FACULTY OF APPLIED SCIENCES

FORENSIC CHEMISTRY (FRS581)

LABORATORY REPORT

EXPERIMENT 5: THIN-LAYER CHROMATOGRAPHY OF

INK

NAME: AHMAD ZAKWAN BIN KASSIM

STUDENT ID: 2021886994
PROGRAMME CODE: AS253
GROUP: AS2535A2
DATE OF EXPERIMENT: 05TH NOVEMBER 2023
DATE OF SUBMISSION: 20TH NOVEMBER 2023
LECTURER’S NAME: MADAM KHAIRULMAZIDAH BINTI
MOHAMED
 INTRODUCTION
These days, ballpoint, fountain pen, and carbon ink are the three most popular varieties.
Ballpoint and fountain pen inks are like gel ink pens in certain aspects. They are often employed
because of their excellent writing. An extensive range of diverse compositions are employed
to create the inks used in gel pens and other writing instruments and printing techniques. These
days, a vast range of synthetic materials, including surfactants, organic and inorganic colours,
resins, and more, can be found in formulations. The main ingredient in ink is dye, which comes
in acidic and alkaline varieties. Sulfonates, chlorosulfonates, and sulfonamides are examples
of common dyes. To extend the life of these inks, extremely thin pigment dispersions of carbon
and cuphthalocyanine are also added. Most ink formulas are kept under wraps. Although
appearance might be a determining element, it is not the sole one. There might be additional
techniques to distinguish between two inks even if they appear identical. Testing is done
without inflicting any damage if possible.

In separation chemistry, one of the most basic methods is thin-layer chromatography

(TLC). It is among the most widely used chromatographic techniques in forensic laboratories
and is both costly and easy to execute. The stationary used in this procedure is a TLC plate,
which is a thin layer of gel adsorbent placed onto a plastic or metal backing. The liquid mobile
phase is absorbed into the gel and is propelled toward the top of the plate by capillary action.
The samples are divided into their component compounds along the way. TLC is used to
compare and identify pharmaceuticals, explosives, dyes, and inks. The sole restriction on the
procedure is that the samples must be soluble. This is a specific problem when looking at pen
inks because of the introduction of gel pens, whose ink is soluble. Gel pen ink cannot be
examined using TLC. Gel pen inks have been proven to be challenging to examine using
standard forensic methods.

MATERIALS AND METHODS

In this experiment, pencil, metric ruler, precoated silica chromatography plate, several
ball-point pens, sampled on white paper, 95% ethanol / pyridine, ethyl acetate, distilled water,
capillary tub and developing chamber were used. 7.5mL ethyl acetate, 3.5mL 95% ethanol and
3.0mL distilled water were added using a 25mL graduated cylinder to a 100mL screw cap
reagent storage bottle to prepare the mobile phase. The cap was affixed tightly and inverted
three times to mix. Stored at room temperature. Mobile phase was added to the chamber to
attain a height of 5 mm. The tank was saturated with mobile phase vapours by adding a piece
of blotter or filter paper to the chamber. Covered with lid and stood at least 15 minutes. The
forged document was examined – do any of the exemplars resemble it either in colour of ink
or handwriting? Preliminary observations were noted. A 0.5cm square from the text of each
document were clipped and each were transferred to a well of the spot plate. Two drops 95%
EtOH / pyridine was added to each well and spot plate was gently swirled to agitate. The solvent
turning blue, or purple was observed, indicating that the ink is dissolving into the liquid. A
pencil line was marked 1cm from the bottom of a short edge of the TLC plate. A clean capillary
tube was used for each sample to spot along the first pencil line. The location of each sample
was sure to note in notebook. Position the TLC plate in the chamber Carefully and quickly and
covered. The progress of the solvent front was checked every 2-3 minutes. The plate was
removed from the chamber and the solvent front was marked with your pencil once the solvent
front reach about ¾ of the TLC plate length. It was allowed to dry completely. A diagram of
the plate was drawn including each band/spot. The plate was photographed to include in your
report. The centre of each band was marked. The Rf value for each band were calculated by
dividing the distance travelled by the band by the distance travelled by the solvent. By
comparing the number of bands and their corresponding Rf values each ink yielded a unique
profile. TLC results were compared between questioned document and exemplars.
 RESULTS
SAMPLE OF PENS SAMPLE OF SIGNATURE DESCRIPTION
SAMPLE 1
BRAND: FABER-
CASTELL
TIP OF PEN: 1.0MM
TYPE OF PEN:
BALLPOINT PEN
COLOUR: BLUE

SAMPLE 2

BRAND: FABER-
CASTELL
TIP OF PEN: 1.0MM
TYPE OF PEN:
BALLPOINT PEN
COLOUR: RED
SAMPLE 3 & 4

BRAND: FABER-
CASTELL
TIP OF PEN: 0.7MM
TYPE OF PEN:
BALLPOINT PEN
COLOUR: BLACK
SAMPLE 5

BRAND: STAEDTLER
TIP OF PEN: 0.7MM
TYPE OF PEN:
BALLPOINT PEN
COLOUR: BLACK
SAMPLE 6

BRAND: SHINE
TIP OF PEN: 0.7MM
TYPE OF PEN:
BALLPOINT PEN
COLOUR: BLACK
Thin layer chromatography plate after being removed from mobile phase

Thin layer chromatography plate under UV light

Calculation for Rf value
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝐶𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡
𝑅𝑓 𝑉𝑎𝑙𝑢𝑒 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑆𝑜𝑙𝑣𝑒𝑛𝑡 𝐹𝑟𝑜𝑛𝑡

SAMPLE 1 (SPOT 1):

1.2
𝑅𝑓 𝑉𝑎𝑙𝑢𝑒 = = 0.39
3.1

SAMPLE RF VALUE (CM)

SAME BRAND DIFFERENT COLOUR
1 SPOT 1 0.39
SPOT 2 0.65
2 (ONLY HAVE 1 SPOT) 0.74
3 SPOT 1 0.65
SPOT 2 0.87
DIFFERENT BRAND SAME COLOUR
4 SPOT 1 0.84
SPOT 2 0.91
5 SPOT 1 0.63
SPOT 2 0.88
6 SPOT 1 0.66
SPOT 2 0.88

DISCUSSION
Five different inks were utilized in this experiment: the blue, red, and black Faber-
Castell Ballpoint Pen, the black Staedtler Ballpoint Pen, and the black Shine Ballpoint Pen.
Three black inks that was different brand, a blue, red, and black ink that was the same brand
were utilized in this experiment. Only dissolved solutions were employed for the thin-layer
chromatography (TLC) plate inspection. Sample 1: Blue ballpoint ink can be divided into two
compounds; sample 2: Red ballpoint ink can only be divided into one compound; sample 3,4,5,
and 6: Black ballpoint ink can be divided into two compounds. Since the band's distance
travelled reached different level for same brand different colour; 1.2cm (sample 1), 2.3cm
(sample 2) and 2cm (sample 3) from the 1cm line at the bottom of the short edge of the TLC
plate), hence the Rf value of the ink is different for samples 1, 2 and 3 which were 0.39, 0.74
and 0.65 respectively. The second spot for sample 1 and 3 went slightly higher (2cm and 2.7cm
respectively from the bottom) than the first, and the Rf value is 0.65 and 0.87 respectively.
Sample 4, 5 and 6 (different brand same colour) has slightly the same distance travelled which
is 2.7cm, 2cm and 2.1cm respectively from the 1cm line at the bottom of the short edge of the
TLC plate, hence the Rf value has slightly same which were 0.84, 0.63 and 0.66 respectively.
It was same for second spot which have slightly same Rf value as shown in the table above.
Additionally, sample 3 and 4 supposedly have same or slightly same Rf value since both are
the same pen. Error may occur during TLC procedure which lead to a big different between Rf
value for sample 3 and 4.

QUESTIONS
1. Apart from TLC, what other methods can be used to differentiate ink?

Gas chromatography (GC) and liquid chromatography (LC), in addition to thin layer
chromatography (TLC), have been effectively employed in the analysis and comparison
of inks. They have two advantages over TLC: they produce data that is both quantitative
and qualitative, and they are both more sensitive than TLC, so that fewer ink plugs are
needed. Furthermore, a lot of gas chromatography-mass spectrometry (GC-MS) was
used. This process made it possible to identify every component of the ink. An infrared
spectrophotometer can also be used to characterize inks. With this method, the
absorption peaks of the solvent, colours, and additives are all shown at the same time
for all the components of the ink. As a result, when comparing two ink samples, IR is
a helpful technique for figuring out whether they could have come from the same
source. The fact that IR requires more sample than traditional chromatographic
techniques is one of its drawbacks.

2. How does ink analysis helps in forensic investigation?

One essential forensic method that might provide important details about a document
under investigation is ink analysis. Modern inks are enhanced with numerous
components to give them better qualities. The colouring substance, which might take
the form of dyes, pigments, or a combination of the two, is unquestionably the most
important element. The liquid body of the ink, sometimes referred to as the vehicle, is
soluble in dye molecules. Pigments, on the other hand, are made of insoluble multi-
molecular granules that have been finely ground and mixed with a medium. Document
examiners are often entrusted with determining the source of the ink as well as the type
of ink found on a contested document. Identifying the source of the ink on a document
can help examiners find forged entries or modifications. To demonstrate that it was not
included at the time the document was first written, a fraudulent entry in a will, for
example, could be added using ink that is chemically different from the rest of the
document but looks the same. While inks can be distinguished from one another by
visual inspection, non-destructive testing, and chemical analysis, it is unlikely that the
ink's source can be positively recognized. It is highly probable that the examiner will
be able to identify the ink precisely in terms of its type, class, and/or brand and conclude
that there is no scientific way to differentiate it from the suspected source.
 3. What are the advantages and disadvantages of using TLC in ink analysis?

TLC is very inexpensive and easy to use. The few supplies needed for TLC are a
chamber, watch glass, capillary, plate, solvent, pencil, and UV lamp. The ideal solvent
can then be used with other methods, like high performance liquid chromatography
once it has been determined. Many compounds can be separated on a TLC plate if the
preferred mobile phase for each molecule is used. The TLC plate's solvents may be
readily switched out, and different solvents can be used depending on the desired
outcomes. As mentioned above, TLC can be used to guarantee a compound's purity. It's
easy to figure out the purity with UV light. The bulk of chemicals may be identified by
checking Rf literature values. It is easy to alter the chromatographic settings to improve
the optimization for resolution of a particular component. However, TLC plates don't
have very long stationary times. As a result, compared to other chromatographic
procedures, the separation time is lower. Furthermore, there is a noticeable increase in
the detection threshold. It would be necessary to use different chromatographic
techniques if a lower detection limit was required. Because TLC is an open system,
temperature and humidity may have an impact on chromatogram findings.

CONCLUSION
The experiment's findings, taken together, demonstrate that the distinctive
characteristics of handwriting on counterfeit documents and the ink analysis using the Thin-
Layer Chromatography technique were studied. Thin-Layer Chromatography can be used to
analyse ink samples, and the brand of pen used can be determined from the Rf values that are
obtained. UV light was used to the 6 samples. Sample 2 only created one distinct ink band on
the TLC plate, whereas sample 1, 3, 4, 5 and 6 produced two. Samples 4, 5 and 6 have slightly
same Rf values which were 0.84, 0.63 and 0.66 respectively for their starting positions,
however Sample 1, 2 and 3’s Rf value were different which were 0.39, 0.74 and 0.65
respectively for their first position is 0.850. Both same brand different colour and different
brand same colour gave different results of Rf values.
 REFERENCES
1. Flournoy, B. (2020, January 21). What is ballpoint pen ink made of? Sciencing.
Retrieved December 30, 2022, from https://sciencing.com/ballpoint-pen-ink-
made-of-10036405.html

2. History of gel pens. Gel Pen – Facts and History of Gel Pens. (n.d.). Retrieved
December 30, 2022, from http://www.historyofpencils.com/writing-
instruments-history/gel-penhistory/

3. Flournoy, B. (2020, January 21). What is ballpoint pen ink made of? Sciencing.
Retrieved December 30, 2022, from https://sciencing.com/ballpoint-pen-ink-
made-of-10036405.html

4. Judge, M. (2019, March 2). Factors that affect RF values in thin layer chromatography.
Sciencing. Retrieved December 30, 2022, from https://sciencing.com/factors-
valuesthin-layer-chromatography-8561359.html

5. Instruments used in ink analysis - forensic articles. Forensic Articles - Observe a Little
More © - Archana Singh. (2022, December 3). Retrieved December 30, 2022,
from https://forensicarticles.com/instruments-used-in-ink-analysis/