FRS581

Laboratory Report

TLC of Pen Ink

Nur Atiqah binti Ahmad
2013574299
Date Performed: 11th May 2015
Date Submitted: 18th May 2015
Group: AS2534B1
Lecturer:
Pn. Khairulmazidah binti Mohamed

Introduction
The ink in modern ball point pens is a mixture of various dyes
components blended together to get the desired colour. Each pen
manufacturer will have a unique formulation for their inks. In forensic
investigation, ink analysis may be an important part of the investigation
of questioned documents, including forged checks, wills, or altered
records. Although all blue or black inks may look the same, there can be
some important differences in their chemical composition. These can be
revealed by laboratory analysis and the results can help assess whether
there have been any additions or alterations to a document.
In todays experiment you will separate the ink pigments using a
technique called Thin Layer Chromatography (TLC). TLC works by moving
the ink along a strip of plastic coated with a thin layer of alumina or silica
gel. The coating on the strip is called the stationary phase. The ink is
moved along the stationary phase by a solvent or a mixture of solvents
called mobile phase. Each pigment will have unique affinity to the
stationary phase as well as a unique solubility in the mobile phase. The
greater the affinity to the stationary phase, the slower the pigment
travels. The greater the solubility in the mobile phase, the faster the
pigment travels.

Objective
To

understand

and

experience

ink

analysis

using

Thin-layer

chromatography technique

Materials
Pencil

Precoated silica chromatography

Metric ruler

plate
Several ball-point pens,

white paper
95% ethanol / pyridine

Distilled water

Ethyl acetate

Capillary tube
Beaker
Aluminium foil
Video Spectral Comparator (VSC)

Procedure
The mobile phase:
The mobile phase solution was prepared using 7.5mL ethyl acetate, 3.5mL
95% ethanol and 3.0mL distilled water to a 100mL screw cap reagent
storage bottle. The solution was mixed to make it homogeneous.
Separation technique;
1. The mobile phase was added to the beaker to attain a height of 5
mm.
2. The beaker was saturated with mobile phase vapors by closing the
beaker with aluminum foil and was left stand at least 15 minutes.
3. Words were written on pieces of paper using pen from different
brands of similar ink colour.
4. 2 drops of 95% EtOH or pyridine was added on each word written
using different pen. The solvent was observed until the ink is
dissolving into the liquid.
5. 1cm from the bottom of a short edge of the TLC plate was marked
using pencil.
6. 1cm before the end of the TLC plate was marked using pencil as an
end line for solvent front.
7. A clean capillary tube was used to spot each sample along the first
pencil line.
8. The location of each sample was noted.
9. The TLC plate was carefully positioned in the beaker and covered
with aluminum foil.
10.

The progress of the solvent front was checked every 2-3

minutes.
11.

The plate was removed from the beaker once the solvent front

reach about of the TLC plate length the solvent front was marked
pencil.
12.

The TLC plate was allowed to dry completely.

13.

The TLC plate was photographed to be included in the report.

14.

The center of each band was marked.

15.

The Rf value for each band was calculated by dividing the

distance travelled by the band by the distance travelled by the

solvent.
16.

Each ink yielded a unique profile by comparing the number of

bands and their corresponding Rf values.

Data and Result:

Figure 1: Dye component of ink samples of different brands. (a) view

under white light (b) viewed under fluorescence light.

Table 1: Dye component of ink samples of different brands

Brand

Ink components

Stabilo

Paper Mate

No brand

Faber Castle

Table 2: Distance traveled by dye component of ink samples of different

brands
Distance travelled, cm

Ink
components

Stabilo

Paper Mate

No brand

Faber Castle

4.65

4.65

4.65

4.50

4.95

4.95

4.90

4.85

5.19

5.15

5.10

5.10

5.20

5.35

5.70

5.60

5.60

6.30

6.25

6.20

6.20

Table 3: The retention factor Rf of the dye component of pen ink samples
Retention factor, Rf*

Ink
components

Stabilo

Paper Mate

No brand

Faber Castle

0.669

0.669

0.669

0.647

0.712

0.712

0.705

0.698

0.747

0.741

0.734

0.734

0.748

0.770

0.821

0.806

0.806

0.906

0.899

0.892

0.892

distance travel by solvent front = 6.95cm

Rf =

distance travel by sample (cm)

distance travel by solvent ( cm)

Discussion
In this practical, black ink samples from different brand were tested
using thin layer chromatography. The ink samples were first dissolved in
pyridine for extraction. The ink samples were spotted on the silica plate
and placed in a closed beaker in mobile phase. The silica plate then was
removed when the solvent reach the end of the plate and dried at room
temperature. The silica plate was observed under fluorescence light.
Sometimes inks of similar colour can look identical to the naked eye
but further tests may be able to distinguish between them. Most
commercial ink is made up of a mixture of several organic dyes. These
dyes can be split up on appropriate developed thin layer chromatography
plate (Saferstein, 2014). Once a TLC plate was run in the mobile phase,
clear bands visible on the plate indicating the separation of different
components of the ink were formed as shown in Figure 1. From the
experiment, it is shown that black ink from Stabilo, Paper Mate, No brand,
and Faber Castle is made up of 5, 5, 7 and 4 different components
respectively. The distance travelled by each component from different ink
samples was recorded in Table 2 above. The retention factor R f calculated
for each component of different samples was recorded in Table 3 above.
Most ink formulation has similar dyes components and some have
different components. This makes a unique characteristic of the ink
samples. In forensic analysis, this characteristic is importance when the
questioned documents need to be compared to the known document. For
example, ink analysis can reveal whether there an addition of number in a
forged cheque or not.

Figure 1 shows the silica plate observed under the white light (a)
and under the fluorescence light (b). Under white light, the true colour of
the component can be distinguished. Under the fluorescence light, all the
dye components appear to be the same and not obviously separated.
Under the fluorescence light, some component cannot be seen. Based on
the Figure 1, the yellow component seen under the white light was not
observed under the fluorescence light but component that are hard to be
observed with naked eye is brightly seen under the fluorescence light.

Conclusion
In conclusion, dye components of the ink samples can be separated and
distinguished using thin layer chromatography technique. This experiment
demonstrates that TLC can he used to identify qualitatively an unknown
compound provided suitable
standards are available. The procedure insures that each student makes
hislher own observations and calculations. Because
the components in the inks are visible, no special visualization techniques
are required. Finally, the experiment is economical
because the TLC plates are prepared in our laboratory; we have found
that 'Iti-in. douhle-strength window pane worksvery
well as the support for the Silica Gel thus eliminating the need to buy
commercial plates
Questions
1. Apart from TLC, what other methods can be used to differentiate ink?
Alterations, obliterations and erasures not visible to the human eye can
often be detected through use of photography and other imaging
devices that utilize ultraviolet and infrared wavelengths of light. Using
radiation filtered at various wavelengths, an imaging instrument such
as a video spectral comparator (VSC) can reveal writing that has been
added with a different ink, or has been altered or removed by
exploiting variations in the way different inks respond to different

wavelengths of light. For example, under certain light sources

combined with an infrared filter, a document containing information
written in ink that has faded over time may be enhanced or processed
to appear darker and therefore more legible.
The main method of non-destructive ink

analysis

is micro-

spectrophotometry. This involves scanning the ink with ultraviolet or

infrared light to record its spectrum, that is, the wavelengths of light it
absorbs. Some inks fluoresce, or emit light, on exposure to ultraviolet,
while others disappear. Each ink should give a distinct pattern or
spectrum on exposure to ultraviolet or visible light. Put simply, this is a
way of discovering the true "color" of the ink. The spectrum of the ink
on the document can therefore be compared with the spectra of
standard inks. Other non-destructive or minimally destructive methods,
such as Raman spectroscopy , can be used to supplement microspectrophotometry. It can be very informative to scan the document
with infrared light because, at high frequencies, ink is invisible but
pencil marks which may lie underneath will show up.
2. How does ink analysis helps in forensic investigation?
Differentiating inks can be important in cases where a document is
suspected of having been altered. Ingredients of the ink which are not
coloured dyes can be seen using ultraviolet or infra-red light.
Comparisons of the ingredients can reveal different pen types. Changes
to the ink 'recipe' by manufacturers can also help to specify a pen type
or sometimes prove when something was written.
3. What are the advantages and disadvantages of using TLC in ink
analysis?
Thin-layer chromatography (TLC) is one of the simplest techniques in
separation chemistry. It is inexpensive, simple to perform and one of
the most common chromatographic methods in the forensic laboratory.
This is one of the few destructive techniques employed by the
document examiner. The main method of destructive testing of ink is
known as thin layer chromatography (TLC). In reality, it is not very
destructive

to

the

document

if

done

with

care.

However,

photographic record of the original document is taken before the

procedure is started. A tiny sample of the inked paper is punched out
using a thin, hollow needle; a hypodermic syringe is ideal. The
investigator avoids places where the pen has changed direction or
where

ink

lines

meet.

This

avoids

any

interference

with

subsequent handwriting analysis. The sample is placed in a test

tube with a solvent that dissolves the ink. Next, a tiny spot of the
sample solution is placed onto a strip of paper, alongside spots from
various reference ink samples. The paper is placed in a beaker
containing a small amount of another solvent. It is positioned so that
the paper dips into the solvent but the spots of sample remain dry. The
solvent is drawn up the paper through capillary action and the sample
spots move up with it.Chromatography means "writing with colors"
and the chemical components of the ink, which are, of course, colored,
travel with the solvent at a speed that depends upon their composition.
The end result with TLC is a pattern of colored spots, known as a
chromatogram,

for

each

ink.

Different

inks

will

have different

chromatograms. If the sample ink has the same chromatogram as one

of

the

reference

inks,

it

suggests

they

are

the

same,

and

so identification can be made. The United States Secret Service has a

reference ink database and the U.S. Treasury has a database of ink thin
layer chromatograms which can be very informative.
Require simple sample extraction and easy to perform; however, it has
disadvantages.

First,

thin-layer

chromatography

is

not

typically

automated; thus the spotting of a slide and the measuring of retardation

factor (Rf) values are difficult to reproduce. In addition, spectroscopic data
is not obtained, although densitometry can be used to analyze spots for
quantitative data when compared to a standard, assuming that the
chemical identity is known (Aginsky 1994). Oftentimes the spots are faint,
rendering them hard to see. In this case, the spots can readily disappear
in a matter of minutes. If an ink sample is composed of two or more
similar dyes, then thin-layer chromatography can result in two or more
unresolved spots with nearly identical colors. This scenario requires

multiple runs with different solvent systems, which is time-consuming.

Thin-layer chromatography reference slides should be stored in a special
environment to prevent spot fading, which requires control of such factors
as

humidity

and

ultraviolet

radiation

exposure.

In

some

cases,

photographs of slides may alleviate the need to store reference slides