Experiment

1
Liquid Chromatography
Introduction:
In this experiment, liquid chromatography is used to separate the substances that are
present in grape flavored Kool Aid®. The dyes responsible for the purple color, FD&C
Blue #1 and Red #40 are separated. In a second experiment, the other components of
Kool Aid®, flavorings and citric acid, are also separated.

Background:
Chromatography is an important analytical tool used to separate the components of a
mixture. The components become separated (or partitioned) between a stationary phase
and a moving phase of the chromatography system. The moving phase is either a gas or
liquid and the stationary phase is usually a solid. The mixture to be separated is combined
with the mobile phase. As the mobile phase solution flows over the stationary phase, the
components of the mixture continually equilibrate between the phases, based upon their
particular affinity for each phase. A higher attraction for the mobile phase leads to a
higher concentration of a component in the mobile phase and a faster rate of transport
through the system. Components with a greater affinity for the stationary phase take more
time to move through the system. The end result of this process is the separation of the
mixture into bands that flow through the system at different rates. If the separation is
sufficient, the bands will exit the system in distinct fractions.

All liquid chromatography systems consist of six basic components:

1. A separation column, usually consisting of a fine granulated solid packed into a
column
2. A solvent that acts as the mobile phase and washes along the column
3. An injection system which is needed to place the mixture on the column
4. A pump or other solvent delivery system that forces the solvent through the
column
5. A detector used to indicate when the components emerge from the column
6. A data recorder

Usually, the solid phase is relatively polar and the solvent is nonpolar in liquid
chromatography. This experiment utilizes reverse phase liquid chromatography (RPC). In
the RPC technique, the stationary phase is a nonpolar solid and the mobile phase is
comprised of a polar solvent.

When a mixture is injected into the RPC column and washed through it, several processes
occur. The more polar components of the mixture are attracted more strongly to the polar
solvent, so they will move more quickly through the column with the solvent. The less
polar components will move more slowly, as they spend more time adsorbed onto the
nonpolar column medium. Ideally, the components should emerge at different times. A
measure of the degree of separation that is achieved is called the resolution of the system.
As the band of each component moves down the column, the band widens due to

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diffusion. As bands widen, they can overlap each other and may prevent clean separation
of the components.

Overview:
The purpose of the experiment is to use liquid chromatography as a tool to separate the
components of unsweetened, grape flavored Kool Aid® or any grape flavored drink.
Miniature liquid chromatography columns called Sep Pak C18 columns are used for the
separation. The Sep Pak column is packed with a silica solid which has a C18 hydrocarbon
bonded to it, ensuring a very nonpolar solid. In Part I, the two dyes in the drink are
separated using dilute isopropyl alcohol as the solvent (a.k.a. eluant). Measurements are
made during the separation that allow for the calculations of the selectivity and the
resolution of the separation process. In Part II, four eluants of different polarities are used
to separate the polar components citric acid and salt, the slightly polar dyes, and the
nonpolar flavoring oils.

Pre-Lab Questions
1. Why is the process of chromatography used?
2. In chromatography, components of a mixture distribute themselves between the
stationary phase and the mobile phase. Explain how the components can be
separated with these two phases.
3. In the liquid chromatography column used in this experiment, the solid has a C 18
hydrocarbon bonded to it. Would a C18 hydrocarbon be a polar or a nonpolar
substance? Justify your answer.
4. Below are typical data for this experiment. 1 mL of a Kool Aid® solution was
loaded on a Sep Pak C18 column. The red and blue dyes were eluted from the
column with a constant flow of 18% isopropyl alcohol. The eluted solution was
collected in a 10-mL graduated cylinder. The volumes of eluant were recorded at
the beginning and end of each color band.

Red Dye Blue Dye

Run #1 Run #2 Run #3 Run #1 Run #2 Run #3
Start of band (mL) 1.0 1.1 1.0 2.0 2.2 2.4
End of band (mL) 1.8 1.8 1.8 3.8 4.2 4.3

This process can also be represented graphically. The x-axis represents the milliliters of
eluant that emerge from the column, and the y-axis represents the concentration of each
dye as it emerges with the eluant.

The first step in calculating the selectivity and resolution of the system is determining the
volumes of eluant corresponding to the bandwidths and band centers for each eluted dye.
a. Bandwidth (W) is the volume, in mL, of eluant containing each dye as it emerges
from the column. Calculate the bandwidth (W) for each dye for each of the three
runs and then determine the average bandwidth (Wave) for each dye.
b. Center of band (a.k.a. average retention volume, VRave) is the volume that
corresponds to the center of each band. The average retention volume is

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calculated by taking the average starting volume for each band and adding ½ of
the corresponding average bandwidth (Wave). VRave = Vstart + ½ Wave
Calculate the average retention volume (VRave) for the red and blue dyes.
c. For each dye, a capacity factor (k’) can be calculated. This term is a relative
measure of the attraction of the dye for the stationary phase as compared to its
attraction for the mobile phase. The equation for capacity factor is:

Where VRave is the average retention volume for each dye and V M is mobile phase
or eluant volume in the cartridge. V M can be estimated to be one half the cartridge
volume, with the stationary phase occupying the other half. For Sep Pak
cartridges, this VM value is 0.49 mL. Calculate k’ for each dye.
d. A selectivity or separation factor, , can now be calculated. This is the ratio of the
k’ values for each dye, with the larger value in the numerator. For good
separation, a mobile phase is usually chosen that gives an  value between 2
and 10. Calculate  for this separation.

e. The resolution, R, a measure of how well the two dyes are separated by the
column and eluant, is given by the equation

Where the numerator is the volume between the band centers and the denominator
represents the average bandwidth. The greater the selectivity, the larger the
numerator and therefore the greater resolution. The resolution can also increase as
the efficiency of the column increases, since this will result in a lower average
bandwidth. Calculate R for this separation.

Materials: isopropyl alcohol (70%, 28%, 18%, and 5% dilutions), distilled water, grape
Kool Aid® solution, Sep Pak C18 cartridge, syringe (10 mL) with male Luer tip, beaker
(100 mL), graduated cylinders (10-mL and 25-mL), syringe (3 mL) with male Luer tip

Safety
Isopropyl alcohol is a flammable liquid and a fire hazard. Do not use near open flames or
other ignition sources. It is slightly toxic by ingestion and inhalation. Wear appropriate
lab safety clothing. Wash your hands prior to leaving the laboratory.

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Procedure

Part I: Isocratic Separation (flow rate and solvent concentration are constant)

Pretreat the Sep Pak C18 cartridge.

1. To help eliminate remixing of closely eluting bands in the cartridge, cut off the
exit tube of the cartridge (the shorter end) at the point where it meets the body of
the cartridge.
2. Fill the syringe with 10 mL of the 70% isopropyl alcohol.
3. Attach the tip of the syringe to the long end of the Sep Pak cartridge and pump the
isopropyl alcohol through the syringe cartridge at a rate of 5-10 mL per minute.
4. Collect the eluted alcohol in a 10 mL graduated cylinder to monitor the flow rate.
5. Repeat steps 2-4 using distilled water.

Inject the sample.

1. Use the 10 mL syringe to slowly inject 1 mL of the Kool Aid® sample onto the
column.
2. Discard the column effluent (the portion washed out as the sample was injected).
3. Remove the cartridge from the syringe.
4. If the same 10 mL syringe was used as in step 1, rinse the syringe 3 times with 10
mL of distilled water to ensure all traces of Kool Aid® are removed.

Elute the sample.

1. Use the 10 mL syringe to slowly elute the dyes. Fill the syringe with the 18%
isopropyl alcohol eluant and attach the syringe to the Sep Pak cartridge.
2. Pump the 18% isopropyl alcohol through the cartridge at a steady rate of 5-10
mL/min.
3. Collect the column effluent in a 10 mL graduated cylinder.
4. Record, using a graduated cylinder, in the Part I data table, the volume of effluent
collected as the first and last colored drops of each of the dyes emerge. If there is
not a perfect separation between the blue and red colored bands, record data for
the beginning and end of the intermediate purple band. The center of the purple
band will serve as the end of the first band and beginning of the last.

Regenerate the column and repeat the measurements.

Repeat the measurements two more times. Between injections, wash the column with 10
mL of distilled water at the same flow rate of 5-10 mL/min. If colored material builds up
on the column, repeat the pretreatment procedure (step 2).

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Part II: Step Gradient Separation

In this procedure, the composition of the eluting liquid is changed. Since the column is
nonpolar, first a very polar solvent, water, is used. Then its composition is changed to less
polar by adding increasing amounts of isopropyl alcohol. This procedure allows the
separation of the citric acid and flavoring oils as well as the dyes.

Pretreat the cartridge.

Follow the same procedure as in Part I.

Inject the sample and elute the components.

1. Slowly inject 1 mL of the Kool Aid® sample onto the column.
2. Elute the polar components of the mixture (citric acid and any sugar present) by
passing 5 mL of water through the column.
3. Collect the effluent in a small graduated cylinder.
4. Elute the red dye by passing 10 mL of 5% isopropyl alcohol through the column.
Note that large amounts of the 5% isopropyl alcohol can be used without eluting
the blue dye, Collect this effluent in a separate graduated cylinder.
5. Use 10 mL of the 28% isopropyl alcohol to elute the blue dye. Collect this
effluent in a third graduate cylinder.
6. Use 10 mL of the 70% isopropyl alcohol to elute the nonpolar flavor oils and
other nonpolar additives. Collect this fraction in a fourth graduated cylinder.
7. Record the color of each eluted fraction in the Part II data table.

Evaporate the solvents and examine the components.

1. Allow the solutions to evaporate by leaving them in the fume hood overnight. Be
sure to label the solutions containing isopropyl alcohol as the solvent.
2. Observe and describe the contents of each of the beakers. Look for color, odor,
and appearance. Enter these observations in the Part II data table.

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Data Tables
Part I: Isocratic Separation
Red Dye Blue Dye
Run #1 Run #2 Run #3 Run #1 Run #2 Run #3
Start of band (mL)
End of band (mL)
W (mL)
VRave (mL)
k’

Calculated values:  ________

R ________

Part II: Step Gradient Separation

Beaker Eluant Observations – Eluted Fractions

1 H2O

2 5% isopropyl alcohol

28% isopropyl
3
alcohol

70% isopropyl
4
alcohol

Calculations
Determine the following values and show calculations. Refer to question four in the Pre-
Lab Questions. Enter the results in Part I of the data table.
1. Bandwidth (W) for each dye.
2. Average retention volume (VRave) for each dye.
3. Capacity factor (k’) for each dye.
4. Selectivity () for the two dyes with this isocratic separation.
5. Resolution (R) for the two dyes with this isocratic separation.

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Post-Lab Questions
1. What is meant by polarity of molecules? What causes differences in polarity?
2. In discussing solubility, the rule “like dissolves like” is frequently used. What
does this phrase mean?
3. Draw the structural formula of isopropyl alcohol. Explain how it differs in
polarity from water.
4. For good separation of the dyes, the resolution should be greater than 1. What was
the value you calculated? Did the two dyes overlap as they emerged from the
column, or was the separation a good separation?
5. In the step gradient separation, four separate fractions were collected. How were
these related to the polarities of the column and of the eluting solvent?
6. Write a concluding paragraph evaluating your execution of the procedure. Include
sources of error, the effects of the error on your results, and improvements that
should be included to increase the accuracy of your results.

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