RACE TO FIND THE CURE – INTRODUCTION - University of Arizona

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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 pa
per 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 s
ynthetic 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.


Updated June, 2005

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 writ
ing”


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 en
d 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 che
micals (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 c
hemicals. 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
chr
omatogram
.


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 pap
er.


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.

Updated June, 2005


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. Th
e 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 b
e 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 $80
0 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 Preparat
ion:

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 vial
s.

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 v
ial 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




Updated June, 2005

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 W
hatman #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 m
easures 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 lin
e 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

Sampl
e 1

Strip D

Sample 2


7.

Hang each chromatography strip from the string using a paper clip; be sure to
center each strip over a weigh boat.

8.

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!)

9.

Remove the weigh boats when the water reaches the top (4 cm) line on th
e
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.

10.

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!


Updated June, 2005

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 poten
tial “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"

B
LTC 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 bee
t 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 chem
ical.
Groups can then compile their data and calculate the average Rf for each
standard.


Student Worksheet Answer Key:

11.

Discuss the following questions with the members of your pharmaceutical
company while waiting for your chromatograms to finish.

Updated June, 2005

a.

Why d
o you think the chemicals separate?

Different molecular weights; differing solubilities of the chemicals


b.

How do you think this technique can be useful?

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


c.

Why wou
ld 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 retain
ed 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 protei
ns 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:
2
nd

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. Wha
t 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?

In unregulated industries such as herbal products, you do not know how

much
of the “active” chemical you are getting within a single brand or between
brands. In regulated industries where there are standards, such as
pharmaceuticals, this variation can affect prices. This is why plants are bred
for maximum yield and consis
tency.

Updated June, 2005


Group Members:__________________________________________________________

Pharmaceutical Company:__________________________________________________


Race to Find the Cure

Student Instructions and Questions


Summary:

This activity simulates the ext
raction, identification and separation of
chemicals in or on plants using chromatography. Students work in groups representing
different pharmaceutical companies racing to find the miracle cure for cancer.


Materials (per group):

1 sheet Whatman #1 filter

paper



3 weigh boats

2 pencils






distilled water

2 rulers






100 mL beaker

2 pairs of scissors





2 ring stands

3 vials containing unknown extracts



string

3 plastic pipettes





3 paper clips



Background Information:

Chromatography is a physica
l method to separate the parts of a mixture. There are a
variety of types of chromatography using gases, gels, electric charges, glass tubes, etc.,
but this activity uses a simple type called
paper chromatography
.


You will use filter paper and put a sm
all amount of each of your samples near the end of
the paper strip. The tip of the paper will be placed in water. The water acts as a
solvent

and the sample dissolves into it. As the water moves up the filter paper because of
capillary action, the sampl
e will percolate up the filter paper as well. The filter paper is
called the
stationary phase
. The sample dissolved in the water is called the
mobile
phase
.


Your group will receive three samples to test. Each sample contains different chemicals.
These
chemicals will show up as different colors in today’s simulation. A sample may
contain one or more chemicals. In your samples today, you will be looking for mixtures
with one or two different chemicals. The chemicals will be represented by different
col
ors on the strips of chromatography paper. 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 than others? 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.


Updated June, 2005

Directions:

1.

Break in
to small groups representing different pharmaceutical companies.


2.

Make your own chromatography strips using one sheet of Whatman #1 filter
paper. You will cut 3 strips that are each 9 cm long x 2 cm

wide. Use a pencil
and ruler for accurate measurements.


3.

Using a pencil and ruler, mark each blank chromatography strip with two
horizontal lines. One line should measure 2 cm from the bottom; the other line
should measure 4 cm from the top.


4.

At the t
op of each strip, use pencil to label one sample number from each of your
vials of unknown extracts.


5.

Place two ring stands far enough apart so that 3 weigh boats can fit in between
with some space (~1”) between each weigh boat.


6.

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.


7.

Apply the unknown extracts (your three samples) using pipettes along the bottom
(2cm) line on the chromatography paper, one sample per strip. Be

sure to use a
different pipette for each vial so there is no cross
-
contamination.


8.

Hang each chromatography strip from the string using a paper clip; be sure to
center each strip over a weigh boat.


9.

Fill each weigh boat with approximately 50 mL of distill
ed water, just enough to
allow only the tip of each strip to dip into the water. (Do
not

allow the sample
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!)


10.

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.


11.

While waiting for your chromatograms to finis
h, discuss the following questions
with the members of your pharmaceutical company. Write the answers on this
instruction sheet (as you will discuss them with the class later).

a.

Why do you think the chemicals separate?





b.

How do you think this technique ca
n be useful?


Updated June, 2005



c.

Why would we want to separate chemicals?





d.

*Advanced Question
*
-

Based on what you know about molecules, what
are some other potential chromatographic or separation techniques?






12. Your samples each contain one or two chemicals (i.
e. A, B, A/B, C/D, etc.). You will
try to identify them by comparing your chromatograms to the standards already prepared
by your teacher (chemicals A, B, C, and D). But, some samples may contain a chemical
that does not match the standards. This is the

“unknown” and could be a cure for cancer!
Do you think you have the “unknown chemical”?


Sample #____ contains chemical(s)___________________.


Sample #____ contains chemical(s)___________________.


Sample #____ contains chemical(s)___________________.


13. After you have determined the chemicals on each of your strips, you will isolate the
chemicals on the strip. You do this by extracting the separated chemicals from the
chromatography paper. Cut out the “individual” colors/chemicals from the
chromatog
ram. Place each fraction into a separate weigh boat containing distilled water.
Observe the chemical dissolve into the water.
You have now isolated a chemical!


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

a.

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




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 t
hink of that
can affect the amount of chemical a plant produces?




c.

How can this variation affect you as a consumer?

Updated June, 2005

BLTC Co.

(Better Living
Through Chemistry)



Updated June, 2005

We Save U
Pharmaceuticals
Updated June, 2005

Mighty Meds Co.


Updated June, 2005

Mortar and Pestle,
Inc.


Updated June, 2005

Fix U Up Company


Updated June, 2005

Pha
rmaceuticals ‘R’
Us