removing the strip from the flask. Allow the strip to dry.
7. It is a good idea to carefully mark the location of each of the separated bands on the strip and the final solvent front, again using a pencil. This is done because some of the color and brightness of each of the spots is lost over time.

Student Worksheet PDF

12602_Student1.pdf