Who Wrote the Ransom Note?

(adapted from Analysis of Ink by Thin-Layer Chromoatography or Paper Chromatography by

Professor Lawrence J. Kaplan, Williams College)
Introduction:
Chromatography is a term that is widely used to describe a family of closely related
separation methods. There are many separation methods, but the feature that
distinguishes chromatography from other physical and chemical methods of separation is
both a stationary and mobile phase; the sample to be separated interacts numerous times
with these phases. The sample is carried through the system via the mobile phase, and
the interactions that occur are due to the differences in the physical and chemical
properties. These differing affinities govern the rate at which the individual components
of the sample pass over the stationary phase under the influence of the mobile phase.

Thin layer chromatography (TLC) is a type of chromatography where the stationary

phase is a thin layer of adsorbent particles attached to the solid plate. A small amount of
sample is applied (spotted) near the bottom of the plate, and the plate is placed in the
mobile phase. This solvent is drawn up by capillary action to a predetermined height.
Each component, being different in chemical and physical composition, will interact with
the stationary phase at a different time (retention time), thereby creating the individual
bands on the plate. The retention time or retention factor (Rf) is used to characterize and
compare components of various samples. The same basic procedure is used for paper
chromatography, however, with paper chromatography, regular or specially treated paper
is used instead of a solid plate.

__distance from origin to center of spot_____

Rf = distance from origin to solvent front
This experiment involves the chromatographic techniques that forensic scientists have
developed for identifying pen inks. The need for differentiating inks arises when dealing
with fraudulent documents, such as those that have been altered or back-dated and in
attributing a particular document, such as a letter, to a particular individual. Until
recently, the color of ink was due to a single component, a dye. Therefore, when this ink
was analyzed, only one spot would be observed. Inks manufactured more recently
contain many components with the color being due to a mixture of dyes. The ability to
distinguish between inks will often allow the forensic scientist to determine the number
and identity of the pens used in preparing a document as well as the year in which these
pens were manufactured.

Since the color of ink is due to a mixture of dyes, it is necessary to separate the dyes from
one another in order to characterize the ink. These dyes are large organic molecules
having a variety of functional groups which give them different properties and allow
them to be resolved by thin-layer or paper chromatography. As an example of this, the
separation of the dyes in the inks in some common pens. With their Rf value in the
solvent system used in the thin-layer chromatography part of this experiment, is
illustrated in Table 1.

Table 1: Color of Dyes and Rf of Dyes from Various Inks

Ink Color observed Rf

Bic (ball point) Blue 0.59

Pink 0.44
Yellow 0.18

Flair (felt tip) Purple 0.48

Yellow 0.35
Purple 0.30
Blue-green 0.20

Pilot Oasis (felt tip) Purple 0.29

Yellow 0.24
Turquoise 0.12
Purple 0.07

Equipment:
Chromatography jar Ruler
Capillary tubes Tweezers
Test tubes: 10x75 mm, 15x100 mm Developing solvent:
Scissors For TLC: mixture of n-butyl
Water bath alcohol: ethyl alcohol: water:
TLC plates or chromatography paper acetic acid: :60:10:20:0.5
Suspect pens For paper chromatography:
Ransom note Acetic acid, 5%
Ethanol, 95% Small vial or tube
Methanol Pencil
Paper Pasteur pipette

Safety:
 Always wear safety goggles.
 Wear gloves and wash thoroughly with soap and water if you get solvent on you.
 Only shine the UV lamps toward the tabletops. UV light is harmful to eyes and skin.

Procedure:
Preparing the samples:
1. Prepare a developing chamber by adding about 0.5 cm of developing solution to
the chamber, and closing the jar until needed.
2. Remove the appropriate sections from the suspect document with a pair of
scissors, cutting them into a few pieces and placing them in a 10x75 mm test tube.
If variations of ink on one document are suspected, be sure to keep the different
sections separate and well labeled.
3. Extract (remove) the ink from the paper using 2-3mL of methanol. The test tubes
can be heated in a water bath (a beaker with water placed on the hotplate; don’t let
the water get too hot or all of the methanol will evaporate) to increase the
solubility of the ink. To aid the removal of the ink from the pieces of the paper,
squirt the hot methanol over the pieces of the paper using a Pasteur pipette.
4. When the methanol solution is fairly dark from the ink, transfer the ink solution
from the test tubes to a small vial or small tube.
5. Evaporate most of the methanol with a stream of N2 to concentrate the ink
sample. If all of the methanol is evaporated, redissolve the ink residue in a few
drops of methanol.

Spotting of the sample:

1. Obtain a TLC plate. Handle the plate by the edges only, making sure to avoid
touching the white silica layer (the side you are using).
2. With a pencil (not pen), use the ruler to gently draw a line about 1.5 cm from the
bottom of the plate. Be careful not to chip off the silica layer. Beginning about
0.5 cm from the side edge of the plate, draw between 3 and 9 evenly spaced
crosshairs–one for each sample (see diagram on following page).
3. Immerse the capillary tube into the sample tube until some of the liquid is drawn
into the capillary.
4. Very gently press the small end of the capillary tube at a crosshair on the TLC
plate. Keep the spot small by touching the capillary quickly to the plate and then
removing it immediately. Allow the spot to dry and repeat the spotting directly
over your original spot.
5. Use the UV-lamp (set on short wave) to ensure you have some sample on the
plate. The plate should glow green, except where you used the capillary tube to
deposit the sample. If no spots appear, use the capillary to apply more sample
 directly over the original spot.
6. Record which spot represents which sample on the diagram on the following
page.
7. Repeat steps 3-6 for the other samples.

Development of the sample:

1. To be sure that the position of the sample spots on the thin-layer plate or the paper
and the solvent level in the chromatography jar are correct, hold the spotted thin-
layer plate or the paper alongside of the jar containing the solvent. The solvent
level must be below the spots and the sides of the plate or paper must never touch
the jar.
2. Place the plate or paper vertically in the developing jar with the upper edges only
resting against the jar.
3. Reseal the jar and allow the solvent to rise until it is about 2-3 cm from the top of
the plate or paper. This will take much longer for the TLC plate than the paper.
If the solvent does not rise far enough before the end of the lab period, your
instructor will remove the plate from the jar and mark the solvent front.
4. Using tweezers, remove the TLC plate and immediately mark where the solvent
stopped rising with a pencil. This is called the solvent front. Allow the TLC plate
to dry.
5. Visualize the spots by illumination under the UV lamp.
6. Mark the center of each spot with a pencil and then measure the distance traveled
by each component. Using the TLC plate diagram below, draw what your plate
looks like. In the table on the following page record the distances from the origin
(original location of the spot) to the solvent front and the distance from the origin
to the center of each spot.

Clean-up
1. Dispose of all liquids as instructed by the teacher.
2. Wash your hands with soap and water.

Data:
s o l v e n t
f r o n t

1 2 3 4 5 6 7 8 9
N a m e o f M e d ic a tio n
Analysis and Calculations:
1. Calculate the Rf for each spot by dividing the distance the spot traveled by the
distance the solvent traveled. The Rf value is characteristic of each substance and
may be used for identification of the substance.

# Sample Distance traveled by Distance traveled by

solvent front (cm) sample (cm) Rf
1

9
2. What causes the inks to move at different rates?

3. Which ingredient has the strongest attraction for the stationary phase? Which has
the weakest?

4. Compare the results of the suspect ink samples to the results of the ransom note
ink. What do the results indicate about the ransom note ink?