A Separation of Inks
By: Elizabeth N. Timblin
Partner: Tim Thomas
Chem 113 Section 103
February 21, 2013
TA: James Morse
Matthew Langston
TABLE OF CONTENTS
Introduction ......................................................................................................................................3
Procedure .........................................................................................................................................6
Results ..............................................................................................................................................9
Conclusion .....................................................................................................................................12
References ......................................................................................................................................13
2
What is chromatography?
In a mixture, two or more substances are combined but are not chemically altered. 1
Because these substances are not chemically altered, the mixture can be separated back into its
component parts. The process of separating a mixture into its component parts is called
chromatography. These component parts are separated between a stationary phase and mobile
phase. The stationary phase is a porous substance, either solid or liquid, that has an attraction for
the components as the mobile phase, either gas or liquid, moves through it.2 Different
components will move at different rates based on their affinity for the stationary phase. The less
attraction a component has for the stationary phase, the faster the liquid mobile phase will pass
through.3 The component parts can then be analyzed and identified.
Chromatography was invented by Russian botanist Mikhail Tswett in the late 1800s
while working with plants. In an attempt to separate different chlorophylls, he seeped a mixture
of dissolved plant material through a glass tube packed with powder. Bands of color formed
down the column as the liquid flowed through it. Each band represented a different type of
chlorophyll. Despite Tswett’s publication in the early 1900s, the work was ignored partly for its
simplicity. German organic chemist Richard Martin Willstatter rediscovered the technique in the
1930s when he was also studying chlorophyll. Willstatter was responsible for introducing a
simple and inexpensive process for separating and analyzing complex mixtures to the world of
science. In 1944, the biggest advance in chromatography took place when paper chromatography
was discovered by two British biochemists, Archer John Porter Martin and Richard Laurence
Millington Synge.4 Martin and Synge were trying to determine the amino acids present in a
specific protein when they invented paper chromatography and won a Nobel Prize in 1952 for
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their discovery.5 Chromatography has been instrumental in the synthesis of insulin, penicillin and
other antibiotics, and was used to reveal the four nitrogenous bases that form DNA.4
How does paper chromatography work?
In paper chromatography, an absorbent paper such as cellulose paper is used as the
stationary phase. Cellulose has many hydroxyl groups available for hydrogen bonding which
helps to form a water layer.5 A liquid solvent with desired properties, such as polarity, forms the
mobile phase. A small sample, i.e. pen ink, is placed on the paper and the paper is suspended in
the solvent so that the mobile phase will migrate up the paper. The different components will
move at different rates and will leave different colored spots on the paper as they separate.6 The
distance traveled by the component compared to the mobile phase is distinct to a particular
component and can be given an Rf value or retention factor.
Rf = distance travelled by component
distance travelled by solvent
Comparisons may be made visually, with UV light and/or with Rf values.
Paper chromatography is just one method for ink separation and identification. Another
method is electrophoresis which is the separation of particles with the use of an electric field.7
While paper chromatography and electrophoresis can be destructive techniques, Raman
microspectroscopy/microscopy is a non-destructive technique8 that observes how photons from a
light source, such as a laser, interact with the sample.9
The purpose of this experiment was to identify four unknown pen inks by first creating
trials of known inks with varying solvents. A previous experiment had been done using FD&C
food dyes with Kool-Aid dyes and pen inks. The solvent mixture used was a 2:1 ratio of 1-
4
propanol and water.5 The polarity of 1-propanol is 4.3 and the polarity of water is 9.0. The total
polarity of this solvent mixture, 5.87, was calculated using the following formula.
Total
Polarity
=
(
polarity of
x
solvent 1
solvent 1 volume
mixture volume
)+(
polarity of
solvent 2 volume
x
solvent 2
mixture volume
Whereas a polarity of 5.87 was sufficient to separate Kool-Aid dyes and compare with FD&C
dyes, it was not sufficient for separating pen inks. See Table 1 for a list of samples tested and
Figure 1 for the corresponding chromatogram.
Table 1
Samples Tested as Compared to FD&C Food Dyes
Spot #
Dye/Ink Identification
1
FD&C Red #3
2
FD&C Red #40
3
FD&C Blue #1
4
FD&C Yellow #5
5
FD&C Yellow #6
6
FD&C Green #3
7
Cheddar Cheese
8
Grape Kool-Aid
9
Orange Kool-Aid
10
Lemon-Lime Kool-Aid
11
My Dye Mixture
12
Blue Bic pen
13
Blue Paper Mate Pen
14
Red Bic Pen
15
Red Paper Mate Pen
16
Black Bic Pen
17
Black Paper Mate Pen
5
)
Figure 1
Chromatogram of Samples Tested
as Compared to FD&C Food Dyes
Looking at the pen ink spots 12 through 17 on the chromatogram in Figure 1, very little
separation occurred and Rf values are all the same. There is no way of telling the inks apart using
this solvent.
Based on the finding that the polarity of this solvent mixture was not adequate in
performing separation of pen inks, the hypothesis was that if solvent mixtures could be created
with varying polarities, the most polar solvent would allow for the best component separation.
Experiment Procedure
Following the given procedure, fourteen known pen inks, five black, five blue, and four
red, were spotted on four different chromatography papers. Starting on the far left of the
chromatograms, the pen inks spotted are as listed in Table 2. Four different solvent mixtures
ranging in polarity were created. See Table 3 for solvent mixtures.10
6
Table 2
Table 3
Trial #
Inks Spotted on Trial Chromatograms
Spot #
Pen Ink Color
Pen Identification
1
Black
Staples
2
Black
Paper Mate
3
Black
Pilot VBall
4
Black
Pilot Easy Touch
5
Black
Bic Round Stic
6
Blue
Staples
7
Blue
Paper Mate
8
Blue
Pilot VBall
9
Blue
Pilot Easy Touch
10
Blue
Bic Round Stic
11
Red
Staples
12
Red
Paper Mate
13
Red
Pilot VBall
14
Red
Pilot Easy Touch
Solvent Mixtures for Paper Chromatography Trials
Solvent 1
Solvent 2
Ratio
Total
Polarity
Name
Polarity
Name
Polarity
1
1-Propanol
4.3
Water
9.0
3:1
5.5
2
Ethanol
5.2
Water
9.0
3:1
6.2
3
Methanol
6.6
Water
9.0
3:1
7.2
4
1-Propanol
4.3
Ethanol
5.2
4:1
4.5
Rather than using a simple solvent of 1-propanol, a mixture of 1-propanol and water was
made in trial #1 to create a solvent with a higher polarity in order to follow the hypothesis. The
7
same was done with ethanol and methanol in trials #2 and 3 respectfully. Water was not used
directly as to avoid too much spreading. For trial #4, a mixture was created from two solvents as
to make a smaller polarity. These solvent mixtures created a three point range in polarity to test
each chromatogram. Each marked chromatogram was stapled in a cylindrical shape and then
placed in a different solvent mixture where as the solvent would travel up the paper through the
samples. A plastic cup was placed over each chromatogram to prevent the vapors from the
solvent from evaporating.6 As the solvents neared the tops of the chromatography paper, the
chromatograms were pulled out of the solvents and unstapled. The solvent lines were marked
with pencil and the chromatograms were laid to dry. Each chromatogram was compared both
with the naked eye and with the assistance of a UV light. Once it was determined which mixture
appeared to create the best separation in the ink pigments, Rf values were calculated for that
chromatogram and a chromatogram of unknown inks was placed in the solvent and covered. The
initial observation of the unknown inks is listed in Table 4. As was done with the trials, the
unknown ink chromatogram was removed from the solvent and unstapled. The solvent line was
marked with pencil and the chromatogram was laid to dry. Once dry, Rf values were calculated
for the unknown ink samples and the unknown ink chromatogram was compared to the trial
chromatogram that used the same solvent. The unknown inks were identified with comparisons
of observation made with the naked eye, observations made with the use of a UV light, and R f
values.
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Table 4
Initial Observations of Unknown Inks
Spot #
Pen Ink Color
1
Red
2
Black
3
Black
4
Blue
Experiment Results
Figure 2 shows chromatograms with the fourteen ink samples processed with the four
different solvent mixtures. See Table 2 for the list of ink samples and Table 3 for the list of
solvents used.
Figure 2
Trial Chromatograms
#1
#2
#3
#4
9
Trial #3 appeared to have the most distinguishable ink separation when viewed with the
naked eye and with UV light. Trials #1, 2, and 4 appeared to separate only one or two inks of
each color. Some noted observations as well as calculated Rf values can be seen in Table 5.
Table 5
Observation of Trial #3
Spot
#
UV Observation
Rf
Value
Sample ID
1
Black Staples
N/A
0.93
2
Black Paper Mate
dark blue spot above yellow
spot with faint purple tail
N/A
0.80
3
Black Pilot VBall
faint yellow at top with dark
grey tail
N/A
0.73
4
Black Pilot Easy Touch
very faint yellow glow
over whole streak
0.68
5
Black Bic Round Stic
N/A
1.00
6
Blue Staples
N/A
0.93
7
Blue Paper Mate
medium blue spot at top
8
Blue Pilot VBall
9
Blue Pilot Easy Touch
large bright blue spot at top
small large bright blue spot at
top with very light fading tail
10
Blue Bic Round Stic
11
Red Staples
12
Red Paper Mate
13
Red Pilot VBall
14
Red Pilot Easy Touch
Visual Observation
faint purple head at top with
very faint purple tail
light purple above yellow spot
and light purple tail
dark purple spot at top with
very faint yellow spot just
below
medium purple fades almost
evenly from top to bottom
dark blue head with
diminishing blue fade
bright pink spot at top
bright pink spot at top with
yellow fading tail
bright pink spot above bright
orange spot with
orange/yellow tail
light pink head w/ pale yellow
tail
very faint dark blue
glow
N/A
1.00
1.00
N/A
1.00
N/A
1.00
bright orange at top
1.00
bright orange at top
1.00
bright orange at top
with bright green
fading tail
green dot at above
bright orange w/ faint
green tail
0.73
1.00
10
The unknown inks were then tested with the solvent mixture used in Trial #3. Once dry,
observations were made with the naked eye, UV light, and Rf values. See Figure 3 for the
chromatograms of unknown inks and Table 6 for observations of the unknown ink
chromatogram.
Figure 3
Unknown Ink Chromatograms
Table 6
Unknown Ink Observations
Spot #
Initial
Ink Color
Visual Observation
UV Observation
Rf Value
1
Red
light pink head w/ pale
yellow tail
green dot at above bright
orange w/ faint green tail
0.97
2
Black
dark blue spot above yellow
spot with faint purple tail
N/A
0.78
3
Black
faint purple head at top with
very faint purple tail
N/A
0.97
4
Blue
dark blue head with
diminishing blue fade
N/A
1
11
By comparing observations and Rf values for Trial #3 and the unknown inks, the
unknown inks were identified as they are listed in Table 7. The solvent mixture used allowed for
correct identification of the unknown sample.
Table 7
Unknown Ink Spot #
1
2
3
4
Unknown Ink Identification
Trial #3 Spot #
14
2
1
10
Ink Identification
Red Pilot Easy Touch
Black Paper Mate
Black Staples
Blue Bic Round Stic
Conclusion
Polarity of solvents determined whether or not inks separated quickly. Solvents with a
range in polarity were created and tested. The hypothesis stated that the solvent with the highest
polarity would be most effective in ink separation. As per the hypothesis, the solvent with the
highest polarity was chosen to test the unknown ink samples. Based on observations, this
procedure correctly identified the inks. Although this procedure was successful, it may have
clearer results if a solvent or solvent mixture with a high polarity but excluding water could be
used as to reduce streaking. It may also be necessary to use a taller piece of chromatography
paper.
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References
1
Keiser, Joseph T. Chemistry 113 Student Packet; Hayden McNeil Publishing: Plymouth, 2012;
p 17.
2
Encyclopedia Britannica. Stationary Phase.
http://www.britannica.com/EBchecked/topic/564121/stationary-phase (accessed February 16,
2013).
3
Rensselaer Polytechnic Institute. Intro to Chromatography. http://www.rpi.edu/dept/chemeng/Biotech-Environ/CHROMO/chromintro.html (accessed February 16, 2013).
4
Discoveries in Medicine. Chromatography. http://www.discoveriesinmedicine.com/BarCod/Chromatography.html (accessed February 16, 2013).
5
Keiser, Joseph T. PSU Chemtrek: Small Scale Experiments for General Chemistry. Hayden
McNeil Publishing: Plymouth, 2012; p 17.2-17.21.
6
Chemguide. Paper Chromatography.
http://www.chemguide.co.uk/analysis/chromatography/paper.html (accessed February 16,
2013).
7
Chen, Hu-Sheng Ph.D; Meng, Hxien-Hui Ph.D; Cheng, Kun-Chi, M.Sc. A Survey of Methods
Used for the Identification and Characterization of Inks Forensic Science Journal. 2002;1:1-14.
8
Chalmers, John M.; Edwards, Howell G. M.; Hargreaves, Michael D. Infrared and Raman
Spectroscopy in Forensic Science, John Wiley & Sons, Ltd: West Sussex, 2012; p 138.
9
Craic Technologies. Science of Micro Raman Spectroscopy.
http://www.microspectra.com/support/technical-support/raman-science/35-technicalsupport/126-science-of-micro-raman-spectroscopy (accessed February 16, 2013).
10
Timblin, Elizabeth N. Chem 113 Lab Notebook; p 3-9.
Thomas, Tim. Chem 113 Lab Notebook; p 10-13.
Codrops. Principles of Color and the Color Wheel.
http://tympanus.net/codrops/2012/02/28/principles-of-color-and-the-color-wheel/ (accessed
February 16, 2013).
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