MODULE FORENSIC CHEMISTRY AND TOXICOLOGY

CHAPTER 5: MICROSCOPY, CHROMATOGRAPHY

AND SPECTROSCOPY

Objectives:
a) Demonstrate knowledge of microscopy, chromatography, and
spectroscopy
b) Apply skill in proper utilization of forensic tools and equipment

ANALYTICAL METHODS / INSTRUMENTATION

IN FORENSIC CHEMISTRY

❖ Because of the increase of the number of substances being analyzed in a forensic

laboratory, more analytical method required to enhance the capability of examiner to
discriminate and identify one substance to another including those represents trace
amount.

❖ There are three (3) basic types of analysis usually classified into:

Physical require investigation of physical property of any

Examination compound of interest

Chemical requires utilizing chemicals in order to chemically

Examination identify any substance

Biological identification of any biological samples from bacteria

Examination to human internal organs, etc.

• The three basic examination utilized two types of examination that falls into qualitative
analysis. This is the screening and confirmatory examination.
SCREENING VS. CONFIRMATORY
▪ Screening: Low cost, fast, semi quantitative, high sensitivity and low
specificity.
▪ Confirmatory: High cost, slow, quantitative, higher sensitivity and high
specificity.

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o Sensitivity – minimum detectable amount of sample.

o Selectivity – distinguishing property by differentiating one substance from another.

❖ Qualities of confirmatory method:

o Utilize the most accurate (specific) testing method available.
o Have sensitivity equal to or better than the screening method.
o Be economically feasible.
o Be simple enough to standardize across many laboratories.
o Produce results that are legally defensible.

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MICROSCOPY

❖ Microscope – is an optical instrument that uses lens or combination of it to magnify and

resolve the fine details of an object.
- It is the most useful and earliest instrument commonly used in a typical
crime laboratory for forensic examinations and still used today for most of the forensic
analysis.

❖ TYPES OF A MICROSCOPE

1. Magnifying glass – the earliest and simplest single lens

microscope. The magnified image is viewed by looking in the lenses.
The image viewed under lens is called virtual image. While the object
appear without the handheld lens is called real image. The lens can
magnify 10x than the real image.

2. Compound microscope – composed of two lenses, the objective

and the eyepiece lens, mounted at each end of a hollow (open) tube. The
two lenses can magnify up to 1500x the real image.
▪ Objective lens – the lower lens where the object to be magnified is
placed. It forms a real, inverted and magnified image.
▪ Eyepiece lens – the inverted magnified object from objective lens
is viewed through this upper lens which further magnifying it into a virtual
image.
The principle of the compound microscope: the optical principle of the
compound microscope includes the passage of light into two lenses to
form a virtual image.

❖ Two Main Part of Compound Microscope

o Mechanical system composed of six parts:
▪ Base – the stand or support of the instrument.
▪ Arm – the C-shape upright structure, connected to the base and use as
handle for carrying.
▪ Stage – a horizontal plate where samples are place for viewing. Sample
was usually mounted on a glass slide and held firmly in place using a spring
clips attached on the stage.
▪ Body tube – is a cylindrical hollow tube where the eyepiece and objective
lenses attached at opposite ends.

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▪ Coarse adjustment – use to focus the lenses by raising or lowering the

tube for better adjustment.
▪ Fine adjustment – also use to focus the image but the movement was
smaller compare to coarse adjustment.

o The optical system has four main parts:

▪ Illuminator – a light bulb that is used to supply light to view the specimen.
• Transmitted illuminator – if the light is directed toward and
through the specimen. (e.g. crystal, from the illuminator to the base
of the specimen).
• Reflected illuminator – for non-transparent or opaque object, the
light source was placed above the specimen’s surface and into the
lens system of the microscope.
▪ Condenser – collects the light rays from the base illuminator and
concentrates them to the specimen. The condenser includes an iris
diaphragm that can be opened or to control the passage of light into the
condenser.
▪ Objective lens – the positioned closes to the specimen. There are several
objectives mounted on the
revolving nosepiece containing
different magnification. The low
power, high power and oil
immersion objective.
▪ Eyepiece or ocular lens –
the lens closest to the eye.
Monocular (one eyepiece) and
Binocular (two eyepiece).

o Total magnification =
magnifying power of objective
x magnifying power of
nosepiece lens

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o Function of Magnifying Power

o Field of view – the area of the specimen that can be seen after it is magnified.
Field of view is inversible proportional to magnifying power.
o Depth of focus – the thickness of a specimen entirely focusses under a
microscope.

3. Comparison microscope – is the combination of

two compound microscope attached to one unit. Use in side
by side comparison of a given sample to the standard
specimen. It uses bridge containing a series of mirrors and
lenses to join two independent objective lenses into a single
binocular unit.

When a viewer looks through the eyepiece lenses of the

comparison microscope, a circular field, equally divided into
two parts by a fine line, is observed.

The specimen is mounted in the left of the objective is seen in the left half of the field, and
the specimen under the right objective is observed on the right half field.

Adjustment of both objective lenses must be matched to assure both specimens are seen
equal magnification with minimal or less distortion.

To compare bullets, cartridge cases and other opaque objects uses vertical or reflected
illumination device, while hair examination uses transmitted illumination.

4. Stereoscopic Microscope – lower magnification for

specimens which do not requires fine details to characterize the
structure. Magnifying power = 10x to 125x
The microscope produces a three-dimensional image of the
specimen magnified used to examine trace evidence. This
becomes the most frequently used and versatile microscope found
in the crime laboratory.
The wide field of view and great depth of focus makes it an ideal
for examining big, bulky trace evidences such as debris, garments,
for gunshot residue, tools and weapons. When fitted with vertical
illumination. It can be used to examined paints, soil and plants.

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5. Polarizing Microscope – devise that uses polarizer to

magnify objects and uses a second polarizing crystal called
analyzer.
o Plane polarizer – light that is confined to a single
vibration.
o Polarizer – the devise that polarize light.
o Polaroid sunglasses – when sunlight passed through
this glass, the light is transmitted as vertical plane only
thus reducing the light glare.
o Glare – are series of light vibrating in horizontal plane
unpleasant to the eye.

Polarizer and analyzer should be aligned parallel to each other so that will be passed
through to be seen by the eye or else you only see darkness. Mechanism of polarizing
microscope.

Polarizing microscope is a modified compound or stereoscopic microscope, wherein

polarizer is placed between the light source and sample stage to polarize light before it
passes through the specimen.

The light that passes through the specimen must be further passed through the analyzer
before it reaches to the eyepiece and finally to the eye.

6. Microspectrophotometer – it is an instrument that links

microscope into a spectrophotometer. This instrument can be
used to view a particle under a microscope while at the same
time, a beam of light is directed into the particle to measure its
characteristics absorption.

it uses different light such as ultraviolet, visible and infrared

light. This microscope is used for color comparison in
examination of pint, fiber and ink. Then the absorption of
radiation by the color of the specimen produced a spectral
characteristic of a compound.

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7. Scanning Electron Microscope – instead of light, the image is seen by aiming a beam
of electrons onto the specimen and studying the electrons emission and seen in the
monitor as virtual image.

The beam was emitted using a hot Tungsten

filament and is focused by means of
electromagnets onto the surface of the
specimen.

The primary electron beam causes the emission

of electrons from the elements that make-up the
upper layers of the specimen.

The emitted electron is collected and amplified

the signal as displayed on a cathode-ray or TV
tube.

The process is to convert the primary electron beam or emitted electrons into the image
of the specimen as displayed on cathode-ray tube.

SEM-EDX is used to determine the elemental composition of the specimen under

examination. When an electron strikes a specimen, an x-ray is generated and analyzes
using an X-ray analyzer as each element produces characteristic peaks in the
spectrogram. Thus, the intensity of x-ray emission is specific for a given element in the
specimen.

Gunshot residue examination was conducted by lifting the trace residue using an adhesive
tape and analyze under SEM. The trace residue or the particle is characterized by their
size shape and elemental composition.

CHROMATOGRAPHY

❖ Chromatography is defined as the physical method of separation in which the

components to be separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile phase) moves in a definite
direction.

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References:
Saferstein, Richard, Criminalistics: An Introduction to Forensic Science, 2nd edition, Prentice
Hall New York, 2001.
Hans Determann and Friedrich Lepusch, The Microscope and its Application.
https://www.sciencedirect.com/topics/chemistry/chromatography

LINKS

TOPIC LINK FOR VIDEO

MICROSCOPE WORKING IN
https://www.youtube.com/watch?v=b2PCJ5s-iyk
ANIMATION

Chromatography | https://www.youtube.com/watch?v=PvHvx7k7UPU

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