LAB – LIQUID CHROMATOGRAPHY
The process of chromatography is a method by which scientists are able to separate mixtures of
substances in a solution by understanding the nature of their polarities. In any chromatography process, the
first necessary component is a stationary phase which is able to attract certain components of the mixture.
For example, if your medium is made up of a substance with polar molecules, it will attract polar molecules
in the mixture, and therefore it will take longer for those polar molecules to travel through the medium.
The second necessary component is the solvent, which must have a polarity different than the medium. As
the solvent travels through the medium, the components of the mixture will be adsorbed by the medium
depending on their polarity. In this lab we will use a C18 Sep-Pak® cartridge. The medium is an 18carbon hydrocarbon, which is very nonpolar. The solvent we will use is water, which is very polar. As a
result, the first component we will see come out of the column will be the most polar component of the
mixture. We will inject and transport the mixture through the medium by using a plastic disposable
syringe.
Purpose: to separate the substances that are present in Grape Kool-Aid® by the process of liquid
chromatography. The dyes that will be separated are FD & C Blue #1 and Red #40.
Chemicals: Isopropyl alcohol, 70% or 91%, colorless, unscented; unsweetened Grape Kool-Aid®
Materials: Sep-Pak® C18 cartridge, 10-mL and 25-mL graduated cylinders, 2 x 50-mL beaker,
2 x 10-mL syringes
Safety Alert: isopropyl alcohol is flammable. Keep it away from excess heat or open flame.
Procedure:
1) Prepare the Kool-Aid® solution as directed on the package, omitting the sugar.
2) Prepare the 18% (by volume) isopropyl alcohol solution by combining 13 mL of 70% isopropyl alcohol
with 37 mL of distilled water (10 mL of 91% / 40 mL of water). This will be the solvent in the
procedure (mobile phase).
3) Cut off the exit tube of the C18 cartridge (the shorter end) where it meets the body of the cartridge (see
Fig. 1). Fill the syringe with 10 mL of undiluted (70%) isopropyl alcohol and attach it to the long
end of the C18 cartridge. Pump the alcohol through the cartridge at a rate of 5-10 mL per minute
Collect the eluted alcohol in a 10 mL graduated cylinder to help you monitor the flow rate.
4) Fill the syringe with 10 mL of distilled water and wash the cartridge by pumping the water through at
same rate as Step #3.
Figure 1
blue layer
injected sample
purple layer
red layer
red drops emerging
LAB – LIQUID CHROMATOGRAPHY
5) Using a different syringe, inject 1mL of the Kool-Aid® solution onto the column. Discard the column
effluent (the portion that washed out as you injected the sample).
6) Fill your original syringe with the 18% isopropyl alcohol solution you made, and pump it through the
cartridge at a rate of 5-10 mL per minute. Collect the column effluent in a 10-mL graduated
cylinder. Make sure to record the total volume of effluent at the first, VR (start), and last,
VR (end), drops of each colored dye. If there is not a good separation of the red and blue dye (you
get a few purple drops), record the beginning and final volume for the intermediate purple band.
The center of this band will serve as the end of the first band and the beginning of the last band.
For visual reference, look at Figure 1.
7) Wash the cartridge with 10 mL of distilled water, pumping at the same rate as the elution step (#6). D
Discard the effluent from this step.
8) Repeat steps 6 & 7 with two more samples. Calculate the averages for each measurement, and use those
numbers in your data table for the calculations.
Data:
Red Dye
Blue Dye
Experimental System
VR (start)
VR (end)
W = VR (end) – VR(start)
VR avg = VR (start) + ½ W
L
1.25 cm
r
0.5 cm
VM = ½ πr2L
0.49 cm3
k’ = (VR avg – VM) / VM
α = k’2 / k’1
N = 16 (VR avg (blue) / Wblue)2
R=
VRavg ( red ) − VRavg ( blue )
1 (W + W )
blue
2 red
Calculations:
Show the work for your calculations in your lab report.
W is the band width, or the volume in mL of each dye as it emerges from the column.
VR avg represents the center of the band for each color dye.
L is the length of the column. r is the radius.
LAB – LIQUID CHROMATOGRAPHY
VM represents the mobile phase (solvent) volume. This represents about 50% of the total empty column
volume.
k’ is the capacity factor. This is a unitless measure of the retention of the dyes. The optimum range is
between 1 and 10.
α is the selectivity factor. This is the ratio of the separation of the k’ values. k’2 represents the larger k’
factor between the blue and the red dye. The ideal range is between 2 and 10. A value of 2 means the dye
that elutes last likes to bond to the C18 medium twice as much as the first dye.
N represents the number of theoretical plates in the column. This is essentially the number of times the dye
is exchanged back and forth between the medium and the solvent. We use the dye that eluted last’s (blue)
VR avg (from the 4th row of the data table) for this calculation.
R is the resolution. This is the major goal of the experiment. This is a unitless number which represents
how well the two components were separated by the column. For VR avg, select the appropriate value from
the 4th row of the data table. As resolution increases above a value of 1, there is a much greater separation
of the dyes.
Disposal: All of the solutions may be safely flushed down the sink. Cartridges may be disposed of or
reused.
Conclusions:
1)
2)
3)
4)
In discussing solubility the rule “like dissolves like” is often used. What does this mean?
Draw the Lewis Structure of isopropyl alcohol. Explain how its polarity differs from that of water.
Which dye is more polar, red or blue? Explain.
Discuss your resolution factor (R) you calculated in the last calculation. In your opinion, was the
separation satisfactory?
5) What possible corrections would you propose to improve your results?