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 inexpensive, simple to perform and one of the most common
chromatographic methods in the forensic laboratory. This methods stationary
phase is a thin layer of gel adsorbent coated onto plastic/metal backing, called a
TLC plate. As the liquid mobile phase is drawn into the 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.

Materials
Pencil

95% ethanol / pyridine

Metric ruler

Ethyl acetate

Precoated silica chromatography

plate

Distilled water

Several ball-point pens, sampled on

white paper

FRS 581 FORENSIC CHEMISTRY

Capillary tube
Developing chamber

Procedure
To prepare the mobile phase:
Using a 25mL graduated cylinder, add:
7.5mL ethyl acetate, 3.5mL 95% ethanol and 3.0mL distilled water to a 100mL
screw cap reagent storage bottle. Affix the cap tightly and invert three times to
mix. Store at room temperature.
1. Add mobile phase to the chamber to attain a height of 5 mm.
2. Saturate the tank with mobile phase vapors by adding a piece of blotter or
filter paper to the chamber. Cover with lid and stand at least 15 minutes.
3. Examine the forged document do any of the exemplars resemble it either in
color of ink or handwriting? Make note of your preliminary observations.
4. Clip a 0.5cm square from the text of each document and transfer each to a
well of the spot plate.
5. Add two drops 95% EtOH / pyridine to each well and gently swirl spot plate to
agitate. You should observe the solvent turning blue or purple, indicating that
the ink is dissolving into the liquid.
6. Mark a pencil line 1cm from the bottom of a short edge of the TLC plate.
7. Use a clean capillary tube for each sample to spot along the first pencil line.
8. Be sure to note the location of each sample in your notebook.
9. Carefully and quickly position the TLC plate in the chamber and cover.
10.Check the progress of the solvent front every 2-3 minutes.
11.Once the solvent front reach about of the TLC plate length, remove the
plate from the chamber and mark the solvent front with your pencil.
12.Allow it to dry completely.
13.Draw a diagram of the plate, including each band/spot. You might want to
photograph the plate to include in your report.
14.Mark the center of each band.
15.Calculate the Rf value for each band by dividing the distance travelled by the
band by the distance travelled by the solvent.
16.Each ink should yield a unique profile by comparing the number the number
of bands and their corresponding Rf values.
17.Compare your TLC results between questioned document and exemplars.

Questions
1. Apart from TLC, what other methods can be used to differentiate ink?
2. How does ink analysis helps in forensic investigation?
3. What are the advantages and disadvantages of using TLC in ink analysis?

Experiment 6: Paint Chip Analysis

Objective
To morphologically and spectroscopic differentiate paint chips layers from
different cars

Materials
FTIR Spectroscopy, Stereomicroscope, plasticine, paint chip samples.

Procedure
Part A
1. Mount the paint chip samples on the plasticine sideways.
2. View the layers of the paint chip using stereomicroscope.
3. Note the wideness or thinness of each layer, colour and the overall number
of layers for each paint chip sample.
4. Draw each of the paint chip samples provided.
Part B
1. Paint chips are mounted on FTIR microscope for mapping.
2. ATR mapping measurements were performed using a 20 x ATR-objective in
combination with a motorized sample stage.
3. Measurement positions and sampling areas were set specifically for each
layer.
4. An acquisition time of 14 sec at a spectral resolution of 4cm -1 was used.
5. During the infrared measurement, the sample is brought into contact with
the tip of the Germanium (Ge) crystal (100m in diameter) of the ATRobjective on all predefined sampling positions.

6. The effective field of view at all sampling positions is adjusted automatically

by a motorized aperture.
The example of paint chips analyzed using ATR FTIR mapping is shown in figure
1b.

Figure 1b Overview image A shows measurement positions with individual aperture

size. B shows corresponding spectra of 6 layers.

Questions
1. What is the advantage of using mapping in FTIR spectroscopy?
2. What does each layer in the paint chips represent?
3. What are other techniques for analysis of paint chip in forensic
investigation?
4. What are the choices of analysis that you will you do if the paint is in the
form of smear?

Experiment 7: Serial Number Restoration

Objective
To demonstrate the obliteration done on chasis/engine number and restore the
original number

Material
Steel plate, Metal stamp, Hammer, Sand paper, Frys reagent, Cotton bud,
Alcohol/acetone

Procedure
1. Each student takes a piece of steel plate.
2. A set of 6 combinations of numbers and alphabets were stamped onto the
steel plate using the metal stamp.
3. These set of numbers and alphabets are then obliterated using sand papers.
4. A new set of 6 combinations of numbers and alphabets are then stamped
onto the same steel plate.
5. Wipe the plate with acetone using the cotton bud.
6. Take a photograph of the plate before the etching process.
7. Moisten cotton tip applicators (swabs) with the Frys reagent solution and
apply to the obliterated area.
8. After a few seconds, wipe off the solution and inspect for visible numbers.
Repeat as necessary. This process may take several hours.
9. Alternate between swabs saturated with the Frys Reagent and the 25% Nitric
Acid solution.
10.To speed up the process, the examiner may wish to use an electrochemical
process. The positive (+) terminal of a standard lantern battery or other
source is connected to the obliterated item, and the negative terminal (-) is
connected to the cotton swab.
11.If any characters become visible note these characters.
Questions
1. What are other reagents and their respective suitable surface for etching
process?
2. Explain the principle of serial number restoration.
3. What are the forensic cases that require etching to be done in such cases?