Biotechnology Curriculum
Freshman Exploratory
What's in Kool-Aid?
Introduction:
When we look at a solution that is colored, we assume that it is made up of only
one substance, one color. But this may not be true. In today’s activity you will be trying
to separate different substances from each other using a process called chromatography. Certain molecules stick to the paper or column bed material better because they
do not dissolve well in water. Once they are placed onto the paper or column, they can
then be rinsed off by increasingly higher concentrations of alcohol. This is one way to
separate molecules from each other. Another method includes electrophoresis which
you will see in a separate activity.
Materials;
Chromatography paper
Assorted different colored pens and markers
15 ml of 70% isopropyl alcohol
80 ml of distilled water
various food dyes
capillary tubes
a screw cap jar such as an empty peanut butter jar
2 graduated cylinders
4 beakers 25, 50, 100ml
ruler
pencil
hair dryer
Paper Chromatography
1.
2.
3.
4.
Obtain a piece of paper 10 cm x 20 cm
Draw a line with a pencil about 2 cm above the bottom edge
Obtain four black ink pens from different sources
Obtain a piece of chromatography paper and draw a line 2 cm from its bottom edge
See below
A.
Place a circle of ink from each pen equally across the line
Do not allow the liquid to go above the line!!!!
B.
Allow the paper to sit in the jar for 30 minutes.
C. Remove the paper and view where the dye has moved to.
Questions
1. Do you see more then one color for any of the pens?
2. Draw what you see for the position of the different colors in the space below..
B. Adsorption Chromatography of Kool-Aid
Materials:
Sep-pak 18, see teachers notes for source of columns.
5 ml syringe
14% isopropyl alcohol
35% isopropyl alcohol
70% isopropyl alcohol
20 ml of grape, orange, lemon-lime Kool-aid into each separate beaker
prepared by adding 20 ml of water (dd) to a beaker and adding the package of sugar-free
kool aid into the beaker and labeling it.
Waste beaker
Beaker of water (dd)
Test tubes + test tube rack
Procedure:
1. Obtain 14%, 35% and 70% alcohol solutions plus other materials from your
teacher.
2. Wash Sep-Pak cartridges in water by first measuring out 3 ml of water and
then placing Sep-Pak onto the column onto the syringe. Push the water
through the sepak into a waste beaker.
Wash Sep-Pak one more time with water.
3. Measure out 2 ml of Kool-Aid of your choice in your syringe and replace SepPak on syringe.
4. Rinse the sep-pak column with 3 ml of water collecting the liquid that comes
out of the syringe, and capture it in a test tube any colors. Remove Sep-Pak
from the syringe and measure out 3 ml of 14% alcohol with the syringe.
5. Replace the Sep-Pak on the syringe
6. Rinse the column with the 14% alcohol while collecting the eluate in a clean
test tube as in step 4 but using 14% alcohol.
Repeat steps 5,6,7 with 35% alcohol and 70% alcohol
7. Record the Kool-aid colors which you removed from the column in the table
below.
Flavor of
Kool-Aid
Water
14%
Alcohol
35%
Alcohol
70%
Alcohol
Grape
Orange
Lemon-lime
Questions:
1. How could you use a Sep-Pak column if you want to investigate a solution for how
pure it was?
2. Is a colored solution always made up of only one chemical?
3. Why do different color dyes separate on the column?
Teacher Notes:
1. This activity will take one to two hours for both procedures. Sepak cartidges can be
ordered from Millipore Corporation, Bedford, MA.
2. It is important to discuss the idea of a pure substance and a mixture of
Substances and why you might want to know if something is pure.
An example, like a medicine, can be used.
The basis for both separation techniques relies on the different
solubilities of substances. Some like to mix with water ( polar )
some like to mix with alcohol (non-polar).
Theory of Absorption Chromatography
Chemicals, such as dyes, are made of either polar or non-polar
substances.
Paper chromatography will separate these chemicals because of the
differences in solubility of the two types in alcohol or water.
Polar (hydrophilic) substance = water
Non-polar (hydrophobic) substance = oil
Principles of Extraction of Kool-Aid
Sep-Pak columns are composed of non-polar hydrophobic beads that attract non-polar
compounds.
Like solvents dissolve like compounds.
Water = polar compound that dissolves polar and ionic compounds.
Oil = non polar compound that dissolves non-polar compounds.
Alcohol = polar and non-polar compound that dissolves in water, but can dissolve nonpolar compounds as well.
The more non-polar the molecules adhering to the beads, the higher the concentration of
alcohol is needed to remove it from the beads
Weak bonding requires lower concentrations of alcohol. How much chemistry you decide
to discuss with students is up to you and depends on their background.
Note: Sep-Pak cartridges can be ordered from Millipore, Bedford
Alcohol used in this activity is isopropyl alcohol obtained from the grocery store
Grape Kool-Aid will give blue and red dyes, plus a yellow faint dye if done well. The
70% eluate gives a grape like flavor.
Other Kool-Aid flavors that work well are orange and lemon-lime
You can use food coloring dyes as well.