Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Obtain a 100-mL graduated cylinder and measure 100 mL of acetone.
2. Transfer the acetone to a clean 250-mL beaker.
3. Using a graduated cylinder measure 100-mL of hexanes.
4. Add the hexanes to beaker containing acetone.
5. Cover with Parafilm to prevent evaporation.

Safety Precautions

Acetone and hexanes are flammable liquids and dangerous fire risks. Acetone is also slightly toxic by ingestion and inhalation. Hexanes are a respiratory irritant. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

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. Dispose of remaining acetone or hexane by evaporating in a fume hood. Dispose of the dry spinach powder, sand, aluminum oxide, and the chromatography column in the regular trash according to Flinn Suggested Disposal Method #26a.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. The experiment can reasonably be completed in one 50-minute class period. The pre-laboratory assignment should be completed before coming to lab to ensure that students have read the procedure and understand the general design of chromatography.
• This lab may be expanded by performing column chromatography on other plant extracts such as tomatoes, carrots, and other leaves.

Teacher Tips

• Chromatography is ideally studied while learning about elements, compounds and mixtures.
• Radial chromatography and thin layer chromatography (TLC) are also means of separating mixtures using a slightly different technique. Below is a list of the kits Flinn offers that study each method. Radial Chromatography— Radial Chromatography T-Shirts! The Experiment You Can Wear! Flinn Catalog No. AP8686. Radial Chromatography, Chemistry with an Artistic Flare, Flinn Catalog No, AP8687. Thin-Layer Chromatography— Introduction to Thin Layer Chromatography, Flinn Catalog No. AP4504 Thin Layer Chromatography Kit, Flinn Catalog No. AP9095

Answers to Prelab Questions

1. What is the purpose of conducting column chromatography?

   The purpose of column chromatography is to separate and identify the components in a mixture.

2. What are the eluents used in this laboratory activity? Note: Reference the Procedure section.

   The eluents used in this laboratory are hexane, 50/50 hexane–acetone mixture and acetone.

3. Why is it important to pack the solid in the column and keep it wet throughout so that there are no holes, spaces or cracks in the adsorbent phase?

   Holes or spaces in the packing of the solid phase will leave channels for the substances to travel through and lead to poor separation.

Sample Data

Answers to Questions

1. How did conducting column chromatography confirm the presence of accessory pigments in spinach leaves?

   Although the plants appear green because they are most abundant in chlorophylls they do indeed contain accessory pigments which were viewed and collected in two additional bands.

2. Use an additional reference source such as a textbook or the Internet to rank the eluents used in this lab according to their polarity. List the nonpolar eluent first.

   Hexane, 50/50 hexane–acetone, and acetone.

3. Given the order which the bands exited the column and the polarity of each eluent, which color pigment was the most polar?

   Traditionally nonpolar eluents are added to the chromatography column first. This is true in this experiment as nonpolar hexane was added first. The dark green pigment is the most polar as it didn’t travel through the column until the polar eluent acetone was added.

4. 1. Column chromatography is performed on a leaf extract from a maple tree. Chromatography produces a green band, as expected, but also separates red, orange, and yellow pigments. Use additional resources to predict the identity of these pigments.

      In photosynthetic organisms the red pigment present is likely anthocyanin, the orange colors come from carotene and the yellow pigments originate from xanthophylls.

   2. Relate this phenomenon to the color of leaves produced in the fall.

      Often times these pigments become visible when leaves begin to die in the fall. The majority of the time the chlorophyll pigment masks the accessory pigments. However, in the fall, as the chlorophylls begin to break down and lose their color, the accessory pigments are still active and give autumn leaves their characteristic colors.

References

Cesa, I. Flinn ChemTopic^™ Labs, Volume 2, Elements, Compounds and Mixtures; Flinn Scientific: Batavia, IL; 2005; pp 25–36.

Introduction

Spinach is a leafy green vegetable rich in iron. However, besides green chlorophyll, additional pigments that are not visible to the naked eye are also present in spinach. The presence and identity of additional accessory pigments in spinach may be determined using column chromatography.

Concepts

• Chromatography
• Adsorption
• Eluents
• Polarity

Background

The word chromatography is derived from two Greek words meaning color (chroma) and to write (graphein)—“color writing.” The term was coined by the Russian chemist Michael Tswett in 1903 to describe a new technique he had invented to separate the pigments in green plant leaves. Tswett found that in addition to chlorophyll, the main green pigment, plant leaves also contained red and yellow secondary pigments. The results were literally “written in color” when a plant extract was passed through a column containing a clay-like adsorbent solid.

More than 100 years after Tswett’s discovery, chromatography has evolved into the most important tool chemists have for separating the components in a mixture. Some examples of different types of chromatography and their uses include:

• Gas chromatography, which is used in forensics and toxicology to analyze drugs and other substances in blood samples.
• Gel-permeation chromatography, which is used to separate and purify proteins.
• Ion-exchange chromatography, which is used to remove ions from water, also known as water softening.

Column chromatography is an example of a more general type of chromatography called adsorption chromatography. The column contains a solid, such as aluminum oxide, Al₂O₃, which acts as the adsorbent. A thin layer of the mixture to be separated is placed on top of the adsorbent. A flow of a liquid eluent or solvent is washed through the column, carrying the components of the mixture to be separated down the solid column.

The rates at which the components travel down the column depend on their relative affinity for the adsorbent versus the eluent. Those components that have a greater affinity for the adsorbent will remain in the column longer, traveling at a slower rate. On the other hand, those components that have a lesser affinity for the adsorbent will not interact with the adsorbent. The components of the mixture that have little affinity for the solid adsorbent will not bind as strongly to the solid phase and will travel through the column at a faster rate with the liquid mobile phase. As the components in the original mixture travel down the column at different rates, they begin to separate into distinct bands. Ideally, each band will contain only a single component from the original mixture, resulting in separation of the mixture.

Successful separation of substances via column chromatography is based on two properties of the substance being separated— its adsorptivity on the solid and its solubility in the eluent. Adsorptivity is the adhesion of the molecules in the substance being separated to the molecules on the surface of the adsorbent. The adsorbent gets its name because it has the ability to bind and hold certain molecules in the mixture to be separated. Different materials with different polarities or other chemical properties may be used as the adsorbent. Aluminum oxide, Al₂O₃, and silica gel are commonly used as adsorbents.

The affinity with which molecules in the mixture being separated will “stick” to the adsorbent particles depends on intermolecular forces. Intermolecular forces are the relatively weak interactions that occur between molecules. The types of intermolecular forces present depend on the nature of both the adsorbent and the substances in the mixture. Nonpolar compounds generally exhibit only weak dispersion forces. Polar compounds display weak dispersion forces and stronger dipole–dipole forces. Traditionally, the adsorbent is a relatively polar material and the eluent is rather nonpolar. Therefore, the more polar the compound in the mixture being separated, the stronger its intermolecular forces to the adsorbent will be, resulting in the compound being very slow to travel through the column.

The choice of the eluent is critical to the success of the separation in column chromatography. Very rarely will a single solvent be able to separate all the components in a mixture. Typically a single solvent may not move the mixture at all or it will carry all of the components at once. To compensate, the composition of the eluent is varied during the process. First a nonpolar solvent is used to remove or carry the nonpolar components through the column. Then solvents with gradually increasing polarity are used until all the components have been removed from the column.

Experiment Overview

The purpose of this experiment is to learn the general technique of column chromatography by separating spinach pigments. Spinach pigments will be separated and collected by the use of multiple eluents that vary in polarity.

Materials

Prelab Questions

1. What is the purpose of conducting column chromatography?
2. What solvents will be used as the eluents in this laboratory activity? Note: Read through the Procedure section.
3. Why is it important to pack the solid in the column and keep it wet throughout so that there are no holes, spaces or cracks in the adsorbent phase?

Safety Precautions

Acetone and hexane are flammable liquids and dangerous fire risks. Acetone is also slightly toxic by ingestion and inhalation. Hexanes are a respiratory irritant. Wear chemical splash goggles, chemical-resistant gloves, and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

Preparation of the Chromatography Column

1. Attach the clamp to the support stand.
2. Place the tip on the bottom of the chromatography column. Pour about 2.5 mL of hexane into the column so that the liquid fills the narrow portion of the column.
3. Slowly pour 2 g of aluminum oxide into the column. Fill the narrow portion of the column until it is about ¾ full. Tap the tip of the column on the lab bench while pouring the aluminum oxide—tapping the column sufficiently will eliminate holes or spaces. Having holes or spaces in the packing of the solid will leave channels for the substances to travel through and lead to poor separation.
4. Place the column in the clamp and tighten until the wide portion of the column is held firmly by the clamp.
5. Place an empty 250-mL beaker underneath the column.
6. Carefully pour a small amount of sand into the column so it forms a 2-mm layer on top of the aluminum oxide (see Figure 1).
   {12043_Procedure_Figure_1}

Preparation and Separation of Spinach Extract

9. Obtain 10 mL each of the following solvents—hexane, 50/50 hexane–acetone, and acetone. Place each solvent in a clean 50-mL beaker or flask.
10. Weigh 0.5 g of spinach powder and place it in a 13 x 100 mm test tube. Add 1 mL of the 50/50 hexane–acetone mixture.
11. Insert a stopper into the test tube and shake vigorously. Continue shaking until the liquid (extract) begins to turn a dark green. Place the test tube in a test tube rack.
12. Remove the tip from the chromatography column and allow the hexane in the column to drain into the beaker below. Allow it to drain until the liquid level is just above the sand layer. Replace the tip on the column.
13. Shake the test tube containing the spinach extract and allow the solid to settle. Place a clean pipet into the test tube and fill the pipet with only the liquid portion of the extract. Avoid obtaining solid in the pipet.
14. Remove the tip from the column and add 5 drops of the spinach extract to the top of the column by running it down the inside of the column in a circular fashion. Do not simply squirt it into the center of the column as this will disturb the sand layer.
15. As soon as the extract is absorbed into the sand layer, carefully add a pipet-full of hexane to the column by running it down the inside of the column in a circular fashion. Continue adding hexane in this manner until the entire column is full.
16. As the solvent moves through the column, it will begin to carry one of the pigments with it. This will take several minutes as the pigments interact with the adsorbent phase. Colored bands should become visible in the column.
17. Label three clean test tubes pigment 1, pigment 2, and pigment 3.
18. As the first pigment band begins to exit the tip of the column, place the appropriate test tube underneath the column to collect the solvent containing this pigment. As soon as the band has completely exited the column, remove the test tube, stopper it and set it aside. Record observations in the data table. The same general procedure will be used to collect additional pigments as they pass through the column. Different solvents, first 50/50 hexane–acetone and then acetone, will be needed to collect pigments 2 and 3, respectively. In each case the same general procedure should be followed. Never allow the top of the column to run completely dry. Add each solvent carefully by running it down the inside of the column in a circular fashion so as not to disturb the sand layer. Collect each pigment band solution in a different test tube. Replace the test tube with a beaker to collect eluent that does not contain a pigment.
19. Collect remaining hexane in the 250-mL beaker. Do not allow column to go dry.
20. Using a pipet, add the 50/50 hexane–acetone mixture to fill the entire column.
21. As the hexane–acetone mixture begins to flow down the column, it will carry another pigment band with it. Collect the eluent in the 250-mL beaker until the colored band is at the bottom of the column.
22. Collect the solvent containing the second band in the appropriate test tube. Record observations in the data table.
23. Allow any remaining hexane–acetone to drain into the 250-mL solvent beaker. Leave a thin layer so the column remains wet.
24. Using a clean pipet, add acetone to fill the column.
25. As the acetone begins to flow down the column, it will carry the last spinach pigment with it. Add more acetone as needed to make sure the column does not dry out until the third band has almost reached the bottom.
26. Collect acetone eluent in the 250-mL solvent beaker until the third band approaches the bottom of the column. Replace the beaker with the third test tube to collect the last pigment band. As soon as the band has completely exited the column, remove the test tube, stopper it and set it aside. Record observations in the data table.
    {12043_Procedure_Figure_2}

Student Worksheet PDF

12043_Student1.pdf