LAB MANUAL

FRS 581
FORENSIC CHEMISTRY

AS 253 BACHELOR OF SCIENCE

CHEMISTRY (FORENSIC ANALYSIS)

FRS 581 FORENSIC CHEMISTRY 1

Table of Contents

Experiment 1: Forensic Fiber Analysis Page 3

Experiment 2: Forensic Explosive Analysis Page 8
Experiment 3: Soil Profile Examination Page 11
Experiment 4: TLC of Steroids in Herbal Medicine Page 14
Experiment 5: Thin-Layer Chromatography of Ink Page 16
Experiment 6: Paint Chip Analysis Page 18
Experiment 7: Serial Number Restoration Page 20

FRS 581 FORENSIC CHEMISTRY 2

Experiment 1: Forensic Fiber Analysis

Objective
To analyze and experience fiber analysis methods in forensic investigations.

Material:
1. compound light microscope
2. prepared wet mounted of self fibers
3. candle
4. one-hole rubber stopper
5. fibre samples
6. forceps

Procedure:
Part A
1. Study the known fiber samples under 50x and 100x low power microscopes bright
field and fluorescence field. Do not use coarse adjustment when using the 10x
objective.
2. Note any pits or striations on the fibers.
3. Determine the visual differences between the manmade (polyester) fibers and
natural (cotton) fibers. Note the color of each as part of this investigation.
4. Study the unknown fiber samples under both objectives. Note any pits or striations
on the fibers.
5. Match the known samples to the unknown samples based on these comparisons.

Part B
1. Place the candle in the hole of the stopper and light it.
2. Note the burning characteristics of each of the known fibers as well as the
unknown fiber. Record your observations in the data table.
a. Holding the fiber in the forceps, bring it close to, but not touching the
flame. Describe the fiber’s behavior as it approaches the flame. Does it
ignite, melt or curl?
b. Holding the fiber in the forceps, touch the fiber to the flame. Does it ignite
quickly or slowly? Does it sputter, drip or melt?

FRS 581 FORENSIC CHEMISTRY 3

c. Remove the fiber from the flame and describe how it behaves. Does it
self-extinguish, continue to burn or glow?
d. Note any odor associated with the fiber in a flame. Does it smell like
vinegar or hair?
e. What kind of residue is left after the fiber is removed from the flame?
Does the fiber leave a white, fluffy ash, a hard bead or a melted blob?

Data Table Part A

Examination of Known Fibers Under a Microscope
Visual Description under bright Sketch
Known Fibers
field and fluorencence field

Data Table Part B

Behavior of Fibers in a Flame
Type of Fiber Unknown

Approaching
Flame
In the Flame

Removed from
the Flame

Odor

Residue

Part C
Objective
To examine tears and damages in the clothes and to relate them with the type of
weapon that has caused them.

FRS 581 FORENSIC CHEMISTRY 4

Materials
Stereomicroscope, biological microscope, glass slides, a pair of scissors, chopper
knife, razor blade, a ough granite stone, a nail, a wodden block, forceps, tweezers
and a needle.

Principle
Tears are caused in a woven fabic by the snapping of the warp and weft fibes in the
yarns. These tears acquire significance since the nature of the severance of the
fibers and their subsequent change in the orientation can provide the cue for
diagnosing the type of the weapon that has caused the tears.

a) Procedure for examining tear caused by a chopper knife

Stretch a piece of cloth onto a wooden block and chop with a chopper knife.

b) Procedure for examining tear caused by a single edged knife

Stretch a piece of cloth on a sand bag or a sand filled container and create a tear
by stabbing with a knife.

c) Procedure for examining tear caused by a double edged knife

Stretch a piece of cloth on a sand bag or a sand filled container and create a tear
by stabbing with a knife having sharp edges on both sides.

d) Procedure for examining tear caused by a sharp nail

Place a piece of cloth on the tip of a sharp nail projecting from a wooden plank
and stretch the cloth until a tear is created.

e) Procedure for examining tear caused by a razor blade

Stretch a piece of cloth leaving its lower side unsupported. Make a slashing
movement with a razor blade and create a tear.

f) Procedure for examining tears caused due to forcible contact with a rough
object- a stone.
Stretch a piece of cloth onto a smooth wooden plank and smash the cloth using a
ough granite stone until a ragged a tattered tear is produced.

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g) Procedure for examining tears caused due to overstretching of cloth
The two ends of a sufficiently long piece of cloth is held by two individuals who
would forcibly stretch the cloth until a tear is produced.

FORENSIC SIGNIFICANCE OF TEARS AND DAMAGES IN CLOTHES

Analysis of tear and damage in the clothes can acquire significance during
reconstruction by offering clue for diagnosing the type of weapon and the manner
of the assault. Logically such a study would be possible only when these
examinations are initiated in the crime scene itself.

Questions
Answer the following based on your observations:
1. Describe the characteristic(s) made the the known fibers under both fields.
2. From the burning tests, which type of fiber is most similar to the unknown fiber?
Describe the characteristics they had most in common.
3. Why might an investigator want to identify unknown fibers from a crime scene?
4. What must scientists be able to do in order for fiber evidence to be useful in a
crime scene investigation?
5. From where do we get the materials to make natural fibers?
6. How are manmade fibers most different from natural fibers?
7. Why is it important to identify cuts and damages in fabrics?

FRS 581 FORENSIC CHEMISTRY 6

 FIBER FLAME ODOR RESIDUE

does not shrink away, burns similar to light, feathery,

COTTON quickly and has glowing burning light to charcoal
natural cellulosic ember after burning. paper. gray in color.

does not shrink away, burns similar to light, feathery,

LINEN quickly, glowing ember after burning light charcoal
natural cellulosic
fire extinguished. paper. gray in color.

WOOL curls away from flame, burns similar to brittle, small black
natural protein slowly, self-extinguishing. burning hair. bead.

curls away from flame, burns

SILK slowly and sputters, self-
similar to bead-like,
natural protein burning hair. crushable black.
extinguishing.

RAYON does not shrink away, burns similar to light, fluffy

manufactured quickly, continues to burn burning residue, very small
cellulosic with glowing ember. paper. amount.

ACETATE fuses and melts away from

similar to hot irregular shaped,
manufactured flame, burns quickly and
vinegar. hard, black bead.
cellulosic continues to burn.
melts, ignites readily, burns
ACRYLIC rapidly and sputters while similar to hot hard, black,
manufactured burning, will drip while vinegar. irregular bead.
burning.
melts away from flame and
NYLON burns slowly, fuses and melts,
similar to hard, tough, gray
manufactured celery. or tan bead.
self-extinguishing.
melts away from flame and hard, tough,
POLYESTER burns slowly, fuses and melts,
chemical
black or brown
manufactured odor.
self-extinguishing. bead.

SPANDEX fuses but does not melt away chemical soft, sticky, and
manufactured from flame. odor. gummy.

FRS 581 FORENSIC CHEMISTRY 7

Experiment 2: Forensic Explosive Analysis
Part A
Sample:
Several types of firecrackers, low and high explosives samples.
Method:
Griess test
1. Mix equal volume of 1x Griess Reagent and samples (working range: 0.43-65
µmolar nitrite).
2. Observe the color changes.

Diphenylamine test
1. Prepare diphenylamine solution by adding 10ml sulphuric acid and 2 ml
distilled water with constant stirring. After few minutes, add 0.05g
diphenylamine powder into the mixture.
2. Dissolve10 mg of solid explosive sample in 10 ml of the sulfuric acid solution,
and add 10 µl of the resultant solution to 1 ml of diphenylamine reagent.
3. Maintain the test tube containing the mixture in a boiling water bath
(approximately 98° C) for approximately 1 hr. Observe the color changes.

Alcoholic KOH
1. Prepare alcoholic KOH by mixing 10g KOH with 100ml absolute alcohol.
2. Add sample into the alcoholic solution in equal volume.
3. Observe the color changes.

Colour changes
Substance Griess Diphenylamine Alcoholic KOH
Chlorate No color Blue No colour
Nitrate Pink to red Blue No colour
Nitrocellulose Pink Blue-black No colour
Nitroglycerin Pink to red Blue No colour
PETN Pink to red Blue No colour
RDX Pink to red Blue No colour
TNT No colour No colour Red
Tetryl Pink to red Blue Red-violet

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Part B

Objective:
To understand some of the main materials used in detonation and how to analyse it.

Materials:
Fireworks/Firecrackers, iron (II) sulphate, concentrated sulphuric acid, 4M sulphuric
acid, 0.1 M iron (II) sulphate, litmus paper, test tubes.

Principle:
An explosive is defined as a substance or mixture of substances that is capable of
producing an explosion by itself, without the need for an outside source of oxygen.
An explosion is a rapid oxidation reaction that liberates a large quantity of energy
and is accompanied by the evolution of a large volume of hot gases and a loud
noise.

Black powder or also called gunpowder is normally a mixture of potassium nitrate,

carbon and sulphur in the ratio 75:15:10. The fuel in black powder is charcoal and
sulphur, while the oxidizer is potassium nitrate (KNO3).

Black powder will simply burn rather than explode unless packed into an
appropriate casing, such as the thick paper shell of a firecracker.

Method:
Brown Ring Test
1. Add iron (II) sulphate to the solution.
2. Slowly add concentrated sulphuric acid such that the sulphuric acid forms a
layer above the aqueous solution.
3. A brown ring will form at the junction of the two layers, indicating the
presence of the nitrate ion

*Note that the presence of nitrite ions will interfere with this test.

The overall reaction is the reduction of the nitrate ion by iron (II) which is oxidised to
iron (III) and formation of a nitrosyl complex.

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 NO3- + 3Fe2+ + 4H+ → 3Fe3+ + NO + 2H2O
[Fe (H2O)6]2+ + NO+ → [Fe(H2O)5(NO)]2+

Nitrite Test
1. Add 4 M sulphuric acid to the sample until acidic.
2. Add 0.1 M iron (II) sulphate to the solution.
3. A positive test for nitrite is indicated by a dark brown solution, arising from the
iron-nitric oxide complex ion.

Test for Sulphur

1. Examine the powder provided under the microscope.
2. Sulphur incorporated in the black powder can be identified by its yellow
colour.

Forensic Significance
Black powder burns rapidly, producing a volume of hot gas made up of carbon
dioxide, water, and nitrogen, and a solid residue of potassium sulphide. Because of
its burning properties and the amount of heat and gas volume that it generates,
black powder has been widely used as a propellant in firearms and as a
pyrotechnic composition in fireworks.

Questions
1. Define explosives and describe the types of explosives and its use.
2. Discuss the commonly used explosives in the military.
3. Black powder is also previously known as.....?
4. What are the materials incorporated in black powder and in what ratio?
5. Why explosive investigation is important in forensic cases?

FRS 581 FORENSIC CHEMISTRY 10

Experiment 3: Soil Profile Examination

Objective:
Study soil characteristics and match samples taken from a suspect to samples found
at a crime scene.

Materials:
(to share by teams of four)
1. 5 hand lenses or compound microscopes or stereomicroscope with 40x
magnification
2. 5 graduated cylinders, 250 mL
3. 5 soil samples, four from suspects and a crime scene sample
4. 5 rubber bands
5. 5 beakers, 250 mL
6. 5 pieces of cheese cloth approximately 8" x 8"
7. 5 teaspoons
8. 5 pieces universal range pH paper
9. 5 watch glasses
10. 5 ultraviolet lights
11. 5 flat toothpicks

Safety Precautions:
Proper eye protection is needed for the UV light.

Background:
A robbery has occurred, and four suspects have been questioned. Soil samples were
taken from the wheel wells of each of the suspects’ vehicles. Do any of the soil
samples match soil from the crime scene?

Procedure:
Part A: Microscopic Examination
Obtain four soil samples (1 to 4) and a crime scene sample.
1. Examine a dry soil sample 1 (from Suspect 1) using a hand lens orlow power
(40x ) on amicroscope. Because soil changes somewhat in appearance as it
dries out, make sure each sample is dry before beginning your examination.
2. Record all information requested on Table 1.
3. Describe or sketch any organisms found in the soil.
4. Describe the color, texture, odor, and overall appearance of the soil.

Overall
Describe or Odor of
Color of Sample appearance of Appearance
sketch any sample
dry sample texture sample under
Soil organisms or (smells
(black, (crumbly, (sandlike, UV light
Sample foreign like ?
brown, gritty, organic, (glows, no
objects or no
gray, etc.) loose, sticky) rocks, glow)
found odor)
minerals)
1

FRS 581 FORENSIC CHEMISTRY 11

3

Crime
Scene

Part B: UV Examination
5. View the sample in a darkened room using a UV light and describe what you
see.
6. Repeat the procedure and examine each of the other suspect samples and
the crime scene sample.

Part C: Determination of pH of the Soil Sample

The pH value is a measure of whether something is acidic, neutral, orbasic.
7. Place a piece of cheesecloth on a clean surface. Place two spoonfuls of soil
from Sample 1 in the center of the cheesecloth. Gather the sides of the
cheesecloth and place a rubber band around the cheesecloth, capturing
the soil sample in a ball within the cloth.
8. Place 50 mL of distilled water in a 250-mL beaker and label it Suspect 1.
9. Place the ball of soil in the water and leave it undisturbed for 10minutes.
10. Repeat steps 7 to 9 for suspect samples 2, 3, 4, and the crimes cene sample.
Let samples sit for 10 minutes.
11. Using the pH paper, determine the pH of the water for each of the soil
samples and complete the chart. If the pH value is less than 7,the soil is acidic.
The lower the number, the more acidic. If the pH equals 7, the soil is neutral. If
the pH is greater than 7, the soil is basic. The higher the number, the more
basic the sample.
12. Record your results on Table 2.

Soil Sample pH Acidic or Basic? pH paper

Crime Scene

Part D: Sedimentation of Soil Samples

Soil samples that seem identical can be further examined by creating a
sedimentation column. As shaken materials separate, they will layer, with the
densest materials settling to the bottom first.
13. Into a 100-mL graduated cylinder, add one heaping teaspoon of Soil Sample
1.
14. Cover the top of the cylinder; shake the contents for 30 seconds.

FRS 581 FORENSIC CHEMISTRY 12

15. Repeat steps 13 and 14 for each of the other samples.
16. Allow all samples to settle overnight and compare the overallappearance of
each sample. Note any floating material in yourdescription.
17. Record your results in Table 3.

Sample 1 2 3 4 Crime Scene

Using colored
pencils, draw
a sketch to
scale of the
layers in the
column
Number of
Distinct
Layers
Description
of
Floating
Material

Questions:
Compare your results from Parts A to D of this activity.

1. The crime scene sample and sample ____ is a match. Justify your results.
2. How could they be used in a court of law?
3. Research what other types of analysis might be performed to compare soil
samples.

References:
Forensic Science: Fundamentals and Investigations, 1st edition. Anthony J. Bertino

FRS 581 FORENSIC CHEMISTRY 13

Experiment 4: TLC of Steroids in Herbal Medicine

Objectives

To detect and visualize the presence of steroid in traditionally prepared herbal

medicine.

Introduction

Steroids are organic compounds with a set chemical structure. Some steroids, such
as cholesterol, estrogen and testosterone, are quite common. There are also
hundreds of others found in animals, plants and even fungi, and each of these
reacts a different way within the organism, leading to a variety of effects. Human
bodies naturally produce and use steroids to aid in reproduction, regulate
metabolism, and enhance muscle and bone growth. As such, they are very useful
chemicals and, if used properly, can have numerous benefits. Within the media,
most negative publicity goes to steroid hormones, which can be used to trigger
certain bodily effects if taken. These substances can be classified into the following
types:

* Sex steroids (or gonadal steroids)

* Corticosteroids
* Anabolic steroids

Most stories of steroid abuse or misuse typically involve anabolic steroids, although
there have been some cases of where corticosteroids are the substance of choice
as well.

Materials

Mortar and pestle Capillary tube

Medicine samples 1% w/v methanolic tetrazolium blue

solution
Steroid standard
Cyclohexane
Dichloromethane
ethyl acetate
Vortex
methanol
TLC plate
10% w/v sodium hydroxide solution

Procedure

1. Using a mortar and pestle, each sample is powdered.

2. All samples must be protected from light and they will be kept in desiccators at
room temperature until analysis.

FRS 581 FORENSIC CHEMISTRY 14

3. A reference standard of steroids at 0.5 mg/mL which is the stock standard
solution is prepared in dichloromethane.
4. By dilution of the standard solution in dichloromethane, a working solution at 0.1
mg/mL is prepared.
5. 1 g of each powdered sample is extracted with 5 mL of dichloromethane for
preparation of sample solutions.
6. To improve the dissolution of extract, the mixture will be mixed thoroughly using
vortex at a suitable speed.
7. Extraction solutions of samples and standard solutions of steroid of 20 µL will be
separately spotted using capillary tube onto 20x20 cm TLC plates containing a
250 µm thick silica gel stationary phase.
8. Prepare mobile phase A that is consisted of dichloromethane : methanol (9:1
v/v) and mobile phase B that is consisted of cyclohexane : ethyl acetate (1:3
v/v).
9. The spotted TLC plate is then placed in a closed chamber containing a layer of
mobile phase for approximately 1 hour.
10. Before the solvent front reached the end of TLC plate, the plate is removed and
will then be dried at room temperature.
11. The resulting TLC pattern will be viewed under shortwave at 254 nm ultraviolet
light. Steroids which absorb shortwave UV at 254 nm will be detected due to
quenching.
12. Then the plate will be sprayed with 10% w/v sodium hydroxide solution and 1%
w/v methanolic tetrazolium blue solution which will be freshly prepared
respectively.
13. After heating, the plate will visualized the violet spots of steroid compounds
under daylight.
14. The 𝑅𝑓 values of steroid compounds are measured and calculated.

Questions

1. Discuss the fundamental principle of thin layer chromatography.

2. What is the justification for using 𝑅𝑓 for comparison purpose?

3. What is the danger of steroid in day to day life?

4. Apart from TLC, how steroid can be detected and analyzed?

5. What are the most common drugs that are abused in Malaysia? Name a few.

FRS 581 FORENSIC CHEMISTRY 15

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 method’s 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 Distilled water

plate
Capillary tube
Several ball-point pens, sampled on
white paper 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.

FRS 581 FORENSIC CHEMISTRY 16

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 ¾ 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?

FRS 581 FORENSIC CHEMISTRY 17

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 (100μm in diameter) of the ATR-objective 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.

FRS 581 FORENSIC CHEMISTRY 18

 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?

FRS 581 FORENSIC CHEMISTRY 19

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, Fry’s 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 Fry’s 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 Fry’s 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?

FRS 581 FORENSIC CHEMISTRY 20