I. Title: Separation of Food Dyes Via Column Chromatography
II Introduction:
RQ. What colored dyes are found in grape flavored Kool-Aid?
Which dye is more nonpolar?
III. Method:

Equipment
10-mL syringes with luer lock tip
4 large test tubes and beaker to hold them.

Reagents
Sep-Pak® C18 cartridge, grape flavored Kool-Aid® or grape soda, 70% isopropyl alcohol
solution , 25% isopropyl alcohol solution, 5% isopropyl alcohol solution , deionized water

Hazards
Isopropyl alcohol is flammable and should be kept away from ignition sources, also
EXPOSURE TO HIGH VAPOUR CONCENTRATIONS MAY CAUSE EYE IRRITATION.
EXPOSURE TO HIGH VAPOUR CONCENTRATIONS MAY CAUSE RESPIRATORY
TRACT IRRITATION, HEADACHE, DIZZINESS, NAUSEA, INCOORDINATION,
DROWSINESS AND LOSS OF CONSCIOUSNESS. ALTHOUGH INGESTION IS
UNLIKELY, LIQUID WOULD IRRITATE UPPER DIGESTIVE TRACT IF
SWALLOWED.INGESTION OF THIS PRODUCT MAY CAUSE HEADACHE,
DIZZINESS, FATIQUE AND CENTRAL NERVOUS SYSTEM DEPRESSION.
Preparation
1.
2.
3.
4.
5.
6.
7.
8.
9.
Obtain 10ml of the 70 % isopropyl alcohol in a syringe.
Put the long end of the column into the syringe.
Rinse the column with the 10 ml of 70% alcohol.
Remove column
Rinse the syringe with 10 ml of distilled water.
Obtain 10ml more distilled water.
Put the column back on the syringe.
Rinse the column with 10ml of distilled water
Remove the column.
Separation
10. Obtain four 20ml test tubes ( large )
11. Obtain 10ml of the "grape" drink and inject into one of the test tubes, note the color.
12. Obtain 10ml of the“grape” drink
13. Add the column to the syringe ( large end into the syringe.)
14. Inject the grape drink into the column and collect the product.(eluent). Note the color
of the product. Note the color of the column.
15. Remove the column
16. Rinse the syringe with 10 ml of distilled water.
17. Obtain 10ml of the 5 % Isopropyl alcohol in the syringe.
18. Add the column to the syringe ( large end into the syringe.)
19. Inject the 10ml of the 5 % Isopropyl alcohol into the column and collect the
product.(eluent). Note the color of the product. Note the color of the column.
20. Remove the column
21. Rinse the syringe with 10 ml of distilled water.
22. Obtain 10ml of the 25 % Isopropyl alcohol.
23. Add the column to the syringe ( large end into the syringe.)
24. Inject the 25 % Isopropyl alcohol into the column and collect the product.(eluent).
Note the color of the product. Note the color of the column.
25. Remove the column.
26. Obtain 10ml of the 70 % isopropyl alcohol in a syringe.
27. Put the long end of the column into the syringe.
28. Rinse the column with the 10 ml of 70% alcohol.
29. Remove column
30. Rinse the syringe with 10 ml of distilled water.
31. Obtain 10ml more distilled water.
32. Put the column back on the syringe.
33. Rinse the column with 10ml of distilled water
34. Remove the column.
Results Table: The following can be used to determine
Test tube
Contents
Step #
Color of column
after liquid is
injected into the
column
Color of the liquid that
passes through the
column or collected in
the test tube
(eluent)
11
14
19
24
Grape control
Grape in Column
Grape in column +
10ml 5 %
isopropyl alcohol
Grape in column +
10ml 25 %
isopropyl alcohol
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
Discussion
The ingredients listed on the Kool-Aid package are: citric acid, clacium phosphate, salt,
maltodextrin, modified corn starch, artificial flavor, ascorbic acid, FD&C red 40 and FD&C
blue 1. The last two ingredients are the interesting components for this demonstration. This
combination of red and blue dyes gives the grape drink its characteristic purple color. The
structures of these dyes can be seen below.
These are relatively non-polar molecules, even allowing for some charge separation from the
partial ionization of the sodium ions. Red 40 is a somewhat more polar molecule than is blue 1.
This can be explained by the sizes of the molecules. Each molecule will have a similar amount
of charge from ionization, but since blue 1 is a larger molecule the resulting charge will be
spread out over a larger molecule. The contents of the Sep-Pak column (stationary phase) are
very non-polar. If the dye molecules are dissolved in a relatively polar solvent (mobile phase)
such as water and the resulting solution is forced through the column, The dye molecules will
preferentially associate with the stationary phase. This results in a colorless liquid (eluent)
exiting the column and the column takes on the purple color from the dyes. If we decrease the
polarity of the mobile phase, it should be possible to cause the dye molecules to leave the
stationary phase and thus remove them from the column. A 5% isopropyl alcohol and 95%
water mobile phase will remove the red 40 dye from the stationary phase and the resulting
eluent will be red from the dye and the column will appear blue from blue 1 that remains on the
column. A slighly less polar mobile phase consisting of 25% isopropyl alcohol and 75% water
will remove the blue 1 dye from the stationary phase and the resulting eluent will be blue from
the dye and the column will appear white or colorless. There may be some slight blue color if
all of the dye has not been removed. The more polar molecule, red 40 is removed with the more
polar mobile phase and the less polar moleculem blue 1, is removed with the less polar mobile
phase.
VI.
Conclusion:
1. Answer the research question.

References
1. Vonderbrink, S.A., Laboratory Experiments for Advanced Placement Chemistry, Flinn
Scientific: Batavia, IL, 1995, pp 149-153.
2. Bidlingmeyer, B.A., Warren, F.V., J. Chem. Educ., 1984, 61, 716-720.
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