Experiment 17: Paper, Thin Layer, Liquid Chromatography

Vlad Stefanovich
24 March, 2016
Chemistry 113-Section 103
Christopher Bailey
Kelli Steinert, Hagan Synnestvedt, Mike Sunyak

Introduction
Most people see the world as a vibrant palette comprised of an infinite number and
variety of colors, hues, and shades. However, few people appreciate the inherent complexity of

colors and that a specific red color, for example, could be a unique blend of blue, red, and yellow
inks. Color is so ubiquitous that the dyes commonly used as additives in everyday consumer
products are carefully defined and regulated by the United States Food and Drug Administration
(FDA). These regulations apply to a group known as Food, Drug, and Cosmetic (FD&C) dyes
which are divided into those that can be added to food and those that are purely used for
industrial means (U.S Department of Health & Human Services, 2013).
Since most compounds are a blend of unique colors, it is difficult to ascertain their true
composition unless a technique was implemented to separate the individual colors.
Chromatography is a useful separation technique by which a mixture is passed through a medium
so that its components travel at different rates. This is done by dissolving the mixture in a
solution called the mobile phase - which carries the mixture through another material called the
stationary phase (Ettre et al., 1993). Chromatography was first used procedurally at the turn of
the 20th century by Russian scientist Mikhail Tsvet (which fittingly means color in Russian) in
experiments dealing with the separation of plant photopigments like chlorophyll A (Tsvet, 1906).
However, it wasnt until the 1940s that Cambridge scientists Archer Martin and Richard Synge
set the ground-work for basic separation techniques like paper chromatography, liquid
chromatography, and gas chromatography with their Nobel-prize winning contributions to
developing chromatogram theory (Martin & Synge, 1941).

Gas chromatography (GC) is a technique where the mobile phase is gas that is passed
through a silica-based stationary phase affixed within a column. This technique is widely used in
biochemical, petrochemical, and industry-chemical fields although the high temperatures at
which the gas chromatogram operates often make proteins denature - a consideration which
somewhat limits the GCs capabilities (Pavia et al., 2006).

Liquid chromatography (LC) is yet another separation technique where the mobile phase
is a liquid that can work with both a planar and column stationary phase.
The focus of this paper will be on the technique known as paper chromatography, which
falls under the category of liquid chromatography. In this process, the experimenter applies
various inks or dyes to the chromatography paper and establishes an axis across which they will
travel when the mobile phase is applied. Note that it is important to create a reference
chromatogram that allows for easy identification of mixtures and their respective placements on
the paper. In this case, the mobile phase solvent is the liquid that you place the chromatogram in
- causing the component dyes to travel up the chromatography paper a distance based on their
relative affinity to the mobile phase. The relative affinity between the mobile phase and the
sample dyes is based on polarity. If the solvent is very polar, it is likely that the nonpolar sample
dyes will not travel that far because they are not attracted to it. Conversely, if the solvent is
nonpolar, then the nonpolar component dyes will travel to the top of the paper due to their
affinity to the mobile phase. The limit of where the mobile phase travels is called the solvent
line. Because of the relationship between sample dyes and the mobile phase , it is crucial to
deduce the right solvent for a group of samples so that the components of every sample can be
identified. One of the ways this is done is by calculating the retention factor (R f values) of each dye
by calculating the quotient of the distance the sample traveled by the distance the solvent
traveled (the solvent line). This value allows for a relative estimate of the rate of flow of each
sample dye with respect to the solvent. Another way of distinguishing between a set of sample
dyes is to look at their interactions with the mobile phase under a UV light as some dyes will
produce tell-tale hues.

Since the aim of the experiment is to determine the components of twelve different pens
(three colors, four brands per color), and because an initial interaction with a mobile phase
consisting of a 2:1 propanol-water mixture caused all of the inks to reach the solvent line, it was
hypothesized that a more polar mobile phase would achieve better separation of the ink
components.
Procedure
The following procedure was taken from the PSU Chemtrek for Chem 111/113
(Keiser et al., 2015). The experiment was conducted with partners Kelli Steinert, Mike Sunyak,
and Hagan Synnestvedt (Steinert, Sunyak, & Synnestvedt, 2016).
The lab was initiated by identifying the polarity of the 2:1-propanol:water mixture used
as the first, standard mobile phase. This was done using the Snyder Polarity Index, a reference
table stating known polarity values of many substances - including water, propanol, methanol,
and ethanol (Solvent Properties, 2002). Knowing the relative polarity of the propanol mixture
was crucial for testing the hypothesis as a more polar mobile phase needed to be synthesized.
The next component was setting up the paper chromatograms for testing of multiple
different polar solvents. Each group member created their own for this purpose. The
chromatogram was set up by drawing a line 0.5 cm in height from the base across the entire
chromatography paper. Twelve, evenly-spaced, intervals were measured out at about 1.0 cm
apart from each other. These were then marked by the twelve pens in the order depicted in Table
1.
Table 1. Key for Standard Placement of Inks on Chromatogram
Spot on paper
1

Pen Color

Pen Brand
Papermate

Black

Pilot (easy touch)

Bic

G2

Papermate

Red

Pilot (easy touch)

Bic

G2

Papermate

10

Blue

Pilot (easy touch)

11

Bic

12

G2

Based on the results of Trial 1 which used a mobile phase of 2:1-propanol:water, it was
deduced that a more polar solvent was required but not quite so polar as distilled water. Each
group member made their own mobile phases within those constraints, then poured a thin layer
in a petri dish, placed the cylindrically-stapled chromatograms within, and covered them with a
plastic cup so that moisture would not escape the experiment. The progression of the mobile
phase was observed up until it was approximately 0.5 cm from the top of the chromatography
paper - at which point each group member marked their solvent lines in pencil before they
evaporated and set their chromatograms out to dry.
This portion of the experiment was repeated as many times as it was necessary to obtain a
usable mobile phase with the optimal polarity for a clean separation of inks. The polarities of
each synthesized solvent were calculated and recorded in order to narrow down the polar range
until this was achievable.

After a standard mobile phase was decided upon, individual group members were given
five unknowns with the task of identifying which pen corresponded with each one. To perform a
comparative analysis of the unknowns, another chromatogram was run with the unknown inks
using the standard mobile phase. Retention factor values were calculated and the two
chromatograms were further compared under an ultraviolet light to distinguish between the
unknown inks.
Results and Discussion
A calculation was performed, according to the Snyder Polarity Index, for the given values
of propanol and water, while also accounting for the quantity of parts propanol per part water.
Propanol = 4.3 polarity
Water = 9.0 polarity
2:1-propanol:water = [2(4.3) + 9.0] /3 = 5.9 polarity
Since it was decided that the ideal mobile phase needs to be between a polarity of 5.9 and
9.0, each group member ran a chromatogram with a mobile solvent varying in polarity.

Figure 1: Trial 1. 2:1-propanol:water

Table 2: Mobile Phases used in Trial 2

Mobile Phases

Polarity

Observations

1:1 methanol-water

7.8

Several inks run close to solvent front, more np ones

stay close to baseline

1:1 ethanol-water

7.1

Every ink except one ran to solvent front

2:1 methanol-water

7.4

Even distribution of ink across solvent front

1:2 methanol water

8.2

Many inks didnt run further than baseline, a few

reached solvent front

Figure 2: Trial 2. 1:1 methanol-water

Based on the results of Trial 2, it was decided to run a single trial using a mobile phase
solvent with a polarity of around 7. Although trials with that polarity had already been run with
little result, another factor came into play in the amount of ink that was deposited on each mark
on the chromatogram and, as result, the next trial was to put more emphasis on a reasonably
standardized amount of ink deposited at each interval. A solution of 3;1 methanol-water was
chosen for Trial 3 as it had a polarity of 7.2 and would test the theory about the positive
correlation between ink deposition and quality of separation.

Figure 3: Trial 3. 3:1 methanol-water

Table 3: Rf values for 3:1 methanol-water mobile phase
Spot on paper

Identity of pen

Rf values

Black papermate

1.0

Black pilot

1.0

Black Bic

0.875

Black G2

0.70

Red papermate

1.0

Red pilot

0.95

Red Bic

1.0

Red G2

1.0

Blue papermate

1.0

10

Blue pilot

1.0

11

Blue Bic

1.0

12

Blue G2

1.0

Figure 4: Trial Chromatogram of Unknown Inks

The unknown inks were primarily determined through a comparative analysis of

the unknown chromatogram and the 3:1 methanol-water experimental trial. Unknown 1
was easiest to decipher as no other black ink except the G2 had an Rf value as low as 0.7.
Unknowns 2 and 4 were determined by observing the pattern of color left behind when
the selected mobile phase was run through it. Both pilot black and bic blue had distinctive
separations that were unmistakable when compared side by side with the unknown inks.
The remaining unknowns proved the most difficult to identify as they left very similar
color patterns and their Rf values showed little variation - thus giving little indication of
their identities. It took exposure to UV light to determine that papermate red fluoresces in
a more distinctive way than pilot red. A comparison of the two chromatograms

showcased these differences and allowed for a successful identification on the first
attempt.
Table 4: Rf values for Unknown Inks
Spot on paper

Identity of Pen

Rf values

Black G2

0.7

Black pilot

0.89

Red pilot

0.94

Blue bic

0.92

10

Red papermate

Overall, this experiment was more of a process than a procedure - where cumulative trials
using different mobile phase solvents narrowed down the probable range in polarity where the
majority of inks would show ample separation for identification. Through a group effort, the best
mobile phase was narrowed down after only three trials. Trial 3 also confirmed the idea that
depositing more ink than in previous trials would lead to improved separation of inks. Given that
the correct identifications were made on the first try, it can be reasonably implied that the correct
polarity for the mobile phase solvent was chosen.
Conclusion
After reviewing the experiment, it can be concluded that it was a success because
solutions of an approximate 7.2 polarity work rather well at deciphering the identity of unknown
inks based on their distinctive separations. This polarity is probably not the only one that could
help discover it, but it yielded good results for the experiment group. By conducting multiple
trials, the right parameters for identifying unknowns were established. As a result, the hypothesis
was correct based on the polarity of the standard that was picked as it was indeed greater than the

first trial which had a mobile phase solvent polarity of 5.9. There is not much that could be done
to improve the setup of the lab as it promotes trial and error so well. The only consideration I
would have is to standardize the amount of ink that is deposited at each mark on the
chromatogram as results suggested that this was indeed a factor in separation.

References
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Adsorption." (Physical-chemical studies of chlorophyll. Adsorption.) Berichte der
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