CHE 230
Lab #9
Chromatography of Kool-Aid
Objectives:
 Understand the basic principles behind column chromatography.
 Separate the dyes in Kool-Aid using column chromatography.
 Understand the relative polarity of the dyes based on their elution.
Tools of the Trade:
 Miniaturized column chromatography set-up
Techniques:
 Reverse-phase chromatography
 Gradient elution
Procedure:
 Prepare solutions:
o 25 mL of each: deionized water, 5% isopropanol (IPA), 25% IPA (v/v % in water)
o 1 packet of grape Kool-Aid dissolved in approximately 200 mL of water
 Connect the mini-column to the syringe as directed
 To perform separation, use a disposable pipet to add solution to (above) the column and pull the solution
SLOWLY through the column using the syringe, always keeping some amount of liquid above the column.
Note: never let the column go “dry”! Always keep a layer of mobile phase above the column.
 Equilibrate the column by passing the following solutions:
o 5-10 mL of 25% IPA
o 5-10 mL of water
 Add the sample by passing 5 mL of the Kool-Aid solution through the column
 Perform separation by passing the following sequence of solutions:
o 5-10 mL of water
o 5-10 mL of 5% IPA
o 5-10 mL of 25% IPA
Note: more volume may be needed to elute the dyes; record volume used
 Collect each “fraction” (each syringe-full or when the color changes) in a separate test tube
 Record observations
Discussion:
 Describe how you think the separation is occurring. What is the mechanism of the separation?
 Why do you think the consistency of the solution added to the column was changed throughout the
separation? This process is called gradient elution.
 All chromatography systems consist of a mobile phase and a stationary phase. Define the system: what
is/are the stationary phase(s), mobile phase(s), sample(s), analyte(s)?
 Rank each colored component from least polar to most polar. Justify your ranking based on the order of
elution.
o Use chemfinder to find the chemical structures of the dyes. Can you justify your ranking also based
on their structures?
 Many analytical procedures require a separation (chromatography) step prior to the analysis. Why?
 This lab is a “qualitative” lab. It could easily be modified to be a “quantitative” lab. Briefly describe how
you could make this a quantitative lab, i.e., how could you determine the concentration of the dyes in the
Kool-Aid sample?
 Compounds need not be colored to be analyzed using chromatography. How could colorless compounds be
detected in a chromatography system?