Brian Gross

Effects of the Polarity of the Mobile Phase in Paper Chromatography

Penn State Chemistry 113 Section 103
Group Members: Molly Grove, Connor Donley, Katie Del Guercio, Shahkila Daniels
TA: Ally Marianelli
February 27, 2014


Introduction
Chromatography is a process used to separate a mixture or solution into its components
using a mobile phase and a stationary phase.
1
The name chromatography, translated from the
Greek roots chroma and graphein, means to write with colors. The process was developed in
1903 by Mikhail Tswett, a Russian botanist who separated plant pigments using a column of
calcium carbonate.
2
This specific method of separation became known as column
chromatography. Since then, many other forms of chromatography have emerged. In 1941,
British chemists Archer J.P. Martin and Richard L.M. Synge developed a process called partition
chromatography in their study of the amino acid composition of wool.
3
Soon after, Martin and
his coworkers sought to develop a process with simpler, more replicable results. They revised
their method, using a sheet of filter paper as the stationary medium to separate the amino acids in
wool. This became known as paper chromatography, and in 1952, Martin and Synge earned a
Nobel Prize for their work on the subject.
3

Paper chromatography, like all other forms of chromatography, involves the separation of
a mixture or solution into its components using a mobile phase and a stationary phase. In paper
chromatography, the stationary phase is a porous filter paper and the mobile phase is a solvent
mixture. The mixture or solution that is to be separated is applied to a small spot near the edge
of the paper. That edge is then placed in the solvent mixture, and the mobile solvent phase is
pulled through the paper by capillary action.
1
The components of the test mixture move with the
solvent up the paper. Separation of the test mixture occurs based on the relative solubilities of its
components. The chromatography paper contains cellulose, making it a very polar stationary
phase. Because of this, more polar components are more soluble in the paper and do not travel
as far as non-polar components, which do not bond as easily to the paper.
4



The polarity of the mobile phase plays a key role in how far the components travel as
well. The more polar the solvent is, the shorter distance the components of the test sample will
travel because the solvent itself has an affinity for the chromatography paper.
5

Paper chromatography has a variety of uses that stem into a wide range of fields. Its
original use was to separate amino acids from a protein, but many other applications have since
been discovered. One major use is in the forensic science field. Paper chromatography can be
used for small samples of DNA found at crime scenes to create a DNA fingerprint, which aids
forensic scientists in identifying potential criminals.
6
In pathology, paper chromatography can
be used to detect alcohol, narcotics, hallucinogens, aspirin, and other chemicals in the blood.
7

Medical companies use paper chromatography to obtain pure compounds for use in developing
medicine.
6

One of the most common uses of paper chromatography, the one used in this lab, is the
separation of colored pigments. Specifically, it was used to separate the inks of a pen. However,
there are other processes for doing this. One such method is capillary electrophoresis. In this
process, ink samples are placed into a porous gel, and a current is run through the gel. The
current causes the inks to move through the gel, and components of the ink become separated
based on their relative sizes. Larger components do not travel as far as the smaller components.
This creates a fingerprint for the ink mixture, which can be used as a key for identifying other
ink mixtures.
8
Another process, called thin layer chromatography, is similar to paper
chromatography, except a very thin layer of material is placed on top of a rigid support, such as
glass or plastic. Samples are applied as in paper chromatography and the sample is placed in a
developing chamber. When the sample is finished, the component separations can be analyzed


and compared under an ultraviolet light.
1
While these two methods can be used to separate
mixtures, paper chromatography is cheap, simple, and quick, and is often equally effective.
8
In our experiment, we were given a sheet of chromatography paper with 5 out of 15
mystery pens spotted on the paper. Our goal was to identify the mystery pens by creating
reference chromatograms suitable for distinguishing between each of the pens by varying the
solvent mixture. In a previous experiment, we used a 2:1 propanol to water mixture as the
solvent, and the nonpolar components of the pen inks were not separated. Since this solvent did
not separate the nonpolar components, we hypothesized that a higher polarity solvent would be
needed. By using a more polar mixture, we anticipated that the mobile phase would have a
higher affinity for the stationary phase, so both the polar and nonpolar components of the ink
would separate within the length of the chromatogram.
Procedure
The procedure for the experiment our group developed was partially adopted from PSU
Chemtrek and can be found in greater detail there.
1
We were tasked with designing an
experiment to identify unknown pens. This stemmed from a previous experiment, which I will
refer to as the original experiment. In the original experiment, we had created a chromatogram
by placing small dots from a variety of pens onto a piece of chromatography paper. Refer to
Table 1 for the dotting order for the pens in the original run. The chromatogram was placed in a
solution of 2:1 1-propanol:water, covered with a plastic cup, and allowed to run until the solvent
front approached the top edge of the paper. However, the original run did not effectively
separate the inks in the pen, and we were tasked with developing a procedure to separate these
inks in order to identify mystery pens.


The first step was to create and test various solvent combinations to see if they could
effectively separate the inks of the pens. The substances available for use were propanol,
isopropanol, ethanol, methanol, and water. Each substance had a different polarity, as indicated
by the Snyder Polarity Index, which ranks the polarity of compounds on a scale of 0-9. For the
polarities of the compounds available to us, see Table 5. Substances were combined to create
solvents of varying polarities. Each combination created was used as the solvent for a different
chromatogram, generating a key which for potential comparison on the mystery runs. The
chromatograms were created by placing a sample dot from each of 15 pens near the bottom edge
of a piece of chromatography paper and placing that edge in the solvent solution. For a list of the
pens used, see Table 3. The solvents were allowed to ascend the paper via capillary action and
the pen inks were separated into their respective components.
Solvents were tested until 2 were found that were thought to achieve good separation and
thought to be good for distinguishing between the pens. After finding these combinations, each
group member was given two pieces of chromatography paper. Group members received
different combinations of pens, but each individual was given 2 chromatograms with the same
sequence of 5 pen spots. One chromatogram was run through each of the 2 selected solvents and
compared to the key that had been created for it. Based on the ink compositions determined
from the mystery test and our comparisons, our task was to identify which mystery pen was used
to create each of the 5 mystery spots.





Results
Table 1: Pen Layout for Brians Original Run
9
Spot # 12 13 14 15 16 17
Pen
Papermate
Blue
Papermate
Red
Papermate
Black
BIC Blue BIC Red BIC Black
*All numbering for the chromatogram spots increases from left to right (i.e. Pen #1 is the farthest
to the left, pen #15 is the farthest to the right)
**Pen layouts for chromatograms that were made by group members that did not factor into the
mystery runs are not listed.

Figure 1: Brians Original Run - 2:1 1-propanol:Water Snyder Polarity of 5.87
Table 2: Mobile Phases Tested by Group Member
9
Group Member Test 1 Solvent Test 2 Solvent Test 3 Solvent
Brian Gross 2:1 Methanol:Water 4:1 Methanol:Water 3:2 Methanol:Water
Connor Donley 1:1 Methanol:Water 1:1 Water:Ethanol N/A
Katie Del Guercio 2:1 Water:Methanol
2:1:1
Water:Propanol:Methanol
N/A
Molly Grove 1:1 Isopropanol:Water 3:1 Water:Isopropanol N/A
Shahkila Daniels 9:1 Methanol:Ethanol 2:1 Water:Propanol N/A





Table 3: Pen Layout for Brians First and Second Test Runs
9

1 Pilot Vball Black 6 Pilot Vball Blue 11 Pilot Vball Red
2 Papermate Black 7 Papermate Blue 12 Papermate Red
3 BIC Black 8 BIC Blue 13 BIC Red
4 Pilot Easytouch Black 9 Pilot Easytouch Blue 14 Pilot Easytouch Red
5 Staples Black 10 Staples Blue 15 Staples Red

Figure 2: Brians First Test Run 2:1 Methanol:Water Snyder Polarity of 7.4
9

Figure 3: Connors First Test Run 1:1 Methanol:Water Snyder Polarity of 7.8
10



Figure 4: Katies First Test Run 2:1 Water:Methanol Snyder Polarity of 8.2
11

Figure 5: Mollys First Test Run 1:1 Isopropanol:Water Snyder Polarity of 6.65
12
Pen layouts for Figure 6 can be found in Shahkilas Laboratory Notebook
13

Figure 6: Shahkilas First Test Run - 9:1 Methanol:Ethanol Snyder Polarity of 6.46
13



Figure 7: Brians Second Test Run - 4:1 Methanol:Water Snyder Polarity of 7.08
9

Figure 8: Connors Second Test Run 1:1 Water:Ethanol Snyder Polarity of 7.1
10

Figure 9: Katies Second Test Run 2:1:1 Water:Propanol:Methanol Snyder Polarity of 7.23
11


Figure 10: Mollys Second Test Run 3:1 Water:Isopropanol Snyder Polarity of 7.8
12


Figure 11: Shahkilas Second Test Run 2:1 Water:Propanol Snyder Polarity of 7.43
13
Table 4: Pen Layout for Brians Third Test Run
9
1 Staples Red 6 Pilot Easytouch Blue 11 Pilot Vball Black
2 Pilot Easytouch Red 7 Pilot Vball Blue 12 Pilot Easytouch Black
3 Papermate Red 8 Staples Blue 13 Papermate Black
4 Pilot Vball Red 9 BIC Blue 14 Staples Black
5 BIC Red 10 Papermate Blue 15 BIC Black



Figure 12: Brians Third Test Run 3:2 Methanol:Water Snyder Polarity of 7.56
9

Figure 13: Mystery Run #1 Using a 3:2 Methanol:Water Solvent
9
Figure 14: Mystery Run #2 Using a 9:1 Methanol:Ethanol Solvent
9





Table 5: Snyder Polarity Index Values for Available Compounds
9
Solvent Polarity Index
1-propanol
4.3
Isopropanol
4.3
Ethanol
5.2
Methanol
6.6
Water
9
The polarity of a solvent can be considered as an average of the Snyder Polarity Index value of
its components.
Sample Solvent Polarity Calculations:
Polarity
2:1:1 Water:Propanol:Methanol
= [2(9) + 1(4.3) + 1(6.6)]/4 = (28.9)/4 = 7.23
Polarity
3:2 Methanol:Water
= [3(6.6) + 2(9)]/5 = (19.8 + 18)/5 = 37.8/5 = 7.56
Discussion
Before we started the experiment, our group concluded that a more polar solvent would
be needed to separate the inks in each of the pens. In the original experiment, 2:1 1-
propanol:water was used as the solvent to create the chromatogram. However, this
chromatogram, which had a Snyder Polarity Index value of 5.87, was ineffective and the inks
from the pens ran all the way up the chromatography paper. This meant that the polarity of the
solvent was too low because it was not binding to the polar chromatography paper. As a result,
the nonpolar dyes ran all the way to the solvent front, and they were not separated.
For our groups solvent mixtures, we decided a solvent with a polarity higher than 5.8 on
the Snyder Index would be necessary to separate the inks and allow us to distinguish between the
pens. For my first solvent, I made a mixture that was 2 parts methanol to one part water,
resulting in a Snyder Polarity Index value of 7.4. However, the inks from the pens still ran all
the way to the solvent front. There was some visible separation in the different pens, but the


results were not definitive enough (see Figure 2). There were not enough distinctive
characteristics to tell apart the pens. While there was a small amount of spread between
components, there was not as much as we would have liked. One positive aspect of this test was
that it was somewhat effective at distinguishing between the black and blue pens, a feature that
the other first-run chromatograms did not accomplish very well. We determined a polarity
higher than 7.4 would be needed for the next runs.
Connor, Katie, and Molly experienced similar results on their initial runs, and they also
determined a new solvent combination would be necessary. Shahkilas first chromatogram, on
the other hand, had a favorable result. Despite having a relatively low polarity of 6.46, her first
run produced a chromatogram that provided a pleasant amount of detail in telling apart the red
pens, and a moderate amount of detail for the blues and blacks. Different shades of pink and
tails of orange and yellow appeared from the red pens on the chromatogram, and our group
decided the 6.46 Snyder Polarity Index solvent would be valuable for use in the unknown tests.
Despite the majority of our group having a failure to produce a solvent for use in the
mystery tests, we were able to successfully determine that our hypothesis was correct: a higher
polarity solvent than that of the original experiment would be needed to separate the components
of the pens.
For my second test, I tried to create a solvent with a Snyder Polarity Index value higher
than 7.4. To accomplish this, I did a qualitative analysis (I did not make any formal
calculations), and I determined a 4:1 methanol:water solvent would have a higher polarity than
my previous run. However, I made a mistake in my analysis. Using more methanol for every
part of water decreased the Snyder Polarity Index value from 7.4 to 7.08. A better test would
have been decreasing the amount of methanol for every part of water, as this would have


increased the solvents polarity. I did not notice this mistake until after the second
chromatogram was finished and I examined the results. My second test was not nearly as
effective as the first one in distinguishing between the black and blue pens, although it was a
little better at telling the reds apart (see Figure 7). Unfortunately, we already had a test that we
figured would be sufficient to differentiate between red pens, so we did not use this 7.08 mixture
in our final runs.
After everyone completed 2 tests, our group decided definitively that Shahkilas first
solvent mixture would be used in our mystery runs due to its ability to discern the red pens.
Despite having run 8 total chromatograms, we were still not confident in our previous tests
ability to differentiate between the black and blue pens. We decided my first run, with a Snyder
Polarity value of 7.4, did not separate the inks enough, so a more polar solvent would be
necessary. Fortunately, Connors first run provided us with an upper boundary. He used a
solvent mixture of 2 parts water to 1 part methanol, and we determined the inks did not travel far
enough up the chromatography paper, meaning the polarity of the solvent, 7.8, was too high (see
Figure 3). We decided a solvent polarity of approximately 7.6 would be sufficient to tell apart
the blacks and blues.
I ran a 3
rd
test using a solvent that was 3 parts methanol to 2 parts water to obtain a
Snyder Index value of 7.56, which was very close to our target value of 7.6. This test yielded
favorable results. There were visible differences in the dye compositions of the black and blue
pens, including yellow tails on the black streaks and noticeable shading variances among the
blues (see Figure 12). Due to these advantages, we decided this solvent, combined with
Shahkilas first run for differentiating between the red pens, would be our second combination
for use in running the unknown pens.


After receiving my two mystery chromatograms, I ran one in the 3:2 methanol:water
solvent (Figure 13) and the other in the 9:1 methanol:ethanol solvent (Figure 14). I called the
7.56 Snyder Polarity Index mixture mystery test 1 and the 6.46 unknown test mystery test 2.
I compared these results to the keys created for each solvent combination (Figures 12 and 6
respectively). I noticed the first dot was clearly Pilot Vball black from mystery test 1 due to its
characteristic black color after comparison with the key. I was also able to determine the fourth
dot shared a resemblance with the sixth dot on the key: they were the same color and they were
both characterized by a more dense coloration close to the original dot and a lighter blue coloring
near the solvent front with a gap between the two zones. From this analysis, I determined dot
four was Pilot Easytouch blue. My mystery test 1 was not as effective as I had hoped in
determining the identity of the 2
nd
dot, which was either black or blue. I noticed a slight yellow
coloring at the top of the streak on the test chromatogram, but four different pens shared this
quality. Not enough of the ink made it all the way to the solvent front, which is where I made
many of my distinctions between blacks and blues, so I had to examine mystery test 2.
Mystery test 2 did not yield the definitive results for the red pens that I had hoped for. It
was able to show me that the 3
rd
mystery pen was Staples red due to the absence of a yellow tail.
However, it was not very effective for the 5
th
mystery pen. There was a long yellow streak
tailing a bright pink pigment that ran all the way to the solvent front. Unfortunately, three pend
shared this property. Additionally, this test did not clear up my uncertainty about the 2
nd
mystery
pen, so I was forced to make an educated guess. For the 2
nd
pen, I guessed Papermate blue and
for the 5
th
pen, I guessed Pilot Easytouch red.
My guesses for mystery pens 2 and 5 were incorrect, but the three pens I confidently
identified were correct. Pen 2 turned out to be BIC black and pen 5 was Papermate red. In total,


I identified 3 out of the 5 mystery pens correctly. The main problem I ran into during the
mystery runs was that the amount of ink used during our test runs was more than the amount of
ink used in the mystery run. Because of this, our test runs yielded darker and more visible
results, and there were more identifiable characteristics for each pen. In the mystery run, we
were not able to dot the mystery pens ourselves, and the initial dots were lighter than in our tests.
This made it more difficult to identify the unknown pens in cases where very subtle differences
were the only distinguishing factors between pens.
Conclusion
Our hypothesis that a more polar solvent than the one used in the original experiment
would be needed to separate the components of the pens was correct. We found that a more
polar solvent had a higher affinity for the polar chromatography paper, and therefore did not
carry the ink components all the way to the solvent front. There was a greater separation with
increasing solvent polarity up to a certain point. At this point, which we determined to be
roughly 7.8 on the Snyder Polarity Index, the solvent was too polar and the components of the
pens did not travel far enough or they did not achieve ideal separation. From our groups
findings, I was able to identify 3 of the 5 mystery pens assigned to us.
For future studies of this sort, even more solvent combinations should be tested. Due to
our limited class time, our group was only able to test a limited number of solvent polarities. It
is likely that a different solvent would be more effective in generating distinctions between the
pens. However, due to lack of time, we were not able to find such a solvent. The fact that I
could only identify 3 of the 5 pens reflected this. Despite my shortcomings in distinguishing


between the mystery pens, my overarching hypothesis held true: a more polar solvent was
needed to improve the chromatographic separation of the components of the pens.
References
1. Thompson, S. PSU Chemtrek: Small-Scale Experiments for General Chemistry 2013-2014;
Hayden McNeil Publishing: Plymouth, MI, 2013; pp 17-1 17-22.
2. "History of Chromatography." History of Chromatography. University of Michigan, n.d.
Web. 21 Feb. 2014.
3. chromatography. Encyclopaedia Britannica. Encyclopaedia Britannica Online.
Encyclopdia Britannica Inc., 2014. Web. 23 Feb. 2014.
<http://www.britannica.com/EBchecked/topic/115917/chromatography>.
4. Chen, Hu-Sheng, Ph.D, Hsien-Hui Meng, Ph.D, and Kun-Chi Cheng, M.Sc. "A Survey of
Methods Used for the Identification and Characterization of Inks." Forensic Science
Journal 1.1 (2002): 1-14. Forensic Science Journal. Web. 22 Feb. 2014.
<http://fsjournal.cpu.edu.tw/content/vol1.no.1/p1.pdf>.
5. paper chromatography. Encyclopaedia Britannica. Encyclopaedia Britannica Online.
Encyclopdia Britannica Inc., 2014. Web. 23 Feb. 2014.
<http://www.britannica.com/EBchecked/topic/441999/paper-chromatography>.
6. Mukherjee, Mukulika. "Paper Chromatography Uses." Buzzle.com. Buzzle.com, 06 July
2012. Web. 25 Feb. 2014. <http://www.buzzle.com/articles/paper-chromatography-
uses.html>.


7. Allison, Keith. "Re: What Are the Different Uses for Paper Chromatography in a Society?"
MadSci Network: Chemistry. N.p., n.d. Web. 25 Feb. 2014.
<http://www.madsci.org/posts/archives/2000-10/971129675.Ch.r.html>.
8. Egan, James M., Kristin A. Hagan, and Jason D. Brewer. "Forensic Analysis of Black
Ballpoint Pen Inks Using Capillary Electrophoresis." FBI. FBI, 28 Feb. 2011. Web. 25
Feb. 2014. <http://www.fbi.gov/about-us/lab/forensic-science-
communications/fsc/july2005/research/2005_07_research01.htm>.
9. Gross, Brian. CHEM 113 Laboratory Notebook, Spring 2014, pp. 6-13.
10. Donley, Connor. CHEM 113 Laboratory Notebook, Spring 2014, pp. 12-14.
11. Del Guercio, Katie. CHEM 113 Laboratory Notebook, Spring 2014, pp. 6-12.
12. Grove, Molly. CHEM 113 Laboratory Notebook, Spring 2014, pp. 11-15.
13. Daniels, Shahkila. CHEM 113 Laboratory Notebook, Spring 2014, pp. 16-21.










2 = http://www.umich.edu/~orgolab/Chroma/chromahis.html
3 = http://www.britannica.com/EBchecked/topic/115917/chromatography
4 = http://fsjournal.cpu.edu.tw/content/vol1.no.1/p1.pdf
5 = http://www.britannica.com/EBchecked/topic/441999/paper-chromatography
6 = http://www.buzzle.com/articles/paper-chromatography-uses.html
7 = http://www.madsci.org/posts/archives/2000-10/971129675.Ch.r.html
8 = http://www.fbi.gov/about-us/lab/forensic-science-
communications/fsc/july2005/research/2005_07_research01.htm
9 = My Notebook
10 = Connors Notebook
11 = Katies Notebook
12 = Mollys Notebook
13 = Shahkilas Notebook