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Chromatography Lab Report

Hunter Miller
March 24th, 2016
Chem 113 Section 003
TA Anthony Katona
Partner Augustey Mongia

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Introduction:
Around every corner and across every landscape the world is full of colors. These colors
could range from the bright and beautiful colors of spring to the dark and dreary colors of a
storm overhead. The one thing that nearly every object has in common is that they have color. In
todays world artificial colors have even become a large part of our world. The color of posters
or pens are all artificial. This is not necessarily a bad thing, after all it gives the world a sense of
colorful beauty. However, some artificial colors may not be what they appear. Often in industry
today, artificial colors, like those in pen ink, are not simply made out of black, blue, or red.
Instead they are made up of a combination of variations of that particular color as well as some
different colors all together.
With all this mixing of colors occurring, it can often be hard to discern what dyes/colors
are present in a certain product and what arent. In order to solve this problem the process of
chromatography is used. Chromatography at its base definition is defined as a method of
separating substances that make up a liquid or gaseous mixture1. However, this in itself raises
infinitely many questions such as what is the purpose of chromatography in everyday life? Who
invented chromatography and why? Also, what are the various techniques used to perform
chromatography? All of these are very valid questions and all require their due diligence to be
answered.
It only makes sense that in order to explain chromatography, the history of the process
itself must first be explained. Chromatography was invented sometime during the early 1900s. It
is currently disputed as to whether it was invented in 19012 or 19033. Regardless of the time, it
was first used be a Russian botanist named Mikhail Tswett. Tswett intended to use the process to
separate plant pigemnts.3 Specifically, he used a column filled with calcium carbonate and added

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plant pigments to the top of it. From there the pigments were able to travel through the column
and were able to separate into the various pigments. This was the first known use of
chromatography and was actually given that particular name during this instance. The term
chromatography was actually derived from Greek words meaning color writing.2 The process
itself has since been adapted and updated due to the advance of technology. However, the general
principle is still the same.
Nevertheless, chromatography is simply more than just a historic process. In order to
understand it fully, its purpose and procedure must be explained. As stated before,
chromatography is a method of separating substances. It does by using what is called a mobile
phase and a stationary phase. The stationary phase can be thought of as the platform on which
the separation will occur. One example of a stationary phase could be a piece of porous paper
such as that used in paper chromatography, which will be described later. Once the stationary
phase is obtained, the sample to be separated is placed on the stationary phase. From here the
mobile phase is implemented. The mobile phase is a fluid that is allowed to flow through the
stationary phase. As it flows it will carry certain components of the sample with it various
distances according to the polarity or non-polarity of each component. The more polar a
component is the less it will travel with the mobile phase. It will have a higher affinity for the
stationary phase. Conversely, if a component is non-polar it will travel a great length with the
mobile phase.4 In general this process can be used for more than just separating plant pigments or
pen inks. Two of its main purposes are to identify concentrations of pollutants in air or water and
to separate out impurities during chemical reactions.1
However, there are various methods for performing chromatography. Each method is
unique and each method can be used for various and more specific purposes. The first type of

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chromatography that should be looked at is paper chromatography, which is the method used in
this particular lab. Paper chromatography has a stationary phase that utilizes a porous and
specially prepared piece of paper known as chromatography paper. This paper is spotted with the
sample to be separated near the bottom. From there a mobile phase is applied to the paper. The
mobile phase travels up the paper and reacts with the sample in order to separate out the various
components of it. Due to varying degrees of polarity, some components will travel further with
the mobile phase because they are more attracted to it. Others will separate out of the mobile
phase into the stationary phase rather quickly indicating a dislike in terms of polarity for the
mobile phase.5 The separated components then appear to be individual pieces instead out one
mixture of the sample.
From here there is one important value that can be calculated for each component in
order to relate the various components to each other. This value is called an Rf value. This value
is calculated as follows:
Rf = distance traveled by component/distance traveled by solvent6
The distance traveled by the solvent is referred to as the solvent front. This is how far the mobile
phase traveled up the stationary phase before the experiment is ended. Therefore, for each
component of the sample, the distance traveled is measured and placed over the solvent front.
This provides the Rf value which is always less than or equal to 1.0.
In the real world paper chromatography actually has many uses. It can be used for
separating amino acids, RNA fingerprinting, and even antibiotics.7 Additionally, it can even be
used to separate candy coatings.8
Nevertheless, there are also several other types of chromatography. One of them is
referred to as liquid column chromatography. In this example, a column, which is usually

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comprised of glass, is used. A stationary phase material is placed inside this column. The sample
and the mobile phase are then applied to the top of the column and allowed to run down the
column until they separate.1 This method is beneficial in testing water samples for pollution.7
Another method that can be used is thin-layer chromatography. In this process a thin plate
is used, which is often a piece of glass. A liquid substrate is then placed on the glass. Then the
sample is placed on one end of the plate and stood upright in a solution that contains the mobile
phase, which will travel up the plate. This causes the sample to separate.1 The practical uses of
this method include helping to determine if there is pesticide residue in food and analyzing the
dye composition of ffibers.7
One final method that can be used is gas chromatography. This method uses a column of
adsorbent material to separate gases. In this process the mobile phase is injected into the column
in a gaseous form. This prevents any gases from escaping the system.1 This can be used to detect
bombs in airports and identify certain drugs.7
Overall, there are several methods to performing chromatography. However, the most
efficient one for the purpose of this lab will be paper chromatography.

Procedure:
The overall goal of this experiment was to determine the identity of a series of unknown
pen inks by separating their components and analyzing them comparatively with a baseline
chromatogram. In order to do so, several steps needed to be taken that were found in a literature
procedure.4
The first part of this experiment was determining the polarity of the baseline 2:1 1propanol:water. In order to do this, the use of the Snyder Index of Polarity was implemented.
Once the polarity was calculated, the decision had to be made on whether or not to choose a

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more polar or less polar solvent as the mobile phase in order to create maximum separation of
the pen ink components.
From there it was necessary to set up the chromatograms to follow in order to determine
the best mobile phase. In order to set up a chromatogram, a piece of chromatography paper was
used. A line was drawn 0.5 inch above the bottom of the horizontal side of the paper. From there
the line was marked 15 times every 1 cm with a dash in order to indicate that this is where each
pen would be spotted. Subsequently, the various pens were each spotted at the indicated dashes.
The order of the pens for every baseline chromatogram were as follows:
Table 1: Order of Pen Inks on Chromatography Paper
Spot Number on Paper
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Color of Pen
Black

Red

Blue

Brand
Pilot Easy Touch
Pilot G2
Pilot Vball
Papermate Inkjoy
Bic Ultraround
Pilot Easy Touch
Pilot G2
Pilot Vball
Papermate Inkjoy
Bic Ultraround
Pilot Easy Touch
Pilot G2
Pilot Vball
Papermate Inkjoy
Bic Ultraround

Different mixtures of solvents were then used to carry out the experiment. In total, the use
of 3 different mixtures, other than the original 1:2 1-propanol:water, were used. These mixtures
were all prepared in the same fashion. After the paper had been spotted, the mixture was created
using the various ratios and solvents. The mixture was then placed in the bottom of a petri dish.
The chromatography paper was then stapled together at the top and the bottom so that it formed a

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cylinder. This cylinder was then placed in the petri dish with the solvent. The paper was then
covered with a clear plastic cup to prevent evaporation, but still allow a visual view of the
experiment.
Following these steps, the chromatogram was allowed to sit until the solvent had crept up
near the entirety of the paper. Once it was close to the top, the chromatogram was removed from
the solvent and set out to dry. This procedure was repeated for every trial.
Once the correct mixture with the correct polarity was found, it was necessary to use this
chromatogram to compare to the unknowns. This designated mixture was again placed in a petri
dish and the unknown chromatogram was placed in the solution. The same procedure was carried
out as previously stated. Once the chromatogram was removed and dried it was compared to the
chromatogram utilizing the same mixture. From here the identity of the unknowns was attempted
to be determined. In order to do so visual observation, Rf values, and UV light observation were
all utilized.
All results were recorded in the notebooks of myself9 and my partner Augustey Mongia.10

Results:

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Image 1 - 2:1 1-propanol:water

The baseline chromatogram of 2:1 1-propanol:water was carried out and its polarity was
measured. The calculation used was as follows:
(2/3 x 4.3) + (1/3 x 9.0) = 5.9 polarity
These calculation incorporated in the polarity values of the individual pieces of the mixture and
their ratios. The mixture was 2/3 1-propanol. The polarity value of propanol is 4.3. The mixture
was also 1/3 water. Water has a polarity index of 9.0. Once these ratios were multiplied by their
respective polarity indexes, they were then added together. This resulted in the polarity value of
the baseline 2:1 1-propanal:water to be 5.9. However, since the pen inks did not completely
separate it was determined that this was not the optimal polarity value. Something more polar
was needed for the mobile phase in order for the various components of the pen inks to enter the
stationary phase faster. Therefore, the polarity value of the solution must be somewhere between
5.9, which was the polarity value of the baseline mixture indicated above, and 9.0, which is the

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polarity index of water. Water is extremely polar so the mixture used should not fall above it due
to the fact that this would result in a mixture that is simply too polar. Overall, the range was
narrowed down to between 5.9-9.0.

Trial 1
Image 2 - 2:1 Ethanol:water

The first mixture that was created was a 2:1 Ethanol:water mixture. Ethanol has a polarity
index of 5.2. This is comparatively higher than 1-propanol, which was used in the baseline and
has a value of 4.3. Therefore, the addition of ethanol should increase the overall polarity value of
this mixture and provide a better mobile phase for the reaction. The 2:1 ration indicates that the
mixture was 2/3 ethanol and 1/3 water. The polarity of the mixture was calculated using the
following equation:
(2/3x5.2) + (1/3 x 9.0) = 6.5
This calculation indicated that the solution had a polarity index value of 6.5. The process
used to calculate tis value was the same as for the baseline calculation except for the fact that the
respective value of ethanol was used. This is an overall increase of 0.6 from the original baseline
mixture. This benefited the separating of the pen ink components. The overall increase in
polarity of the mobile phase caused some of the pen ink components to separate out sooner.

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Therefore, it was easier to identify these various components. However, the mixture was simply
not polar enough. The red and blue dyes in particular did not separate completely. They went
entirely to the solvent front and in doing so made it unable to determine the various components
and Rf values of those components. Therefore, the mixture again needed to be more polar. After
this the range for the polarity of the mixture could now be limited to between 6.5-9.0.

Trial 2
Image 3 - 2:1 Methanol:water

For the second trail a mixture of 2:1 Methanol:water was used. Methanol has a polarity
value of 6.6. This is an increase from the previous mixture, which used ethanol that had a value
of 5.2. This addition of methanol was able to increase the overall polarity of the mobile phase.
The polarity value for it was calculated as follows:
(2/3 x 6.6) + (1/3 x 9.0) = 7.4
This indicates that the polarity value for the second trail mixture was 7.4. This is an
increase of 0.9 from the previous mixture. This was positive because an increased polarity
mixture was required. This overall benefited in the separation of both the black and red inks.
Nearly all components of the black and blue inks were able to be determined from this mixture.
However, the blue inks still had components that caused them to travel all the way with the

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mobile phase to the solvent front. Therefore, this indicates that even at a value of 7.4 the mixture
was still not simply polar enough. As a result, an increased polarity mixture was necessary for
our next trial. Now the scope of that mixture in terms of polarity could be narrowed down ever
further. The mixture now needed to be between 7.4-9.0.

Trail 3
Image 4 - 1:1 Methanol:water

For the third trial a mixture of 1:1 Methanol:water was used. In comparison to trial 2, the
components of this mixture are still the same. However, the ratio is now 1:1. This means that the
solution is now half water and half methanol. Since water is extremely polar this means that the
overall polarity of this mixture will be relatively high. The polarity of this mixture was calculated
as follows:
(1/2 x 6.6) + (1/2 x 9.0) = 7.8
At a polarity of 7.8, this was the most polar mixture that was produced. Therefore, it
turned out to be an excellent mobile phase for our experiment. As shown above, the various
components of the inks nearly all separated out. The only fault that this mixture had was that it
was perhaps a little too polar for the black inks. They did not separate out as well in this mixture.
However, they still separated to a good degree and overall this mixture provided the best results

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in terms of the separation of the pen inks. This mixture was subsequently chosen as the one that
would be most beneficial to use when testing unknowns.

Unknowns:
Image 5 - Unknowns

Once it was determined which mixture would be the best mobile phase, the goal of this
experiment could now be carried out. The mixture could now be used as the mobile phase in
testing unknown pen inks to determine which ones they are based on comparison with all 15 of
the pen inks in that same mixture. Therefore, the same process was used as in the previous trial.
The only difference was that there were now 5 unknown inks on the chromatography paper
instead of 15 known ones. Overall, the polarity of this mixture still remained at 7.8. After the
chromatogram was allowed to dry the following observations were recorded in order to help
identify the unknowns:
Table 2: Observations of Unknowns
Unknown Spot

Details

Rf values

Number
1

Light pink thin streak, appeared

0.67

yellow in UV light
Thick purple streak that turned

0.55

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yellow near solvent front
Thin faded purple streak that

0.83
0.38

turned to yellow near the solvent

0.88

front
Thick faded light pink streak,

0.95

appeared orange in UV light

Thick bright pink streak that

1.0

turned to orange near the solvent

front, appeared dark and yellow
under UV light
Based upon these results the unknown pen inks were able to be comparatively analyzed
by looking at all 15 of the pen inks in the 1:1 Methanol:water mixture, carried out in trial 3. The
ones that appeared similar would be the matches and would help to determine the identity of the
unknowns. The following spots from trial 3 were analyzed in detail and compared to the
unknown inks to determine the identities:
Table 3: Observations of Selected Knowns
Spot Number

Brand Name and

Details

Rf Values

Color
Papermate inkjoy

Light pink streak,

0.60

Red

appeared mostly

Bic Ultraround

yellow in UV light
Purple thick streak

0.55

Black

that turned to yellow

0.83

Papermate Inkjoy

near solvent front

Purple thin streak that 0.40

Black

turned to yellow near

0.83

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Pilot Easy Touch

the solvent front

Light pink streak that

Red

appeared faded,

0.90

looked orange under

10

Bic Ultraround

UV light
Thick bright pink

Red

streak that turned to

1.0

orange near solvent

front
Sample Calculation for Rf value = distance traveled/solvent front = 2.5/4.2 = 0.60
These observations and calculations gave great insight into the identity of the unknowns. From
this information the unknowns were able to be determined with relative ease. The unknowns
were designated as follows:

Table 4: Identification of Unknowns

Unknown Spot Number
1
2
3
4
5

Brand Name and Color Determined

Papermate Inkjoy Red
Bic Ultraround Black
Papermate Inkjoy Black
Pilot Easy Touch Red
Bic Ultraround Red

Discussion:
It did not take long to determine the correct mixture that was necessary to determine the
identity of the unknowns. Only 3 different mixtures were tested before the correct one was
decided on. This was positive because it was efficient in terms of time.

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Once that was determined, the next most difficult part of this experiment was determining
the identity of the unknowns. Many factors went into doing so. The first unknown was perhaps
one of the trickiest to decide. It very closely resembled that of the fourth unknown. Therefore,
based on just visual representation, it was nearly impossible to distinguish the two. The Rf value
of unknown 1 seemed to match up with that of the red Papermate inkjoy and the Rf value of 4
seemed to match up with that of the red Pilot easy touch. However, neither of the matches were
exact. The true factor that helped in distinguishing these to was the use of UV light. Once the
unknowns were placed under UV light, the first unknown appeared yellow while the fourth
appeared orange. This distinction matched the first unknown with the red Papermate inkjoy and
the 4th unknown with the Pilot easy touch.
Now that 1 and 4 had been determined it was time to look at unknown 2. However,
unknown two was very similar in color to that of unknown 3. Additionally, the two had relatively
close Rf values. Unknown 2 had Rf values of 0.55 and 0.83 whereas unknown 3 had values of
0.38 and 0.88. Therefore, it was difficult to determine their identity based off of these values.
Furthermore, the black ink showed no data under the effects of the UV light. As a result, just
looking at them visually seemed to be the best option. The real distinction between the two lied
in the thickness and brightness of the components. Unknown 2 had a very thick and vibrantly
colored streak. Unknown 3 had a faded and seemingly thin streak. Based off of this information,
it was determined that unknown 2 was the black Bic ultraround and unknown 3 was the black
Papermate inkjoy.
Following this determination, there was only one unknown left to determine. Unknown 5
was another red ink pen. However, it was very distinctive from the previous two. In all categories
it was clear which pen this unknown was. Visually, the streak was distinctive by the fact that it

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had an orange circle near the solvent front. Additionally, its Rf value was 1.0, which is very
unique. This means the components travelled to the solvent front. Also, upon UV examination,
the unknown appeared to be dark and yellow. All of these factors combined led to the conclusion
that unknown 5 was indeed the red Bic ultraround.
After taking all of these facts into consideration, it was determined that the correct
identification of all 5 unknowns had been made. Therefore, the experiment was a success.
Overall, the experiment relied heavily on polarity. The concept of polarity came into play
when deciding which mobile phase to use when testing the unknowns. The basis of this lies
within the idea of intermolecular forces. Whenever two polar solvents encounter each other a
dipole-dipole bond. This bond dissolves the two into each other. This is where the phrase like
dissolves like comes from. This bond allowed certain components of the inks to travel further
with the mobile phase or less depending on their polarity. Its one of the main reasons why this
experiment is even able to succeed in the first place.

Conclusion:
Overall, the experiment was very successful. The identification of all the unknowns was
made on the first attempt. The mixture of 1:1 methanol:water seemed to work out well as the
mobile phase. The unknown inks all separated to near completeness.
Throughout the experiment the major keys were choosing the correct mixture and
identifying the unknowns. After trying combinations of 2:1 ethanol:water and 2:1
methanol:water, it was determined that a more polar solvent was still necessary. This was found
in the 1:1 methanol:water mixture. By using this he unknowns were able to be determined using
visuals, Rf values, and UV light.

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One thing that would be beneficial to the overall success of the experiment would be if
there were less known pen inks to choose from. By using less known pens, this would decrease
the amount of time that the experiment takes while still allowing the point of the entire
experiment to get across.
Overall, this lab effectively communicated the purpose and usefulness of
chromatography.

References:
1

Hartman, Marvis E. "Chromatography." World Book Advanced. World Book, 2016. Web. 15
Mar. 2016.

"Chromatography". Encyclopdia Britannica. Encyclopdia Britannica Online. Encyclopdia

Britannica Inc., 2016. Web. 15 Mar. 2016.

"History of Chromatography." Umich.edu. Web. 15 Mar. 2016.

Thompson, Stephen. PSU Chemtrek: Small-Scale Experiments for General Chemistry.

Plymouth: Hayden McNeil, 2015. Print.

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5

"Paper Chromatography". Encyclopdia Britannica. Encyclopdia Britannica Online.

Encyclopdia Britannica Inc., 2016. Web. 15 Mar. 2016.

Clark, Jim. "Paper Chromatography." Chemguide.co.uk. 2007. Web. 15 Mar. 2016.

"Chromatography." Soinc.org. Web. 15 Mar. 2016.

Burdge, Julia, and Jason Overby. Chemistry Atoms First. Boston: McGraw-Hill, 2012. Print.

Miller, Hunter. Chem 113 Notebook, Spring 2016. Pgs. 26-30.

10

Mongia, Augustey. Chem 113 Notebook, Spring 2016. Pgs. 64-65, 21-23.