HIGH PERFORMANCE LIQUID CHROMATOGRAPHY LAB (HPLC)
Separation of the Dyes in Grape Kool-Aid
PRE-LAB
Use the information you read in the introduction to complete the pre-lab below.
Purpose/Objectives:
1.
2.
3.
4.
Background: Define the following
MixtureSolventMoleculeAdsorbentPolarQuestions:
1.) Why does the mixture in the tube separate? Explain….
2.) What substances make up Kool-Aid?
3.) What molecule will flow out of the tube first?
4.) Is the Sep-Pak cartridge polar or non-polar?
5.) What factor causes molecules to attract or repel other molecules?
6.) Give 2 everyday examples of molecular interactions that are attracted to one another and 2 that
repel.
Safety:
Check test tube and beaker labels at the beginning of the lab to make sure they can be clearly read.
Keep the plastic beakers of Isopropanol and Methanol separate so they do not get mixed up.
DO NOT lean the syringe on the test tubes when you are pushing the alcohol solution through!
Empty and rinse your waste container after you have completed the lab. Wipe your tables with Lysol and clean
up around your sink.
Conclusion Questions:
1. Which alcohol gave you more concentrated dye samples?
2. List all the solvents and their concentrations used in the separation according to their relative polarity.
Hint: Compare the chemical structures of the two alcohols and the percentage of water in each mixture.
3. What would have happened if you had used the HPLC solvents in reverse order? (60% alcohol first and
water last)
4. Was the separation a physical process or did a chemical reaction take place?
5. When you poured the two fraction together (Final steps), you probably observed a layered effect. What
property(ies) of the solutions caused the layering?
6. If two substances have exactly the same polarity, could they be separated using HPLC? Explain.
7. a. If two colorless substances differed in polarity, could they be separated by HPLC?
b. If you answered yes to 7a. describe two different methods you could use to find the colorless substances
once they were separated.
INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY LAB
(HPLC)
Separation of the Dyes in Grape Kool-Aid
OBJECTIVES:
1.
2.
3.
4.
To understand the usefulness of separating mixtures in order to analyze them.
To separate the different dyes that make grape Kool-Aid purple.
To compare the use of methyl and isopropyl alcohols as solvents.
To compare some properties of molecules you use in the separations.
BACKGROUND:
Many times a complicated problem can often be solved by isolating and analyzing the parts that make up the
whole. In this lab, the work usually done by expensive pumps and automated equipment in a HPLC laboratory
will be completed by you. In this experiment, you will use the same sorts of solvents and the same carbon-18
adsorbent that over 90% of the scientists use for HPLC. Using your hands, eyes, and brain you will measure,
mix, and propel liquid through a tube packed with solid adsorbent. Separation of a mixture added to the tube
occurs because some molecules in the mixture are more attracted to the solvent moving through the tube while
other molecules in the mixture are more attracted to the solid stationary adsorbent. The molecule that is most
attracted (most easily dissolved) to the solvent, and least attracted to the solid adsorbent, will flow out of the
tube first.
EVERYDAY EXAMPLES:
When two molecules are attracted to each other, they tend to stick to one another. Interactions like this make
transparent tape stick to paper and food stains stick to clothing. Conversely, if molecules repel one another, the
substances do not stick. A ball-point pen fails to write on greasy paper; water rolls off a waxed car; oil and
vinegar separate in salad dressing. Chemical properties determine which atoms join together in each molecule
and how molecules will attract or repel other molecules.
In a separation similar to liquid chromatography, spaghetti sauce on clothing may partly wash out with water.
The rest of the stain can be removed with a different solvent, such as soapy water or dry-cleaning fluid, which
attract the oily part of the stain away from the cloth. The stain is separated into two parts each dissolved by a
different solution.
SUMMARY:
Kool-Aid is composed of several substances, including sugar, acids, dyes and flavoring. These substances are
differentially soluble in various solvents. Water is a highly polar molecule and alcohol solutions show a range
of polarities. The adsorbent in the Sep-Pak C-18 cartridge is extremely non-polar, as you would expect with its
18-carbon-long hydrocarbon chains. HPLC takes advantage of these molecular qualities to separate molecules
in a mixture.
MATERIALS:
Check your lab station to make sure there is all the equipment listed below. You will have either methanol or
isopropanol.
 Sep-Pak C18 cartridge
 Methanol solution: 100%, 60%, 20% and 5%
 Isopropyl alcohol solution: 100%, 60%, 20% and 5%
 Distilled water
 10 ml syringe with luer tip
 Container for liquid waste
 Ten Test tubes to collect the fractions you want to keep and Test tube holder
 Grape drink (Kool-Aid) dissolved in water
 1.0 ml pipet for loading the Kool-Aid onto the cartridge
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY LAB (HPLC)
Separation of the Dyes in Grape Kool-Aid
Procedure:
One lab pair will use the methyl alcohol solutions while their neighboring lab pair will use the isopropyl
alcohol. Each lab pair will share their data so the characteristics of the two types of alcohol can be
compared. Enter your data on the data sheet at each step.
1. Preparing the Sep-Pak: Use only your groups alcohol.
A. Label the 5 small test tubes Kool-Aid, water, 5% alcohol, 20% alcohol, and 60% alcohol on the tab of a
piece of clear tape so it can easily be removed during clean up.
Attach the cartridge onto the syringe the way your teacher demonstrated.
B. Precondition the C-18 adsorbent with 10 ml of the 100% solution of your alcohol by removing the
plunger from the syringe and pouring in 10 ml of the alcohol. Return the plunger to the syringe and push
the solution through a drop at a time into the waste container.
NEVER draw up the solutions through the syringe….ALWAYS pour solutions into the top.
C. Rinse the C-18 adsorbent by pushing through 10 ml water into the waste container.
D. Remove the plunger from the syringe and add 1 ml of the Kool-Aid with the pipet. Return the plunger
and push the solution through the cartridge into the test tube labeled Kool-Aid. DO NOT lean the
syringe on the test tube while pushing the solution through. Save test tube and record data on table.
2. Separation Steps:
A. Push 10 ml of water through the cartridge into the test tube labeled water. This fraction may contain
Kool-Aid flavorings. Save test tube and complete that section of the data table.
B. Push 10 ml of the 5% alcohol through the cartridge into the test tube labeled 5%. Save test tube and
complete data table.
C. Push 10 ml of the 20% alcohol through the cartridge into the test tube labeled 20%. Save test tube and
complete data table.
D. Push 10 ml of the 60% alcohol through the cartridge into the test tube labeled 60%. Save test tube and
complete data table.
E. Clean the cartridge by pushing 10 ml of your 100% alcohol solution into the waste cup.
Final step:
Using only the tubes from your alcohol that you saved, carefully pour the blue fraction onto the red as your
teacher demonstrated. (holding the test tube of red at a 90 degree angle, slowly pour the blue down the side
of the test tube trying not to mix the colors) Observe and describe what happens in the data table.
Answer all the conclusion questions on the pre-lab handout.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY LAB (HPLC)
Data Tables (copy into your lab book)
Type of alcohol our group used _____________________________________
Lab Partners names _________________________________________________________________________
STEP
1D. Preparing the
Sep-Pak
Isopropanol
2A. Separation
Isopropanol
Liquid put
through the
cartridge
1 ml Kool-Aid
COLOR of
liquid collected
in test tube
10 ml water
Was the color most
attracted to the
water or to the c-18
adsorbent?
2B. Separation
Isopropanol
Sketch of Sep-Pak showing
location of colors
Did the color in the cartridge move?
Describe movement.
10 ml 5% alcohol
Did the color in the cartridge move?
Explain.
2C. Separation
Isopropanol
10 ml 20% alcohol
What happened to the color in the
cartridge? Explain.
2D. Separation
Isopropanol
10 ml 60% alcohol
What colors are still in the cartridge?
Final step - in the
spaces  describe
what happens
Isopropanol
STEP
1D. Preparing the
Sep-Pak
Methanol
2A. Separation
Methanol
Liquid put
through the
cartridge
1 ml Kool-Aid
COLOR of
liquid collected
in test tube
10 ml water
Was the color most
attracted to the
water or to the C-18
adsorbent?
2B. Separation
Methanol
Sketch of Sep-Pak showing
location of colors
Did the color in the cartridge move?
Describe movement.
10 ml 5% alcohol
Did the color in the cartridge move?
Explain.
2C. Separation
Methanol
10 ml 20% alcohol
What happened to the color in the
cartridge? Explain.
2D. Separation
Methanol
10 ml 60% alcohol
What colors are still in the cartridge?
Final step - in the
spaces  describe
what happens
Methanol