Updated June, 2005

Race to Find the Cure

Isolation of Chemicals from Plant Leaves

Modified July 2005 by Debbie Gevirtzman,

Southwest Environmental Health Sciences Center, University of Arizona

Based on a modification of “Isolation of Synthetic Chemicals from Plant Leaves” (From Chemicals in the Environment Activities, Project LABS - Learning About Basic Science, Original Developers: Andrea Martin, Abington Friends School, Jenkintown, PA; Dr. Paul Reibach and Dr. Diana Bender, Rohm and Haas Company, Spring House, PA). The original modification was done by Stefani D. Hines, Southwest Environmental Health Sciences Center, University of Arizona, August 2000.

Summary: This activity simulates the extraction, identification and separation of chemicals in or on plants using paper chromatography. Students work in groups representing different pharmaceutical companies who are racing to find a miracle cure for cancer.

Application:

·  use of chemicals in plants as pharmaceutical drugs

·  qualitative analysis techniques

·  isolation of synthetic chemicals such as pesticides for plants

Materials:

Fresh spinach Rulers

Fresh beet leaves Scissors

Distilled water Plastic pipettes

Blender (or mortar and pestle) Weigh boats

Strainer and container to fit below Ring stands

18 small vials with lids* String

Red, green, blue and yellow food coloring Paper clips

Whatman #1 filter paper 100 mL beaker

Pencils

Copies of “Student Instructions and Questions”**

*Film canisters work nicely. Photo shops will often give them away for free!

**The number of copies will vary depending on class size, number of pharmaceutical companies, one set per group vs. one set per student, etc.

Background Information:

In this activity, students will simulate being pharmacists from different pharmaceutical companies trying to find a cure for cancer. Some types of medication are derived from plants. Students will do a simulation of real work done by scientists by using pureed plant leaves and the technique of paper chromatography.


Chromatography

Chromatography was developed in the early 1900’s by Alexandr Tswett, a Russian botanist trying to extract the different pigments in leaves that were changing color in the fall. The word “chromatography” originates from Greek = “color writing”

Chromatography is a physical method to separate the parts of a mixture. The mixtures in this activity are plant leaves pureed in a blender with a little bit of water (but the teacher should not tell the students the types until the end of the lab).

There are a variety of types of chromatography using gases, gels, electric charges, glass tubes, etc., but we will do a simple type called paper chromatography.

Paper Chromatography

  1. A small amount of the pureed plant matter is placed near the bottom end of a strip of filter paper.
  2. Only the tip of the bottom end of the paper will be placed in water.
  3. The water acts as a solvent and the pureed plant dissolves into the water as it moves up the paper.
  4. Water moves up the filter paper because of capillary action, causing the pureed plant matter to percolate up the filter paper with the water.
  5. The filter paper is called the stationary phase.
  6. The pureed plant dissolved in the water is called the mobile phase.

The pureed plant samples contain different chemicals (in this lab, chlorophyll and added food coloring). These chemicals will appear as different colors in this experiment. A plant may contain one or more chemicals. In these samples, students will be looking for mixtures with one or two different chemicals. The different colors that represent each chemical will move along the filter paper at different rates. Some colors travel faster and farther than others, so the different colors spread out along the filter paper. The end result is called a chromatogram.

Why do some colors travel faster and further?

·  Some pigments are more soluble in the water, so they move at a faster rate.

·  The size of the pigment molecule is also a factor. Larger molecules travel more slowly, thus less far up the filter paper.

Chromatography is special because:

·  It can separate very complex mixtures with great precision – ie. it can separate proteins that may only vary by a single amino acid.

·  It can be used to separate delicate products.

·  It can separate very small quantities of substances from each other.

·  Chromatography can be used in many different fields of science besides pharmacology – microbiology, molecular biology, chemistry, biotechnology…. It can be used to determine the ingredients that make up a flavor, or to test for drugs in a urine sample, to determine if pesticides are in soil or water, or to separate proteins.

Comparing the Results

Once students have made their chromatograms, they will compare them to the standards for chemicals A, B, C and D that the teacher will have already made. Students will try to identify which chemicals are in their samples. They may have a single chemical or combinations of two chemicals (ie. B/C or A/D). They may have an unknown chemical that does not match any of the standards. The unknown chemical is natural red coloring in the beet leaves. This is the chemical the “pharmacists” are looking for, because it perhaps could be a cure for cancer (or some other disease). The motivating aspect of the experiment is the competition among the students to answer the question, “Which pharmaceutical company will find the cure?”.

In the Real World…
Although a pharmaceutical company may think it has found a cure for a disease, it may take a great deal of time and money until the medicine may be used in patients.
On average, only five out of 5,000 medicines are tested in clinical trials (the rest fail in lab or animal studies). Only one of these five is eventually approved for use in patients. A pharmaceutical company spends approximately $800 million during the 10-15 years it takes to bring a new medicine from the lab to your pharmacy. (Davis, A. Findings. National Institute of General Medical Sciences. NIH Publication No. 04-4932, February 2004.)
Teacher Preparation:

A. Sample Preparation:

1.  Place spinach and a very small amount of water in a blender and blend. You want the liquid to be very concentrated and dark green.

2.  Pour the mixture into a strainer with a container below.

3.  Distribute the liquid in the container into 16 of the vials.

4.  Repeat steps 1 and 2 for the beet leaves, and put the liquid into the remaining 2 vials.

5.  Add 2 drops of food coloring to each vial according to the table below. For samples with two different colors, add 2 drops of each color to the vial. Label each vial with the sample number according to the table below. You will end up with two sets of nine vials.

Sample # / Color / Standard ID
1 / red / C
2 / green / D
3 / blue / A
4 / yellow / B
5 / unknown (beet)
6 / red/green
7 / yellow/red
8 / blue/green
9 / blue/yellow


B. Standards Preparation:

Standards A, B, C, and D (samples 1-4) will be used by your students to determine which samples they have. The directions below are for one set of standards, but you may choose to make several sets of standards so that they are more easily shared by a large class. You do not need to make a complete set of all nine samples, although you may choose to so that students can see the complete set of samples at the end of the experiment.

  1. Make your own chromatography strips using Whatman #1 filter paper. You will cut 4 strips that are each 9 cm long x 2 cm wide. Use a pencil and ruler for accurate measurements.
  2. Mark each blank chromatography strip with two horizontal lines. One line measures 2 cm from the bottom; the other line measures 4 cm from the top. Use a pencil and a ruler. See diagram in the appendix.(Coming soon)
  3. At the top of each strip, use a pencil to label A, B, C, and D.
  4. Place two ring stands far enough apart so that 4 weigh boats can fit in between the two stands, with some space (~1”) between each weigh boat.
  5. Tie a piece of string to each rod approximately 10 cm up from the base of the ring stand. If the string slips down, secure with tape.
  6. Using a different pipette for each chromatography paper strip, apply a line of the monochromatic (single color) samples to the penciled line drawn at 2cm on each strip of chromatography paper. Be sure to use a different pipette for each vial so there is no cross-contamination.

Strip A / Sample 3
Strip B / Sample 4
Strip C / Sample 1
Strip D / Sample 2
  1. Hang each chromatography strip from the string using a paper clip; be sure to center each strip over a weigh boat.
  2. Fill each weigh boat with approximately 50 mL of distilled water, just enough to allow only the tip of each strip to dip into the water. (Do not allow the sample placed along the 2 cm line to be placed directly in the water, or it will dissolve into the water, rather than move up along the strip!)
  3. Remove the weigh boats when the water reaches the top (4 cm) line on the chromatography strip. This will occur after approximately 10-15 minutes. Allow the strips to hang to dry for a few minutes. Then, lay the strips flat.
  4. Mount the strips onto a piece of cardstock. You might consider laminating the cardstock or putting into a clear pocket to protect the chromatograms from your students’ wet hands!


Student Activity:

1.  Break up the students into six groups, each representing a different pharmaceutical company. Each company is competing to find a plant containing the potential “miracle cure” for cancer. (Six company name sheets are included with this activity, or have your students create their own names.)

Note: This activity can actually be done in groups as small as two students, allowing for more hands-on experience. For large class sizes, this would mean creating more than 6 pharmaceutical companies and making more than 2 vials of each sample.

2.  Distribute the samples to each company according to the table below.

PHARMACEUTICAL COMPANIES /
SAMPLES
/
Answer "CHEMICALS"
BLTC Co. / 1 / C
5 / unknown
8 / AD
We Save U Pharmaceuticals / 2 / D
6 / CD
9 / AB
Mighty Meds Company / 3 / A
4 / B
7 / BC
Mortar and Pestle, Inc. / 4 / B
5 / unknown
9 / AB
Fix U Up Pharmaceutical Company / 2 / D
6 / CD
8 / AD
Pharmaceuticals R Us / 1 / C
3 / A
7 / BC

Note: Some blue food coloring will have blue and red lines and some green food coloring may have blue and yellow lines, so answers may vary slightly.

3.  Have students follow the directions on the student worksheets.

4.  Extensions:

·  Grow spinach and beet plants with food coloring in the water, or spray food coloring onto the plants to simulate pesticides.

·  Have each group calculate the Rf (ratio of fronts) for each standard (Rf = dye distance/solvent distance). The Rf is characteristic for any given chemical. Groups can then compile their data and calculate the average Rf for each standard.

Student Worksheet Answer Key:

  1. Discuss the following questions with the members of your pharmaceutical company while waiting for your chromatograms to finish.
  1. Why do you think the chemicals separate?
Different molecular weights; differing solubilities of the chemicals
  1. How do you think this technique can be useful?

Can be used to physically separate/isolate chemicals; can be used to identify chemicals

  1. Why would we want to separate chemicals?

Application of a specific chemical for its properties – e.g. pharmaceuticals, pesticides; its behavior can be affected in the presence of other chemicals

d.  *Advanced Question* - Based on what you know about molecules, what are some other potential chromatographic or separation techniques?

SIZE: Gel-filtration chromatography – Separates proteins based on size and shape; uses carbohydrate polymer beads in a column; smaller molecules are diffused into the beads and are retained in the column longer than larger molecules

CHARGE: Ion-exchange chromatography and electrophoresis – Separate molecules based on the charge they carry. Electrophoresis uses electrodes and is a very powerful technique. More than 1,000 different proteins have been extracted from one species of bacterium in just one experiment!

BONDING AFFINITY: Affinity chromatography – separates molecules based on hydrogen bonds and other attractive forces

From Vollhardt, K.P.C. and Schore, N.E. Organic Chemistry: 2nd Edition. W.H. Freeman and Company, New York. 1997.

14. Discuss the answers to the following questions with the members of your pharmaceutical company and write down your answers.

a. What are some challenges you can see from trying to extract/isolate a chemical?

Still may not have isolated a specific chemical, i.e. could be a complex mixture; get a very small amount of the chemical per plant

b.  Plants can vary in the amounts of compounds they contain within a species or even with in a given plant. What are some factors you can think of that can affect the amount of chemical a plant produces?

Growth conditions; genetics; time of year

c.  How can this variation affect you as a consumer?