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Using Paper Chromatography to Identify Unknown Inks
Samar Almarzooqi
2/21/13
Partners: Mariam Ahmed
CHEM 113 Section 103
TA: Steve Kennedy
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Introduction
Chromatography is a way to separate mixtures of a solution into separate
components. The technique is often used in a variety of different settings, like scientific
research, medicine, and industrial processes, because it allows for the analysis of the
components of many mixtures.1 Paper chromatography specifically is a type of
chromatography that “separates dried liquid samples with a mobile and stationary
phase.”2 Paper chromatography uses special cellulose paper that acts as a stationary
phase. The different samples act as a mobile phase when dissolved in a solvent. The
sample is originally at one side of the chromatogram paper, and it is pulled through the
stationary phase through “capillary action.” The components of the sample are then
separated through the movement of the samples through the stationary phase. The
differing components of samples can be separated into their different pigments, and each
pigment signifies another component of the sample.3
Russian botanist, Mikhail Tsvet, first created chromatography in 1906 in an
experiment that separated chlorophyll pigments. He did so by using organic solvents to
extract green material from the plant, and he then allowed the material to filter through a
solid powder. Using polar solvents, the extract was moved through the powder. He then
observed different bands of color across the column, which he deduced showed the
different types of chlorophyll within the extract. His approach signified a novel way to
separate and “investigate the chemistry of complex natural mixtures.”2 The scientific
community did not agree with chromatography as the best means of separating
components of a sample, and it was not until the 1930s when the uses of chromatography
were readdressed. In a type of chromatography termed partition chromatography,
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chemists Martin and Synge analyzed the structure of proteins through paper
chromatography. Today, chromatography is a technique used in many different settings
as a means for separating mixtures into their separate components. The different types of
chromatography include: Liquid Chromatography, Gas Chromatography, Thin-Layer
Chromatography, Ion-Exchange Chromatography, and Paper Chromatography.2
Chromatography separates mixtures into their components by utilizing the idea
that different chemicals move at different rates through the liquid or solid stationary
phases. Because the individual components of a mixture move at different rates, they will
become separated from each other.4 In Paper Chromatography, the stationary phase is a
liquid held within a solid, most often it is the cellulose paper and water complex. The
mobile phase is the sample tested, and it travels through the stationary phase by placing
the stationary phase in the mobile phase. The mobile phase is then pulled up through the
stationary phase through capillary action, which allows for the separate components to be
pulled up to different distances. In chromatography, the objective is to separate the
individual components as much as possible, which is possible by maximizing the
differences in the component migration and minimizing the spreading of the component
process.3
Depending on the interactions between the stationary phase and mobile phase, the
migration distances can be optimized. Using polar compounds as the mobile phase will
not have as large of a migration distance because the polar compounds will be attracted to
the polarity nature of the water. By using nonpolar compounds, the distance the molecule
migrates will be farther because it will not react with the water in the stationary phase.
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Making sure both the solvent and the molecules have similar polarities will maximize the
distance the components will travel.1
Paper Chromatography has a limited use in the effective application of the
technique, mainly because it cannot separate complex mixtures and cannot lead to a
quantitative analysis of the concentrations of individual components.5 The advantages of
using Paper Chromatography, though, are that it is inexpensive and easy to reproduce.
Also, because the chromatography paper is manufactured in a uniform structure, the
chromatography for a particular sample can be compared to another without many
variables affecting the difference in component migrations.3
In Forensic Science, chromatography can be used to separate unknown
compounds and identify them based on the distance each component travels. In drug
tests, the urine is tested through chromatography to identify drugs. In pathology,
chromatography can be used to identify possible poisonous substances in dead bodies to
determine if a possible murder took place.6 In cases where advanced technology cannot
be used, chromatography can be used to analyze mixtures of unknown substances and is
effective in doing so. Applicable in the use of chromatography is the use of paper
chromatography to separate individual portions of candy coatings to determine the type
of food dyes present.7 Paper chromatography is an inexpensive technique that identifies
unknown solutions, so it is easy to use in practical applications where time and energy are
limited.8
In the lab conducted, paper chromatography was used to identify both different
color of inks (red, black, and blue), and brand of the ink (BIC Round Stic, Paper Mate,
Staples, Pilot Easy Touch, and Pilot V Ball). The variability in the mobile solvent used
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was exploited in order to maximize the distance of the migration of the ink and be able to
distinguish the different brands for the same color from each other. Previously in the first
experiment, a 2:1 1-propanol/water solvent was used to separate the components of the
six ink samples.3,10 Because all of the ink samples traveled the same distance at a very far
distance along the chromatograph paper and 2:1 1-propanol/water is less polar than
water, it could be concluded that the dyes were non-polar.3 Using the principle of “like
dissolves like”, the assumption can be made that substances of more similar polarities
would not travel as far as substances of extremely different polarities.11
In order to maximize the distance the components of each ink traveled, using
solvents that are more nonpolar will result in the ink traveling a farther distance.
Therefore, less polar solvents were used in order to maximize the separation of the
individual components of the ink. Because methanol is amphipathic, it was predicted that
the individual components of the ink would spread more depending on the polarity of the
individual components. Our hypothesis was that less polar solvents would be more
effective in separating the individual components of the ink samples into different colors
due the nonpolar nature of the inks. Therefore, spreading of the different components
shown by different colors along the chromatogram would help distinguish different
brands of ink from each other. In order to test the hypothesis and determine the identity
of different ink samples, different samples of solvents with different degrees of polarities
were used in order to find the best possible solvent mixture.
Procedure
Following the procedure from the PSU Chemtrek, students determined which
solvent systems would be most effective in identifying unknown ink brands.3 Paper
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Chromatography is an easy and quick way to separate components of a solute, so in order
to maximize the amount of samples of different solvent systems in separating the
components of ink, a group of two people in the lab each conducted paper
chromatography on two different solvent types for each color and brand of ink, 15 in
total.9,10 The goal of the lab was to improve the separation of the individual components
in each type of ink in order to determine the identity of the ink by comparing known ink
Paper Chromatography samples to the unknown ones. Each student was given two
similar chromatography papers, each with four different ink dots on it. In order to create a
sample chromatogram that successfully separated the components of each ink to
differentiate between the different brands, each student then made chromatograms with
the fifteen different colored inks and brands (five of each color, each a different brand). A
line was drawn across chromatograph paper 0.5 cm from the bottom, and each ink dot
was placed at an equal distance away from each other. In order to avoid confusion for the
order and identity of the fifteen known inks along the chromatogram, a number system
was used where each dot was given a number from 1-15 and then in the lab notebook,
their identity was accounted for. On the chromatograms, the order of the inks were:
Blue
Black
Red
1 Pilot VBall BG05
2 Paper Mate
3 BIC Round Stic
4 Pilot Easy Touch
5 Staples 1.0
6
7
8
9
10
11
12
13
14
15
The chromatograms then needed to be rolled into a cylindrical shape and then
stapled so that the end of the two sides were not touching.
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Different solvents were available to use, and using 1-propanol, ethanol, and 1:1
methanol-ethanol solutions to coat the bottom of each individual petri dish, the two
different chromatograms were placed in the petri dish. A plastic cup was then placed over
the chromatogram to limit the evaporation of the solvent used. A waiting time period of
about 15 minutes was needed to provide ample time for the individual components to
either travel or spread depending on their polar characteristics. Once the Paper
Chromatography method was finished for each chromatogram, they were taken out of the
petri dish and a line was drawn across the “solvent front line”.3 After being set down to
dry, they were analyzed in order to determine if the separation of the components of the
ink allowed for the distinction of the different types of ink. For the red ink, an ultraviolet
light was provided in order to further analyze the type of red ink it was, but our lab group
did not utilize it.
From comparing the individual chromatograms, it was determine that the 1propanol solvent better separated the components of the black and blue inks and allowed
for the distinction of the different types, and that the 1:1 methanol/ethanol solvent
allowed for better separation of the components.3 Therefore, for the two unknown
chromatograms given, both solvents were used, one for each chromatogram. The same
steps for preparing the chromatogram for the fifteen known inks and colors were then
conducted again, this time for the unknown inks. The unknown inks were lettered A-D in
order to avoid mistaking the numbers from the previous chromatograms for the unknown
chromatogram in the Discussion section. Afterwards, a comparison between the
chromatograms with the known identities of the ink and the chromatograms with the
unknown was done in order to successfully determine the identity of the inks.3
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Results
Figure 1: Original Chromatogram with 2:1 1-propanol/water 10
This figure is the original chromatogram using the solvent 2:1 1-propanol/water using six
inks (#12-16).
Figure 2: Chromatogram to Determine Unknown Inks-1:1 Methanol/Ethanol 9
Figure 3: Chromatogram to Determine Unknown Inks- 1-propanol10
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Figure 4: Chromatogram to Determine Unknown Inks-ethanol10
Skewed color because the chromatogram was not placed in the petri dish so that the
whole bottom was touching the same amount of solvent. Therefore, the mobile phase did
not travel up the paper in a straight vertical line.
Figure 5: Unknown Chromatogram-1:1 Methanol/Ethanol10
Figure 6: Unknown Chromatogram – 1-propanol10
Skewed color because the chromatogram was not placed in the petri dish so that the
whole bottom was touching the same amount of solvent.
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Discussion
The goal of the lab was to distinguish between different brands of ink by
maximizing the separation of each component of the ink through the experimentation
with different solvents as the mobile phase. Because the chromatograms were
distinguished depending on the color spread of the ink, finding a solvent that resulted in a
variety of distinguishing colors for an ink was important. Using multiple solvents for the
unknown chromatograms was done because the polarity of the components of each
unknown ink was not known. Therefore, choosing solvents with different polarities was
ideal in determining which solvent helped separate the colors within the ink. Therefore,
the solvents 1:1 methanol/ethanol, 1-propanol, ethanol, were used to determine which
was most effective. As shown in the Results section, the 1:1 methanol/ethanol and 1propanol were used because they showed the best results.10 The chromatograms
successfully showed the separation of each ink into distinct colors that could be
characterized to one brand of ink. Both the chromatograms done using the ethanol
(Figure 4) and 1-propanol (Figure 6) showed lines of ink migration that were not vertical,
but skewed to a side. This probably occurred as a result of the way the chromatogram
was stapled on either end, and due to the unleveled ends, not every surface of the bottom
received the same amount of the solute. Though the Figure 4 was not beneficial in
determining the distance each ink would have migrated, it was used in determining the
ineffectiveness of the solvent ethanol in producing distinct colors for each ink. Figure 6
could also still be used in observing the color of the spreading due to the migration of the
mobile phase.
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Before any Paper Chromatography was conducted, an observation of the ink
stains on the chromatogram showed that each ink dot for a color looked the same except
for the Pilot VBall brand, which was very dark. Therefore, an observation of the
unknown dots revealed that A, the red ink, was most likely Pilot VBall because the ink
dot was very dark and prominent.10
The chromatograms for the 1:1 methanol/ethanol showed a distinct color
separation for the red inks, so the solvent was used to confirm the assumption of the red
ink being Pilot VBall. The chromatogram for the 1-propanol showed a distinction for
both the blue and black inks in the range and spread of the color, so it was used in order
to confirm the identities of the two unknown blue colored and the one unknown black
colored inks.
From the Snyder Polarity Index, I did expect the 1-propanol solvent to be more
effective than the 1:1 methanol/ethanol solvent in separating the individual components
of the ink because the polarity of 1-propanol is 4.3 compared to 5.2 for ethanol and 6.6
for methanol12. As stated in the Introduction, the more nonpolar the solvent is, the greater
the migration distance for the ink. Therefore, I expected the migration distance and the
separation of the 1-propanol to be more prevalent than those of 1:1 methanol/ethanol
because the nonpolar components of the ink would migrate a further distance while the
polar components would not migrate as much of a distance.
Once the unknown chromatograms were completed, it took quite a bit of time in
determining the identities of unknown inks B (Blue) and C (Black) because for both inks,
two different brands showed similar migration distances and spreading in the colors of
the components of the ink. For both colors, both BIC and Paper Mate showed the similar
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patterns on the chromatograms. Each had minimal color spreading near the bottom, and a
heavy dark color near the top.10 For Unknown A-Red, the identity was confirmed to be
Pilot VBall because the spreading of the color of the ink started right at the original dot
and continued to the top in a orange to pink color range, which was only unique to the
Pilot VBall (Figures 2 and 3). The Unknown C- Black was determined to be Staples 1.0
because when comparing it to the chromatogram (Figure 2), #10 resembled the color
range and spreading of the ink with minimal spreading near the bottom and a heavy
purple-black color near the top. Therefore, the first guess made for the identities was: A
Red-Pilot VBall, B Blue-Pilot Easy Touch, C Blue-Staples 1.0, and D Black-BIC.
Unknowns B and D were wrong, so it was easy to determine that B was therefore the
only other option, BIC, and D was also the only other option, Paper Mate.
Conclusion
As determined before in a previous experiment, the pen inks observed were
nonpolar due their farther distance migration when using less polar solvents. Rather than
just relying on their nonpolar qualities, our lab group determined that the individual
components of the ink were at different ranges in polarity. Therefore, choosing solvent
systems that were different in their polarities would yield different results for the
different inks depending on their components. Therefore, the 1-propanol solvent with of
4.3 and a 1:1 methanol/ethanol, polarity index of 5.2 and 6.6 respectively, were used in
order to maximize the possible identification of each ink.12 The hypothesis from the
beginning of the experiment that the less polar solvents would results in a farther
migration distance of the inks was accepted, but an addition to the hypothesis is the
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possibility of polar solvents allowing for better identification of the unknowns due to
individual polarities of the components of the ink being at different polarities.
For future experiments, an improvement for identifying the unknowns would be
studied to further separate the components of the brands BIC and Paper Mate since the
chromatograms showed similar spreading patterns for both brands for both blue and black
inks. Also, a further study on natural dyes, as done in the first part of the experiment,
could be done so that the polar-nonpolar interactions of Paper Chromatography could be
applicable in the biochemical sense. This way, the results from the chromatograms of that
experiment would be relevant because food dyes are prevalent in almost every food
product. Finally, calculating Rf values for each unknown ink in further experiments could
yield quantitative evidence to support whether an ink was most likely a specific brand.3
Works Cited
1
Clark, Jim. "Paper Chromatography." Paper Chromatography. N.p., 2007. Web.
20 Feb. 2013.
2
"Paper Chromatography." Partnerships for Environmental Education and Rural
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3
Thompson, Stephen. PSU CHEMTREK: Paper, Thin Layer, and Liquid
Chromatography. New Jersey: Hayden McNeil, 2012. Print
4
"Chromatography." HowStuffWorks. HowStuffWorks, Inc., n.d. Web. 20 Feb.
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<http://www.123HelpMe.com/view.asp?id=122366>.
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6
Chubb, L. G. "Application of Paper Chromatography to Avian Pathology."
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Science Association Inc. Web. 13 Feb. 2013.
<http://ps.fass.org/content/38/3/668.abstract>.
7
Burdge, Julia R. Chemistry: Atoms First. New York, NY: McGraw-Hill, 2012. Print.
8
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2013. <http://www.tutorvista.com/biology/uses-of-paper chromatography>.
9
Ahmad, Mariam, Chem 113 Laboratory Notebook, pp. 14-15.
10
Almarzooqi, Samar, Chem 113 Laboratory Notebook, pp. 14-15.
11
"Like Dissolves Like and Molecule Ion Attractions." Vanguard Group, Inc., n.d.
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12
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