ABSTRACT

The purpose of this investigation is to know how to

separate molecules using chromatography. In
chromatography, there are two phases, stationary
phase and mobile phase. This depends on whether the
molecule has greater affinity for paper or the solvent.
If the molecule has greater affinity for paper, it stays in
stationary phase. On the other hand, if it has greater
affinity with the solvent, it goes to mobile phase. To
separate the food dye, we tested using
chromatography in water, isopropyl alcohol, acetone,
and ethanol.

Anthony Hower (Argumentation and

Documentation, Pre-Lab part 1, editing), Min
Su Jeon (Data Collection and Computation,
Pre-Lab part 2), and Julia Huh (Abstract,
Procedure and Materials, Post-Lab)

INVESTIGATION 5 AP Chemistry

Separating molecules that are attracted to each other

Contents
Pre-Lab Questions ......................................................................................................................................... 1
Materials ....................................................................................................................................................... 3
Procedure...................................................................................................................................................... 3
Argumentation and Documentation............................................................................................................. 7
Post-Lab Assessment .................................................................................................................................... 7

PRE-LAB QUESTIONS
Part I
1. Why does the water creep up the paper? Explain this. Draw the molecular interactions
associated with the macroscopic observation.

Water creeps up paper because of capillary action due to cohesion and adhesion. Water
has hydrogen bonding, so it attracts itself strongly, and it is also attracted to other polar
molecules, such as the fibers in the paper. Because the water molecules are so attracted
to the molecules in the paper, the attraction between them is enough that it will pull the
water molecules upward against the force of gravity.

2. In your kitchen, there is a mixture that is usually listed as a single ingredient in recipes:
baking powder. Using the common names for the chemicals, baking powder is actually a
mixture of sodium bicarbonate, cream of tartar (sodium bitartrate), and cornstarch (see
Figure 1). Looking at the three molecules in Figure 1, how are they similar and how are
they different? Draw a picture of how each molecule would interact with a bunch of water
molecules. (this can be done with ChemSketch)

They are all similar because they are all organic molecules mostly made out of carbon,
hydrogen, and oxygen, but they are different because they are different sizes and have
different numbers of hydrogen bonding sites. The Cornstarch polymer is the biggest, and
thus would have more London dispersion forces, but also by far has the most number of
places where hydrogen bonding can occur, and thus has a strong attraction to the water
molecules. Its presence is mostly used to absorb water when stored to keep the powder
from reacting before so desired. However, corn starch has poor solubility in water
because it is a long glucose chain, but it can remain in a suspension. A big difference
between the starch and the other two molecules is that it is a covalently bonded
molecule, but the other two are salts, which means that the sodium splits from the rest
of the molecule when dissolved in water. Sodium bitartrate is bigger than sodium
bicarbonate and has more hydrogen bonding sites, so it would be more attracted to the
water molecules. It is a sodium acid salt of tartaric acid. Sodium bicarbonate is the
 smallest molecule in the mixture, and has only one hydrogen bonding site, therefore, it
would be the least soluble molecule. It is a salt composed of sodium and bicarbonate ions.

3. How would you separate this baking powder mixture into the three parts?

The cornstarch polymer can be removed from the other two molecules by putting the
baking powder in water, then running it through a filter paper because the cornstarch will
not dissolve. The other two chemicals can be separated using chromatography. Because
sodium bitartrate is more attracted to water, it will be pulled up farther than sodium
bicarbonate, leading them to be separated as they get pulled up the paper different
distances.
Part II
1. How can molecules attract each other when they are in a mixture? Predict how ethanol
would interact with those molecules. Draw a picture illustrating the interactions between
the components of the mixture and the solvent, ethanol. The intermolecular forces are
different because they involve different chemical structures. They are also attractions
based on different distributions of electrons (electronegative elements or low
electronegativity). Student drawings will vary.

If they interact with ethanol, the hydroxyl group of ethanol will form hydrogen bonds with
the carbonyl groups of sodium bitartrate and sodium bicarbonate. With Cornstarch
polymer, hydrogen from hydroxyl group of ethanol will form hydrogen bonds with the
oxygen in hydroxyl group of cornstarch.

2. What does the Rf value describe on a microscopic level? Why is this important? The Rf
value is a ratio that shows the distance a molecule has traveled divided by the distance
the solvent has traveled. It is the relative distance traveled when compared to the solvent.
It creates an observable quality that is related to the level of attraction between the
molecule and the solvent.

Rf value shows how much a molecule have moved. In microscopic level, it shows whether
a molecule moved or not, giving us a measurement of molecule attraction.

3. If the molecule had a very high affinity for the stationary phase, how would this affect the
Rf value? Explain. A molecule with a very high affinity for the stationary phase would have
a very low Rf value, because it would not travel very far at all in comparison to the solvent.

The Rf¬ value will be really small because the attraction will keep the molecules together,
preventing them from moving.
 4. What role does the mobile phase play in the distance a molecule travels in
chromatography? What does the mobile phase describe? The mobile phase is the solvent
and if the molecule has attractive forces for the solvent that are greater than the
attraction for the stationary phase, it would have a very high Rf value.

The mobile phase is the rate at which the materials are being separated. As it is stated in
the question, if the molecule has attractive forces for the solvent that are greater than
the attraction, it would have a high Rf value, which means that the distance a molecule
travels is very distant.

5. If you combined a polar solvent with a molecule that has a carbonyl group (carbon with a
double-bonded oxygen), would it have a high or low Rf value? Justify your answer with
what you understand of intermolecular forces.

If I combined a polar solvent with a molecule that has a carbonyl group, I would have a
very high Rf value. This is because carbon with a double bonded oxygen are non-polar and
thus have a very weak attraction between the polar solvent. This will increase the Rf value.

MATERIALS
4 Cylindrical glass with lids, pencil, ruler, tape to make labels, chromatography paper, sample
dye, distilled water, isopropyl alcohol, acetone, ethanol, tweezers.

PROCEDURE
After you put the dye on the chromatography paper, put each paper in to a cylindrical glass that
is filled with water, isopropyl alcohol, acetone, and ethanol, respectively. After the solvent seems
to reach almost the top of the paper, take it out and measure the distance from base line to the
molecule that has been separated.
Data Collection and Computation
1. Why did you select the solvents that you tested? Did your data support your hypothesis
or disprove your hypothesis?

The solvents we selected were Distilled Water, Acetone, Isopropyl Alcohol and Ethanol.
We chose these because they were the solvents available to us. Our hypothesis was that
the water will separate more than other solvents. The data supports our hypothesis.

2. What explanations can you provide for your separation of the three molecules? How was
the choice of the solvent connected to the separation process?
 The separation of the three molecules is caused by each molecule’s different amount of
attraction to the solvent. For example, Blue 1 is attracted more to water molecules
because of more possible hydrogen bonds it can have with water than any other
molecules. This would separate the Blue 1 molecules from other ink molecules and move
the blue 1 ink up the paper along with the water that is attracting the ink. The choice of
solvent is connected to the amount of separation by the ability of how many Inter
Molecular Forces it can make with the inks.

3. What part of the chromatography setup did the molecules interact with, stationary or
mobile phase? How would you explain this interaction using intermolecular forces?

First, during stationary phase, the molecules interacted with the paper and each other.
The molecules first spread a bit, but did not go further because of interaction with each
other. During the mobile phase, the water molecules interacted with the solvents. As the
solvent crept up the paper, the molecule’s interaction with the solvent allows the ink to
creep up along with the solvent. The interactions between the solute and the solvent is
most likely to be London Dispersion Force and Hydrogen Bonds. Solvents that have
hydroxyl group or atoms that can form hydrogen bonds will most likely have more
interaction with the ink because it can form stronger intermolecular forces such as
hydrogen bonds.

4. Draw a picture of how the chromatography worked. Explain your picture using the
following terms: stationary phase, mobile phase, and intermolecular forces.

Figure 1
In the picture, a picture of the chromatography setup is shown. A paper with a dot of ink is dipped
into solvents. During stationary phase, the solvent starts creeping upwards, but the dot of solute
is not moved.

Figure 2

In the picture, the paper is shown. We can see that the ink is carried upward with as the
solvent creeps upward. During the mobile phase, the solute is carried upward as seen in
Figure 2. This upward movement is caused by intermolecular forces between the solvent
and the solute. During the stationary phase, the solute is stationary because the IMF with
the paper is stronger than IMF with solvent. But as solvent touches the solute, the strong
IMF with the solute and solvent pulls the solutes apart from each other and the paper so
that the ink separates and seeps upward.

5. Evaluate which solvent is the one with the best “green chemistry” rating (using the
reference in the background section). What intermolecular forces would this solvent form
with the three molecules in the mixture?

Distilled water would have the best “green chemistry” rating. Distilled water was most
effective and is not harmful. Water forms hydrogen bonds or at least dipole-dipole
attractions with each molecule.

6. Which molecule spent the most time in the stationary phase and why?
 The Red and Yellow spent the most time in the stationary phase because they are not as
big as the blue molecule, which contributes to weaker London Dispersion Forces.

7. Calculate the Rf values for each chromatography trial that you completed and include it
in the data table.

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑇𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒

𝑅𝑓 value =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑇𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
6 𝑐𝑚
𝑅𝑓 value Water: Yellow = = 0.75
8 𝑐𝑚
6.6 𝑐𝑚
𝑅𝑓 value Water: Red = = 0.835
8 𝑐𝑚
7.4 𝑐𝑚
𝑅𝑓 value Water: Blue = = 0.925
8 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Isopropyl Alcohol: Blue = = 0
4.8 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Isopropyl Alcohol: Red = = 0
4.8 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Isopropyl Alcohol: Yellow = = 0
4.8 𝑐𝑚
0.7 𝑐𝑚
𝑅𝑓 value Acetone: Blue = = 0.17
7.2 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Acetone: Yellow = = 0
7.2 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Acetone: Red = = 0
7.2 𝑐𝑚
4.2 𝑐𝑚
𝑅𝑓 value Ethanol: Blue = = 0.7
6 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Ethanol: Yellow = = 0
6 𝑐𝑚
0 𝑐𝑚
𝑅𝑓 value Ethanol: Red = = 0
6 𝑐𝑚

Rf Values
Distilled Water Isopropyl Acetone Ethanol
Alcohol
Blue 1 0.925 0 0.17 0.7

Red 40 0.835 0 0 0

Yellow 5 0.75 0 0 0

Figure 3: Data Table

ARGUMENTATION AND DOCUMENTATION
Write an explanation of how intermolecular forces and the molecular structure of the molecules being
separated determine the ideal solvent for use in chromatographic separation. Be sure to cite specific
evidence from this experiment to support your explanation.

Intermolecular forces and the molecular structure of the molecules being separated determines the ideal
solvent for chromatographic separation because the size and shape of the molecule affects the strength
of the intermolecular forces between the molecules, which determines how attracted the solute
molecules are to the solvent molecules. Their level of attraction is crucial because it determines how much
they will be “pulled” up the chromatography paper. A solvent that is attracted to all molecules being
separated is essential for success, especially if the level of attraction varies depending on the solute. In
our experiment, we found that water was by far the best solvent to use for chromatography because its
strong hydrogen bonds were attracted to the dipole moments in the dye molecules. What made it better
was that it was attracted to the blue the most, thus “pulling” it up higher than any other molecule we
tested, followed by red and yellow. This differential in attraction allowed for the separation of colors into
zones, where we could identify each molecule group. However, in most of our other tests, with the
exception of blue, our solutes were essentially not very attracted to the solvents, leading to little or so
movement up the paper. The dye molecules were more attracted to the paper than to the solvents.
However, due to the shape of blue 1 and its multiple oxygen-sulfur dipole moments, and its larger size
(and thus larger London-dispersion forces), the molecule was attracted enough to Ethanol’s single
hydrogen bond enough to move up the paper, and attracted to acetone enough to move a little bit from
the original point. However, Isopropyl alcohol’s attractions towards the dyes were not strong enough to
pull them form the paper. In conclusion, the shape and size of the solute molecules as well as the number
of dipole moments they have affect their attraction towards certain solvents, which determines the ideal
solvent for chromatographic separation. In most cases, water seems to be the best solvent because of its
strong hydrogen bonding, which explains why it is often named the “universal solvent”.

POST-LAB ASSESSMENT
Explain why pyridine is soluble in water and benzene is not.
Hydrogen bonding. Pyridine (C6H5N) is more electronegative than Benzene. Pyridine has a
nitrogen in the center and lone pairs around the ring that is available for hydrogen bonding. On
the other hand, benzene is non-polar, and it cannot form strong bonds with the water molecule.