The Use of Paper Chromatography to Analyze and Identify Pen Inks

Maya Evanitsky* and Matt Easton

CHEM 113, Sec. 103

TA: Katie Haas

21 February 2013

IntroductionPaper chromatography is a technique for used to separate out components of a mixture

based on the amount of the components distributed between a stationary phase (a porous

substance) and a mobile phase, a fluid that flows through the stationary phase1. This technique

was first invented in the late nineteenth century by Mikhail Tswett, who used it to separate

different chlorophylls, the pigments plants use to absorb sunlight. In this new method, he filtered

a mixture of dissolved pigments through a glass tube packed with calcium carbonate powder. As

the solution progressed downward, each pigment was absorbed by the powder with differing

degrees in strength, forming a series of colored bands with each band of color representing a

different substance. Tswett called the colored bands a chromatogram and recommended the

technique, now known as adsorption chromatography, be used to separate colorless substances.

Despite a published report in 1905, Tswett’s work was largely ignored. It wasn’t until the early

1930s that chromatography eventually gained notice due to the work of British biochemists,

Archer John Porter Martin and Richard Laurence Millington Synge. Later, Martin and Synge

developed paper chromatography as a way to analyze proteins in the 1940s and received the

Nobel Prize in chemistry in 1952 for their work1.

Paper chromatography works by using a highly porous paper that is six percent water so

that water acts as the stationary phase rather than the actual paper2. The mixture is dotted in small

samples at the starting place before the paper is placed inside a glass or plastic tank along with

the liquid mobile phase. As the mobile phase is absorbed, it travels up the paper, taking the

components of the mixture along with it. The components separate based on their affinity to the

stationary and mobile phases. Substances that are more attracted to the mobile phase will move

farther than substances more attracted to the stationary phase.

Other types of chromatography used include gas chromatography, liquid column

chromatography, and thin layer chromatography. Gas chromatography separates out compounds

based on how volatile they are3. The mobile phase is an inert gas, usually helium or nitrogen, and

transports the vaporized mixture through a column. The mixture is then forced through the

column containing the liquid stationary phase, and the compounds are separated out based on

their boiling points3. Liquid chromatography, similar to paper chromatography, features a liquid

mobile phase and a stationary phase that consists of small materials packed together in a column.

First the mixture, then the mobile phase flows through the material to separate out the

components of the mixture2. In thin layer chromatography, a thin layer of material (usually gel)

on a glass or plastic support is used for the stationary phase2. Small samples are placed on the

material and the apparatus is placed in a developing chamber along with the liquid mobile phase.

There are several advantages to using chromatography as a process of analysis.

Chromatography allows for identification of unknown substances based on their components in a

relatively straightforward and quick process. Also, several different samples can be compared to

each other, or to reference standards, in order to properly identify them. In paper and thin layer

chromatography, the retention factor (Rf value) can be calculated by measuring the distance the

substance travelled from its starting place and dividing this value by the distance travelled by the

solvent front2. The Rf values should always be the same for a specific substance when using a

certain solvent, but due to differences in the sample size placed on the chromatography paper,

they are not exactly reproducible4. Nevertheless, they are reasonably good guides and can be

used to identify the components of a sample4. However, paper chromatography can only

qualitatively determine the amount of components, not quantitatively. Therefore, it cannot be

used to determine the empirical or molecular formula of the substance being analyzed.

In this experiment, paper chromatography was used to separate and analyze fifteen

different pen inks in order to distinguish them and find the mobile phase that worked best. This

mobile phase was then used to identify four unknown pen inks selected from the fifteen used by

comparing them to the previous trials. This process has many forensic applications, specifically

the identification and differentiation of inks. This could be used to match the ink on a note found

at a crime scene to ink from a pen found elsewhere, such as the suspect’s house. It can also be

used to examine the ink of documents involved in forgery cases5. Other methods that can be used

to analyze and differentiate inks are thin layer chromatography and capillary electrophoresis5.

Thin layer chromatography is similar to paper chromatography and achieves similar results, but

is more expensive due to the materials involved2. Capillary electrophoresis has the issue with a

high probability that many ink formulas are too similar to be differentiated without specific

information known prior to analysis5. Another approach of capillary electrophoresis involving

ultraviolet-visible spectral collection on reference standards was able to distinguish the pens

further, yet implementation of this process would be difficult due to the demanding requirements

for preparation of sample and buffer solutions5. Overall, paper chromatography is efficient, cost-

effective, simple, and ensures reliable results and is thus a prime choice for ink analysis.

Based on evidence obtained from a previous experiment involving paper

chromatography, pens with blue and black ink will require a significantly more polar mobile

phase than a 2:1 1-propanol/water solution, as will the red inked pens, but less so than the blue

and black inks. This is due to the fact that the yellow component is more polar than the

components of the other inks. Still, all three inks reached the solvent front in the previous

experiment. The mobile phase in the previous experiment had a 5.87 Snyder Index polarity

value6 and the mobile phase with an index value of 5.5 separated the red unknown into its

components, but failed to separate the blue and black inks. A mobile phase with an index value

of 7.12 managed to separate the blue and black inks to a greater degree. Thus, the part of the

hypothesis concerning the blue and black inks was supported by the results while the part

involving the red ink was rejected.

Procedure

The experiment was developed based on the procedure found in a literature source2 and

the stated hypothesis. The materials used were chromatography paper, fifteen different pens, four

different solvents with varying polarities, Petri dishes, and plastic cups. The first step was to set

up all four chromatograms by placing the pen inks on the indicated spots (see Tables 1-2). After

this, chromatogram 1 (see Figure 1) was placed in a Petri dish containing a mobile phase

consisting of a 1:1 water/2-propanol combination with a Snyder index value of 6.65 and covered

with a clear plastic cup. For the second trial (see Figure 2), a mobile phase of a 1:1

methanol/water mixture with an index value of 7.8 was used based on the results of

chromatogram A, in which all the inks travelled to the solvent line. The third trial (see Figure 3)

involved a 3:1 2-propanol/water mixture with an index value of 5.5, which separated the reds and

blacks fairly well, unlike the second trial. The last trial (see Figure 4) used 3:2 water/2-propanol

solvent with an index value of 7.12 and worked well to separate and distinguish all the pens.

For the two unknown chromatograms (see Figures 5-6), 3:1 2-propanol/water and 3:2

water/2-propanol solvents were used as the mobile phases. Table 3 shows the mobile phases and

their corresponding Snyder index polarity values6 for each chromatogram.

Table 1. Key for Chromatograms B and D7

Table 3. Mobile Phases Used7 and Corresponding Polarity Index Value6 Chromatogram Mobile Phase Used Polarity Index Value1 (from previous experiment) 2:1 1-propanol/water 5.872 1:1 water/2-propanol 6.653 1:1 methanol/water 7.84 3:1 2-propanol/water 5.55 3:2 water/2-propanol 7.12

Results

The chromatogram from the previous experiment has been included as Figure 1 as a

reference for the trials completed. For that experiment, only two six pens were analyzed, a red,

black, and blue for Papermate and Bic. While the red pens separated well, the black inks did to a

lesser degree and the blue inks did not separate at all. All six pens travelled to the solvent front

and were difficult to distinguish.

Number on Chromatogram

Pen Brand/ Ink Color

1 Papermate – blue2 Papermate- black3 Papermate – red4 Bic – blue5 Bic – black6 Bic – red7 Pilot V-Ball - blue8 Pilot V-Ball - black9 Pilot V-Ball - red10 Pilot Easy Touch - blue11 Pilot Easy Touch - black12 Pilot Easy Touch - red13 Staples - blue14 Staples - black15 Staples - red

Table 2. Key for Chromatograms A and C8

Number on Chromatogram

Ink Color/Brand

1 Blue Papermate2 Blue Bic3 Blue Pilot V-Ball4 Blue Pilot Easy Touch5 Blue Staples6 Black Papermate7 Black Bic8 Black Pilot V-Ball9 Black Pilot Easy

Touch10 Black Staples11 Red Papermate12 Red Bic13 Red Pilot V-Ball14 Red Pilot Easy Touch15 Red - Staples

The chromatogram from trial one (see Figure 2) shows the results of using the 1:1

water/2-propanol solution as the mobile phase. All fifteen pens travelled to the solvent front and

had long trailing edges, which were relatively faint for the red inks. While the red inks separated

quite well, the blue and black inks were not as successful. For the second trial, a more polar

mobile phase consisting of a 1:1 mixture of methanol and water was used (see Figure 3). While

not all the inks travelled to the solvent front and separated into their components fairly well,

most of the blue and black pens had similar results and were hard to distinguish. For the third

trial, a significantly less polar mobile phase was used. The chromatogram in Figure 4 shows that

while all inks travelled to the solvent front, the red and black inks divided out well, while the

blues did not. For the last trial, a 3:2 mixture of water and 2-propanol was used, which had the

second highest polarity out of all the trials. Figure 5 indicates that all pens travelled to the solvent

front and separated into their components sufficiently so as to be distinguishable from one

another.

For the set of unknown inks (two blue pens, one black pen, and one red pen) two

chromatograms were set up and analyzed with two different mobile phases. For the first

chromatogram, a mobile phase of 3:1 2-propanol/water was used. The red pen divided into its

components, while the black pen did so slightly. The blue pens did not separate, and surprisingly

did not travel to the solvent front. For a better result for the blue and black inks, a mobile phase

of 3:2 water/2-propanol was used on the second chromatogram. This distinguished the blue pens

more, as well as the black ink, but did not have as successful results as the fourth trial in which

the same mobile phase was used. The inks were identified from left to right as Pilot Easy Touch

blue, Papermate blue, Staples red, and Pilot V-ball black.

Figure 1. Chromatogram 1 shows the results of using 2:1 1-propanol/water as the mobile phase7.

Figure 2. Chromatogram 2 shows the results of using 1:1 water/2-propanol as the mobile phase8.

Figure 3. Chromatogram 3 was used for trial two and demonstrates the effects of a solution of 1:1 water/methanol as the mobile phase7.

Figure 4. Chromatogram 4 shows the results of trial three, which used a 3:1 2-propanol/water solution as the mobile phase8.

Figure 5. Chromatogram 5 shows the effects of using 3:2 water/2-propanol as the mobile phase for the last trial7.

Figure 6. Chromatogram A features the unknown inks which were analyzed with a 3:1 solution of 2-propanol/water7.

Figure 7. Chromatogram B features the results of using 3:2 water/2-propanol to analyze the

unknown inks7.

Discussion

In the first trial, a mobile phase consisting of one part water and one part 2-propanol with

a polarity Snyder index value6 of 6.65 was used. The fact that all fifteen pen inks reached the

solvent front indicates the samples all had a polarity closer to this than that of the stationary

phase, water. Based on their separations, the black and red pens had more polar components,

such as the yellow dye, in addition their less polar ones. Additionally, the blue and black pens

left much longer trailing edges than the red pens. While this trial worked well to distinguish the

red pens, the blue and black inks did not separate well and could not be distinguished from one

another.

The next trial featured the use of equal parts water and methanol as the mobile phase,

which had a polarity index value of 7.86. Most of the inks were concentrated near the spotting

line, except the three Pilot V-ball pens, indicating this brand in particular uses less polar inks

than the others. Most of the black pens separated into purple and yellow components; only the

pilot V-ball stayed black. This suggests the pilot V-ball components all have a similar polarity

and thus travelled roughly the same distance on the chromatogram. While the black and red pens

were differentiable, most of the inks failed to separate even to a small degree. The blue pens all

had similar interactions and so overall this trial failed.

Since such a high polarity seemed less successful than the lower polarity used in trial one,

a mobile phase with a polarity index value of 5.56 and made up of three parts 2-propanol and one

part water was applied. This separated the red and black inks extremely well, but the blue inks

separated only slightly. This indicated that the black and red inks had more polar components

than the blue inks. With the aid of a UV light, the blue and red inks were made more

distinguishable and overall this trial was successful.

For the last trial, a mobile phase comprised of three parts water and two parts 2-propanol

with a polarity of 7.12 was used in order to separate the less polar blue inks. While this was not a

high enough polarity to keep the inks from reaching the solvent front, it did separate each of the

inks into at least some of their components. The blues were divided into several different blue

and purple components while the red inks were separated into red and yellow. All the black inks

separated into purple and black, except for the Pilot V-ball which stayed black. Even though

much of the components for each ink blended into their corresponding components trailing

edges, the pens were easily distinguishable from one another.

The results of the experiment show that a less polar mobile phase, or one of a similar

polarity to 2:1 1-propanol/water solution, was needed to sufficiently separate the red inks while a

more polar mobile phase was necessary for the blue and black inks. This is consistent with the

part of the original hypothesis that the blue and black inks would require a much more polar

mobile phase, but rejects the part stating that the red inks needed a more polar mobile phase as

well, albeit to a lesser degree than the other two ink colors. Thus the hypothesis was partially

correct for the first four trials. The use of two different mobile phases to identify the four

unknown pens, the first of a polarity index of 5.56 and the second had a polarity index of 7.126,

reflected this observation and the consequent change in the hypothesis.

The second hypothesis predicted that a mobile phase of a similar or lesser degree in

polarity would be successful in analyzing the red inks while a more polar mobile phase would be

successful for the blue and black inks. The first mobile phase, a 3:1 2-propanol/water mixture,

was used to examine the red unknown, and successfully separated the ink into its components of

red and yellow. This was then compared to the second trial and properly identified as the Staples

red pen. However, the black and blue pens did not separate. Afterwards, the second mobile

phase, a 3:2 mixture of water/2-propanol, was employed to analyze the two other unknown

colors. This did not separate the blue and black inks as well as during the fourth trial, yet, some

separation did occur. This was sufficient to compare the unknowns to Chromatogram 4 and

identify them as Pilot Easy Touch, Papermate, and Pilot V-ball from left to right.

While the experiment was successful in identifying mobile phases to analyze and identify

the ink samples, there were still several sources of error. An example of this would be that the

paper was handled with bare hands, no gloves or tongs were used. While this was minimized and

kept to the edges of the paper, it is a possibility that oil could have been transferred from hands

to the paper and somehow affected the results. Another issue is that the liquids used to make the

mobile phases might have been contaminated or diluted during the processing or packaging

stage. In addition to this, an exact ratio was not prepared when using the mobile phases. While

accurate procedures were used to mix the solutions, there is always a small margin of error. Also,

an unknown contaminant may have interacted with the chromatography paper used and thus

affected the results. All of these are examples of random error9 and thus could have affected the

results in a multitude of ways, such as increasing or decreasing the distance travelled by the

samples, or changing how the samples diffused through the paper.

Conclusion

The results indicate that a more polar mobile phase worked well to separate and identify

the blue and black inks, while a less polar mobile phase worked for the red inks. This supported

the section of the null hypothesis which stated that the blue and black inks would require a

significantly more polar mobile phase than the one used in the previous experiment, yet

contradicted the section that stated the red inks would need a more polar mobile phase. Overall,

the data demonstrated that the inks were considerably less polar than the stationary phase, water,

and thus reached the solvent front for mobile phases that were only slightly polar. However, in

two of the trials all of the pens reached the solvent front, but still separated and distinguishable

from one another. This indicates that the components of the inks each had different, albeit

similar, polarities.

In the future, it would be practical to wear gloves while handling the chromatography

paper so as to prevent any chance of contamination from the oil present on one’s fingers.

Further studies involving paper chromatography can be found in the areas of both

chemistry and biology. One such experiment could be using paper chromatography to obtain a

qualitative analysis of the ions present in tap water. Another use of this technique is to study the

path of carbon as photosynthesis occurs in a plant10.

References

1. Liquid Chromatography. Encyclopedia of Analytical Chemistry: Applications, Theory

and Instrumentation, 1st ed.; Wiley: New York, 2011; Vol. 1, pp 305-310.

2. Thomspon, S.; Keiser, J.T. PSU Chemtrek. 2012. Hayden-McNeil. pp. 17.2-17.22

3. The Chemistry Hypermedia Project. Gas Chromatography (GC).

http://www.files.chem.vt.edu/chem-ed/sep/gc/gc.html (accessed Feb 10, 2013)

4. Macalester College. Separation of Amino Acids by Paper Chromatography.

http://www.macalester.edu/~kuwata/Classes/2001-02/Chem%2011/Revised%20Amino

%20Acids%20(9%201%2001).pdf (accessed Feb 12, 2013)

5. Egan, J. M.; Hagan, K. A.; Brewer, J. D. Forensic Analysis of Black Ballpoint Pen Inks

Using Capillary Electrophoresis. Forensic Science Communications. [Online] 2005, 11.

http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july2005/

research/2005_07_research01.htm. (accessed Feb 10, 2013)

6. Sanderkok.com. Properties of Solvents on Various Sorbents.

http://www.sanderkok.com/techniques/hplc/eluotropic_series_extended.html (accessed

Feb 10, 2011)

7. Evanitsky, Maya. CHEM 113 Laboratory Notebook, pp 10-13.

8. Easton, Matt. CHEM 113 Laboratory Notebook, pp. 6-11.

9. University of Maryland. Random vs. Systematic Error.

http://www.physics.umd.edu/courses/Phys276/Hill/Information/Notes/ErrorAnalysis.html

(accessed Feb 13, 2013)

10. Calvin, M.; & Benson, A.(2008). The Path of Carbon in Photosynthesis IV. The Identity

and Sequence of the Intermediates in Sucrose Synthesis. Lawrence Berkeley National

Laboratory: Lawrence Berkeley National Laboratory. Retrieved from:

http://escholarship.org/uc/item/1kg564fz