Experiment 5: Thin Layer Chromatography of Ink

Objective

To understand and experience ink analysis using Thin Layer

Chromatography technique

Introduction

Thin Layer Chromatography (TLC) is one of the simplest techniques in

separation chemistry. It is expensive, simple to perform and one of the
most common chromatographic methods in the forensic laboratory. This
methods stationary is a thin layer of gel adsorbent coated onto
plastic/metal backing, called a TLC plate. As the liquid mobile phase is
drawn into gel, it is carried by capillary action up toward the top of the
plate. Along the way, the samples are separated into their constituent
chemicals. TLC is used for the identification and comparison of drugs,
explosives, inks and dyes. The technique is limited only by the need for
samples to be soluble. This is a particular issue when investigating pen
inks since the development of gel pens, the ink of which is insoluble.
Therefore gel pen ink cannot be analyzed via TLC. In fact gel pen inks have
proved difficult to analyze via typical forensic techniques.

Procedure

Preparation of the mobile phase

7.5 ml ethyl acetate, 3.5 ml 95% ethanol and 3.0 ml distilled water were
added in 25 ml graduated cylinder. Parafilm was used to close tighly the
beaker with this volatile solvent.

Analysis of ink
1. Mobile phase was added into the beaker to attain a 5 mm height.
2. The constant signatures with same size, same pressure with
different type of pen were prepared
3. The signatures were examined under compound microscope and
visible light comparison (VLC) for uv, visible and fluorescence light.
4. The information was recorded and observation was made.
5. 1 cm square of paper was colored with 4 different type of pen and
dissolved in 2 ml of ethanol placed in test tube.
6. The color changes of the test tubes were observed. The blue solvent
indicate that the ink was dissolved into the liquid.
7. A pencil line 1 cm from the bottom and above of a short edge of the
TLC plate was marked.
8. A clean capillary tube was used for each of the sample to spot along
the first pencil line.
9. The location of each samples were noted down in the notebook.
10. The TLC plate was carefully and quickly placed in the beaker
with mobile phase and covered.
11. The progress of the solvent front was checked every 2 3
minutes.
12. The plate was removed from the beaker once the solvent front
reach about of the TLC plate length and the solvent front was
marked using a pencil.
13. The TLC plate was allowed to dry completely.
14. A diagram of the plate included each band/spot was drew and
photographed.
15. The center of each band was marked and the R f value for each
band was calculated.
Results

Description about the sample use

Sample Descriptions
Brand - Fiber Castell (CX7)
Type - Roller pen
1 Size - 0.7 Fine
Color Super smooth blue
Made in Malaysia
Brand - Fiber Castell (CX5)
Type - Roller pen
2 Size - 0.5 S-Fine
Color Super smooth blue
Made in Malaysia
Brand Pilot Rexgrip
Type Roller pen
3 Size - 0.7
Color Blue
Made in Japan
Brand Pilot A21
Type - Roller pen
4 Size - 0.7
Color Blue
Made in Japan
Dissolving in ethanol

Sample ink 1 Sample ink 2 Sample ink 3 Sample ink 4

TLC plate and Rf value

Sample RF value

Compound (cm)
1 1 2.5
2 2.8
3 3.25
4 3.55
5 4.5
2 1 2.6
2 2.8
3 3.3
4 3.5
5 4.5
3 1 2.6
2 2.8
3 3.3
4 3.5
5 4.3
4 1 2.6
2 2.9
3 3.3
4 3.6
5 4.3
Under compound microscope

Sample Picture Description

1 Smooth
signature
Uneven surface
Have striation
The first layer
of the ink not
been
transferred well
2 Very smooth
signature
Have striation
The ink was
transferred very
well along the
signature

3 Not smooth
signature
Uneven surface
Have striation
The ink not fully
transferred on
several part of
signature
4 Not smooth
signature
Uneven surface
Have striation
The ink not
transferred well
on the whole
signature
Under VLC

Type of Examination Result Descriptions

Fluorescence
Ink sample 1 and ink
sample 2 show about
90% fluorescence.
Ink sample 2 and ink
sample 3 does not
fluorescence.

UV (254 nm)
All of the ink sample
not effective in UV.
UV (312 nm)
All of the ink sample
not effective in UV.

VIS (530 nm)

All of the ink sample
can see through this
wavelength.

VIS (668 nm)

Ink sample 1 and 2
were very blurred
compared to ink
sample 3 and 4 under
this wavelength.
VIS (714 nm)
Ink sample 1 and 2
only can saw about 10
% through this
wavelength.

VIS (725 nm)

Ink sample 1 and 2
only can saw about 5 %
through this
wavelength.

VIS (778 nm and

above) Ink sample 1 and 2
were totally
disappeared through
this wavelength.

Discussion
In this experiment, rolling ball marker ink was used based on 4 brands of
pen. These inks were water based and usually contain organic liquids such
as glycols and formamide to retard the drying of the ball point. The dyes in
these inks were water soluble or acidic dye salts. The light fastness of
these dyes range from good for the metal-ized acid dyes to poor for some
of the basic dye salts. Water fastness was usually poor, except that some
of these dyes have an affinity for cellulose fibers in paper which produces
a degree of water fastness. Water-resistant rolling ball marker inks were
also available. These inks were totally insoluble in water and can only be
dissolved in strong organic solvents, such as pyridine or dimethylsulfoxide
(DMSO).

All of the 1 cm square paper that colored with 4 types of blue ink samples
was dissolved in ethanol and turns to blue solution. This is because all of
the inks were dissolving into the liquid. The solutions were used for the
Thin-layer chromatography (TLC) examination. As the result, all of the ink
samples were separated into 5 compounds. Since ink sample 1 and 2 from
the same brands of pen which Faber Castell, the R f value were quite same.
There also slightly no different for R f value of ink sample 2 and 3 because
the ink came from Pilot brands.

In fluorescence examination, ink sample 1 and 2 showed a positive result

compared to another 2 samples. The effectively of fluorescence of ink
sample 1 and 2 was based on the same volume of the signatures
fluoresce. Both 254 nm and 312 nm wavelength of ultraviolet were not
effective to examine the signature from all of ink samples. However, the
signatures were effective in different filter of wavelength in visible. In fact,
the wavelength for visible examination was about 380 nm until 780 nm. In
this experiment, the filter of wavelength was started at 530 nm and all of
the signatures still can see through this wavelength. At 668 nm, ink
sample 2 and 3 became very blurred and only can see about 5% at 725
nm. When the filter of wavelength was change at 778 nm and above, they
were totally disappeared.
Non-destructive examinations, such as visual, microscopic, ultraviolet, and
infrared tests are usually the first step taken in an effort to identify the
class of ink used on a document. Further destructive testing may also be
necessary to discriminate among ink types. Liquid chromatography may
be conducted in order to determine the chemical composition of inks used
in a document. As one of the few destructive techniques employed by
forensic investigators, a small piece of the document containing the ink is
cut away, dissolved in solvent and analyzed. The inks composition can
then be compared to the International Ink Library, a database of more than
9,500 inks that is maintained by the U.S. Secret Service.

Besides thin layer chromatography (TLC), both gas chromatography (GC)

and liquid chromatography (LC) have been successfully employed in the
analysis and comparison of inks. Their advantage over TLC was that they
yield quantitative as well as qualitative data, and they were both generally
more sensitive than TLC, which means that they required fewer plugs of
ink. In addition, gas chromatography-mass spectrometry (GC-MS) was
often used. This methods allowed the identification of each of the
components in the ink. A newer technique in chromatography, capillary
electrophoresis, has also been applied to ink analysis, although the
research has been very limited thus far. Capillary electrophoresis was
similar to HPLC but it used tiny columns for separation, so that as little as
one or two plugs may be used for analysis.

Infrared spectrophotometer also can be used for characterizing inks. This

technique shows absorption peaks for all of the components of the ink at
one time, including the solvent, dyes and additives. Because of this, IR can
be very helpful in comparing two ink samples to see whether they could
have originated from the same source. One of the disadvantages of IR is
that it requires more sample than do chromatographic methods in general.
It is difficult to use micro plugs for sampling in IR. Various sampling
methods have been tried, including using a microscope to focus on micro
plugs, with mixed success.
Other than that, mass spectrometry, with and without gas
chromatography, has been used for the analysis of inks. One newer type of
MS, called laser desorption, uses a laser to remove ink from the surface
of paper and analyze it. An advantage of this type of mass spectrometry is
that the ink does not have to be removed from the paper first. A piece of
paper with ink writing on it can be directly introduced into the instrument
and it is also essentially non-destructive. Laser desorption can be used to
track an ink dye as it ages. When dyes age, they undergo chemical
degradation. One popular ballpoint pen ink dye, methyl violet, degrades by
losing CH3 groups, replacing them with hydrogen atoms. This results in the
loss of fourteen mass units from the molecule.

Chemical tests are important in the examination of printing inks because

they also allow examiners to provide an approximation of when the
document was written. This may be particularly salient in the case of
forgeries; for example, it may be discovered that the ink on a document is
much too new for its purported age.

Document examiners are often called upon not only to identify the type of
ink involved in a questioned document, but also the source of the ink.
Determining the origin of the ink on a document may be especially useful
in helping examiners to detect fraudulent entries or alterations in a
document. For example, a fraudulent entry in a will may be added with ink
that is visually similar but chemically different from that of the rest of the
document, indicating that it was not included at the time of its original
writing. While differentiation among inks can be accomplished by visual
examination, nondestructive, and chemical tests, positive identification of
the specific source of the ink is unlikely. More probably, the examiner will
be able to trace the ink back to the specific type, class, and or brand of
ink, and suggest that it is scientifically indistinguishable from the
suspected source. Infrared or ultraviolet examination also may allow the
examiner to differentiate between different types of ink.

TLC is very simple to use and inexpensive. There are little materials
needed for TLC which are chamber, watch glass, capillary, plate, solvent,
pencil, and UV-light. Therefore, once the best solvent is found, it can be
applied to other techniques such as High performance liquid
chromatography. More than 1 compound can be separated on a TLC plate
as long as the mobile phase is preferred for each compound. The solvents
for the TLC plate can be changed easily and it is possible to use several
different solvents depending on the desired results. As stated earlier, TLC
can be used to ensure purity of a compound. It is very easy to check the
purity using a UV-light. Identification of most compounds can be done
simply by checking Rf literature values. The conditions of chromatography
can be modify easily to increase the optimization for resolution of a
specific component.

However, TLC plates do not have long stationary phases. Therefore, the
length of separation is limited compared to other chromatographic
techniques. Also, the detection limit is a lot higher. Other chromatographic
techniques would have to use if a lower detection limit id needed. TLC
operates as an open system, so factors such as humidity and temperature
can be consequences to the results of chromatogram.

Conclusion

In conclusion, the ink sample from Faber Castell (sample 1 and 2) and ink
sample from Pilot (sample 3 and 4) were separated into 5 compound using
TLC. Ink sample 1 and 2 were effective in fluorescence compared to ink
sample 3 and 4. In this experiment, the UV was not effective for all of the
ink samples and only effective in different filter of wavelength for VIS
examination.

References

1. Lectures notes FRS 581 (Ink Analysis), Madam Khairulmazidah

Mohamed.
2. Saferstain.R (2015). Criminalistics-An Introduction to Forensic
Science. Scientific Examination of Documents.(11th ed., pp.467-470).