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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 6 112 Paper Mate 7 123 BIC Round Stic 8 134 Pilot Easy Touch 9 145 Staples 1.0 10 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 1-

propanol 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 1-

propanol 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

1Clark, Jim. "Paper Chromatography." Paper Chromatography. N.p., 2007. Web.

20 Feb. 2013.

2"Paper Chromatography." Partnerships for Environmental Education and Rural

Health (PEER). Partnership For Environmental Education and Rural

Health, n.d. Web. 20 Feb. 2013.

3Thompson, 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.

2013. <http://science.howstuffworks.com/chromatography-info.htm>.

5"Tank Chromatography.." 123HelpMe.com. 20 Feb 2013

    <http://www.123HelpMe.com/view.asp?id=122366>.

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6Chubb, L. G. "Application of Paper Chromatography to Avian Pathology."

Poultry Science 3rd ser. 38.668-676 (1959): n. pag. Poultry Science. Poultry

Science Association Inc. Web. 13 Feb. 2013.

<http://ps.fass.org/content/38/3/668.abstract>.

7Burdge, Julia R. Chemistry: Atoms First. New York, NY: McGraw-Hill, 2012. Print.

8"Uses of Paper Chromatography." TutorVista. TutorVista.com, n.d. Web. 13 Feb.

2013. <http://www.tutorvista.com/biology/uses-of-paper chromatography>.

9Ahmad, 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.

Web. 14 Feb. 2013. <http://www.kentchemistry.com>.

12"Properties of Solvents on Various Sorbents." HPLC. Kok ChemWare, Web.

21 Feb. 2013.

<http://www.sanderkok.com/techniques/hplc/eluotropic_series_extended.html>.

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