Last updated Dec. 2013
Chemistry 151
Lab 4: Chromatography
Introduction
Mixtures, both homo- and heterogeneous, can be separated (or resolved) into substances by physical
means. Common examples of physical separations include filtration (separation by size) and distillation
(separation by boiling point). This lab examines another separation technique that’s commonly used in
science labs: chromatography.
Chromatography usually involves taking a mixture (often referred to as the mobile phase) and passing it
through a stationary phase. The mobile phase is usually in the liquid, gas, or aqueous phase while the
stationary phases is typically a liquid or solid. Depending on the type of chromatography being performed,
there can be various factors that affect how well the mixture in the mobile phase separates (if it separates at
all), but the most common factor is how the components of the mixture interact with the stationary phase.
For example, if a chemical in a mixture interacts strongly with the stationary phase, it will take longer to
pass through another that only interacts weakly.
There are many different types of chromatography, some of which can involve elaborate (and usually
expensive) instrumentation. In this lab, however, we will use a very simple form known as paper
chromatography. As the name implies, the stationary phases is simply a piece of paper (albeit one
designed specifically for this technique). The mixtures being analyzed are usually applied directly
(spotted) at one end of the paper, which is then placed in a solvent that serves as the mobile phase. The
solvent will travel from one end of the paper to the other—just like water rising up a paper towel—
dissolving the mixtures and carrying them across the paper. If a proper solvent is chosen as the mobile
phase, the mixture will resolve as it passes through the stationary phases, allowing one to identify its
components.
The movement of each component in the mixture can be described in terms of its retention factor (Rf),
which is simply a ratio of how far the component traveled relative to the how far the solvent did (often
referred to as the solvent front).
Rf =
distance traveled by component
distance traveled by solvent front
If, for example, a component in a mixture traveled the same distance as the solvent, its R f would be 1.0. If
it only travels half the distance of the solvent, it would have an Rf of 0.5. The retention factor can be a
useful way to quantitatively express how well a mixture resolves in a particular chromatogram. The more
resolved the components of a mixture are, the greater the difference between their R f values.
In this lab, you will analyze mixtures of six common FD&C (food, drug, and cosmetic) dyes: blue 1, blue
2, red 3, red 40, yellow 5, and yellow 6. First you will prepare a set of known mixtures and use these to
determine which of three solvents act as the best mobile phase. This solvent will then be used to help you
identify the dyes present in a set of unknown mixtures
To help you identify the components of your mixtures, each chromatogram you prepare will contain the
mixtures being analyzed as well as the individual dyes. Once the mixtures are resolved, you will compare
the retention factors of their components with those of the individual dyes. However, since most of these
dyes have their own distinct color, that can also be used in your analysis.
Procedure
Part I - Choosing a Solvent
1. Get three beakers, ranging from 250-600 mL in size (they don’t all have to be the same size). Using
masking tape, label each with one of the solvent systems being studied: deionized water, rubbing alcohol,
and 0.10% table salt.
2. Using a graduate cylinder, measure 7 mL of each solvent and transfer it to its respective beaker (for 600
mL beakers, use 10 mL). Cover each beaker with Parafilm to prevent evaporation as you prepare the
chromatograms.
3. Take a 7.5 cm x 13.5 cm piece of chromatography paper and, using a pencil, draw a line approximately
1 cm from the bottom of the long end of the paper. This will be your origin line.
4. Make a series of 10 marks along the line you drew. They should be at least 1 cm apart and the ones on
either end should be at least 1.5 cm from the edge of the paper.
5. Label the markings as shown in Figure 1 below.
B1
1cm
1.5cm
B2
Y5
Y6
R3
R40
B1
R40
B1
Y5
R3
Y6
B2
Y5
1cm
Figure 1
6. Repeat steps 3-5 twice more, giving you three identical chromatograms.
7. Place 2-3 drops of each of the dyes you’ll be using in a well plate (label wells using masking tape if
needed). Take your plate and 10-12 toothpicks back to you work area.
8. To spot your chromatogram, dip a toothpick in a dye for a few seconds to soak up some of the liquid
then gently press the toothpick on the paper at the origin line. Make sure your spots are concentrated
enough that they won’t disappear as they move up the paper (most will stretch out as they travel), but don’t
make the spots too large or else the spots may overlap as the chromatogram develops. For your mixtures
(the last four marks), spot the second dye directly over top of the first.
9. Roll each chromatogram long-ways into a cylinder. Staple the ends so the edges are touching, or as
close as possible, but not overlapping (see Figure 2).
10. Remove the Parafilm and place a chromatogram into each solution, making sure the spots aren’t
submerged into the solution and that the paper doesn’t touch the sides of the beaker.
11. Replace the Parafilm and allow the solution in each beaker to rise up the chromatogram. When the
mobile phase is about 1.5 cm from the top of the paper, remove it, unroll it and to allow it to dry*. As
they’re drying, the solutions will continue to creep up the paper for a couple of minutes. Once it’s stopped
moving, use a pencil to mark the solvent front and allow the paper to finishing drying. Note the solvent
system used in the top right corner.
*At this point, you’ll probably realize that one solvent is definitely not the one you’ll be using for Part II.
You won’t need to wait for this one to reach the top of your chromatogram.
12. Measure the retention factors of each spot and determine which solvent resolved your mixtures the
best. Measure each distance from the origin line to the top of the spot.
·· ··
Figure 2
Part II – Identifying the Dyes in an Unknown Mixture
1. Prepare a fourth chromatogram, similar to ones made in Part A, and spot it with the six individual dyes
plus two unknowns that will be assigned by the instructor, giving you a total of eight spots.
2. Use the solvent you decided worked best in Part I to resolve your chromatogram. Do not use the same
solution you used to develop the previous chromatogram. Dispose of it and measure another 7 mL. Also
make sure you use new toothpicks to avoid contaminating your samples.
Write your and your partner’s names on the four chromatograms and attach them to one of your reports (or
attach two to yours and two to your partner’s; doesn’t matter). If you’ve already tossed your
chromatograms from Part I in the trash, I hope you’ve learned a lesson about reading the entire procedure
before starting a lab.
Waste Disposal
All waste can be poured down the drain with running water.
Name: _____________________________
Section: ________
Data
Part I - Choosing a Solvent
1) Solvent Front
Water
Rubbing alcohol
0.10% table salt
Starting time
__________
__________
__________
Ending time
__________
__________
__________
Time elapsed, min
__________
__________
__________
Distance moved by
solvent, cm
__________
__________
__________
2) FD&C dyes
Water
Distance, cm
Rf
Rubbing alcohol
Distance, cm
Rf
0.10% table salt
Distance, cm
Rf
Blue 1
______
______
______
______
______
______
Blue 2
______
______
______
______
______
______
Yellow 5
______
______
______
______
______
______
Yellow 6
______
______
______
______
______
______
Red 3
______
______
______
______
______
______
Red 40
______
______
______
______
______
______
Blue 1
______
______
______
______
______
______
Yellow 5
______
______
______
______
______
______
Blue 1
______
______
______
______
______
______
Red 40
______
______
______
______
______
______
Red 3
______
______
______
______
______
______
Yellow 6
______
______
______
______
______
______
Blue 2
______
______
______
______
______
______
Yellow 5
______
______
______
______
______
______
B1/Y5 mix:
B1/R40 mix
R3/Y6 mix:
B2/Y5 mix
3) Which solvent system did the best job resolving the dyes? Explain.
Part II – Identifying the Dyes in an Unknown Mixture
1) Unknowns
______________ & ______________
2) Solvent system
______________
3) Distance traveled by
solvent, cm
______________
Time elapsed
______________
4) FD&C dyes
Distance, cm
Rf
Blue 1
______
______
Blue 2
______
______
Yellow 5
______
______
Yellow 6
______
______
Red 3
______
______
Red 40
______
______
5) Unknowns
Color
Distance, cm
Rf
spot 1
______
______
______
spot 2
______
______
______
spot 3*
______
______
______
spot 4*
______
______
______
spot 1
______
______
______
spot 2
______
______
______
spot 3*
______
______
______
spot 4*
______
______
______
Unk # _____
Unk # _____
*If applicable. Otherwise, leave blank
6) Identification of unknowns
Unknown #_____
Dyes present: _____
_____
_____
_____
Unknown #_____
_____
_____
_____
Dyes present: _____
Name: _____________________________
Section: ________
Post-Lab Questions
1. What problems might you have encountered if the following had happened.
a) The solvent moved up the paper too quickly.
b) The chromatogram was curled into a cylinder unevenly, causing it to tilt slightly when placed in the
beaker.
c) The origin line was drawn with an ink pen.
2. The following data was collected:
Solvent A
Solvent B
Solvent C
B1
0.89
0.85
0.87
B2
0.15
0.10
0.18
Retention factors
Y5
0.30
0.38
0.35
R3
0.22
0.23
0.21
R40
0.66
0.60
0.54
a) Which solvent resolves the five dyes the best? Explain.
b) A piece of chromatography paper contains four spots: B1, Y5, R40, and a B1/Y5 mixture. Using your
answer from 2a, sketch the predicted chromatogram (use circles to represent the dyes, as seen in the last
pre-lab question, not the oblong streaks you saw in your actual chromatograms).
solvent front
origin line
3. Two-dimensional chromatography is a technique where the mobile phase is passed through the
stationary phases twice, the second pass being perpendicular to the first.
a) Using your answer from question 2a, sketch a chromatogram that was spotted with a mixture of B1, B2,
and R3 (again, using circles to represent the dyes).
solvent front
origin line
initial
spot
b) Sketch the predicted chromatogram if the paper from question 3a was turned 90 degrees and placed into
the solvent a second time.
solvent front
1st origin
line
initial
spot
2nd origin line
Name: _____________________________
Section: ________
Pre-Lab Questions
1. Define each of the following:
a) Mobile phase
b) Stationary phases
c) Solvent front
d) Origin line
2. Explain why each of the following are important when preparing a chromatogram?
a) Not have the spots too close to each other on the origin line.
b) Keep the origin line above the surface of the solvent
c) Remove the chromatogram before the solvent travels the entire length of the paper.
3. A chromatogram was prepared using a procedure similar to Part I of this lab.
solvent front
origin line
A
B
C
D
E
A/B
C/D
a) Measure the distance traveled by the solvent (cm preferred, but any unit will do). ________
b) Measure distance traveled by substances A-E (in the same unit as the solvent front).
A _______
B _______
C _______
D _______
E _______
D _______
E _______
c) Determine the retention factors for substances A-E
A _______
B _______
C _______
Show you work for calculating the retention factor of A
d) Calculate the retention factors for the two spots in each mixture (A/B and C/D)
A _______
B _______
C _______
D _______
e) If you had a sample that was believed to contain two or three of the five substances measured above,
could the solvent used to prepare the above chromatogram be used to identify the components of your
unknown mixture? Why or why not?