Paper Chromatography of

Standard Inks and Unknown

Inks
Joy Li
March 26, 2015
Lab Partners:
Katherine Li
Sabrina Lemus
Jiarong Lin
Katheryn Penrod
Chem 113 Section 002

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Introduction:
In this study, paper chromatography was explored for chemical analysis and were applied to the
identification of unknown inks. Chromatography is a method used to separate organic and
inorganic compounds for the purpose of analysis and study.1 Mixtures of substances are
separated into their components, through the use of the stationary phase and mobile phase. 2 This
method of chromatography was created by Russian botanist, M.S. Tswett, while studying plant
life coloring materials.1 The stationary phase is typically a solid, while the mobile phase is
typically either a liquid or a gas. The process is carried out by passing the mobile phase through
the stationary phase, resulting in different components of different travel rates separated out. 3
The differential rates of migration are what provide the separation. 4 Solutes spend more time in
the mobile phase if they have a greater affinity for the phase. Solutes spend more time in the
stationary phase if they have a greater affinity for the stationary phase. 1 In this process, the
compound is placed on the stationary phase. The mobile phase is passed through, and the
components of the compound are carried by the mobile phase through the stationary, depending
on the level of affinity to each phase.5 The mobile phase of both paper and thin-layer
chromatography is the solvent.

The paper chromatography stationary phase is considered the paper strip, which is composed of
cellulose. However, cellulose provides hydroxyl groups that result in hydrogen bonding of water,
making up 6% of the weight of the paper, along with any other water absorbed from the process.
This forms a layer that constitutes the stationary phase. The sample is applied to one end of the
paper, and capillary action pulls the mobile phase through the paper. The method of paper
chromatography was developed by Martin Synge, and Consden..6

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Some variation of chromatography is utilized in over 60% of worldwide chemical analysis. 1 The
precision found in chromatography makes it a prime option for analysis, as it can purify nearly
any soluble substance. Additionally, the conditions required of chromatography are not typically
severe, allowing for the separation of delicate products. 4 Examples of chromatography use
include crime scene sample analysis in law enforcement, chemical purification in manufacturing
plants, detection of blood alcohol levels in medicine, and the determination of pollution level in
water supply by environmental agencies.5

In the experiment, different solutions of different polarities were tested for separation ability of
the dyes of various pen inks through paper chromatography. The goal was to design the "perfect"
solution of a certain polarity level to identify unknown inks. The composition found to be most
effective was then used to analyze a set of unknown pen inks and determine their respective
identities.6 The hypothesis of the experiment stated that a more polar solvent would allow for
better separation of the dyes, however, a solution more polar than water would cause the process
to reverse. For the less polar solutions, the stationary phase would be more polar than the mobile
phase. However, as the solution got more polar, the two roles would switch, causing a reverse
process, with a more polar liquid mobile phase. This would be too polar, and would not provide
for the best test for separation.7 Ink analysis has found use in forensics and the investigations of
the legitimacy of official documents. Other potential methods of ink identification include paper
electrophoresis,
electrophoresis.8

luminescence,

microspectrometry,

laser

excitation,

and

capillary

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Procedure:
This procedure was executed as in the literature.6 Eight various solutions were created through
mixtures of substances of known concentration and known Snyder polarity value. These
solutions were tested as mobile phases for the separation of the known pen inks. The solution
that achieved the best separation was to be used in the separation of the unknown pen inks, in
effort to match the unknowns with their identity, based solely on the paper chromatography
results. The different substances were mixed in ratios of either 1:1 or 2:1, and their relative
Snyder Polarity values were calculated based on these ratios.
Snyder Polarity
5.87
6.65
7.40
7.60
7.80
8.07
8.20
8.63
This was calculated following the setup:

Solution
2 1-propanol:1 water
1 1-propanol:1 water
2 methanol:1 water
1 acetic acid:1 water
1 methanol:1 water
2 water:1 acetic acid
2 water:1 methanol
2 acetic acid:1 water

(0.5)(4.3)+(0.5)(9.0)= 6.65
(50%)(polarity propanol)+(50%)(polarity water)
The solutions were (1 1-propanol:1 water) (Snyder Polarity: 6.65); (2 propanol:1 water) (5.87);
(1 methanol:1 water) (7.80); (2 methanol:1 water) (7.40); (2 water:1 methanol) (8.20); (1 acetic
acid:1 water) (7.60); (2 acetic acid:1 water) (8.63); and (2 water:1 acetic acid) (8.07). For the last
three solutions, at the absence of acetic acid, a solution was created that equaled the acetic acid
in terms of Snyder polarity. This was achieved by taking substances of known polarities and
solving for the ratio that would provide a Snyder polarity of 6.2 (equal to that of acetic acid). The
equation set up followed that :

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( 4.3 ) ( x )+ ( 6.6 ) ( 1x )=6.2

where 4.3 was the polarity of isopropanol and 6.6 was the polarity of methanol. "x" represented
the parts of the whole that needed to be isopropanol, and "1-x" represented the remaining part of
the whole that needed to be composed of methanol. "x" was found to be 0.1739 parts isopropanol
to 0.826 parts methanol, or 1.74 mL to 8.26 mL. After the solutions were created, each was tested
in paper chromatography of the various pen inks by marking the paper strip with all pen inks,
rolling the strip into a cylinder, and placing it in a petri dish with a small layer of the solution.
The results for all eight solutions were analyzed for the best separation and the ability to
distinguish/differentiate between the different inks. The best solution was then used on the
unknown pen ink strips, and the results compared to the original chromatography separation with
the chosen solution, through visual comparison and Rf comparison. Paper chromatography often
utilizes Rf values to compare and distinguish different runs from one another. It is known as the
retention factor and is defined as "the fraction of time spent in the mobile phase." This value is
found by dividing the distance moved by the component, by the distance moved by the mobile
phase front. The Rf values of dyes can be used as a tool to compare and match inks in
identification.6

The sample key used was as follows:

Known Ink 1
Known Ink 2
Known Ink 3
Known Ink 4
Known Ink 5
Known Ink 6
Known Ink 7
Known Ink 8
Known Ink 9

1. Pilot Easy Touch (ba)

2. Pilot G-2 07 (ba)
3. Pilot Vball BG (ba)
4. Papermate InkJoy (ba)
5. Pilot Vball BG (r)
6. BIC Ultra Round (r)
7. Pilot G-2 07 (r)
8. Pilot Easy Touch (r)
9. BIC Ultra Round (ba)

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Known Ink 10
Known Ink 11
Known Ink 12
Known Ink 13
ba= black; bu= blue; r= red

10. Pilot G-2 07 (bu)

11. BIC Ultra Round (bu)
12. Pilot Easy Touch (bu)
13. Papermate InkJoy (bu)

Results:
The eight solution tests yielded a few runs that were unusable in ink identification, and a few that
were fairly good potential candidates for the solution of the final procedure. Figure 1 shows a
reference chromatogram in (1 Propanol:1 Water) with polarity 6.65. This solution yielded one of
the better test runs. The inks separated nicely, with distinct changes in the dyes and clear
component zones to use for Rf calculation. Figure 2 shows a reference chromatogram in (2
Propanol:1 Water) with polarity 5.87. The R f value for ink 2 was indeterminable, and it was
difficult to distinguish between inks 4 & 9, 6 & 8, and 10 & 11. Figure 3 shows a reference
chromatogram in (1 Methanol:1 Water) with polarity 7.80. This test was not considered usable as
a reference for identification, as many of the runs were too faint, and a few completely absent (1
& 8). Figure 4 shows a reference chromatogram in (2 Methanol:1 Water) with polarity 7.40. The
nonlinear runs of this test resulted in incomparable references that could not be used to identify
the unknown inks, as the component zones were not identifiable, and the dye separations difficult
to distinguish. Figure 5 shows a reference chromatogram in (2 Water:1 Methanol) with polarity
8.20. Similarly, this run yielded an unusable reference test, with inks 1, 4, 8, & 12 barely
showing up. Figure 6 shows a reference chromatogram in (1 Acetic Acid:1 Water) with polarity
7.60. This test resulted in fairly good separation of the dyes, with the exception of ink 8.
However, those that did separate were pretty clear, and distinguishable from one another. Figure
7 shows a reference chromatogram in (2 Acetic Acid:1 Water) with polarity 8.63. This test was
unacceptable as a reference with many of the runs turning with immeasurable R f's and

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indistinguishable bands. Figure 8 shows a reference chromatogram in (2 Water:1 Acetic Acid)

with polarity 8.07. This test was another that was deemed unusable, with splits in a number of
the runs (2 & 3), and absences of dye separation from 1, 4, & 8.

Ultimately, the mixture of (1 Acetic Acid: 1 Water) was chosen as the solution for separation of
the unknowns. Figure 9 shows an unknown test run chromatogram in (1 Acetic Acid:1 Water)
with polarity 7.60. The test yielded split runs for three out of the four of the group, and the cause
for splits was not known at the time, so the solution was changed to a less polar one, (1
Propanol:1 Water). Figure 10 shows an unknown test run chromatogram in (1 Propanol:1 Water)
with polarity 6.65. This was infinitely more successful, and the separations were distinct, easily
distinguishable from one another. Table 1 showed the Rf values for the reference inks, and Table
2 showed the Rf values for the unknown inks. The comparison of the two, along with the visual
color separation from the physical chromatograms allowed for the identification of the
unknowns. The identified pens were Pilot Vball BG (ba), Pilot G-2 07 (ba), Pilot G-2 07 (bu),
BIC Ultra Round (bu), and Pilot Vball BG (r).

Figure 1. 1 Propanol:1 Water Original Reference Paper Chromatography of Known Inks

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The best solution was determined to be 1 Propanol:1 Water as it was the easiest to distinguish
between the various inks, especially in visual comparison. The separation colors were different
(i.e. #11 and #12), and the runs were distinct.

Figure 2. 2 Propanol:1 Water Original Paper Chromatography of Known Inks

1
0

1
1

1
2

13

The pen inks were harder to differentiate between with solution of 2 Propanol:1 Water (i.e. #6
and #8). Without clear differences from ink to ink, the solution/chromatogram were unable to be
used in identification of the unknowns.

Figure 3. 1 Methanol:1 Water Original Paper Chromatography of Known Inks

10

11

12

13

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Many of the runs for this test were too faint, and a few of the runs had splits in them, making the
determination of the Rf values difficult. Inks 1, 8, 11, and 12 were barely detectable.

Figure 4. 2 Methanol:1 Water Original Paper Chromatography of Known Inks

The test yielded runs that were not straight and curved toward the end, making Rf value
determination impossible. The separations were not distinct bands and were blurry/unclear.
Figure 5. 2 Water:1 Methanol Original Paper Chromatography of Known Inks

This solution resulted in too faint runs for inks 1, 4, 8, 9, 11, 12, and 13, and splits in 2, 3, and 7.

Figure 6. 1 Acetic Acid:1 Water Original Paper Chromatography of Known Inks

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10

11

12

13

The runs of inks 1 and 8 were too faint, and 11 and 12 were indistinguishable from one another.

Figure 7. 2 Acetic Acid:1 Water Original Paper Chromatography of Known Inks

10

11

12

13

This test resulted in runs with immeasurable Rf, and with curved runs.
Figure 8. 2 Water:1 Acetic Acid Original Paper Chromatography of Known Inks

10

11

12

13

The test yielded split and faint runs, at 2, 3, and 7, and 1, 4, and 8, respectively. Rf was
immeasurable.

Figure 9. 1 Acetic Acid:1 Water Unknowns Paper Chromatography Test #1

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Initially, the solution of 1 Acetic Acid:1 Water was found to be the best test, and was used on the
unknown inks. However the splitting of the sample of unknown #2 and the unclear separation of
the sample of unknown ink #4 caused a change in the solution used.

Figure 10. 1 Propanol:1 Water Unknowns Paper Chromatography Test #2

The second test on the unknowns was conducted with a solution of 1 Propanol:1 Water, as it was
determined to be a better solution for separation and distinction among the inks. This test fared
better, with no splitting and more distinct runs. This test resulted in more distinct
bands/separations, especially in the instance of unknown #4.

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Table 1. Samples of Possible Pen Ink Dyes and Distance Travelled with R f values for Paper
Chromatography in 1 Propanol:1 Water Solution
ba=black pen, bu=blue pen, r=red pen
Sample Name
1. Pilot Easy Touch (ba)
2. Pilot G-2 07 (ba)
3. Pilot Vball BG (ba)
4. Papermate InkJoy (ba)
5. Pilot Vball BG (r)
6. BIC Ultra Round (r)
7. Pilot G-2 07 (r)
8. Pilot Easy Touch (r)
9. BIC Ultra Round (ba)
10. Pilot G-2 07 (bu)
11. BIC Ultra Round (bu)
12. Pilot Easy Touch (bu)
13. Papermate InkJoy (bu)
total distance travelled dye
Rf =
total distance travelled solvent

total solvent: 3.81 cm

Distance Travelled (cm)

3.79
2.11
3.78
3.79
2.65
3.75
2.70
3.79
3.79
2.86
3.78
3.79
3.80
2.45
3.79
2.62
3.80
3.80

Pilot Easy Touch (ba):

Rf =

Rf Value
0.99
0.55
0.99
0.99
0.69
0.98
0.71
0.99
0.99
0.75
0.99
0.99
1.00
0.64
0.99
0.69
1.00
1.00

3.79
=0.99
3.81

Table 2. Unknown Pen Samples Rf Values/Distance Travelled for Paper Chromatography in 1

Propanol:1 Water Solution, and Sample Identities
Sample
Distance (cm)
Rf value
Sample Identity
Unknown #1
3.36
0.79
Pilot Vball BG (ba)
Unknown #2
1.34
0.32
Pilot G-2 07 (ba)
Unknown #3
4.13
0.98
Pilot G-2 07 (bu)
Unknown #4
3.09
4.01
0.73
0.95
BIC Ultra Round (bu)
Unknown #5
3.32
3.87
0.79
0.92
Pilot Vball BG (r)
The final solution was determined to be (1 Propanol:1 Water), as it displayed no splits in the
runs, and the dyes were distinguishable from one another, making the identification process
easier. Initially, the (1 Acetic Acid:1 Water) was determined to be the best solution with a Snyder

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polarity of 7.60. However, after a first trial with "splitting" dye trials, the solution was switched
to (1 Propanol:1 Water) with a lower polarity of 6.65.
Discussion:
Chromatograms 1, 2, & 6 (Figures 1, 2, & 6 respectively) were of good quality, while 3, 4, 5, 7,
& 8 were of poorer quality and not in the running for use in the final procedure. From the better
three chromatograms, two of the solutions were used in tests of the unknown inks. The "perfect"
solution was changed from the original (1 Acetic Acid:1 Water) (7.60) to the solution of (1
Propanol:1 Water) (6.65) instead. This change was made due to the unsuccessful first test of the
unknown inks. The group only turned out one successful chromatogram. The other three either
had splits in the runs or slanted runs. These original issues interfered with the accurate
identification of the unknown inks, and in the second trial, the solution was changed. The second
test yielded far more accurate results. The runs were not splitting, and the final separations of the
known dyes were more distinct from one another. The ultimate solution was better in visual
comparison for identification of the pen brands. This change supported the idea that the solution
needed to be a polar one, and also the idea that an effective solution could not be too polar either,
hence the switch to a less polar solution. The final procedure utilized the (1 Propanol:1 Water)
solution to separate the unknown inks into their distinct dye components through paper
chromatography.

The first unknown was determined to be Pilot Vball BG (ba), since it had the strongest looking
color run in comparison to the other black inks, and an R f value near 1. The second unknown was
determined to be Pilot G-2 07 (ba) as it had the shortest distance travelled and smallest R f value
out of the black inks. The third ink was determined to be Pilot G-2 07 (bu), based on the similar

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color trails of the unknown and known test trials, as well as the vibrancy of the
colors/separations. The fourth ink was determined to be BIC Ultra Round (bu), as it was the only
blue ink on the reference that had both two component zones and a purple/violet hue. The final
unknown was determined to be Pilot Vball BG (r) as it separated into a dark pink and a dark,
saturated orange shade. The other two red inks with two component zones were significantly
lighter in hue. This final procedure allowed for the precise and accurate identification of all five
unknowns.

Sources of error that occurred during the experiment included the particular rolling of the
chromatography paper so as to give way to curved test runs that were immeasurable for R f
values. Another error included the particular way of dotting the pen inks onto the origin line.
When the dot was created by circling around the exterior followed through with attempting to
shade in the interior, the lack of ink in the interior caused a splitting of the runs during the
chromatography process. This made it impossible to determine the R f values, and affected the
clarity of the separation of dyes. Also, evaporation may have occurred during the
chromatography, either with the cup or at the lack of it, resulting in faded/blurry runs that were
not clear. All these errors may have contributed to a solution being overlooked for the final
procedure due to an unusable reference trial. The concentration of ink dot of the samples were
likely not equal, with some containing much less ink than others, affecting the quality of the
trials and the distinctions between the inks. However, as reflected by the accurate identification
of inks, the method was still fairly effective, and could be expanded upon for further exploration.

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Future studies could explore the differences of solutions of smaller differences in polarity, for
instance, examining a range of solutions from 6.5 to 8.0 in Snyder polarity, as opposed to the
5.87 to 8.63 of this experiment. Future studies could also explore the threshold at which point the
solution was too polar, and the factors/characteristics of the chromatography subjects that affect
the threshold of reversal.
Conclusion:
The hypothesis that the stationary phase would be more polar than the mobile phase for less
polar solutions was supported by the experiment. However the statement that a solution more
polar than water would cause the process to reverse was not studied in the particular experiment,
so it was neither supported nor rejected. The most effective solution for separation was polar, but
not too polar, allowing for the best differentiation among the various inks, and for the most
accurate identification of the unknowns. The (1 Acetic Acid:1 Water) solution was too polar, at a
Snyder value of 7.60, and the (1 Propanol:1 Water) solution of 6.65 Snyder polarity was much
better suited for distinguishing among the inks. The best solution needed to be fairly polar to
separate the dyes into their distinct components, but not too polar so that the separation could not
be fully completed on the paper. Any solution more polar than water should have reversed the
process, resulting in reference test that would not allow for the accurate identification of
unknowns. Future studies could potentially explore the differences of solutions of smaller
differences in polarity, as well as the threshold of polarity, looking into the factors/characteristics
of the chromatography subjects that affect that threshold.
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