Experiment #6
Chromatography
OBJECTIVES

To become familiar with techniques separating mixtures

To learn about chromatography, specifically paper chromatography
DISCUSSION
The word chromatography means color-writing and the name was chosen
at the beginning of this century, when the method was first used, to
separate colored components from plant leaves.
The name
‘chromatography’ may be misleading, because separation techniques do
not depend on color. Many variations of this technique exist today.
Chromatography is used as a very reliable and fast technique to separate
a variety of natural and synthetic mixtures into their components.
Several types of chromatography exist today, among which are paper
chromatography, thin-layer chromatography or TLC, liquid-liquid
chromatography, gas chromatography, or high performance liquid
chromatography or HPLC.
Chromatography is a separation technique used to separate the
components of a mixture. Many separation methods rely on physical
differences between the components of a mixture. Undoubtedly, you are
already familiar with several means chemists use to effect separations
based on physical differences. These techniques include: Filtration,
where separation may be effected because substances are present in
different states (solid vs. liquid); Centrifugation, where separation is
effected by differences in density; and Distillation, where separation is
effected by taking advantage of differences in boiling temperatures of the
various components. In this laboratory exercise, we will effect a
separation of a mixture of food dyes using paper chromatography.
In a typical experiment, a mixture of different chemicals is partitioned
between a stationary phase and a moving phase (eluent or solvent). The
moving phase may be a liquid or a gas; the stationary phase is typically a
solid. As the mobile phase flows over the stationary phase, the
components in the mixture are carried along. The ability to separate a
mixture into its components basically depends on two fundamental
concepts: solubility and adsorption. The better soluble a component is
in the moving phase the faster it will be transported along the stationary
phase. Adsorption refers to the ability of a substance to ‘stick’ (or be
adsorbed) to the surface. The better the component is adsorbed to the
stationary phase, the slower it will be transported along the stationary
phase. As the mixture moves over the stationary phase, the components
in the mixture move further and further apart into discrete zones.
Paper chromatography uses ordinary filter paper as the stationary
phase,
which
consists
primarily
of
cellulose.
Thin-layer
chromatography (abbreviated TLC) uses a thin glass plate coated with
either aluminum oxide (alumina) or silica gel as the solid phase. The
moving phase is a liquid and is chosen accordingly with respect to the
properties of the components in the mixture.
In a typical experiment, a single drop of the
mixture is applied at one end of a strip of filter
paper. The filter paper is then placed in a shallow
layer of solvent or solvent mixture in a jar or
beaker. Since the filter paper is permeable to
liquids, the solvent begins to rise by capillary
action. As the solvent rises to the level at which
the spot of mixture was applied, the components of
the spot that are completely soluble will be moving
along with the solvent upward (refer to Figure 1).
The rate at which a component will move along
depends on the relative tendency to be dissolved in
the solvent and adsorbed on the paper. By this
means, the original spot of the mixture is spread
out. The net effect is that the components
separate from each other and spread out into a
series of bands or new spots.
Figure 1
Set up for thin layer
chromatography
It is possible to describe the position of spots (so the substances that
have separated) in terms of their retention factor, the Rf value (Figure 2).
The retention factor is defined as:
Rf =
distance traveled by spot
distance traveled by solvent
Because the retention factor for a particular mixture may vary depending
on the conditions, a sample of known composition is typically analyzed at
the same time on the same sheet of paper or slide. The Rf value is a
characteristic property of a given compound in a given solvent in a
chromatography experiment. (See page 76 of your text for more
information on chromatography)
Figure 2: Example for calculating the Rf value
In today’s experiment, you will analyze (not eat; sorry) the shell coating of
M&M candies. Some M & M’s use yellow #5 (tartrazine) as color. This
dye causes allergic reactions in some people and must therefore be
named as an ingredient in products that contain it. The M&M package
states that yellow #5 is one of the colors added. The big question is then:
is the dye present in all candies or only in some of the candies of certain
colors? The answer to this question is important to the person who is
allergic to yellow #5 and wants to know which candies are safe to eat.
In the second part of today’s experiment, you will analyze felt-tip pen
inks. Felt-tip pens come in many different colors but most colors are
often mixtures of primary color inks. We will use water-soluble pens and
further study the importance of the proper solvent or solvent mixture in
paper chromatography for optimal separation.
MATERIALS AND EQUIPMENT
M&M candies
felt-tip pens, both water-soluble and permanent
Whatman #1 chromatography paper or filter paper
250 mL beaker
0.1 % NaCl, acetone, ethanol
butanol : water : ethanol : acetic acid mixture (120:40:20:1)
Optional: Bring in some different M&M candies, or any other color
coated candies.
More info on M&M chromatography can be found at
http://www.pitt.edu/~n3lsk/m-mchromproc.html
http://colossus.chem.umass.edu/genchem/chem122/122_Experiment_
2.htm
PRELABORATORY ASSIGNMENT: Chromatography
Questions
1. Explain in your own words why samples can be separated into their
components by chromatography.
2. Why must the spot applied to a chromatography plate be above the
level of the developing solvent?
3. In a chromatographic experiment, why do some substances move up
the stationary phase faster than others?
4. If the solvent front moves 8.0 cm and a component in a sample being
analyzed moves 3.2 cm from the baseline, what is the Rf value?
EXPERIMENTAL PROCEDURE
Record all data and observations directly into your notebook.
Part I (M&M candies)
1. A 10 x 10- cm strip is cut from Whatman #1 chromatography or filter
paper. Along one side, 1 cm from the edge, draw a straight pencil line,
and make seven marks at about 1 cm intervals. Six of these are
identified by code for the different colored candies, and the seventh for
the standard. The standard will be the compound tartrazine.
2. To separate the dyes used to coat M&Ms, you must first prepare a
solution of the coating dyes. To do this, place four M&M’s of the same
color into a small beaker. Add 3 mL of a 50/50 mixture of water and
ethanol and swirl the solvent until the candy coating has dissolved.
Remove the M&Ms from the solvent before the chocolate center is
exposed. The dye solutions are not very concentrated, so you will need
to spot these dye mixtures several times to obtain a sufficiently dark
spot. Dry each spot between applications of the mixture to maintain as
small and concentrated a spot as possible. Try to keep your spots less
than 4mm in diameter.
3. Repeat the procedure for all color-coated candies. Gently dry the
paper by moving it in the air.
4. Use a 250 mL beaker. Add 0.1% sodium chloride, (solvent or eluting
solution) so that the bottom of the beaker is covered. Place the filter
paper in the eluting solution, with the sample spots down near the liquid
surface, but not in the liquid (Figure 3). Tightly seal the beaker with
clear film and let it stand undisturbed. The solvent will gradually rise by
capillary action, carrying the components in the spots along.
Figure 3: Arranging the filter paper in a circle
5. The paper is taken from beaker when the solvent front is about 2 cm
from the top. Because the paper remains wet for several more minutes
and the solvent continues to rise, it is very important to draw the
solvent line and circle the colored spots immediately.
6. Measure the distance from the straight line on which you applied the
spots to the solvent front. Then measure the distance from the pencil
line to the center of each colored spot and calculate the Rf value from the
two number you obtained. Repeat the procedure for each colored candy
spot that you applied.
7. Compare the Rf values that you calculated to the ones given in the
Table to determine which colors are present in each candy that you
investigated.
Each organic compound, and many inorganic compounds, has a
characteristic Rf value in a certain solvent. The colors of the shells of M
& M candies contain food coloring that are organic dyes and therefore
display characteristic Rf values.
Table I The composition of the candy shells is shown in the Table
below. (J.Chem.Edu, 2002, Vol.79, 847)
Sample
Colors after
Rf values
development
Yellow M&M
yellow
0.50
orange M&M
orange
0.35
Red M&M
red
Green M&M
yellow, blue
0.48, 0.78
Brown M&M
blue, yellow
0.77, 0.48
0.12
orange, red
0.35, 0.13
Blue candy
blue
0.22, 0.82
Food Color
yellow
0.58
Record in your notebook:
Record your solvent, distance solvent moved (in cm), distance color
moved (in cm), and calculate the Rf value for each color.
Compare the Rf values you have obtained with the literature values in
Table I.
Part II (Felt
tip pens)
1. A 10 x 10- cm strip is cut from Whatman #1 chromatography or filter
paper. Along one side, 1 cm from the edge, draw a straight pencil
line, and make four marks at 1 cm intervals. Obtain several watersoluble pens from your Instructor.
2. Apply a single small spot of different ink to each of the marks on the
filter paper. Allow the spots to dry completely. Gently dry the paper
by moving it in the air.
3. Use a 250-mL beaker. Add the butanol : water : ethanol : acetic acid
mixture (120:40:20:1), which is the solvent or eluting solution. Make
sure that the bottom of the beaker is covered. Place the filter paper in
the eluting solution, with the sample spots down near the liquid
surface, but not in the liquid (Figure 1). Tightly seal the beaker with
clear film and let it stand undisturbed. The solvent will gradually rise
by capillary action, carrying the components in the spots along.
4. The paper is taken from the beaker when the solvent front is about 2
cm from the top. Because the paper remains wet for several more
minutes and the solvent continues to rise, it is very important to draw
the solvent line and circle the colored spots immediately.
5. Measure the distance from the straight line on which you applied the
spots to the solvent front. Then measure the distance from the pencil
line to the center of each colored spot and calculate the Rf value from
the two numbers you obtained.
6. Repeat the procedure with several permanent ink markers using the
same solvent.
Record in your notebook:
Record your solvent, distance solvent moved (in cm), distance color
moved (in cm), and calculate the Rf value for each color.
WASTE DISPOSAL
Dispose of the solvents and the used filter papers in the designated
container in the fume hood.
QUESTIONS
1. The plate has been allowed to remain too long in the developing
chamber. How will this influence the Rf value? Comment on all
possibilities.
2. What would be the result of applying too much sample to the TLC
plate?
3. Two components have the same Rf value in a chromatogram. Are they
necessarily identical in structure?