Today you are going to separate the different colored dyes in grape Kool-Aid™ using column chromatography.

Chromatography is all about separation. The word chromatography comes from

chromato- which means "color" and -graphy which means "the field of study"

Chromatography is not only used to separate colored solutions, but components of non-colored solutions can be separated as well.

It will help you understand this experiment if you know something about chromatography. First of all, Grape Kool-Aid is a mixture of carbonated water, sugar, ascorbic acid, citric acid, and dyes.

There are 2 parts to a chromatographic system. The part that stays in one place is called the stationary phase, and the part the moves (a liquid) called the mobile phase. Substances are separated based on which of the 2 parts, which are called phases, they are most attracted.

1. If your substances (the dyes in the Kool-Aid) are attracted to the mobile phase, they will pass through the stationary phase along with the mobile phase.

2. Furthermore, different substances can be attracted to the stationary phase with differing amounts of attraction and this leads to liquid substances passing through the stationary phase at different rates.

Procedure

Part 1
Obtain three (4) 50 mL beakers ( 3 glass and 1 plastic). Label 2 of the glass beakers 5% and 25% alcohol respectively. Pour about 40 mL of the appropriate alcohol dilution into its beaker. Pour one of the two grape Kool-Aid solutions into the plastic beaker. Add distilled water to the third glass beaker.

Take the small cartridge and place the short end snugly onto the tip of the syringe. Use the plunger to pull up 10 mL of the 25% isopropyl alcohol through the cartridge into the syringe. Push the alcohol out through the syringe back through the column into a 100 mL waste beaker.

Next, use the plunger to pull up 10 mL of distilled water through the cartridge into the syringe. Push the water out through the syringe back through the column into the waste beaker.

Remove the cartridge (column) from the syringe and draw up 10 mL of the grape Kool-Aid from the beaker. Place the cartridge (column) back on the syringe and force the Kool-Aid through the column SLOWLY and into a well in a 24-well well plate. When one well fills up move to the next well until all of the first fraction has been pushed out. What color is the solution you've forced into the well?

Again, remove the cartridge from the syringe. Now pull 10 mL of 5% isopropyl alcohol into the syringe and place the cartridge back on the syringe. Push the 5% alcohol through the column into a clean well on the microplate. What color is the solution in the well?

Again, remove the cartridge from the syringe and this time pull 10 mL of 25% isopropyl alcohol into the syringe. Put the cartridge back on the syringe. Push the 25% alcohol through the column into a new well of the plate. What color is the solution in the well?

Part 2:
Use a Pasteur pipet and transfer 5 mL of the first fraction to a test tube. Add 2 drops of concentrated HCl, mix, and place in a boiling water bath for 5 minutes. Remove from the water bath, cool with running water on the outside of the test tube, and then neutralize by adding about 24 drops of the 1 M NaOH. Check with a pH strip to see that the pH is about 7 Add 6 mL of Benedict's Solution, mix well and place the test tube back in the boiling water bath. Watch for a color change. If none after 5 minutes consider that your Kool-Aid was sweetened with an artificial sweetener instead of sugar.

Repeat Part 1 only with the green Kool-Aid. Everything from Part 1 can be poured down the drain. The liquid from Part 2 may be poured down the drain. Dump any solid out onto paper toweling and place in a trash can. Wash up everything with WARM, soapy water and rinse well.

Results and Discussion

We call the portions of the sample that pass through the column at different rates, fractions.

• For what purpose did we use column chromatography?
  
• What are some other uses for column chromatography?
  

Why did you have to add the concentrated HCl before you did the Benedict's Test? What is a reducing sugar? What is the equation for the reaction occuring during a positive Benedict's Test?