Paper Chromatography

Experiment 17

MARCH 7, 2014Alexandria Conti

Chem 113 Section 104Group Members: Jaimie Collo

TA: Steve Kennedy

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Introduction:

Chromatography is used in experimentation to separate mixtures of certain substances

into their components (1). Chromatography works on one principle: that it works in phases (1).

The stationary phase, composed of either a solid or a liquid supported on a solid, is fixed in place

for the chromatography procedure while the mobile phase, composed of either a liquid or a gas,

flows through the stationary phase and carries the components of the mixture with it (1). Given

that the components each travel different distances at different rates, the distances traveled by the

different components may be used to identify unknown substances.

Mikhail Semyonovich Tsvet was born in 1872 and would go on to become a Russian

botanist that would develop a certain scientific technique that would revolutionize the world (2).

Tsvet began investigations on the isolation and chemistry of chloroplast pigments early into his

career (3). In order to isolate these components, Tsvet developed the technique of

chromatography that utilized absorption techniques and column operation (3). Today,

chromatography it has found a special place in the scientific community.

Chromatography gained momentum in the scientific community and quickly expanded

beyond just separating plant pigments. Today, paper chromatography has many important uses,

including obtaining pure compounds from a mixture, qualitative analysis of the different

compounds found in drugs by pharmaceutical companies, DNA and RNA fingerprinting in

forensic science, as well as the detection of alcohol or chemicals in blood (4).

There are many different types of chromatography in the world today. Ion exchange

chromatography, for example, uses resin as the stationary solid phase in order to covalently

attach anions and cations onto it (5). Solute ions of the opposite charge dispersed throughout the

liquid phase are then attracted to the resin by electro static forces (5). Another version of

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chromatography is partition chromatography (5). This form consists of a liquid stationary phase

that forms on the surface of a thin film (5). Solute then equilibrates between the mobile phase

and the stationary liquid (5). For the ease of this lab, paper chromatography will be used because

it is readily available.

The science behind chromatography begins at the stationary phase, paper. The paper used

is made out of cellulose, which is composed of polymer chains that have OH- groups sticking out

all around them (1). These OH- groups attract water, which binds to the surface and becomes the

new stationary phase (1). Either a non-polar or polar solvent will then be used to separate the

components (1). If a non-polar solvent is used, the non-polar molecules in the mixture will have

more affinity for the solvent rather than the polar water, so they will mostly remain dissolved in

the solvent and will not travel very far up the paper (1). The polar molecules, on the other hand,

are extremely attracted to the water and will travel quickly up the paper: the more polar the

component, the further up the paper it will travel (1). This is because of the like-dissolves-like

principle, in which polar compounds are likely to dissolve in other polar compounds and

nonpolar compounds are likely to dissolve in other nonpolar compounds (6). This principle is

why the components travel different distances and, therefore, are easy to identify. If the polarity

of both the mobile and the stationary phase were to be reversed, this process would work in

reverse.

The distances contribute in the form of Rf values. An Rf value is the distance traveled

from the start of the chromatography to the center of the substance spot divided by the distance

from the start to the solvent front at the end of the chromatography (7). Each substance has a

specific Rf value associated with it (7). By measuring the Rf values of certain inks and dyes, an

identity will be able to be given to an unknown by comparing it to the standards.

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This project focuses on developing a procedure that will allow all colors of ink (red, blue,

and black) to be better separated. By clearing establishing sample identity chromatograms,

unknown chromatograms will then be able to be run and compared to the standards in order to

identify these unknowns. The procedure was then followed to determine the identities of

unknown pen samples.

Fig 1: Chromatogram original – 5.87 – 2:1 1-proponol:water (8)

In order to improve the separation of the pen ink, both polar and nonpolar solvents would

be needed in order to better separate the ink samples. Based on previous experimentation

completed in chromatogram #1 (see Fig. 1 above), it seems as though the black pens need a

slightly polar solvent to ensure proper separation because these pen ink components ran to the

solution line, meaning the chromatogram did not fully separate, while the red pens needed a

more polar solvent in order to correctly separate because the red ink components seemed to run

further than the black and blue pens and did not effectively separate either. In order for the

unknown pen samples to be correctly identified, different polar solvents must be created and

analyzed during this experiment. A slightly more polar solvent will be created using a 4:1 mix of

methanol and water, while an even more polar solvent will be made using a 1:1 mix of methanol

and water.

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Procedure (Reference the PSU Chemtrek (9) for complete step-by-step instructions):

A chromatography key was created in which all the chromatography papers followed in

this lab. This allowed for an organized and easy way to maintain each separate chromatogram

completed throughout experimentation. The key that was followed for all chromatography papers

created is seen below:

Table 1: Chromatogram Key for all chromatograms used in experimentation

1 – Papermate

black

2 – Pilot black

(ball)

3 – Pilot black

(easy-touch)

4 – Staples black 5 – Bic black

6 – Papermate

blue

7 – Pilot blue

(ball)

8 – Pilot blue

(easy-touch)

9 – Stales blue 10 – Bic blue

11 – Papermate

red

12 – Pilot red

(ball)

13 – Pilot red

(easy-touch)

14 – Staples red 15 – Bic red

Table 2: Synder Polarity Index (10)

Substance Polarity

1-propanol 4.3

2-propanol 4.3

Ethanol 5.2

Methanol 6.6

Water 9

A chromatography paper was set up and solution of 1:1 methanol: water was prepared by

mixing 5 ml of methanol with 5 ml of water. This created a solution with polarity 7.8 ((6.6+9)

2¿

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(The chromatography paper was labeled with “#1 – more polar – 7.8” in order to keep

organized.) The chromatogram was stapled into a circle so that it could stand by itself in the

solution created.

The chromatography was run by filling a petri dish with the solution created above. The

circular chromatography paper was placed into the petri dish and cover the system with a plastic

cup so that the solution does not evaporate off the paper before the chromatography is run to

completion. The chromatography was given time to run so that the components would separate.

This step was important because the whole point of the lab was to determine the identity of

unknown substances based on the components found in them when separated. In order to

separate the components from the substance completely, the chromatography must run to

completion.

The chromatography was stopped by taking it out of the solution when the solution has

ran up the paper to about 1-2 cm from the top of the page. The ending line of the solution was

marked in order to determine, once the paper dried, which components fully separated and which

did not (those that did not were touching the line). The paper was allowed to dry before any

components were analyzed.

The steps above were followed for a solution of:

1. 2:1 1-propanol: ethanol. Polarity = 4.6 ((4.3+4.3+5.2)

3¿

2. 4:1 methanol: water. Polarity = 7.08 ((6.6+6.6+6.6+6.6+9)

5¿

The solutions above were used to run chromatography papers on the unknowns given and

their identities were determined based on the streaks matched to the full chromatograms ran

above.

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Results:

Fig 2: Chromatogram original – 5.87 – 2:1 1-proponol:water (8)

The orignal chromatogram was made using a soltuion with polarity 5.87. The pens test

(#12-17) did not run to completion given that they all run off the edge of the solution line. It

seems a more polar solution would be needed to separate the pens further due to the components

polarity and the polarity of the stationary phase (water).

Fig 3: Chromatogram #1 – more polar – 7.8 – 1:1 methanol: water (8)

Chromatogram #1 was run using a solution of polarity 7.8. While this chromatogram

shows excellent separation of the red pens, it seems to be too polar for the blue and black pens,

given that they did not run far up the paper at all. It seems a less polar solution will be needed for

these pens. However, this solution could easily be used to identify any unknown red pens.

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Fig 4: Chromatogram #2 – less polar – 4.6 – 2:1 1-propanol: ethanol (11)

Chromatogram #2 was run using a solution of polarity 4.6. This solution was created in

the hopes that a less polar solution would allow better separation of the black and blue pens.

Most of the pens did not separate fully and ran to the end of the solvent line. The streaks created

above are not distinguishable from each other as most of them look similar.

Fig 5: Chromatogram #3 – polar – 7.08 – 4:1 methanol: water (8)

Chromatogram #3 was more polar than #2 (4.6) and slightly less polar than #1 (7.8). This

solution allowed for a distinguishable separation of the blue and black pens. It seems that the

components found in blue and black pens are slightly less polar than the components of red pens,

given the results of these test chromatograms. Nonetheless, chromatogram #3 would be an

excellent determinant of the components of the unknown blue and black pens.

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Fig 6: Unknown pens #1 – Ran with solution #1 – 7.8 (8)

By using solution #1, unknown pen #1 was easily identified as Papermate red based on

the pattern the pink streak with a yellow blotch located near the top made. When compared with

the chromatogram made with solution #1, the unknown pen #1 easily matched the streak made

by Papermate red. However, the rest of the pens are undistinguishable. It seems solution #3 will

be needed to identify them.

Fig 7: Unknown pens #1 – Ran with solution #3 – 7.08 (8)

Solution #3 allowed the rest of the pens to be identified. The blue and black streaks left

by each pen were able to be matched with the pen samples that streaked in chromatogram #3.

Pen #2 was found to be Staples blue based on the long purple streak that contained a hint of blue

near the top, pen #3 was found to be Papermate blue based on the hue of the thick, blue streak

created, and pen #4 was found to be Papermate black based on the purple streak with a yellow

blotch nestled near the top of the streak.

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Fig 8: Unknown pens #2 – Ran with solution #3 – 7.08 (11)

The second set of unknown pens consisted entirely of blue and black pens. Because of

this, solution #3 was able to be used to identify all the pens. The streaks created matched the

streaks the known pens made in chromatogram #3 and all the identities were able to be obtained.

Pen #1 was found to be Bic blue because of the long purple streak created that had a certain blue

middle in it, pen #2 was found to be Pilot blue (easy-touch) because of the hue of the long, blue

streak created, pen #3 was found to be Staples blue due to the long purple streak that had a small,

blue dot nestled near the center of the streak, and pen #4 was found to be Pilot black (easy-touch)

because of the faint purple line created that has a strong, yellow blotch near the top of the streak.

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Discussion:

The pen samples from the chromatography ran earlier with the solution of polarity 5.87

were looked at first. It seems that the solution 2:1 1-proponol: water was not a polar enough

sample to separate the components of the pens, as each ran to the end of the chromatography

paper with no distinguishable streaks made. According to the rules of chromatography based on

using a polar solution, the more polar the substance is the further up the paper it will run due to

the like-dissolves-like principle between the polar substance and the polar stationary phase. This

is due to the adhesive properties of the components and the solution; the more polar a component

is the more attracted it will be to a more polar solution and the polar paper. Therefore, a more

polar substance will be needed to further separate the pen ink components.

A more polar solution was created using a 1:1 mix of methanol: water, creating a solution

with polarity 7.8. It was important to use a mixture because it allowed for the polarity of the

solution to be exactly manipulated and a polarity could be created that best separated the

solutions. This solution allowed the red pen inks to run smoothly, each creating a streak

distinguishable from the other brands of red pen samples. However, the blue and black pens did

not migrate as far. Only the Pilot black (ball) and the Pilot blue (ball) pens made streaks that

were completed in full. The other pens, though, did not fully migrate. This means that the

solution was too polar for the pen components, as they mostly stuck to the bottom of the

chromatogram. Given that the more nonpolar a pen component is, the further it will migrate up

the page given the use of a more nonpolar solvent (due to the adhesive properties between the

two like substances), the blue and black pens were not polar enough to migrate up this extremely

polar solution. A less polar solvent seemed to be needed in order to separate these pens, so a less

polar solvent was next created.

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The less polar solvent was made using a 2:1 mix of 1-proponol: ethanol, which gave a

polarity of 4.6. Once this chromatogram was run, every pen ran to the edge of the chromatogram.

This meant that the pen components were more polar than 4.6, as they easily traveled up the

paper with the solvent. A more polar solvent would be needed to separate the blue and black

pens, but it could not be as polar as the first solution, or the blue and black pen components

would not correctly separate.

The last solution created was of polarity 7.08, which is slightly less polar than the 7.8

solution #1, and was made using a 4:1 mix of methanol: water. Excellent separation of the blue

and black pen components were achieved using this solution, meaning that the polarity of the pen

ink components were just polar enough to match the solution and create distinguishable streaks

on the paper. This last solution was used to identify the unknown blue and black pen samples

because it provided a great separation the standard pen samples.

Solution #1 was first used to identify the unknown red pen samples given in the first set

of unknowns. There was only one red streak made and, when compared with the standard pen

samples ran with solution #1, the unknown streak really matched the streak made by Papermate

red: the light pink streak with a yellow blotch near the top of the unknown exactly matched the

pattern the Papermate red pen made (see Fig. 3). Therefore, unknown pen #1 in the first set of

unknowns was correctly identified to be a pen mark of Papermate red.

Solution #3 was next used to identify all the blue and black pen samples of the first set of

unknowns. The streaks created by the last three pen samples were easily identified when

compared to the standard pen samples ran with solution #3. Unknown pen sample #2 created a

purple streak with a blue blotch right in the middle of the streak, which matched the streak made

by the Staples blue pen from chromatogram #3 (see Fig. 5). Unknown pen #2 was then correctly

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identified to be a Staples blue pen. The long, blue streak created by unknown pen #3 matched the

long, blue streak made by the Papermate blue pen from chromatogram #3, so their identities

were found to be the same (see Fig. 5). Unknown pen #4 made a long, purple streak with a

yellow blotch near the top, which matched the exact streak made by the Papermate black pen

from chromatogram #3 (see Fig. 5). Therefore, these identities were matched and all the

unknown pens from set #1 were identified.

The unknown set #2 of pens consisted entirely of blue and black pens, so only one

chromatography experiment was needed with solution #3 because all pens easily and

distinguishably separated.

Unknown pen #1 created a long, purple streak with a hint of purple in it that matched the

Bic blue streak made in chromatogram #3 (see Fig. 5). Therefore, these identities were matched.

Unknown pen #2 created a thick, blue streak that matched the thick, blue streak created by the

Pilot blue (easy-touch) pen from chromatogram #3 (see Fig. 5). These identities were then

matched. The purple streak with a blue blotch right in the middle of the streak matched a streak

made by the Staples blue pen from chromatogram #3, so these identities were matched (see Fig.

5). The last pen, unknown pen #4 from unknown set #2, made a light purple streak with a yellow

blotch set in the middle of the streak. This pattern matched the same pattern made by the Pilot

black (easy-touch) pen from chromatogram #3 (see Fig. 5). Therefore, these identities were

matched and all pens from unknown set #2 were identified correctly.

Given the relative polarities of each solution used, it seems as though the red pen ink

components were more polar than those of the blue and black pens. This was determined because

the more polar solution created complete chromatograms of the red pens, while the blue and

black pens barely made it halfway up the paper. The more nonpolar the solvent, the further the

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more nonpolar components will travel because these more nonpolar components have an

adhesive attraction to the nonpolar solvent. The polar components do not have this strong

attraction, so they will separate out quickly. The blue and black pens quickly separated out when

the more polar solvent was used while the red pens ran their chromatograms to completion,

meaning that the red ink components were more nonpolar.

The earlier hypothesis made about improving the separation of the pen inks by using

more polar solvents ended up being correct. The more polar solvents of 7.8 and 7.08 ran

chromatograms to completion, rather than the chromatogram made using a solution of polarity

5.87. This is because the pen ink components are relatively nonpolar, so they will not travel

further up a paper if the polarity of the solution used is more polar. The slightly more polar

solvent easily separated the blue and black ink pens while the even more polar solvent used

easily separated the red ink pens. However, the substances traveled less because more polar

solvents inhibit migration of the nonpolar ink of the pens.

By following the procedure and creating solutions of polarities 7.8, 4.6, and 7.08, the

correct identities of the pens were easily obtained. Unknown set #1 consisted of pens Papermate

red, Staples blue, Papermate blue, and Papermate black. Unknown set #2 consisted of pens Bic

blue, Pilot blue (easy-touch), Staples blue, and Pilot black (easy-touch).

A possible source error may include the varying pen mark sizes used in each

chromatogram. Given the amount of ink applied to the paper, the chromatography will look

different as a different amount of dye travels up the page. This error could be accounted for by

exactly applying the same amount of ink to each chromatogram.

The results of this experiment could be improved if more chromatograms on the

unknown ink samples were allowed to be run. This way, we could create several chromatograms

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that, when combined, create an extensive key in which every ink sample could be easily

identified because the marks would be easily distinguishable from each other.

These results could be extended to a forensic study in which pen samples on pieces of

evidence need to be identified. By using solutions to run on sample chromatograms, the piece of

evidence could then be run using the same solutions. Then, the chromatogram containing the

unknown pen sample would be compared to the keys and an identity would be able to be made

and, hopefully, the case could be solved.

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Conclusion:

The identity of all unknown pens were determined through the creation of several

solutions of different polarities and by running chromatograms on each. The separation of the

pen ink was improved upon by increasing the polarity of the original chromatogram’s solution.

In order to separate the blue and black ink pens, a slightly more polar solution was needed

because the components of the pens are nonpolar. Therefore, they will not climb as high up the

stationary phase of the paper as polar substances would because like-dissolves-like and the

nonpolar substances would be attracted to the solution, rather than the paper. The red pens turned

out to be even less nonpolar than the blue and black pens, so an even more polar solution was

needed due to the relative polarities of the mobile phase and the stationary phase. In the future,

this experiment could be carried out better if more solutions of different polarities were included.

This would allow for very distinguishable differences in the distances traveled by the

components, which would allow for easier identification. The experiment was completed

successfully and all unknown pens were successfully identified.

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References:

1. Paper Chromatography. Chem Guide.

http://www.chemguide.co.uk/analysis/chromatography/paper.html (accessed Feb 25,

2014).

2. Mikhail Semyonovich Tsvet. Encyclopedia Brtiannica.

http://library.williams.edu/citing/styles/acs.php (accessed Feb 24, 2014).

3. Williams, Kathryn R. Colored Bands: History of Chromatography. Chemical Education

Today [Online] 2002. http://pubs.acs.org/doi/pdf/10.1021/ed079p922 (accessed Feb 24,

2014).

4. Paper Chromatography Uses. Buzzle. http://www.buzzle.com/articles/paper-

chromatography-uses.html (accessed Feb 25, 2014).

5. Modern Chemical Techniques; the Royal Society of Chemistry: New York, 2006.

6. Smith, Michael B. Organic Chemistry: An Acid-Base Approach;CRC Press: New York,

2011.

7. Rf Equation. University of Wisconsin-Platteville.

http://www.uwplatt.edu/chemep/chem/chemscape/labdocs/catofp/chromato/tlc/tlcq.htm

(accessed March 3, 2014).

8. Conti, Alexandria. Chemistry 113 Laboratory Manual: Pennsylvania State University,

2014.

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

2013-2014; Hayden-McNeil Publsihing: Plymouth, MI, 2013; p 17-20.

10. Synder Polarity Index. Sanderkok.

11. Collo, Jaimie. Chemistry 113 Laboratory Manual: Pennsylvania State University, 2014.