SCHOOL OF CRIMINAL JUSTICE AND PUBLIC SAFETY

FORENSIC INSTRUMENTATION

A Self-regulated Learning Module

A Self-regulated Learning Module 1

 SCHOOL OF NATURAL SCIENCES
General Luna Road, Baguio City Philippines 2600

Telefax No.: (074) 442-3071 Website: www.ubaguio.edu E-mail Address: ub@ubaguio.edu

A Learning Module in

FORENSIC
INSTRUMENTATION
Prepared By: Mark Gideon M. Wallis, RMT

https://www.waynesburg.edu/

www.camdencc.edu www.forensicscienceeducation.org

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Introduction

I. Course Code and Title: FORSCI7 (Forensic Instrumentation)

Course Description and Information: This is a 3-unit course with 2 units lecture and 1 unit laboratory. Forensic
Instrumentation deals to provide the students with a basic understanding of the theory and application of the methods of
modern analysis as applied to forensic problems. It shows how spectroscopy, Spectrophotometry, chromatography and
microscopy can be applied in forensic examinations.

II. Requirements of the Course:

1. Regular Attendance to classes: you must attend online classes and live quizzes regularly by logging in to our
scheduled online activities. Online lectures will be done through Canvas and/or facebook live. Assessments shall
be given through Quizziz, Pear Deck, Canvas and/or Google forms. For offline students, your attendance will be
monitored through your responses to text information and through timely correspondence. Offline students will
also be given quizzes and activities through phone calls and text messages.
2. Submission of required activities: All required activities (assignments, research work, and laboratory
illustrations) should be submitted on or before the given deadline. Deadlines will be posted by the teacher in the
google classroom and messenger group chat. It will also be texted to offline students. Learning output for online
students, submit to the teacher’s email address that will be given during the class orientation; For offline students,
submit via mail or express courier (“padala”) addressed to: Instructor’s name, School of Natural Sciences,
University of Baguio, Baguio City.
3. Study/Learning Guidelines:
a. Manage your time properly. As students of higher education (College), you are expected to be more
responsible in paying attention to course schedules, requirements, and deadlines. Schedule how you will
accomplish all the requirements in all your enrolled courses (reading the modules, reading on research/
enhancement questions, doing assignments and laboratory illustrations) and focus your attention when doing
your tasks.
b. Observe proper conduct. Despite this online mode of learning, you must still maintain appropriate school
behavior at all times. All standards of student conduct outlined in the University of Baguio Student Handbook
remain in full effect during this time of distance learning. Be honest in answering your quizzes and exams.
Work independently in doing your tasks and assignments.
c. Maintain a performance of high standards. Give your best in accomplishing all the assigned tasks. Do not be
complacent with just a 70% passing cut score.
d. Communicate properly. Promptly respond to notifications by regularly visiting our google classroom and
messenger group chat. If you have confusions or queries in any part of this module, I am here to guide you
through. Send your academic concerns using same online platforms. For offline students, text messages and
mobile calls are welcome during scheduled hours of the day and week. Be guided by this schedule when
communicating:
▪ Respect private hours. I do not always open my laptop/email/messenger 24/7. Send your queries and/or
concerns during regular office hours. For concerns that need immediate attention, send through mobile
text.
e. Show mutual support. Support one another. Let us all be responsible and supportive in making this new
learning process more effective.
f. Live lecture/Video conferencing guidelines:
f.1 Be punctual. Live lectures/Video conferences will be scheduled during the official class period/time of this
course. Log in to the platform at least 5-10 minutes before the class period. Prepare your learning
materials such as this module, pens, papers, etc. Attendance will be checked during the lecture/video
conference.
f.2 Maintain professionalism.
- Wear appropriate clothing and set your gadget in an appropriate area. You may be asked to turn on
your video/camera at any time during the lecture.

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 - Log in using your UB gmail account. Unidentified names like nicknames, phone models, etc. will not
be allowed in the video conference.
- Mute your microphone as soon as you log in to the platform to avoid any excess background noise.
Unmute your microphone when instructed to do so.
- Respect privacy. Do not take a screenshot, picture, snapchat, etc. of your teacher or fellow students,
nor make any unnecessary audio or video recordings.
f.3 Remain focused and engaged. Do not be distracted by your gadget. Keep your videoconference

Endorsed by:

Teresa N. Villanueva, RMT, MACT

Dean, School of Natural Sciences

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Study Schedule

Week Lecture Study Guide Laboratory Study Guide

Number
Week No. 1 Chapter I. Introduction to Orientation
Week No. 2 Forensic Instrumentation Activity # 1
Week No. 3 Chapter II: Microscopy Activity # 2
Week No. 4 Activity # 3
Week No. 5 First Grading Examination
Week No. 6 Chapter III: Photometry Activity # 4
Week No. 7 Activity # 5
Week No. 8 Chapter IV. Chromatography Activity #6
Week No. 9 Activity #7
Week No. 10 Midterms Examination
Week No. 11 Chapter IV. Chromatography Activity # 8
Week No. 12 Activity #9
Week No. 13 Chapter V. TOXICOLOGY OF Activity #10
Week No. 14 ALCOHOL Activity #11

Week No. 15 Finals Examination

Note: For offline learners, please feel free to message me through Text SMS for additional
instructions.

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Rubrics

Rubrics for the evaluation of Essay question activities and Questions for research
CRITERIA HIGHEST Outstanding Satisfactory Poor
SCORE
1. Content and Focus 7 Sharp, distinct & Apparent point made No apparent point and no
substantial controlling about a specific topic to minimal evidence of
-Awareness about the
point about a specific or question with awareness and
specific topic
question or topic with sufficient awareness knowledge. (1 point)
-Presence of relevant ideas evident awareness and knowledge. (4
thru tacts, examples, details, and knowledge. (7 points)
opinions and explanation points)
2. Organization 5 Sophisticated Functional Confused or inconsistent
arrangement of arrangement of arrangement no logical
-Order developed and
content with evident content that sustains order or evidence of
sustained with in the
and/or subtle a logical order w/ transition. (1 point)
paragraph
transition (5 points) some evidence of
transition (3 points)
3. Style and Conventions 3 Good grammar, Sufficient grammar Incorrect grammar and
spelling and sentence and minor spelling major spelling errors
-Choice, use and
formation throughout errors and sentence throughout the paragraph.
arrangement of words
the paragraph. (3 formation (1 point) (0 point)
-Grammar, mechanics, points)
spelling, usage & sentence
formation

Rubrics for the evaluation of Illustrations and Diagrams

CRITERIA HIGHEST Outstanding Satisfactory Poor
SCORE
1. General Appearance 3 Lines or patterns are There are smudges Smudges or stray marks
drawn clearly and not or stray pencil marks obscure details of the
- Neatness and labelling
smudged. Minimal but these do not illustration. Over-all quality
erasure or stray significantly affect of the drawing shows
pencil marks. the over-all minimal effort to complete
Labelling is accurate appearance of the careful work. Important
and complete. (3 illustration. Labels labels are missing. (1
points) are included but point)
there are minor
problems in the
accurate
identification of a
part. (2 points)
2. Organization and 5 Provides complete Provides clear Only few important
content and well organized illustration however contents are illustrated (1
illustrations (5 points) some details are point)
-Illustrations and sequencing
missing (3-4 points)
3. Attention to details 2 The illustration is Some minor errors Major discrepancies are
accurately drawn (2 are present however very noticeable in the
points) these do not distract illustration. (no point)
the information
conveyed. (1 point)

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 Chapter I. Introduction to Forensic Instrumentation

Desired learning outcomes:

At the end of this chapter, you should be able to:
a. understand the basics of forensic instrumentation
b. identify and describe each of the different equipment’s used in forensic instrumentation
c. discuss the importance and the role of instrumentation in forensic analysis

FORENSIC INSTRUMENTATION/ANALYTICAL CHEMISTRY

- Deals with the methods for determining the chemical composition of samples (evidences) of matter as applied to
forensic problems.
a. Qualitative Method
- Yields information about the identity of atomic or molecular species or the functional groups in the sample.
b. Quantitative Method
- Provides numerical information as to the relative amount of one or more components of the sample.

Importance of the Study of Forensic Instrumentation

a. Develop an understanding of those instrumental tools and their application to solve important analytical problems;
b. Familiarize with the fundamental principles of operation of modern analytical instrumentation;
c. Appropriate choices and efficient use of measurement tools;
d. Understanding the advantages and limitations of various tools;
e. Knowledge of measurement principles is necessary for calibration, standardization and validation of instrumental
method.

Classification of Methods Used in Forensic Instrumentation

a. Classical Methods
- In the early years of chemistry, most analyses were carried out by separating the components of interest (the
analytes) in a sample by precipitation, extraction or distillation.
- For qualitative analyses, the separated components were then treated with reagents that yield products that
could be recognized by their colors, their boiling and melting points, their solubilities in a series of solvents, their
odors, their optical activities or their refractive indexes.
- For quantitative analyses, the number of analytes is determined by gravimetric or by volumetric
measurements.
- In gravimetric measurements, the mass of the analyte or some compound produced from the analyte is
determined.
- In volumetric, also called titrimetric procedures, the volume or mass of a standard reagent required to react
completely with the analyte is measured.
- The methods for separating and determining analytes are still used in many laboratories. The extent of their
general application is, however, decreasing with the advent of instrumental methods to supplant them.

b. Instrumental Methods
- Early in the 12th century, scientists began to exploit phenomenon other than those used for clinical century,
scientists began to exploit phenomenon other than those used for classical methods for solving analytical
problems.
- Measurements of such analyte physical properties as conductivity, electrode potential, light absorption or
emission, mass to charge ration, and fluorescence began to be used for quantitative analysis.
- Highly efficient chromatographic and electrophoretic techniques began to replace distillation, extraction, and
precipitation for the separation of the components of complex mixtures prior to qualitative or quantitative
determination.

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Types of Instrumental Methods:
Most of the characteristic properties used for instrumental analysis require a source of energy to stimulate a
measurable response from the analyte.

Characteristics Instrumental Methods

Emission of Radiation Emission spectroscopy (X-ray, UV, visible, electron, Auger),
fluorescence, phosphorescence and luminescence (X-ray, UV
and visible)
Absorption of radiation Spectrophotometry and photometry (X-ray, UV, Visible, IR),
photoacoustic spectroscopy; nuclear magnetic resonance and
electron spin resonance spectroscopy
Turbidimetry, nephelometry, Raman spectroscopy
Scattering of radiation Refractometry, interferometry
Refraction of radiation X-ray and electron diffraction methods
Diffraction of radiation Polarimetry, optical rotary dispersion, circular dichroism
Rotation of radiation Potentiometry; chronopotentiometry
Coulometry
Electrical potential Amperometry, polarography
Electrical charge Conductometry
Electrical current Gravimetry (Quartz crystal microbalance)
Electrical resistance Mass spectrometry
Mass Kinetic methods
Mass-to-charge ratio Thermal gravimetry and titrimetric, differential scanning
Rate of reaction calorimetry; differential thermal analyses, thermal
Thermal characteristics conductometric methods
Radioactivity Activation and isotope dilution methods
The second column are based on the various physical and chemical properties. Some instrumental techniques
are more sensitive than the classical techniques, but others are not. Gravimetric or volumetric approach may suffer less
interference.

Instruments for Analysis

An instrument for chemical analysis converts information about the physical or chemical characteristics of the
analyte to information that can be manipulated and interpreted by a human. Thus, an analytical instrument can be viewed
as a communication device between the system under study and the investigator.
To retrieve the desired information from the analyte, it is necessary to provide a stimulus, which is usually in the
form of electromagnetic, electrical, mechanical, or nuclear energy. The stimulus elicits a response from the system under
study whose nature and magnitude are governed by the fundamental laws of chemistry and physics.

RESPONSE
STIMULUS SYSTEM Numerical
UNDER
STUDY Graphical
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SSS

SSTUDY
a. Data Domains
The measurement process is aided by a wide variety of devices that convert information from one form to
another. Maybe broadly classified into:
1. Nonelectrical Domain - Among these characteristics are the length, density,
chemical composition, intensity of light, pressured.
2. Electrical Domain - The modes of encoding information as electrical quantities:
a. Analog domain signal – information is encoded as the magnitude of one of the electrical quantities like
voltage, current, charge or power.
b. Time domain information – information is stored as the time relationship of signal fluctuations rather than in
amplitudes of the signal.
c. Digital domain - data are encoded in a two-level scheme. It may be represented by the state of a light bulb, a
toggle switch or a logic-level

b. Detectors, Transducers, and Sensors

The term detectors, transducer and sensor are often used synonymously, but in fact the terms have somewhat
different meanings. The most general of the three terms, detectors, refers to a mechanical, electrical or chemical device
that identifies, records, or indicates a change in one of the variables in its environment such as pressure, temperature,
electrical charge, electromagnetic radiation, nuclear radiation, particulates or molecules.
The term transducer refers specifically to those devices that convert information in nonelectrical domains and the
converse. It includes photo iodides, photomultipliers and those that produce current.
The term, sensor pertains to the class of analytical devices that are capable of monitoring specific chemical
species continuously and reversibly like glass electrodes and other ion selective electrodes.
Generally, instruments for chemical analysis comprise just a few basic components:
Instrument Energy Analytical Information Input Data Signal
source information Sorter Transducer Domain of Processor
(stimulus) Transduced Readout
Information
Photometer Tungsten Attenuated Filter Photo iodide Electrical Amplitude
lamp light beam Current Digitizer
LED display
Amplifier
Atomic Inductively UV or Visible Monochroma- Photomultiplier Electrical Digitizer
Emission coupled radiations tor Current Digital
Spectrometer plasma display
Amplitudes
Digital timer
Charge Amplitudes
Coulometer Direct required to Cell potential Electrode Time Digital timer
current reduce or
source oxidize
analyte Amplitude
Digitizer
Digital
pH Meter Sample/ Hydrogen Glass Glass – Electrical display
Glass Ion electrode Calomel Voltage Amplitude
Electrode Activity electrode Digitizer
Digital
Mass Ion Source Mass-to- Mass Electron Electrical display
Spectrometer charge ratio Analyzer multiplier Current Computer
system
Electro-
Meter
Computer
Gas Flame Ion Chromato- Biased Electrical System
Chromato- concentration Graphic Electrode current Digitizer
graph with Vs. time Column
flame
ionization
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- The current form the phototransuder is then passed through a resistor R1 which according to Ohm’s law produces a
voltage (V) that is proportional to the intensity of the fluorescence.
- Finally, V is measured by the digital voltmeter to provide a readout proportional to the concentration of the substance in
the sample.

Informati Intensity Electrical

voltage
on flow of current
analyte

readout

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 Experiment No. 1
MATERIALS & INSTRUMENTATIONS

INTRODUCTION:

The forensic laboratory utilizes analytical instrumentation applicable to training, routine and specialized analytical and
forensic case work. This instrumentation provides capabilities in robust and reliable analysis of the varied physical
evidences from the crime scenes and natural products.

I. OBJECTIVE
- Identify the different laboratory instruments and materials essential in the conduct of Forensic Laboratory.
- Determine the different use/s of each material

II. MATERIALS
a. High Precision Analytical Balance S. Serological Pipettes (10,5,1 ml)
b. Graduated Cylinder t. Micropipette and tips (1000 ul)
c. Beaker u. Pipettor
d. Volumetric Flask v. Laboratory centrifuge
e. Stirring rod
f. Liquid dispenser
g. Filter paper (Ordinary and Whatman)
h. Bunsen Burner
i. Iron Stand with Iron Ring
j. Fume hood
k. Iron clamp
l. Safety Cabinet for reagents
m. Wire Gauze
n. Test tubes with Test tube rack
o. Spatula (Wooden and Porcelain)

III. PROCEDURE

1. Research for the actual appearance of each of this instrument used in Forensic Chemistry.
2. Determine the use/uses of these instruments and materials in Forensic Chemistry.
3. Draw neatly each material and instrument demonstrated by your laboratory instructor.

V. ACTIVITIES
- Use separate bond papers, draw the different instruments, glass wares and instruments and label their parts.
- Indicate their use or uses in forensic laboratory.

VI. QUESTIONS OF RESEARCH

1. Give the functions/uses of each of these instruments and materials in Forensic Investigation.

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 Experiment No. 2
AUTOMATIONS A IN FORENSIC INSTRUMENTATION
(GC-MS & FTIR)

INTRODUCTION:

Adding or upgrading a laboratory automation system obviously impacts how laboratory staff perform their jobs,
and it may also provide opportunities for technologists to enhance their knowledge base and skill sets. As
existing responsibilities are reconfigured due to automated work flows, lab technologists must reevaluate
where to reposition team members to maximize their value to the laboratory, promote continued professional
development, and ensure that the laboratory remains compliant. http://www.americanlaboratory.com/913-
Technical-Articles/39230-Laboratory-Automation-Im.

I. OBJECTIVE: At the end of the activity, students are expected to be able to:
- Identify the different automation machines in forensic laboratory;
- Determine the different use/s of each material

II. MATERIALS: For the illustration and diagrams please use electronic sources and books if available.

III. Work Activity:

A. Illustration/Drawing
Illustrate schematically the operational principle of the following instruments.
a. Gas Chromatography – Mass Spectrometer (GC-MS)
b. Fourier Transform Infrared Spectrometer (FTIR)

B. Questions for research

1. What is the principle of Gas Chromatography – Mass Spectrometry?

2. What are the uses of GCMS in Forensic Laboratory?
3. What is the principle of Infrared Spectrometer?
4. What are the uses of FTIR in the Forensic Laboratory?

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 Experiment No. 3
AUTOMATIONS B IN FORENSIC INSTRUMENTATION
(UV-VIS & AAS)

INTRODUCTION:

Adding or upgrading a laboratory automation system obviously impacts how laboratory staff perform their jobs,
and it may also provide opportunities for technologists to enhance their knowledge base and skill sets. As
existing responsibilities are reconfigured due to automated work flows, lab technologists must reevaluate
where to reposition team members to maximize their value to the laboratory, promote continued professional
development, and ensure that the laboratory remains compliant. (http://www.americanlaboratory.com/913-
Technical-Articles/39230-Laboratory-Automation-Im).

I. OBJECTIVE: At the end of the activity, students are expected to be able to:
- Identify the different automation machines in forensic laboratory;
- Determine the different use/s of each material

II. MATERIALS: For the illustration and diagrams please use electronic sources and books if available.

III. Work Activity:

C. Illustration/Drawing
Illustrate schematically the operational principle of the following instruments.
c. Ultra-Violet Visible Spectrometer (UV-VIS Spectrometer)
d. Atomic Absorption spectrometer (AAS)

D. Questions for research

1. What is the principle of Ultra-Violet Spectrometer?

2. What are the uses of UV-VIS in Forensic Laboratory?
3. What is the principle of Atomic Absorption Spectrometer?
4. What are the uses of AAS in the Forensic Laboratory?

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 Chapter II: Microscopy

Earliest Microscopes
A. Antioni van Leeuwenhoek (1632-1723)
•1673 - Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine
assayer, and as a minor city official. Leeuwenhoek is incorrectly called "the inventor of the microscope".
•Created a “simple” microscope that could magnify to about 275x, and published drawings of microorganisms in 1683
•Could reach magnifications of over 200x with simple ground lenses - however compound
microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke
claimed they were too difficult to use - his eyesight was poor.
•Discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells,
microscopic nematodes
•In 1673, Leeuwenhoek began writing letters to the Royal Society of London - published in
Philosophical Transactions of the Royal Society
•In 1680 he was elected a full member of the Royal Society, joining Robert Hooke, Henry
Oldenburg, Robert Boyle, Christopher Wren.

Secondary Microscopes
B. George Adams Sr
• George Adams Sr. made many microscopes from about 1740-1772 but he was predominantly just a good manufacturer
not inventor (in fact it is thought he was more than a copier!)
• Simple microscopes could attain around 2-micron resolution, while the best compound microscopes were limited to
around 5 microns because of chromatic aberration.
C. Chester More Hall
• In the 1730s a barrister names Chester More Hall observed that flint glass (newly made glass) dispersed colors much
more than “crown glass” (older glass). He designed a system that used a concave lens next to a convex lens which could
realign all the colors. This was the first achromatic lens. George Bass was the lens-maker that actually made the lenses,
but he did not divulge the secret until over 20 years later to John Dolland who copied the idea in 1759 and patented the
achromatic lens.
D. Giovanni Battista Amici
• In 1827 Giovanni Battista Amici, built high quality microscopes and introduced the
first matched achromatic microscope in 1827. He had previously (1813 designed
“reflecting microscopes” using curved mirrors rather than lenses. He recognized the
importance of coverslip thickness and developed the concept of “water immersion”

Some Definitions
• Absorption
– When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective
absorption of white light produces colored light.

• Refraction
– Direction changes of a ray of light passing from one transparent medium to another with different optical density. A ray
from less to denser medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths.

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• Diffraction
– Light rays bend around edges - new wave fronts are generated at sharp edges - the smaller the aperture the lower the
definition.
• Dispersion
– Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index
with wavelength, such as the spectrum produced by a prism or a rainbow

Magnification

• An object can be focused generally no closer than 250 mm from the eye (depending upon how old you are!)
• this is considered to be the normal viewing distance for 1x magnification
• Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers
a larger part of the retina - that is it is “magnified” at the place where the image is formed

The applications of microscopy in the forensic sciences are almost limitless.

This is due in large measure to the ability of microscopes to detect, resolve and image the smallest items of
evidence, often without alteration or destruction. As a result, microscopes have become nearly indispensable in all
forensic disciplines involving the natural sciences. Thus, a firearms examiner comparing a bullet, a trace evidence
specialist identifying and comparing fibers, hairs, soils or dust, a document examiner studying ink line crossings or paper
fibers, and a serologist scrutinizing a bloodstain, all rely on microscopes, in spite of the fact that each may use them in
different ways and for different purposes
The principal purpose of any microscope is to form an enlarged image of a small object.
As the image is more greatly magnified, the concern then becomes resolution; the ability to see increasingly fine
details as the magnification is increased. For most observers, the ability to see fine details of an item of evidence at a
convenient magnification, is sufficient.
For many items, such as ink lines, bloodstains or bullets, no treatment is required and the evidence may typically
be studied directly under the appropriate microscope without any form of sample preparation. For other types of evidence,
particularly traces of particulate matter, sample preparation before the microscopical examination begins is often essential.
Typical examples of sample preparation might include: mounting a crystal in index of refraction oils to determine its optical
properties, reconstituting a blood crust particle and staining it for leukocytes and other cells, preparing a cross-section of a
fiber or mounting a specimen in a nonfluorescent mounting medium to observe its autofluorescence.
As a general rule, the type of specimen, the information one wishes to obtain from it and the type of microscope
chosen for the task will determine if sample preparation is required and the type of processing required (Epi-Flour
Microscopes).

Forensic microscope is mainly used by forensic science experts to help them with their crime investigations. It
has the ability to identify and compare the proofs collected to justify that a victim, suspect or the place of crime had contact
on each other. It is a valuable tool for investigators in tracing evidences that are often small and cannot be seen with the
human eye.

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 This type of microscope also works on forensic ballistics which is the study of criminal law and answering
technological questions arising while the investigation of crime is in process. This study is on the objects like handguns,
ammunition, bullet holes and traces of a close shot, thermal effect of powder gases.
Nowadays, forensic microscopy become an exciting field for many forensic science experts, investigators, the law,
and the criminology students. Nevertheless, the most important purpose of forensic microscope is to help investigators to
resolve crime cases and contributes to the accuracy of judgements (Welcome of Forensic Microscope, 20130.

Today, forensic scientists can choose from a variety of techniques to study this
evidence, but perhaps the most important technique has been forensic microscopy. Forensic microscopy encompasses
the identification and classification of a wide range of materials and substances: impressions such as fingerprints and foot
prints, fractured fragments such as broken tools and torn paper, trace evidence such as hairs and fibers, genetic markers,
bullets, and handwriting.

The tools employed in forensic microscopy vary from the basic low power hand magnifiers to high power electron
microscopes:

a. Binocular (stereo) microscopes are commonly used in document examination, as in trying to determine whether
one pen stroke passes over another.
b. Scanning electron microscopes are used for morphological and elemental analysis, and to identify gunshot
residue (GSR).
c. Transmission electron microscopes are used for pathogen analysis and for the examination of paint pigments.
d. Infrared or IR microscopes are used in drug identification, while
e. Phase contrast microscopes are used to characterize materials such as glass and biological fluids.

Types of Microscopes Used in the Forensic Sciences

A variety of microscopes are used in any modern forensic science laboratory. Most of these are:

a. Light microscopes which use photons to form images, but

b. Electron microscopes, particularly the scanning electron microscope (SEM), are finding applications in larger,
full-service laboratories because of their wide range of magnification, high resolving power and ability to
perform elemental analyses when equipped with an energy or wavelength dispersive X-ray spectrometer.

I. Stereomicroscope

a. This is the simplest type of microscope in terms of both construction and use.

b. Consists of two compound microscopes which are aligned side-by-side at the correct visual angle to provide a true
stereoscopic image.

c. The long working distance (space between the specimen and objective lens), upright non-reversed image and large
field of view make these the instruments of choice for performing preliminary examinations of evidence as well as
manipulating small particles and fibers to prepare them for more detailed microscopical or instrumental analyses or
comparisons.

d. Specimens rarely require any sample preparation. The specimen is simply placed under the microscope and observed.

e. The useful magnification range of stereo micro-scopes is typically between 2.5 x and about 100 x .

f. Modern stereomicroscopes have choice of illuminators which can provide brightfield and darkfield reflected,
fluorescence and transmitted light permit the microscopist to visualize microscopic objects and features which might
otherwise appear invisible, and thus escape detection.

g. Attaching the microscope to a boom stand permits it to be swung out over large objects such as clothing, piles of
debris or even entire vehicles.

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h. Both photographic and video cameras can be attached to record images for inclusion in a report, as a courtroom exhibit
or to display to colleagues.

II. Compound microscope

a. Compound microscopes represent a significant step up in magnification, resolution and difficulty of use from the
stereomicroscope.

b. Magnifications range from 2.5 x to about 1300 x with a corresponding increase in resolving power.

c. Most observations transmitted light which places limitations on the specimens which are to be studied.

d. Reflected light instruments, used to study bullets and tool marks, have found limited use in forensic laboratories and are
generally confined to the examination of metals that have been prepared by grinding and polishing.

f. A variety of compound microscopes are available to the forensic microscopist and their selection will depend on the
types of evidence to be studied. These include:

a. Standard brightfield,
b. Phase contrast, comparison, hot stage, fluorescence and polarizing
microscopes.
c. Brightfield microscopy is used to observe and study the morphology of
microscopic specimens. In the forensic laboratory these can include a range of
materials almost too numerous to list...

III. Polarizing microscope

a. Most useful and versatile instrument in the hands of a trained and experienced forensic microscopist.
b. Allows transparent solids to be examined in plane polarized light (to isolate unique vibration directions in a crystal
or crystalline polymer), between crossed polar

Type of
Sample preparation required Identification features observed
evidence

Preparation on glass slides in mounting Color. Overall shape and length. Scale patterns. Cross-
Hairs medium. Scale impressions. Cross-sections. sectional shape and structural features. Pigmentation
Clearing medulla. distribution. Cellular structure of medulla.

Scraping to look for epidermal and vascular General color and texture. Shape of any crystals recovered.
Vegetable tissue and crystals. Maceration into fiber Appearance of epidermis and other cells. Features of fiber
fibers ultimate. Microchemical testing for lignin ultimate: length, structure of lumen, cell walls and pits.
content. Comparison to reference slides.

Presence or absence of bordered pits, Crossfield pits.

Preparation of three sections: transverse, radial
Recognition of arrangements of tracheid’s or fibers and
and tangential. Mounting in glycerin alcohol or
Wood vessel elements. Detailed study of pitting types on vessels
permanent medium and staining. Even slivers
and tracheids. The arrangement of the cells in all three
and sawdust can be sectioned.
sections. Comparison to reference slides and illustrations.

Shape. Type and arrangement of apertures: pores and/or

Acetolysis (boiling in mixture of acetic anhydride
furrows. Structure (e.g., columellae) and sculpturing (e.g.,
Pollen and sulfuric acid) to remove cytoplasm.
echinate) of the exine. Comparison to reference slides and
Mounting in glycerin, glycerin jelly or silicon oil.
atlas figures.

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 Shape of girdle and valve views, size, arrangement of
Boiling in strong acids to destroy organic matter.
Diatoms pores, fine structure of the central araphid in the center of
Mounting in high refractive index medium.
the diatom.

Recognition of leukocytes and other cells which may give

Blood crust Reconstitute in reagent such as toluidine blue.
clues as to other tissues present.

In addition to the microscope itself, a set of calibrated indexes of refraction oils are required to perform optical
crystallographic measurements. Table 2 summarizes the determinative methods used along with some typical
applications.

Table 2 Optical properties and their determination with the polarizing microscope
Optical
Measurement Determinative value
property

Refractive Immersion method. Specimen placed in index of Identification of unknown isotropic crystals.
index refraction oils until it disappears. Comparison of glass particles.

Orientation of principal vibration direction with polarizer of Crystallographic identification of chemical

Refractive
microscope. Matching of two (uniaxial crystals) or three crystals and minerals. Identification and
indices
(biaxial crystals with index of refraction oils. comparison of artificial fibers.

Numerical difference between two principal refractive

Identification of crystalline and semicrystalline
indices. By subtracting larger refractive index from
Birefringence materials. Comparison of certain artificial
smaller or by measurement of retardation with a
fibers.
compensator and measurement of thickness.

Aid in the identification of crystals, minerals

Convention based on relative magnitudes of refractive
and fibers. For artificial fibers the more easily
Optic sign indices. By comparison of values of refractive indices or
determined sign of elongation is the same as
by means of compensators.
the optic sign.

Aid in determining the optical character of

Viewed in convergent light between crossed polar with
Interference crystals as uniaxial or biaxial. Provides optical
objective of high numerical aperture using a Bertrand
figure orientation of crystals without diagnostic
lens or pinhole.
external morphology.

Diagnostic aid in the identification of heavy

Rotation of the crystal, particle or fiber between extinction
minerals and their varieties in soil mineralogy.
Pleochroism positions with only one polarizer inserted in the optical
Comparison aid in the examination of colored
path.
artificial fibers.

IV. Phase Contrast Microscope

a. Used primarily in serological and glass examinations.
b. Its principal use in serology is to observe cells in biological fluids or after reconstitution in aqueous mountants.
c. Uses half-silvered rings and disks placed in the optical system to change phase differences into amplitude differences
which can then be observed and photographed.
d. Spermatozoa, epithelial cells, and other cellular matter can be studied in detail without staining using this technique.
e. The measurement is conducted in a hot stage mounted on a phase contrast microscope.

V. Fluorescence microscopy
a. Based on the property of certain substances to emit light of a longer wavelength after they have been irradiated with
light of a shorter wavelength.
b. This emitted light is called fluorescence and differs from luminescence in that the emission of light stops after the
exciting radiation is switched off.

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c. The fluorescence may originate from fluorescent ‘tags’ attached to proteins or other compounds which cause the
substance they react with to fluoresce after the nonreacting remainder of the reagent is washed away, or it may originate
from autofluorescence.
d. The first technique is the basis for detecting antigen-antibody reactions which occur on a cellular level and has been
applied to a limited extent in forensic serology.

VI. Electron microscope

a. Electron microscopes make use of electrons rather than photons to form their image.
b. The transmission electron microscope (TEM) was developed first, followed some years later by the scanning electron
microscope (SEM).
c. Transmission instruments are generally more difficult to use and require more painstaking sample preparation than
scanning microscopes and thus have found very few applications in forensic science.
d. Specimens for TEM must be extremely thin to permit penetration by the electron beam.
e. The image in an SEM is formed from collected secondary or backscattered electrons emitted from (and just beneath)
the surface of the sample and not by transmitted electrons as in the TEM.
f. Since the SEM only looks at the surface of a specimen, sample preparation is often much simpler and frequently
consists simply of placing the specimen on a piece of conductive carbon tape.
g. It may be necessary to vacuum deposit a layer of carbon or gold over nonconductive specimens to make them
conductive, although the new ‘environmental SEMs’ can image nonconductive samples in a low vacuum.
h. SEMs are now in use in many forensic laboratories around the world. Most of these microscopes are equipped with
energy dispersive X-ray spectrometers for elemental analysis. X-ray spectrometers collect the X-rays which are produced
along with the secondary and backscattered electrons when a specimen is bombarded in a vacuum with electrons.
i. One of the principal uses of analytical SEMs in forensic science laboratories is the detection and analysis of gunshot
residue (GSR) particles.

Epi-Flour Stereoscope (2005). Retrieved on February 13, 2015 at http://what-when-how.com/forensic-

sciences/microscopy/.

Eye on Forensic Microscopy (2005). Retrieved on February 12, 2015 at http://www.rdmag.com/articles/2005/12/eye-

forensic-microscopy.

Welcome to Forensic Microscope (2013). Retrieved on February 13, 2015 at http://www. forensicmicroscope.com/

(Michelle, M. (2007). Uses of Forensic Microscopes. Retrieved on February 13, 2015 at

http://www.ehow.com/about_5523339_uses-microscopes-forensic-science.html

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 Experiment No. 4
FORENSIC MICROSCOPY

INTRODUCTION:

A Forensic Microscope is a device that is used when there is a forensic examination on fibers, tool marks, hairs, body fluid
sample, skin cells, fingerprints, paint chips, soil and plant matter, ink and all other small evidences.

Microscopy and forensic science are two different studies but they can still be interrelated to benefit one another. Forensic
science deals with the use systematic procedure and science itself to answer the doubts in regards to legal substances.
Forensic evidence is anything that you can show in the court to prove that a person is innocent or guilty. Forensic science
used a forensic comparison equipment for various applications such as forensic documentation, forensic entomology, fire,
crime scene auto theft and arson investigation.

Microscopy on the other hand, is named after microscope that is an apparatus and is being widely use on several field of
science. Forensic microscopy then is referred to us the study of forensic science with the use of microscopes.

Forensic microscope is mainly used by forensic science experts to help them with their crime investigations. It has the
ability to identify and compare the proofs collected to justify that a victim, suspect or the place of crime had contact on
each other. It is a valuable tool for investigators in tracing evidences that are often small and cannot be seen with the
human eye.

Nowadays, forensic microscopy become an exciting field for many forensic science experts, investigators, the law, and
the criminology students. Nevertheless the most important purpose of forensic microscope is to help investigators to
resolve crime cases and contributes to the accuracy of judgements ( http://forensicmicroscope.com/).

I. OBJECTIVES: At the end of the activity, the students are expected to be able to:

1. identify the different parts of the microscope.

2. determine the uses of the compound & electric microscopes in Forensic Instrumentation.
3. familiarized with the uses of stereomicroscope and comparison microscope in forensics.

II. MATERIALS

a) Comparison Microscope d. Electric Microscope

b) Reference Materials e. Charts/Illustrations
c. Stereomicroscope f. Gun powder/ prepared slides

III. PROCEDURE

a. The instructor conducts a short lecture and demonstration on the mechanical parts and operation of the electric
microscope, stereo microscope and comparison microscope in forensics will be rendered.

IV. ACTIVITY

A. Draw and label the parts of the:

a. Electric microscope
b. Stereo microscope
c. Comparison microscope

B. Questions for Research

a. Identify the parts of a compound microscope and give the importance of each part.
b. Define the following terms related to Microscopy:
1. Magnification
2. Resolution
3. Parfocal
4. Working Distance

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 Experiment No. 5
FORENSIC MICROSCOPY
(Focusing)

Introduction:

The process of focusing consists of adjusting the relationship between the optical system of the microscope and the
object to be examined so that a clear image of the object is obtained. The distance between the upper surface of the glass
slide on the microscope stage and the faces of the objective lens varies depending upon which of the three objectives is in
the focusing position. It is a good practice to obtain a focus with the low-power objective first, then change to the higher
objective required to avoid accidentally damaging the objective lens, the specimen, or both. Most modern microscopes
are equipped with parfocal objectives (meaning that if one objective is in focus, the others will be in approximate focus
when the nosepiece is revolved). With the low-power objective in focusing position, observe the following steps in
focusing (http://www.tpub. com/corpsman/230.htm).

I. OBJECTIVES: At the end of the activity, the students are expected to be able to:

1) appreciate how to perform accurately the focusing technique.

2) gain knowledge and accuracy in the manipulation of a compound microscope.

II. MATERIALS:
a. Stereo microscope
b. Compound electric microscope
c. Different types of blood specimen

III. PROCEDURE: A recorded video of the procedure will be uploaded by the instructor.

A. Electric Microscope

1. Seat yourself behind the microscope, then lower your head to one side of the microscope until your eyes are
approximately at the level of the stage. Place the slide of specimen in the stage and adjust the slide so that the specimen
is positioned on top of the aperture.

2. Using the coarse adjustment knob, lower the body tube until the face of the objective is within 1/4 inch of the object.
Most microscopes are constructed in such a way that the low-power (green) objective cannot be lowered and make
contact with the object on the stage.

3. While you are looking through the ocular, you should use the coarse adjustment knob to elevate the body tube until the
image becomes visible. Then use the fine adjustment knob to obtain a clear and distinct image. Do not move the focusing
knob while changing lenses.

4. If the high-power objective (yellow) is to be used next, bring it into position by revolving the nosepiece (a distinct "click"
indicates it is in proper alignment with the body tube). Use the fine adjustment knob only to bring the object into exact
focus.

5. If specimen is too dark, you can increase lighting by opening the iris diaphragm of the condenser.

6. The oil-immersion objective (red) is used for detailed study of stained blood and bacterial smears. Remember that the
distance between objective lens and object is very short, and great care must be employed so the specimen is not
damaged. After focusing with the high-power objective and scanning for well-defined cells, raise the objective, place a
small drop of immersion oil, free of bubbles, on the slide, centering the drop in the circle of light coming through the
condenser. Next, revolve the nosepiece to bring the oil-immersion objective into place, and, by means of the coarse
adjustment knob, slowly lower the body tube until the lens just makes contact with the drop of oil on the slide. The instant
of contact is indicated by a flash of light illuminating the oil. The final step in focusing is done with the fine adjustment

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knob. It is with this lens in particular that lighting is important. The final focus, clear and well-defined, will be obtained only
when proper light adjustment is made.

B. Stereo Microscope

1. Set the microscope to the lowest possible power on your microscope. This ensures that you will have the longest
working distance that your microscope is capable of, which is usually provided by your manufacturer in the manual or on
the listing.

2. Adjust the microscope so that it sits to about that distance, then use the eyepieces to fine tune the image to clarity. At
this point, you should see an image with great detail. If not, the distance is not correct, or there is something wrong with
the microscope. For example, AmScope stereo microscopes of the SM series are 7x – 45x out of the box. At 7x, we’re
given it has a working distance of 4″. By setting it to 4″ away from the sample’s surface we want to focus on, I can achieve
focus through the microscope.

3. Adjust the magnification higher, then adjust the working distance lower. Do this slowly to get a feel for the microscope’s
focusing distance. Note that it’s fairly exponential how quickly the working distance drops off, so it’s normal to see that you
have to move the stereo microscope head further down per magnification change as you go. I would try to adjust the
magnification by .25x on the dial (estimate if your dial isn’t labeled to that significant figure).

4. Continue to adjust each part slowly until you reach the maximum magnification for your microscope and are focused.

5. Once max magnification/minimum working distance is achieved, try to do it again in reverse until you reach minimum
magnification/maximum working distance. Remember to take your time to ensure you get each step right, and you’ll be
able to focus a stereo microscope in no time.

6. If you’ve been successful, congratulations! You’ve gotten the basics down of how to focus a stereo microscope!
http://microscopegenius.com/microscope-101-focus-a-stereo-microscope/

IV. ACTIVITY

- Illustrate and describe the appearance/s of the prepared slides using the electric microscope and
stereomicroscope in the prescribed magnification.

Electric Microscope:

LPO (Avian Blood) OIL IMMERSION (Human Blood)

Describe:_________________________________________________________________________________________
_________________________________________________________________________________________________

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_________________________________________________________________________________________________
__________________

Stereo Microscope:

20 Peso Bill

Describe:________________________________________________________________________________________
_________________________________________________________________________________________________
_________________________________________________________________________________________________
__________________

Page | 22
 Chapter III: Photometry

Photometry
Introduction
The productivity of the modern forensic laboratory depends on instrumentation. The high output and accuracy of
results obtained by modern analytical devises could not be achieved by the tedious manual separation and quantitation
methods of classical analytical chemistry. From the simplest colorimeter to the most complex automated system. With an
understanding of basic concepts, one can master the purpose and function of any analytical instrument, in spite of the
flashing lights and complex computer driven components (Calbreath 1992).
Spectrophotometry
Quantitation of a substance in solution by spectrophotometry is accomplished by measuring the amount of light
absorbed by that solution after appropriate treatment. The lighter absorbed, the higher the concentration of the material
under study.
A spectrophotometer allows the application of several different approaches that provide a full analysis of the
materials being studied.
Colorimetry
An associated term and is often used to describe the same technique. It is a simple device and very limited
flexibility.
Theory of Light Waves
a. Light is considered as a wave, although it can also be pictured as a particle in some circumstances;
b. As a wave, the light ray will have a peak and a trough.
c. Each light wave possesses certain characteristic properties.
d. The distance between two successive peaks is defined as the wavelength and gives the light its characteristic
color in a visible region.
e. Wavelength is defined as a distance between two peaks and express it in nanometer (nm). One nanometer is
equal to 10-9 m.
f. Light below about 300 nm is considered to be in the ultraviolet region.
g. If the light has a wavelength between approximately 400 nm (blue) and 800 nm (red), it us in the visible region.
h. When the wavelength is above approximately 1000 nm, we say the light is in the infrared region.
Amplitude – the older term used to define a light wave, is the distance between the peak and trough. The higher
the amplitude, the more intense the light and the lighter energy produced at that wavelength.
Beer-Lambert Law
The basic principle of spectrophotometry involves measuring the amount of light absorbed by a solution and
relating that absorption to the solutions’ concentration.
If we shine light through a liquid material, part of the light energy is absorbed by the molecules in solution. The absorption
of light depends on the structure of the molecule, primarily the types of covalent bonds present. By comparing the amount
of light entering the solution (the incident light) with the amount of light passing through without being absorbed (the
transmitted light), we can calculate the concentration of material in the solution. The difference between the amount of
incident light and transmitted light can be expressed mathematically and is referred to as the absorbance of the solution.
The Beer-Lambert Law (Beer’s Law) express the relationship between concentration and absorbance:
A=abc
Where: A - is measured absorbance;
a - is coefficient of absorptivity (specific for each compound);
b – is path length of cuvette.;
c. – concentration of the solution
Because we usually want to measure absorbance and calculate the concentration from that data, we rearrange the
equation to give

C = A_
Ab
Since a and b are constant, there is a linear relationship between the absorbance of a material and the concentration
between the absorbance of a material and the concentration of that material in a solution. As the concentration increases,
the absorbance increases.

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https://ph.images.search.yahoo.com/images/view;_ylt=A2oKiHC34gZVWi8Ah7K1R

Operation of a spectrophotometer

Any spectrophotometer has the following basic components:

1. Light source
2. Wavelength selector
3. Sample holder
4. Detector system
5. Data readout

https://ph.images.search.yahoo.com/images/view;_ylt=

A specific system may be very simple and involve manual operation or it may be coupled to a computer or microprocessor
that carries out a number of data-acquisition and processing steps automatically. All spectrophotometers operate in the
same fundamental way.

The light source provides the incident light for the system. In most cases we work in the visible range (approximately 400
– 800 nm) and can use a tungsten lamp. For work below about 3oo nm, a different light source is needed, a deuterium
lamp. The deuterium lamp requires a separate power supply and tends to be much more expensive and sensitive than a
tungsten light source.

Page | 24
The light source emits light of a wide variety of wavelength (or at least a very narrow band of wavelength) for our
measurements. The proper wavelength is selected by a prism or grating system (monochromator). Light shines on the
wavelength selector and is selector and is spread into a wide band of rays. By moving the selector, we can direct the
specific desired wavelength through the sample. Less expensive instruments use a filter instead of a monochromator. The
filter allows a wider range of wavelengths to pass through but is much less expensive. Some loss of sensitivity can be
expected if a filter system is used for wavelength selector.

The sample holder is a glass or plastic container (either round or square) called a cuvet, which is designed to pass most
of the incident light through without absorbing it. More sophisticated instruments uses square cuvets to minimize the light
scatter more likely to occur with a round cuvet. The liquid sample is poured into the cuvet, which is then placed in the
sample holder so the light can pass through it to the detector. The low acomposition of the cuvet is important for proper
analysis. Although regular glass cuvets are transparent in the visible region and the near-UV, measurements below about
330 nm require special quartz cuvets that do not absorb UV light strongly. These cuvets are much more expensive that
glass ones.

https://ph.images.search.yahoo.com/images/

Detector is a phototube that responds to light striking it. When the transmitted light hits the tube, an electric signal is
generated, going through the detector system to a readout (a moving needle on a dial or a digital display) to indicate the
amount of light passing through a sample.

Absorbance Measurements

To use the spectrophotometer for quantitative purposes, first ascertain the wavelength of maximum absorbance for the
compound of interest. Each chemical has a region where light is absorbed and the other regions where little or no light is
absorbed.

Often a small amount of light is absorbed simply because the cuvette absorbs (or scatters) some of the light rays. Some
of the absorbance reading may be due to other materials in the reaction system. To compensate for this unwanted light
absorption, reagent blank in the same way sample is measured. The light absorbed by the reagent blank can be
subtracted from the measurement using the sample and a “true” absorbance reading can be obtained. Many instruments
do this blank correction automatically.

Measurement of the reagent blank can provide a good, inexpensive quality control procedure. As long as the absorbance
value of the reagent blank is relatively constant, the reagents may be functioning properly. If the blank value changes
markedly (usually seen as an increase in the blank absorbance), some deterioration of reagent has occurred, and new
material needs to be prepared and employed in the assay.

The sample itself provides some nonspecific absorbance of light not related to the chemical reaction. A serum sample that
has hemoglobin (due to hemolysis)), absorbs some light owing to interfering substances. A sample blank can compensate
for all at least part of this unwanted absorbance. The sample is added to a mixture containing all the components of the
reaction except the reagent, which reacts to form the final-colored product.

Page | 25
Calculation of Concentration from Absorbance Measurements

1. Ratio of Standard to Unknown

The simplest type of concentration measurement involves determination of the absorbance values for a known
concentration of the compound of interest (standard) and the measurement of that parameter in a patient sample
(unknown). If we let As and Au indicate the absorbance values of the standard and unknown respectively and cs and cu
indicate the concentrations of standard and unknown, the following equations are derived:

As = abcs and Au = abcu

Aucs
cu = As

Example: The absorbance of the unknown at 450 nm is 0.428, and the absorbance of 5.0 mg.dl standard is 0.372.
Compute for the concentration of the unknown (cu). Answer: 5.75 mg/dl

2. Use of a Standard Curve

In analytical chemistry, a calibration curve is a general method for determining the concentration of a substance in an
unknown sample by comparing the unknown to a set of standard samples of known concentration. A calibration curve is
one approach to the problem of instrument calibration; other approaches may mix the standard into the unknown, giving
an internal standard.

The calibration curve is a plot of how the instrumental response, the so-called analytical signal, changes with the
concentration of the analyte (the substance to be measured). The operator prepares a series of standards across a range
of concentrations near the expected concentration of analyte in the unknown. The concentrations of the standards must
lie within the working range of the technique (instrumentation) they are using. Analyzing each of these standards using the
chosen technique will produce a series of measurements. For most analyses a plot of instrument response vs.
concentration will show a linear relationship. The operator can measure the response of the unknown and, using the
calibration curve, can interpolate to find the concentration of analyte.

In more general use, a calibration curve is a curve or table for a measuring instrument which measures some parameter
indirectly, giving values for the desired quantity as a function of values of sensor output. For example, a calibration curve
can be made for a particular pressure transducer to determine applied pressure from transducer output (a voltage). Such
a curve is typically used when an instrument uses a sensor whose calibration varies from one sample to another, or
changes with time or use; if sensor output is consistent the instrument would be marked directly in terms of the measured
unit (Wikipedia, the free encyclopedia from http://en.wikipedia.org/wiki/Calibration_ curve).

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A. ATOMIC ABSORPTION SPECTROPHOTOMETERY (AAS)

- Is used to measure concentration by detecting absorption of electromagnetic radiation by atoms rather than by
molecules.

- The usual light source, known as a hallow-cathode lamp, consists of an evacuated gastight chamber containing an
anode, a cylindrical cathode, and an inert gas such as Helium and Argon. When voltage is applied, the filter gas is
ionized. Ions attracted to the cathode collide with the metal, knock atoms off, and the metal atoms to be excited. When
they return to the ground state, light energy is emitted that is characteristic of the metal in the cathode. A separate lamp
generally is required for each metal (eg., a copper hollow cathode lamp is used to measure Cu).

- Electrode-less discharge lamps are a relatively new light source for AAS. A bulb is filled with argon and the element to
be tested. A radio-frequency generator around the bulb supplies the energy to excite the element, causing a characteristic
emission spectrum of the element.

- The sample being analyzed must contain the reduced metal in the atomic vaporized state. This is commonly done by
using the heat of a flame to break the chemical bonds and form free, unexcited atoms. The flame is the sample cell in this
instrument, rather than a cuvette. There are various burner designs, but the most common one is known as a premix long-
path burner. The sample, in the solution, is aspirated as a spray into a chamber, where it is mixed with air and fuel. This
mixture passes through baffles, where large drops fall and are drained off. Only fine droplets reach the flame. The burner
is a long and narrow slit, to permit a longer path length for absorption of incident radiation to occur.

Principle:

Light from the hollow lamp passes through the sample of ground-state atoms in the flame. The amount of light absorbed is
proportional to the concentration. When a ground-state atom absorbs light energy, an excited atom is produced. The
excited atom then returns to the ground state, emitting light of the same energy as it absorbed. The emitted energy forms
the flame will go in all directions, and it will be a steady emission. Because the purpose of the instrument is to measure
the amount of light absorbed, the light detector must be able to distinguish between the light beam emitted by the hollow-
cathode lamp and that emitted by excited atoms in the flame. To do this, the hollow-cathode light beam is modulated by
inserting a mechanical rotating chopper between the light and the flame or by pulsing the electrical supply to the lamp.
Because the light beam absorbed enters the sample in pulses, the transmitted light also will be in pulses. There will be
less light in the transmitted pulses because part of it will be absorbed.

There are two light signals from the flame: an alternating signal from the hollow-cathode lamp and a direct signal from the
flame emission. The measuring circuit is tuned to the modulated frequency. Interference from the constant flame emission
is eliminated electronically by accepting only the pulsed signal from the hollow cathode.

The monochromator is used to isolate the desired emission line form the other lamp emission line. In addition, it serves to
protect the photodetector from excessive light emanating from flame emissions (Bishop, 2000).

Page | 27
B. FLAME PHOTOMETRY

It is used to measure light emitted by excited atoms. It is used primarily to determine concentration of Na+, K+, or Li+. The
sample solution is aspirated into the flame. The purpose of the flame is twofold. Chemical bonds are broken to produce
atoms, and then atoms absorb energy from the flame and enter an excited electronic state. The excited atoms return to
the ground state by emitting light energy that is characteristic for that atomic species. The emitted light, focused by lenses
or mirrors, passes through monochromators that are selected to transmit radiant energy from the specific atoms.

The monochromator for Na+ is set at 589 nm, for K+ at 767 nm, and for Li+ at 671 nm. The emitted light then strikes a
photodetector, which generates an electric signal proportional to the concentration of the atoms in the flame.

Fluctuation in the light source of the flame photometer is generally caused by changes in fuel or air pressure that affect
flame temperature or rate of sample aspiration. Stability is achieved by using an internal-standard system. For sodium or
potassium measurements, lithium can be added in equal amount to all standards and samples to act as an internal
standard. The readout is a ratio comparing the emission intensity for the internal standard with that of the element being
analyzed.

The same amount of lithium is present in the blank, sample, and standard. Any change in aspiration rate or flame
temperature or rate of sample aspiration. Stability is achieved by using an internal-standard system. For sodium or
potassium measurements, lithium can be added in equal amount to all standards and samples to act as an internal
standard. The readout is a ratio comparing the emission intensity for the internal standard with that of the element being
analyzed. The same amount of lithium is present in the blank, sample and standard. Any change in aspiration rate or
flame temperature will affect the sodium, potassium and lithium emissions proportionately. The ratio remains constant,
thus compensating for possible fluctuations (Bishop, 2000).

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C. FOURIER TRANSFORM INFRARED SPECTROSCOPY

Spectroscopic methods are based on the interaction of radiation with matter. In the case of infrared spectroscopy, the
radiation is in the infrared region of the electromagnetic spectrum, as seen in Figure 1, with wavelengths between 0.78
and 300 µm. This region can be further separated into near (0.78-3 µm), mid (3-30 µm), and far (30-300 µm) infrared.

This article focuses on mid-infrared radiation, which can be absorbed by the sample upon interaction with molecular
vibrations. The exact frequency that is required for this interaction depends on numerous factors: the type of vibration,
such as stretching or bending; the strength of the bonds, such as single, double or triple bonds; the atoms and isotopes
involved; the symmetry of the molecule; and the chemical environment. Consequently, every infrared active molecule has
an unique infrared fingerprint; however, not all vibrations can absorb mid-infrared radiation.

While infrared spectroscopy is an old method, it was recently revolutionized and revitalized by the introduction of Fourier
transformation, with the first commercial instruments made available in the early 1980s. As opposed to traditional
dispersive techniques, absorption of light at all frequencies is monitored simultaneously based on interference, and
recorded as an interferogram.

This interferogram is subjected to Fourier transformation, which calculates the contributions of the constituent frequencies
and plots the spectrum as intensity versus wavelength, or wavenumbers (cm-1), as is commonly used in infrared
spectroscopy.

Page | 29
Samples

One of the major advantages of Fourier transform infrared (FTIR) spectroscopy is that it can give detailed qualitative and
quantitative chemical information without destroying the sample. In most cases, the samples used for FTIR spectroscopic
investigations can be completely recovered and used for further analysis elsewhere.

Exceptions include cases when samples are too concentrated, practically absorbing all infrared radiation, and thus must
be diluted by mixing with infrared transparent diluters, such as KBr for diffuse reflectance measurements.

While the sample is not destroyed in this procedure, it is intimately combined with KBr making it unrecoverable; however,
samples in most cases can easily be recovered. This is particularly important when examining the chemical composition
of unique, non-reproducible samples such as in forensic analysis, or in the analysis of artwork and historical artefacts. It
would hardly be worth ruining Mona Lisa’s smile in order to determine its chemical components.

The same is true with most experiments in the laboratory, where further experimentation or characterization needs to be
done on a small quantity of sample.
An additional advantage of FTIR spectroscopy is its versatility. It is a tool that can be used on almost any kind of sample,
regardless of the physical state. Samples may be organic, inorganic, biological, polymers, or any combination of these.

While other techniques such as nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS) may be
able to produce more specific or detailed data than FTIR spectroscopy, they are often considerably more expensive, have
lower throughput, require more sample, labor and training, and can be more difficult to automate. In addition, they may not
be as versatile or allow for spatially resolved studies, such as micro spectroscopy. Ultimately, FTIR spectroscopy can
provide key data at low cost and high speed.

Applications

With a host of techniques available, FTIR spectroscopy can be used to examine almost any problem thrown its way. This
may sound arrogant, but the benefits of FTIR are extremely far reaching.

The techniques described in this article are currently being employed to investigate topics ranging from doping tests in
sports and quality control of Formula One car tires, to medical diagnostics and pharmaceutical identification; quality
control and monitoring in the food, semiconductor, solar energy and pulp and paper industries; identification and
preservation of art and historical artifacts; and the tracking of toxins to determine how long they stay in groundwater.

Measurement techniques

As stated previously, one of the greatest strengths of FTIR spectroscopy is its versatility. Measurements are fully
customizable to suit the samples, needs and preferences of the user.

Transmission

Transmission is the oldest and perhaps most straightforward infrared spectroscopic method: the sample is simply placed
in the beam path of the infrared radiation. Samples can be gases, liquids or solids, with different sample cells available for
each case, such as sealed for gases and volatile liquids studied under different pressures, or open containers for fixed or
Page | 30
variable path lengths.

The advantages of the technique are simplicity, practically no sample preparation and a superior speed of more than 100
spectra per second. In addition to this, the flow through setup means that continuous, real-time monitoring is possible.

The disadvantages include the sometimes difficult handling procedures and maintenance of the sample cells; for example,
cleaning can be challenging as windows must be made of infrared transparent material and not glass.

In addition, the useful concentration range for this technique can be limited by the very short path lengths required for
highly absorbing materials, such as aqueous solutions and the need to dilute highly absorbing samples with infrared
transparent materials.

Application areas of the technique include: continuous or batch gas analysis, which can be used for environmental
monitoring such as car exhaust testing, and continuous or batch water quality monitoring, which can be used to study or
monitor wastewater, groundwater and crude oil quality.

Reflectance

Reflectance methods are based on the interactions of reflected infrared light with the sample. These methods are often
used instead of transmission techniques when transmission is too difficult to implement, or when only specific areas of the
sample need to be analyzed, such as surfaces or thin layers.

The three most common reflection methods described independently below are diffuse reflectance, attenuated total
reflectance and grazing angle FTIR spectroscopy.

Micro spectroscopy

By attaching a microscopy accessory to an FTIR spectrometer, spatially resolved chemical compositional analysis
becomes possible, producing high quality qualitative and quantitative chemical maps of the sample.

The highest achievable spatial resolution depends on a number of factors: the intensity of the infrared light source, the
wavelength of the light and the type of detector, such as single element or focal plane array (FPA).

Modern FPA detectors are capable of recording images at diffraction limited spatial resolution, or, in other words, the
spatial resolution depends on the wavelength of the light, which in the case of mid-infrared radiation means a spatial
resolution in the micron range, consisting of thousands of spectra within a minute. Different accessories are available for
the microscopes as well, allowing for transmission, reflection, ATR or grazing angle measurements.

Analysis can be done on single samples, multiple spots on a single sample or on multiple samples. A flow through setup
for continuous spatial and chemical monitoring can also be implemented. Samples can be solids or liquids, suspensions
or colloids.

The obvious advantage of micro spectroscopy is the possibility to investigate the spatial distribution of chemical changes
and sample heterogeneity, and to follow reaction fronts and reactions at certain sites, as well as precise sampling, such
as when only measuring a certain area in a sample.

Compared to these gains, the disadvantages are minor. Although faster than many other microscopy techniques, FTIR
micro spectroscopy is slower than FTIR spectroscopy. Microscopy is also slightly more expensive than standard FTIR
spectroscopy; however, considering the amount of information gained it is still inexpensive. FTIR micro spectroscopy can
be harder to automate and requires more training than other FTIR methods, especially for data analysis, which is not
always straightforward and may require additional multivariate techniques.

Micro spectroscopy has been developed mostly in connection with biomedical research, which is at the forefront of the
technique; however, other application areas are emerging quickly, ranging from art to forensics, and nanomaterials to
polymers ( AWE International (2012). http://www.awe imagazine.com/article.php?article_id=706).

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D. ULTRAVIOLET VISIBLE SPECTROSCOPY

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-Vis or UV/Vis) refers to absorption

spectroscopy or reflectance spectroscopy in the ultraviolet-visible spectral region. This means it uses light in the visible
and adjacent (near-UV and near-infrared [NIR]) ranges. The absorption or reflectance in the visible range directly affects
the perceived color of the chemicals involved. In this region of the electromagnetic spectrum, molecules undergo
electronic transitions. This technique is complementary to fluorescence spectroscopy, in that fluorescence deals with
transitions from the excited state to the ground state, while absorption measures transitions from the ground state to the
excited state.[1]

Principle of ultraviolet-visible absorption

Molecules containing π-electrons or non-bonding electrons (n-electrons) can absorb the energy in the form of ultraviolet or
visible light to excite these electrons to higher anti-bonding molecular orbitals.[2] The more easily excited the electrons (i.e.
lower energy gap between the HOMO and the LUMO), the longer the wavelength of light it can absorb.

Applications

An example of a UV/Vis readout

UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such
as transition metal ions, highly conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is
commonly carried out in solutions but solids and gases may also be studied.

• Solutions of transition metal ions can be colored (i.e., absorb visible light) because d electrons within the metal
atoms can be excited from one electronic state to another. The color of metal ion solutions is strongly affected by
the presence of other species, such as certain anions or ligands. For instance, the color of a dilute solution of
copper sulfate is a very light blue; adding ammonia intensifies the color and changes the wavelength of maximum
absorption (λmax).
• Organic compounds, especially those with a high degree of conjugation, also absorb light in the UV or visible
regions of the electromagnetic spectrum. The solvents for these determinations are often water for water-soluble
compounds, or ethanol for organic-soluble compounds. (Organic solvents may have significant UV absorption; not
all solvents are suitable for use in UV spectroscopy. Ethanol absorbs very weakly at most wavelengths.) Solvent
polarity and pH can affect the absorption spectrum of an organic compound. Tyrosine, for example, increases in
absorption maxima and molar extinction coefficient when pH increases from 6 to 13 or when solvent polarity
decreases.
• While charge transfer complexes also give rise to colors, the colors are often too intense to be used for
quantitative measurement.

A UV/Vis spectrophotometer may be used as a detector for HPLC. The presence of an analyte gives a response assumed
to be proportional to the concentration. For accurate results, the instrument's response to the analyte in the unknown
should be compared with the response to a standard; this is very similar to the use of calibration curves. The response
(e.g., peak height) for a particular concentration is known as the response factor.

The wavelengths of absorption peaks can be correlated with the types of bonds in a given molecule and are valuable in
determining the functional groups within a molecule. The Woodward-Fieser rules, for instance, are a set of empirical
observations used to predict λmax, the wavelength of the most intense UV/Vis absorption, for conjugated organic
compounds such as dienes and ketones. The spectrum alone is not, however, a specific test for any given sample. The
nature of the solvent, the pH of the solution, temperature, high electrolyte concentrations, and the presence of interfering
substances can influence the absorption spectrum. Experimental variations such as the slit width (effective bandwidth) of
the spectrophotometer will also alter the spectrum. To apply UV/Vis spectroscopy to analysis, these variables must be
controlled or accounted for in order to identify the substances present.[4]

UV-Vis spectroscopy is also used in the semiconductor industry to measure the thickness and optical properties of thin
films on a wafer. UV-Vis spectrometers are used to measure the reflectance of light, and can be analyzed via the Forouhi-
Bloomer dispersion equations to determine the Index of Refraction (n) and the Extinction Coefficient (k) of a given film
across the measured spectral range.[c

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https://ph.images.search.yahoo.com/images/view;_ylt=A2oKiHCWYBNVtEkA5021Rwx

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 Experiment No. 6
SPECTROPHOTOMETRY PART I

Introduction:

A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by
passing a beam of light through a sample and measuring the intensity of light reaching a detector.

The beam of light consists of a stream of photons, represented by the purple balls in the simulation shown below. When a
photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will
absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of
the light beam.

The light source is set to emit 10 photons per second. Watch the motion of the photons and observe how some of the
photons are absorbed (removed) as the beam of light passes through the cell containing the sample solution. The
intensity of the light reaching the detector is less than the intensity emitted by the light source.

I. OBJECTIVES: At the end of the activity, the students are expected to be able to:

1. To know the principles of spectrophotometric operation.

2. To gain the skills of manipulating the spectrophotometer.

II. MATERIALS: Please use any online reference or book material.

d. Spectrophotometer c. Micropipette with tips
e. Cuvettes d. Standards
e. Blank Solution

III. PROCEDURE

A. Examine the spectrophotometer and locate the following parts:

1. Temp. Controlled Cuvette Well 6. Keyboard

2. Cover 7. Power Switch
3. Incubator 8. Power Cord Connection
4. Printer Paper Well 9. Serial Port
5. Alphanumeric Display 10. Voltage Select Switch

IV. ACTIVITY

A. Drawing and Illustration

a. Draw and label the external and Internal parts of Stat Fox (digital)
b. Draw and label the operational system of Simple Photometry

B. Questions for Research

a. What is the use/importance of spectrophotometer in Forensic Science?

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 Experiment No. 7

OPERATIONAL SYSTEM OF SPECTROPHOTOMETER

Introduction:

A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by
passing a beam of light through a sample and measuring the intensity of light reaching a detector.

The beam of light consists of a stream of photons, represented by the purple balls in the simulation shown below. When a
photon encounters an analyte molecule (the analyte is the molecule being studied), there is a chance the analyte will
absorb the photon. This absorption reduces the number of photons in the beam of light, thereby reducing the intensity of
the light beam.

The light source is set to emit 10 photons per second. Watch the motion of the photons and observe how some of the
photons are absorbed (removed) as the beam of light passes through the cell containing the sample solution. The
intensity of the light reaching the detector is less than the intensity emitted by the light source.

I. OBJECTIVES: At the end of the Activity, the students are expected to be able to:

1. To know the principles of spectrophotometric operation.

2. To gain the skills of manipulating the spectrophotometer.

II. MATERIALS: Please use any online reference or book material.

a. Spectrophotometer c. Micropipette with tips
b. Cuvettes d. Standards
e. Blank Solution

III. PROCEDURE: The instructor will be uploading a video recording of the performance of the procedure.

A. Examine the spectrophotometer and locate the following parts:

1. Temp. Controlled Cuvette Well 6. Keyboard

2. Cover 7. Power Switch
3. Incubator 8. Power Cord Connection
4. Printer Paper Well 9. Serial Port
5. Alphanumeric Display 10. Voltage Select Switch

B. Operation of the Spectrophotometer (Stat Fac 1904)

1. To begin, turn the instrument on using the power switch on the rear panel.
2. The lamp begins to warm up as soon as it comes on. While entering parameters, the
45 second warm up is occurring simultaneously. The incubation block and cuvette well
are preset to maintain 37’C +/- 0.3’C. 37’C is attainable in the incubation block for
volumes of 2 ml or less; temperature-controlled assays should therefore use volumes
equal to or below 2 ml to achieve 37’C.
3. Press a mode key to select the desired automatic calculation:
Absorbance Mode ABS key (1)
Standard Mode STND Key (2)
4. The mode designations are located above the number keys. The printer will print the
name of the mode that was selected, and the display will indicate the next instruction.
Before reading begins in each mode, the instrument will momentarily reference air.
After that, it will recognize the insertion of a tube and read automatically. To cancel a
mode of operation at any time, press the CLEAR key twice.
5. Select the wavelength/filter by pressing the assigned number key as follows:

Key 1 340 nm Key 2 405 nm Key 3 450

Key 4 505 nm Key 5 545 nm Key 6 600 nm
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 5, If differential filter is not needed, press the 0 (zero) key, after pressing the key for the
desired differential wavelength, press the ENTER key.

Tips for Tube Reading:

a. Wipe any dust, moisture, or fingerprints from the tubes before using.
b. Do not read tubes that contain bubbles or condensation.
c. Use a blank material with absorbance of less than 0.400 A.
d. Use the same type and size of tubes for the blank as that was used for the samples.

Blanking:

During normal operation of this instrument in every mode, operator prompting via the display will indicate when to read
the blank tube. The blank tube’s absorbance is read and printed relative to air and then subtracted from each specimen.

IV. ACTIVITY

A. Draw/ Illustrate the External and Internal parts of Stat Fox 1904
B. Questions for Research

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 Experiment No. 8

OPERATIONAL SYSTEM OF SPECTROPHOTOMETER

(Enzyme Linked Immuno Sorbent Assay – ELISA)

Introduction:

ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying
peptides, proteins, antibodies and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then
complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme
activity via incubation with a substrate to produce a measureable product. The most crucial element of the detection
strategy is a highly specific antibody-antigen interaction.

ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and
proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the
reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material
during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for
measuring specific analytes within a crude preparation (https://www.bosterbio.com/protocol-and-troubleshooting/elisa-
principle).

I. OBJECTIVES

4. To know the principle of ELISA operation.

5. To gain the skills of manipulating the spectrophotometer for ELISA determination.

II. MATERIALS
f. Spectrophotometer c. Micropipette with tips
g. Cuvettes d. Standards
e. Blank Solution
f. Micro tubes

III. PROCEDURE

ELISA Step-by-step

1. Antibody coating
Specific capture antibody is immobilized on high protein-binding plates by overnight incubation. Plates are blocked
with irrelevant protein e.g., albumin.
This step is omitted when using Mabtech's pre-coated plates.
2. Protein capture
Samples and standard dilutions are added to the wells and will be captured by the bound antibodies.
3. Detection antibody
Specific biotinylated detection antibody is added to the wells to enable detection of the captured protein.

4. Streptavidin-enzyme conjugate
Streptavidin conjugated with alkaline phosphatase or horseradish peroxidase is added to the wells and will bind to
the biotinylated antibody. Learn more about enzymes and substrates in our tutorials and guidelines.
5. Addition of substrate
Colorimetric substrate is added to the wells and will form a colored solution when catalyzed by the enzyme.

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6. Analysis
Absorbance is measured in an ELISA reader and the amount of protein in the samples is determined. Learn more in
our ELISA analysis guidelines (https://www.mabtech.com/ knowledge-center/assay-principles/elisa-assay-principle/elisa-
step-step).

IV. ACTIVITY: The instructor will be giving a recorded video of the demonstration measurement the concentration of
thyroxin (T4) from human blood applying the principle of ELISA technique.

1) Draw and Label the External and Internal parts of Stat Fax for ELISA Determination.
2) Draw Schematically the Operating Principle involved in ELISA reaction.

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 Chapter IV. Chromatography

I. CHROMATOGRAPHY

Chromatography as a technique for purifying substances is particularly useful for analyzing the multicomponent
specimens that are frequently received in the crime laboratory. For example, illicit drugs sold on the street are not
manufactured to meet government labeling standards; instead, they may be diluted with practically any material at the
disposal of the drug dealer to increase the quantity of product available to prospective customers. Hence, the task of
identifying an illicit-drug preparation would be arduous without the aid of chromatographic methods to first separate the
mixture into its components.

The theory of chromatography is based on the observation that chemical substances tend to partially escape into
the surrounding environment when dissolved in a liquid or when absorbed on a solid surface. This is best illustrated by a
gas dissolved in a beaker of water kept at a constant temperature. It will be convenient for us to characterize the water in
the beaker as the liquid phase and the air above it as the gas phase. If the beaker is covered with a bell jar, some of the
gas molecules escape from the water into the surrounding enclosed air. The molecules that remain are said to be in the
liquid phase; the molecules that have escaped into the air are said to be in the gas phase. As the gas molecules escape
into the surrounding air, they accumulate above the water; here, random motion carries some of them back into the water.

Eventually, a point is reached at which the number of molecules leaving the water is equal to the number
returning. At this time, the liquid and gas phases are in equilibrium. If the temperature of the water is increased, the
equilibrium state readjusts itself to a point at which more gas molecules move into the gas phase. This behavior was first
observed in 1803 by a British chemist, William Henry. His explanation of this phenomenon, known appropriately as
Henry’s law, may be stated as follows:

When a volatile chemical compound is dissolved in a liquid and is brought to equilibrium with air, there is a fixed ratio
between the concentration of the volatile compound in air and its concentration in the liquid, and this ratio remains
constant for a given temperature.

The distribution or partitioning of a gas between the liquid and gas phases is determined by the solubility of the
gas in the liquid. The higher its solubility, the greater the tendency of the gas molecules to remain in the liquid phase. If
two different gases are simultaneously dissolved in the same liquid, each will reach a state of equilibrium with the
surrounding air independently of the other. For example, gas A (green balls) and gas B (blue balls) are both dissolved in
water. At equilibrium, gas A has a greater number of molecules dissolved in the water than does gas B. This is so
because gas A is more soluble in water than gas B. Now return to the concept of chromatography. both phases—liquid
and gas—were kept stationary; that is, they were not moving. During a chromatographic process, this is not the case;
instead, one phase is always made to move continuously in one direction over a stationary or fixed phase.
Simply, we can think of chromatography as being analogous to a race between chemical
compounds. At the starting line, all the participating substances are mixed together; however, as

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the race progresses, materials that prefer the moving phase slowly pull ahead of those that prefer to remain in the
stationary phase. Finally, at the end of the race, all the participants are separated, each crossing the finish line at different
times.

The different types of chromatographic systems are as varied as the number of stationary and moving-phase
combinations that can be devised. However, three chromatographic processes—gas chromatography, high-performance
liquid chromatography, and thin-layer chromatography—are most applicable for solving many analytical problems in the
crime laboratory.

A. Gas Chromatography (GC)

Gas chromatography (GC) separates mixtures on the basis of their distribution between a stationary liquid phase
and a moving gas phase. This technique is widely used because of its ability to resolve a highly complex mixture into its
components, usually within minutes.
In gas chromatography, the moving phase is actually a gas called the carrier gas, which
flows through a column constructed of stainless steel or glass. The stationary phase is a thin film of liquid within the
column.

Two types of columns are used:

1. Packed Column - With the packed column, the stationary phase is a thin film of liquid that is fixed onto small granular
particles packed into the column. This column is usually constructed of stainless steel or glass and is 2 to 6 meters in
length and about 3 millimeters in diameter. Capillary columns are composed of glass and are much longer than packed
columns—15 to 60 meters in length. These types of columns are very narrow, ranging from 0.25 to 0.75 millimeter in
diameter.
2. Capillary Column - can be made narrower than packed columns because their stationary liquid phase is actually
coated as a very thin film directly onto the column’s inner wall. In any case, as the carrier gas flows through the packed or
capillary column, it carries with it the components of a mixture that have been injected into the column. Components with
a greater affinity for the moving gas phase travel through the column more quickly than those with a greater affinity for the
stationary liquid phase. Eventually, after the mixture has traversed the length of the column, it emerges separated into its
components.

A simplified scheme of the gas chromatograph is shown in Figure 5–5. The operation of the instrument can be
summed up briefly as follows: A gas stream, the so-called carrier gas, is fed into the column at a constant rate. The carrier
gas is chemically inert and is generally nitrogen or helium. The sample under investigation is injected as a liquid into a
heated injection port with a syringe, where it is immediately vaporized and swept into the column by the carrier gas. The
column itself is heated in an oven in order to keep the sample in a vapor state as it travels through the column. In the
column, the components of the sample travel in the direction of the carrier gas flow at speeds that are determined by their
distribution between the stationary and moving phases.

If the analyst has selected the proper liquid phase and has made the column long

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enough, the components of the sample will be completely separated as they emerge from the
column. As each component emerges from the column, it enters a detector. One type of detector uses a flame to ionize
the emerging chemical substance, thus generating an electrical signal. The signal is recorded onto a strip-chart recorder
as a function of time. This written record of the separation is called a chromatogram. A gas chromatogram is a plot of
the recorder response (vertical axis) versus time (horizontal axis). A typical chromatogram shows a series of peaks, each
peak corresponding to one component of the mixture. The time required for a component to emerge from the column from
the time of its injection into the column is known as the retention time, which is a useful identifying characteristic of a
material. However, because other substances may have comparable retention times under similar chromatographic
conditions, gas chromatography cannot be considered an absolute means of identification.

Conclusions derived from this technique must be confirmed by other testing procedures.
An added advantage of gas chromatography is that it is extremely sensitive and can yield quantitative results. The amount
of substance passing through the GC detector is proportional to the peak area recorded; therefore, by chromatographing
a known concentration of a material and comparing it to the unknown, the amount of the sample may be determined by
proportion. Gas chromatography has sufficient sensitivity to detect and quantitate materials at the nanogram
(0.000000001 gram or 1 × 10–29 gram) level.1

An important extension of the application of gas chromatography to forensic science is the technique of pyrolysis
gas chromatography. Many solid materials commonly encountered as physical evidence—for example, paint chips, fibers,
and plastics—cannot be readily dissolved in a solvent for injection into the gas chromatograph. Thus, under normal
conditions these substances cannot be subjected to gas chromatographic analysis. However, materials such as these can
be heated or pyrolyzed to high temperatures (500–1000°C) so that they will decompose into numerous gaseous products.
Pyrolyzers permit these gaseous products to enter the carrier gas stream, where they flow into and through the GC
column. The pyrolyzed material can then be characterized by the pattern produced by its chromatogram or pyrogram.

B. High-Performance Liquid Chromatography (HPLC)

Recall that a chromatographic system requires a moving phase and a stationary phase in contact with each other.
The previous section described gas chromatography, in which the stationary phase is a thin film and the moving phase is
a gas. However, by changing the nature of these phases, one can create different forms of chromatography. One form
finding increasing utility in crime laboratories is high-performance liquid chromatography (HPLC). Its moving phase is a
liquid that is pumped through a column filled with fine solid particles. In one form of HPLC, the surfaces of these solid
particles are chemically treated and act as the stationary phase. As the liquid moving phase is pumped through the
column, a sample is injected into the column. As the liquid carries the sample through the column, different components
are retarded to different degrees, depending on their interaction with the stationary phase. This leads to a separation of
the different components making up the sample mixture. The major advantage of HPLC is that the entire process takes
place at room temperature.

With GC, the sample must first be vaporized and made to travel through a heated column. Hence, any materials
sensitive to high temperatures may not survive their passage through the column. In such situations, the analyst may turn

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to HPLC as the method of choice. Organic explosives are generally heat sensitive and therefore more readily separated
by HPLC. Likewise, heat-sensitive drugs, such as LSD, lend themselves to analysis by HPLC.

C. Thin-Layer Chromatography (TLC)

The technique of thin-layer chromatography (TLC) uses a solid stationary phase and a moving liquid phase to
separate the constituents of a mixture. A thin-layer plate is prepared by coating a glass plate with a thin film of a granular
material, usually silica gel or aluminum oxide. This granular material serves as the solid stationary phase and is usually
held in place on the plate with a binding agent such as plaster of Paris. If the sample to be analyzed is a solid, it must
first be dissolved in a suitable solvent and a few microliters of the solution spotted with a capillary tube onto the granular
surface near the lower edge of the plate. A liquid sample may be applied directly to the plate in the same manner. The
plate is then placed upright into a closed chamber that contains a selected liquid, with care that the liquid does not touch
the sample spot.
The liquid slowly rises up the plate by capillary action. This rising liquid is the moving phase
in thin-layer chromatography. As the liquid moves past the sample spot, the components of the
sample become distributed between the stationary solid phase and the moving liquid phase. The components with the
greatest affinity for the moving phase travel up the plate faster than those that have greater affinity for the stationary
phase. When the liquid front has moved a sufficient distance (usually 10 cm), the development is complete, and the plate
is removed from the chamber and dried

Because most compounds are colorless, no separation will be noticed after development
unless the materials are visualized. To accomplish this, the plates are placed under ultraviolet
light, revealing select materials that fluoresce as bright spots on a dark background. When a
fluorescent dye has been incorporated into the solid phase; non-fluorescent substances appear as dark spots against a
fluorescent background when exposed to the ultraviolet light.

In a second method of visualization, the plate is sprayed with a chemical reagent that reacts with the separated
substances and causes them to form colored spots. Figure 5–10 shows the chromatogram of a marijuana extract that has
been separated into its components by TLC and visualized by having been sprayed with a chemical reagent.

Once the components of a sample have been separated, their identification must follow. For this, the questioned
sample must be developed alongside an authentic or standard sample on the same TLC plate. If both the standard and
the unknown travel the same distance up the plate from their origins, they can tentatively be identified as being the same.
For example, suppose a sample suspected of containing heroin and quinine is chromatographed alongside known heroin
and qui nine standards, as shown in Figure 5–11. The identity of the suspect material is confirmed by comparing the
migration distances of the heroin and quinine standards against those of the components of the unknown material. If the
distances are the same, a tentative identification can be made. However, such an identification cannot be considered
definitive, for numerous other substances can migrate the same distance up the plate when chromatographed under
similar conditions.

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 Thus, thin-layer chromatography alone cannot provide an absolute identification; it must
be used in conjunction with other testing procedures to prove absolute identity.
The distance a spot has traveled up a thin-layer plate can be assigned a numerical value
known as the Rf value. This value is defined as the distance traveled by the component divided
by the distance traveled by the moving liquid phase. For example, in Figure 5–11 the moving
phase traveled 10 centimeters up the plate before the plate was removed from the tank. After
visualization, the heroin spot moved 8 centimeters, which has an Rf value of 0.8; the quinine migrated 4 centimeters, for
an Rf value of 0.4. Thousands of possible combinations of liquid and solid phases can be chosen in thin-layer
chromatography. Fortunately, years of research have produced much published data relating to the proper selection of
TLC conditions for separating and identifying specific classes of substances—for example, drugs, dyes, and petroleum
products. Th ese references, along with the experience of the analyst, will aid in the proper selection of TLC conditions for
specific problems.

Thin-layer chromatography is a powerful tool for solving many of the analytical problems
presented to the forensic scientist. The method is both rapid and sensitive; moreover, less than
100 micrograms of suspect material are required for the analysis. In addition, the equipment necessary for TLC work has
minimal cost and space requirements. Importantly, numerous samples can be analyzed simultaneously on one thin-layer
plate. The principal application of this technique is in the detection and identification of components in complex mixtures.

II. ELECTROPHORESIS

Electrophoresis is somewhat related to thin-layer chromatography in that it separates materials according to

their migration rates on a stationary solid phase. However, it does not use a moving liquid phase to move the material;
instead, an electrical potential is placed across the stationary medium. The nature of this medium can vary; most forensic
applications call for a starch or agar gel coated onto a glass plate. Under these conditions, only substances that possess
an electrical charge migrate across the stationary phase (see Figure 5–12). The technique is particularly useful for
separating and identifying complex biochemical mixtures. In forensic science, electrophoresis finds its most successful
application in the characterization of proteins and DNA in dried blood.

Because many substances in blood carry an electrical charge, they can be separated and identified by
electrophoresis. Forensic serologists have developed several electrophoretic procedures for characterizing dried blood.
Many enzymes present in blood are actually composed of distinct proteins that can be separated by electrophoresis on
starch gel. These proteins migrate on the plate at speeds that vary according to their electrical charge and size. After
completion of the electrophoresis run, the separated proteins are stained with a suitable developing agent for visual
observation. In this manner, characteristic band patterns are obtained that are related to the enzyme type present in the
blood. Likewise, , mixtures of DNA fragments can be separated by gel electrophoresis by taking advantage of the fact that
the rate of movement of DNA across a gel-coated plate depends on the molecule’s size. Smaller DNA fragments move at
a faster rate along the plate than larger DNA fragments.

III. MASS SPECTROMETRY

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 This instrument is one of the most important tools in a crime laboratory. Its ability to separate the components of a
complex mixture is unsurpassed. However, gas chromatography (GC) does have one important drawback—its inability to
produce specific identification. A forensic chemist cannot unequivocally state the identification of a substance based solely
on a retention time as determined by the gas chromatograph. Fortunately, by coupling the gas chromatograph to a mass
spectrometer this problem has largely been overcome.

The separation of a mixture’s components is first accomplished on the gas chromatograph. A direct connection
between the GC column and the mass spectrometer then allows each component to flow into the spectrometer as it
emerges from the gas chromatograph. In the mass spectrometer, the material enters a high-vacuum chamber where a
beam of high-energy electrons is aimed at the sample molecules. The electrons collide with the molecules, causing them
to lose electrons and to acquire a positive charge (commonly called ions). These positively charged molecules or ions are
very unstable or are formed with excess energy and almost instantaneously decompose into numerous smaller fragments.
The fragments then pass through an electric or magnetic field, where they are separated according to their masses. The
unique feature of mass spectrometry is that under carefully controlled conditions, no two substances produce
the same fragmentation pattern. In essence, one can think of this pattern as a “fingerprint” of the substance being
examined. The technique thus provides a specific means for identifying a chemical structure. It is also sensitive to minute
concentrations. At present, mass spectrometry finds its widest application in the identification of drugs; however, further
research is expected to yield significant applications for identifying other types of physical evidence. The combination of
the gas chromatograph and mass spectrometer is further enhanced when a computer is added to the system. The
integrated gas chromatograph/mass spectrometer/ computer system provides the ultimate in speed, accuracy, and
sensitivity. With the ability to record and store in its memory several hundred mass spectra, such a system can detect and
identify substances present in only one-millionth-of-a-gram quantities. Furthermore, the computer can be programmed to
compare an unknown spectrum against a comprehensive library of mass spectra stored in its memory. The advent of
personal computers and microcircuitry has made it possible to design mass spectrometer systems that can fit on a small
table.

IV. ULTRAVIOLET, VISIBLE, AND INFRARED SPECTROPHOTOMETRY

ULTRAVIOLET AND VISIBLE SPECTROPHOTOMETR measure the absorbance of UV and visible light as a
function of wavelength or frequency. For example, the UV absorption spectrum of heroin shows a maximum absorption
band at a wavelength of 278 nanometers. This shows that the simplicity of a UV spectrum facilitates its use as a tool for
determining a material’s probable identity. For instance, a white powder may have a UV spectrum comparable to heroin
and therefore may be tentatively identified as such. (Fortunately, sugar and starch, common diluents of heroin, do not
absorb UV light.) However, this technique will not provide a definitive result; other drugs or materials may have a UV
absorption spectrum similar to that of heroin. But this lack of specificity does not diminish the value of the technique, for
the analyst has quickly eliminated thousands of other possible drugs from consideration and can now proceed to conduct
other confirmatory tests, such as thin-layer or gas chromatography, to complete the identification.

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 In contrast to the simplicity of a UV spectrum, absorption in the infrared region provides a far more complex
pattern. Example, the IR spectra of heroin and secobarbital shows that
the absorption bands are so numerous that each spectrum can provide enough characteristics to identify a substance
specifically. Different materials always have distinctively different infrared spectra; each IR spectrum is therefore
equivalent to a “fingerprint” of that substance and no other.

This technique is one of the few tests available to the forensic scientist that
can be considered specific in itself for identification. The IR spectra of thousands of organic
compounds have been collected, indexed, and cataloged to serve as invaluable references for
identifying organic substances.

SUMMARY

The proper selection of analytical techniques that will allow the forensic scientist to identify or compare matter can
best be understood by categorizing all substances into one of two broad groups: organics and inorganics. In general,
organic substances contain carbon. Inorganic materials encompass all other known chemical substances. Another
consideration in selecting an analytical technique is the need for either a qualitative or a quantitative determination. The
former relates just to the identity of the material, whereas the latter requires the determination of the percent composition
of the components of a mixture.

Chromatography, spectrophotometry, and mass spectrometry are all readily used by a forensic scientist to identify
or compare organic materials. Chromatography is a means of separating and tentatively identifying the components of a
mixture. Spectrophotometry is the study of the absorption of light by chemical substances. Mass spectrometry
characterizes organic molecules by observing their fragmentation pattern after their collision with a beam of high-energy
electrons. Gas chromatography (GC) separates mixtures on the basis of their distribution between a stationary liquid
phase and a mobile gas phase. In GC, the moving phase is actually a gas called the carrier gas, which flows through a
column. The stationary phase is a thin film of liquid contained within the column. After a mixture has traversed the length
of the column, it emerges separated into its components. The written record of this separation is called a chromatogram.
A direct connection between the GC column and the mass spectrometer allows each component to flow into the mass
spectrometer as it emerges from the GC. Fragmentation of each component by high-energy electrons produces a
“fingerprint” pattern of the substance being examined.

Other forms of chromatography applicable to forensic science are high-performance liquid chromatography
(HPLC) and thin-layer chromatography (TLC). HPLC separates compounds using a stationary phase and a mobile liquid
phase and is used with temperature-sensitive compounds. TLC uses a solid stationary phase, usually coated onto a glass
plate, and a mobile liquid phase to separate the components of the mixture. A technique analogous to TLC is
electrophoresis, in which materials are forced to move across a gel-coated plate under the influence of an electrical
potential. In this manner, substances such as proteins and DNA can be separated and characterized.

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 Most forensic laboratories use ultraviolet (UV) and infrared (IR) spectrophotometers to characterize chemical
compounds. In contrast to the simplicity of a UV spectrum, absorption in the infrared region provides a far more complex
pattern. Different materials always have distinctively different infrared spectra; each IR spectrum is therefore equivalent to
a “fingerprint” of that substance.

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 Experiment No. 9

POLYMERASE CHAIN REACTION

(PCR)

Introduction:

Polymerase chain reaction (PCR) is a method of DNA replication in which a target DNA sequence is amplified to several
million copies in just a few hours. PCR method relies on thermal cycling which exposes the reactants to varying
temperature conditions to allow temperature – dependent reactions to occur (Molecular Biology, UST Biology Teachers.

I. OBJECTIVES

1. To know the principle of PCR operation.

2. To gain the conceptual skills in amplifying the gene of interest
Eg. Apolipoprotein B (ApoB)

II. MATERIALS

a. Text books c. E-books

b. Illustrations d. Library work

III. PROCEDURE

a. Each group will perform a PCR reaction on the extracted DNA using ApoB specific primer.
b. When all components of PCR are combined, assemble such preparation in the thermal cycler.
c. In the thermal cycler, enter the PCR conditions indicated in the table below:

PCR Conditions

Temperature Step Time Cycle

50’C incubation 2 minutes 40
95’C Pre-denaturation 10 minutes 40
95’C Denaturation 15 sec 40
60’C Annealing 60 sec 40
72’C Extension 30 sec 40
72’C Final extension 30 sec 40
4’C

d. Load the 0.2 ml tubes with the PCR mix in their thermal cycler. Run the protocol

IV. ACTIVITY:

3) Draw and Label the External and Internal parts of a thermal cycler.
4) Draw Schematically the Principle involved in PCR reaction.

Page | 47
 Experiment No. 10

SEPARATION TECHNIQUES USED FORENSIC SCIENCE

GEL ELECTROPHORESIS

Introduction:

Gel electrophoresis is a method of separating DNA according to molecular size. The DNA molecules are separated by
passing an electrical field through a porous gel (agarose). The one end of the electrical field is negatively charged and the
other end is positively charged. Since DNA is negatively charged, DNA will be pulled towards the positively charged end
of the gel. The travel speed of the DNA molecules through the porous membrane is inversely proportional to its length
(DNA). Meaning the smaller molecules of DNA travel faster compared to larger molecules.

I. OBJECTIVES

a. To evaluate the importance of Gel Electrophoresis as a separation technique

b. To display patience in performing an activity to determine if the gene of interest has been amplified.
..
II. MATERIALS

a. Reference Materials
b. Books
c. Library Work

III. PROCEDURE

1. Run the agarose gel at 100 V for 1 hour and 25 minutes.

2. Visualize the DNA bands using a gel documentation system (gel doc).

IV, ACTIVITY

a. Schematic illustration of the DNA bands using gel documentation system (gel doc).
b. Illustrate the picture of Electrophoresis apparatus with DNA gel stain, loading dye and DNA ladder.

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 Experiment No. 11

SEPARATION TECHNIQUES USED FORENSIC SCIENCE

PAPER CHROMATOGRAPHY

Introduction:

Few methods of chemical analysis are truly specific to a particular analyte. It is often found that the analyte of interest
must be separated from the myriad of individual compounds that may be present in a sample. As well as providing the
analytical scientist with methods of separation, chromatographic techniques can also provide methods of analysis.

Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a
gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase.
The phases are chosen such that components of the sample have differing solubilities in each phase. A component which
is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in
the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample
components will become separated from each other as they travel through the stationary phase.

Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas Chromatography)
use columns – narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is
transported through the column by continuous addition of mobile phase. This process is called elution. The average rate
at which an analyte move through the column is determined by the time it spends in the mobile phase.

Chromatography is a method for analyzing complex mixtures (such as ink) by separating them into the
chemical from which they are made. Chromatography is used to separate and identify all sorts of substances in police
work. Drugs from narcotics to aspirin can be identified in urine and blood samples, often with the aid of chromatography.

The relative degrees of separation of the components of a mixture may be expressed by comparing their rate
factors or ratio of front or Rf values. To compute the Rf value, use the following formula:

Rf value = Distance the compound traveled

Distance the solvent traveled

I. OBJECTIVES

a. To evaluate the importance of paper chromatography as a separation technique

b. To display patience in performing an activity
c. To separate components of a mixture using paper chromatography
..

II. MATERIALS
1. Four Test tubes Felt-tipped pen
2. Aluminum foil 95% ethyl alcohol
3. Four strips of Whatman filter paper Distilled water
4. Corks 70% isopropyl alcohol
5. Test tube rack Ether
6. Cutter scissors
7.

III. PROCEDURE
Page | 49
1. Preparation of the paper chromatogram

a. Using your scissors, cut the filter paper in such a way that it will fit the test tubes without touching the sides and
the bottom

b. Using a pencil, lightly draw a line 1 cm or less above the edge of the Whatman filter paper
c. Gently and quickly touch the center of the line with the point of your felt-tipped pen. The area touched should
not exceed the width of the filter paper
d. Repeat step ‘b’ by applying the ink on the same mark 4-6 times allowing the spots to dry before each
application
e. Prepare the other three filter papers similarly.
f. Get four corks (it should fit your test tubes)
g. Slit the bottom of the cork carefully with a cutter
h. Insert the paper chromatogram in the slit, securing the paper firmly

2. Preparation of the developing chambers

a. Prepare four test tubes and label them according to the solvent inside

b. Place 1 mL of 95% ethyl alcohol on the first test tube

c. Place 1 mL of Distilled water on the second test tube
d. Place 1 mL of 70% isopropyl alcohol on the third test tube
e. Place 1 mL of ether on the fourth test tube

3. Development of the spot

a. With careful handling inserts the chromatogram in your developing chambers using the cork as handle
b. Make sure that the chromatogram is vertical and does not touch the side of the test tube
c. Only the edge of the filter paper should touch the solvent and the solvent should not touch the spot in any
way!
d. Secure the cork on top of the test tube and let stand undisturbed for 30 – 45 minutes or until the solvent front
is 1 cm away from the upper edge and there is already visible separation of the ink

Note: Because molecules in ink and other mixtures have different characteristics (such as size and solubility),
they travel at different speeds when pulled along a piece of paper by a solvent. For example, black ink contains
several colors. When the solvent flows through a word written in black, the molecules of each one of the colors
behave differently, resulting in a sort of “rainbow” effect.
Many common inks are water soluble and spread apart into the component dyes using water as a solvent. If
the ink you are testing does not spread-out using water, it may be “permanent” ink. In such cases, you will have to
use a different solvent.

a. Carefully remove the paper from the test tube

b. Mark the position of the solvent before it dries up
c. Dry the developed chromatogram completely
d. Using a scotch tape, attach your developed chromatogram at the back of this page and label properly
e. Draw the set-up at the back of this page

e. Calculation of the Rf value

a. Measure the distance of each spot in cm from the origin of the sample
b. Measure the distance traveled by the solvent

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 c. Calculate the Rf value using the above formula for each chromatogram
d. Show your calculations for the four different solvents at back of this page.

f. Observations:
a. Compare the four chromatograms
________________________________________________________________________________
________________________________________________________________________________
________________________________

b. To which solvent was a good separation achieved?

___________________________________________________________

IV, ACTIVITY

c. Schematic illustration of the procedure.

d. Illustrate each result in different solutions before and after chromatography

1. The name chromatography indicates that originally it was a method having something
to do with color. Amino acids are colorless compounds. How can they be separated?
using paper chromatography?
2. Describe the mobile phase and the stationary phase of chromatography
3. State the Rf value and give its importance in chromatography
4. Differentiate Absorption from Adsorption and give an example of each process.

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 Chapter V. TOXICOLOGY OF ALCOHOL

The Fate of Alcohol in the Body

The subject of the analysis of alcohol immediately confronts us with the primary objective of
forensic toxicology—the detection and isolation of drugs in the body to determine their influence
on human behavior. In the case of alcohol, however, the problem is further complicated by practical considerations. The
predominant role of the automobile in our society has mandated the imposition of laws to protect the public from the
drinking driver. This has meant that toxicologist shave had to devise rapid and specific procedures for measuring the
degree of alcohol intoxication. The methods used must be suitably designed to test hundreds of thousands of motorists
annually without causing them undue physical harm or unreasonable inconvenience, while at the same time providing a
reliable diagnosis that can be supported and defended within the framework of the legal system.

Alcohol, or ethyl alcohol, is a colorless liquid normally diluted with water and consumed as a
beverage. Logically, the most obvious measure of intoxication would be the amount of liquor a
person has consumed. Unfortunately, most arrests are made after the fact, when such information is not available to legal
authorities; furthermore, even if these data could be collected, numerous related factors, such as body weight and the rate
of alcohol’s absorption into the body, are so variable that it would be impossible to prescribe uniform standards that
would yield reliable alcohol intoxication levels.

Like any other depressant, alcohol primarily affects the central nervous system, particularly
the brain. The extent of the depression is proportional to the concentration of alcohol within the
nerve cells. The nerve functions most susceptible to alcohol are found in the surface areas of the forebrain. Later, as the
person absorbs alcohol to a greater extent, the functions of the central and rear portions of the brain are affected. The
nerve functions that are most resistant, and the last to fail, are centered in the brain’s medulla, which regulates such vital
functions as respiration and heart activity.

Theoretically, for a true determination of the quantity of alcohol impairing an individual’s

normal body functions, it would be best to remove a portion of brain tissue and analyze it for alcohol content. For obvious
reasons, this cannot be done on living subjects. Consequently, toxicologists concentrate on the blood, which provides the
medium for circulating alcohol throughout the body, carrying it to all tissues, including the brain. Fortunately, experimental
evidence supports this approach and shows blood-alcohol concentration to be directly proportional to the concentration of
alcohol in the brain. From the medicolegal point of view, blood-alcohol levels have become the accepted standard for
relating alcohol intake to its effect on the body.

Alcohol appears in the blood within minutes after it has been consumed and slowly increases
in concentration while it is being absorbed from the stomach and the small intestine into the
bloodstream. When all the alcohol has been absorbed, a maximum alcohol level is reached in the blood, and the post-
absorption period begins. Then the alcohol concentration slowly decreases until a zero level is again reached.

Many factors determine the rate at which alcohol is absorbed into the bloodstream, including
the total time taken to consume the drink, the alcohol content of the beverage, the amount consumed, and the quantity
and type of food present in the stomach at the time of drinking. With so many variables, it is difficult to predict just how
long the absorption process will require. For
example, beer is absorbed more slowly than an equivalent concentration of alcohol in water, apparently because of the
carbohydrates present in beer. Also, alcohol consumed on an empty
stomach is absorbed faster than an equivalent amount of alcohol taken when there is food in the
stomach. The longer the total time required for complete absorption to occur, the lower the peak
alcohol concentration in the blood. Depending on a combination of factors,
maximum blood-alcohol concentration may not be reached until two or three hours have elapsed from the time of
consumption. However, under normal social drinking conditions, it takes anywhere from thirty to ninety minutes from the
time of the final drink until the absorption process is completed.

During the absorption phase, alcohol slowly enters the body’s bloodstream and is carried to
all parts of the body. When the absorption period is completed, the alcohol becomes distributed
uniformly throughout the watery portions of the body—that is, throughout about two-thirds of
the body volume. Fat, bones, and hair are low in water content and therefore contain little alcohol, whereas alcohol
concentration in the rest of the body is fairly uniform. Hence, if blood is not available, as in some postmortem situations, a
medical examiner can select a water-rich organ or fluid—for example, the brain, cerebrospinal fluid, or vitreous humor—
for determining the body’s alcohol content to a reasonable degree of accuracy.
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As the alcohol is circulated by the bloodstream, the body begins to eliminate it. Alcohol is
eliminated through two mechanisms—oxidation and excretion. Nearly all of the alcohol (95–98
percent) consumed is eventually oxidized to carbon dioxide and water. Oxidation takes place almost entirely in the liver.
Here, in the presence of the enzyme alcohol dehydrogenase, the alcohol is converted into acetaldehyde and then to
acetic acid. The acetic acid is subsequently oxidized in practically all parts of the body to carbon dioxide and water.
The remaining alcohol is excreted unchanged in the breath, urine, and perspiration. Most significantly, the amount of
alcohol exhaled in the breath is in direct proportion to the concentration of alcohol in the blood. This observation has had a
tremendous impact on the technology and procedures used for blood-alcohol testing. The development of instruments to
reliably measure breath for its alcohol content has made possible the testing of millions of people in a rapid, safe, and
convenient manner.

The fate of alcohol in the body is therefore relatively simple—namely, absorption into
the bloodstream, distribution throughout the body’s water, and finally, elimination by oxidation and excretion.
The elimination or “burn-off” rate of alcohol varies in different individuals; 0.015 percent w/v (weight per volume) per hour
seems to be average once the absorption process is complete. However, this figure is an average that varies by as much
as 30 percent among individuals.

Alcohol in the Circulatory System

The extent to which an individual may be under the influence of alcohol is usually determined by
measuring the quantity of alcohol present in the blood system. Normally, this is accomplished in
one of two ways: (1) by direct chemical analysis of the blood for its alcohol content and (2) by
measurement of the alcohol content of the breath. In either case, the significance and meaning of the results can better be
understood when the movement of alcohol through the circulatory system is studied.

Humans, like all vertebrates, have a closed circulatory system, which consists basically of a
heart and numerous arteries, capillaries, and veins. An artery is a blood vessel carrying blood
away from the heart, and a vein is a vessel carrying blood back toward the heart. Capillaries are tiny blood vessels that
interconnect the arteries with the veins. The exchange of materials between the blood and the other tissues takes place
across the thin walls of the capillaries.

Let us now trace the movement of alcohol through the human circulatory system. After alcohol
is ingested, it moves down the esophagus into the stomach. About 20 percent of the alcohol
is absorbed through the stomach walls into the portal vein of the blood system. The remaining
alcohol passes into the blood through the walls of the small intestine. Once in the blood, the alcohol is carried to the liver,
where its destruction starts as the blood (carrying the alcohol) moves up to the heart. The blood enters the upper right
chamber of the heart, called the right atrium (or auricle), and is forced into the lower right chamber of the heart, known as
the right ventricle. Having returned to the heart from its circulation through the tissues, the blood at this time contains very
little oxygen and much carbon dioxide. Consequently, the blood must be pumped up to the lungs, through the pulmonary
artery, to be replenished with oxygen.

The respiratory system bridges with the circulatory system in the lungs, so that oxygen can
enter the blood and carbon dioxide can leave it. The pulmonary artery branches into capillaries lying close to tiny pear-
shaped sacs called alveoli. There are about 250 million alveoli in the lungs, all located at the ends of the bronchial tubes.
The bronchial tubes connect to the windpipe (trachea), which leads up to the mouth and nose. At the surface of the
alveolar sacs, blood flowing through the capillaries comes in contact with fresh oxygenated air in the sacs. A rapid
exchange now proceeds to take place between the fresh air in the sacs and the spent air in the blood. Oxygen passes
through the walls of the alveoli into the blood while carbon dioxide is discharged from the blood into the air. If, during this
exchange, alcohol or any other volatile substance is in the blood, it too will pass into the alveoli. During breathing, the
carbon dioxide and alcohol are expelled through the nose and mouth, and the alveoli sacs are replenished with fresh
oxygenated air breathed into the lungs, allowing the process to begin all over again.

The distribution of alcohol between the blood and alveolar air is similar to the example of a
gas dissolved in an enclosed beaker of water. Here again, one can use Henry’s law to explain how the alcohol divides
itself between the air and blood. Henry’s law may now be restated as follows:
When a volatile chemical (alcohol) is dissolved in a liquid (blood) and is brought to equilibrium with air (alveolar
breath), there is a fixed ratio between the concentration of the volatile compound (alcohol) in air (alveolar breath)
and its concentration in the liquid (blood), and this ratio is constant for a given temperature.

Page | 53
The temperature at which the breath leaves the mouth is normally 34°C. At this temperature,
experimental evidence has shown that the ratio of alcohol in the blood to alcohol in
alveoli air is approximately 2,100 to 1. In other words, 1 milliliter of blood will contain
nearly the same amount of alcohol as 2,100 milliliters of alveolar breath. Henry’s law thus
becomes a basis for relating breath to blood-alcohol concentration.

Now let’s return to the circulating blood. After emerging from the lungs, the oxygenated
blood is rushed back to the upper left chamber of the heart (left atrium) by the pulmonary vein.
When the left atrium contracts, it forces the blood through a valve into the left ventricle, which is
the lower left chamber of the heart. The left ventricle then pumps the freshly oxygenated blood
into the arteries, which carry the blood to all parts of the body. Each of these arteries, in turn,
branches into smaller arteries, which eventually connect with the numerous tiny capillaries embedded in the tissues. Here
the alcohol moves out of the blood and into the tissues. The blood then runs from the capillaries into tiny veins that fuse to
form larger veins. These veins eventually lead back to the heart to complete the circuit.

During absorption, the concentration of alcohol in the arterial blood is considerably higher
than the concentration of alcohol in the venous blood. One typical study revealed a subject’s arterial blood-alcohol level to
be 41 percent higher than the venous level thirty minutes after the
last drink.2 This difference is thought to exist because of the rapid diffusion of alcohol into the
body tissues from venous blood during the early phases of absorption. Because the administration of a blood test requires
drawing venous blood from the arm, this test is clearly to the advantage of a subject who may still be in the absorption
stage. However, once absorption is complete, the alcohol becomes equally distributed throughout the blood system.

A breath test reflects the alcohol concentration in the pulmonary artery. Breath-test results
obtained during the absorption phase may be higher than results obtained from a simultaneous
direct analysis of venous blood. However, the former is more reflective of the concentration of
alcohol reaching the brain and therefore more accurately reflect the effects of alcohol on the subject. Again, once
absorption is complete, the difference between a blood test and a breath test should be minimal.

Blood Alcohol Concentration

Blood alcohol concentration (BAC) depends on the number of drinks, the alcoholic content of the drink, the period in which
these drinks were consumed the time of the first drink and the time of the last drink, body weight, sex, age, and food in the
stomach. In addition, BAC is influenced by health, medications, and co-abuse of drugs.

It is accepted scientifically that a 150-lb man will have a BAC of 0.025% after drinking 1 oz of 100 proof (50%) alcohol.
This assumption is accurate under almost all circumstances. BAC easily can be calculated as follows.

BAC = 150 ÷ Body weight (lb) × % ethanol content ÷ 50

× Ounces of alcohol consumed × 0.025

One drink gives a BAC of 0.02% in a 200-lb man. Thus, after consuming four drinks his BAC would reach 0.08%, the legal
limit in most U.S. states. Approximately 0.02% of blood alcohol is dissipated from this man. ADH is low in infants,
neonates, and females. For this reason, females generally reach the legal limit with fewer drinks than males do.

For medical management of the patient, serum ethanol concentration is determined. For forensic purposes, blood ethanol
concentration is required. For this reason, serum ethanol levels need to be converted to blood ethanol levels and vice
versa. The average value for a serum-to-blood ratio was found to be from 1.04 to 1.26 with a mean value of 1.14.
Therefore, by dividing serum value by 1.14, blood alcohol concentration is obtained (6). The level of
ethanol reaching the brain and its effect on the CNS is very well correlated 30 Forensic Toxicology: Medico-Legal Case
Studies with the level of intoxication (6). For this reason, BAC is always correlated with symptoms of intoxication.

Blood Ethanol (%) Intoxication

1. 0.01–0.05 There is only slight physiological impairment.

2. 0.05–0.07 Euphoria; increased self-confidence, impairment of reaction responses.
3. 0.07–0.10 Impairment of reaction sponsors, attention, visual acuity, and judgment.
An individual may appear sober.
4. 0.10–0.20 Increased impairment of a sensory motor activity. Reaction times, attention,
Visual acuity, and judgment progress to increase in drowsiness, disorientation,
Page | 54
 and emotional liability.
5. 0.20–0.30 Staggering, drunk, lethargic, sleepy, or hostile and aggressive.
6. 0.30–0.40 Unconscious and stupor.
7. +0.4 Coma, and possible death.

Breath-Test Instruments

From a practical point of view, the idea of drawing blood from a vein to test motorists suspected
of being under the influence of alcohol simply does not provide a convenient method for monitoring alcoholic drivers.
Having the suspect transported to a location where a medically qualified person can draw blood would be costly and time
consuming, considering the hundreds of tests that the average police department must conduct every year. Thus, breath
analysis serves a very useful purpose in providing an easily obtainable specimen along with a rapid and accurate result. A
breath tester is simply a device for collecting and measuring the alcohol content of alveolar breath.

The first successful commercial breath-test device, known as the Breathalyzer, was developed
in 1954 by R. K. Borkenstein, who was a captain in the Indiana State Police. The
Breathalyzer required the subject to blow into a disposable mouthpiece that led into a metal cylinder. The last portion of
breath (alveolar breath) was trapped in the cylinder. The amount of
breath collected in this manner was 52.5 milliliters, or 1/40 of 2,100 milliliters.3 We have already
seen that the amount of alcohol in 2,100 milliliters of alveolar breath approximates that in 1 milliliter of blood. Hence, in
essence, the Breathalyzer was designed to measure alcohol concentration present in 1/40 of a milliliter of blood. The
quantity of alcohol in the trapped breath was measured by passing the breath into a glass ampoule containing potassium
dichromate, sulfuric acid, and water. Any alcohol in the breath immediately dissolves in the dichromate solution and is
oxidized to acetic acid. In the oxidation process, potassium dichromate is also destroyed. The extent of this destruction is
measured by the Breathalyzer and is related to the quantity of alcohol passed into the ampoule.

Basically, the Breathalyzer is a spectrophotometer (see Chapter 5) designed to measure the

absorption of light passing through the potassium dichromate solution at a single wavelength. To better understand its
operation, let’s examine what is happening in the ampoule when alcohol is converted to acetic acid. Whenever a chemical
reaction occurs between two or more substances, chemists use a chemical equation as a shorthand method to describe
the changes taking place. The equation serves two purposes: it identifies the participants, and it describes the quantitative
aspects of the reaction.

The following equation depicts the chemical reaction taking place in the ampoule

2K2Cr2O7 + 3C2H5OH + 8H2SO4 →potassium dichromate ethyl alcohol sulfuric acid yields
2Cr2(SO4)3 + 2K2SO4 + 3CH3COOH + 11H2O chromium sulfate potassium sulfate acetic acid water

From this chemical equation, we can see that there is always a fixed relationship between the
number of potassium dichromate molecules reacting with the alcohol. Two molecules of potassium dichromate always
combine with three molecules of ethyl alcohol. Hence, determining the amount of potassium dichromate consumed is an
indirect way of determining the quantity of alcohol originally present. Silver nitrate is also present in the Breathalyzer
ampoule; however, this substance acts only as a catalyst to speed up the rate of reaction between potassium dichromate
and ethyl alcohol. As a catalyst, silver nitrate undergoes no net change itself during the reaction.

Starting in the 1970s, the Breathalyzer was phased out and replaced by the computerized
breath-alcohol instruments that dominate the field today. Interestingly, these instruments still
have one thing in common with the old Breathalyzer: they measure the alcoholic content of alveolar breath. Like the
Breathalyzer, they assume that the ratio of alcohol in the blood to alcohol in alveoli air is 2,100 to 1 at a mouth
temperature of 34°C. Unlike the Breathalyzer, modern breath testers are free of chemicals. Most of these devices aim
beams of infrared radiation at the sample cell containing the alveolar breath to detect and measure alcohol. In principle,
these instruments operate no differently from the spectrophotometers. Any alcohol in the subject’s breath is passed into
the instrument’s breath chamber. A beam of infrared light is aimed through the chamber. A filter is used to select a
wavelength of infrared light at which alcohol will absorb. As the infrared light passes through the chamber, it interacts with
the alcohol and causes the light to decrease in intensity. The decrease in light intensity is measured by a photoelectric
detector that gives a signal proportional to the concentration of alcohol present in the captured breath sample.

This information is processed by an electronic microprocessor, and the percent blood-alcohol

concentration is displayed on a digital readout. Also, the blood-alcohol level is printed on a card

Page | 55
to produce a permanent record of the test result. Most infrared breath testers aim a second infrared beam into the same
chamber to check for acetone or other chemical interferences on the breath. If the instrument detects differences in the
relative response of the two infrared radiations that does not conform to ethyl alcohol, the operator is immediately
informed of the presence of an “interferant.”

Another approach for measuring alcohol in breath is to use a fuel cell detector. A fuel cells
converts a fuel and an oxidant into an electrical current. In evidential breath-testing devices that
use this concept, breath alcohol is the fuel and atmospheric oxygen is the oxidant. Alcohol is
converted in the fuel cell into acetic acid, generating a current that is proportional to the quantity
of alcohol present in the breath.

Infrared and fuel-cell-based breath testers are microprocessor controlled so that all an operator
has to do is press a start button and the instrument automatically moves through a sequence
of steps that produce a printout of the subject’s test results. These instruments also perform self-diagnostic tests to
ascertain whether the instrument is in proper operating condition.

Considerations in Breath Testing

An important feature of these instruments is that they can be connected to an external alcohol
standard or simulator in the form of either a liquid or a gas. The liquid simulator comprises a
known concentration of alcohol in water. It is heated to a controlled temperature and the vapor
formed above the liquid is pumped into the instrument. Dry-gas standards typically consist of a
known concentration of alcohol mixed with an inert gas and compressed in cylinders. The external standard is
automatically sampled by the breath-test instrument before and/or after the subject’s breath sample is taken and
recorded. Thus the operator can check the accuracy of the instrument against the known alcohol standard.

The key to the accuracy of a breath-testing device is to ensure that the unit measures the alcohol in the alveolar breath
(deep-lung breath) of the subject. This is typically accomplished by
programming the unit to accept no less than 1.5 liters of breath from the subject. Also, the subject must blow for a
minimum time (such as 6 seconds) with a minimum breath flow rate (such as 3 liters per minute).
Another feature of these instruments is the slope detector. As the subject blows into the instrument, the breath-alcohol
concentration initially will rise steadily as a function of time. The
instrument accepts a breath sample only when consecutive breath measurements show little or no rate of change in
breath alcohol concentration. This approach ensures that the breath sample being measured is alveolar or deep-lung
breath and thus most closely relates to the true blood alcohol concentration of the subject being tested.
A breath-test operator must take other steps to ensure that the breath-test result truly reflects
the actual blood-alcohol concentration of the subject. A major consideration is to avoid measuring “mouth alcohol”
resulting from regurgitation, belching, or recent intake of an alcoholic beverage.

Also, the recent gargling of an alcohol-containing mouthwash can lead to the presence of
mouth alcohol. As a result, the alcohol concentration detected in the exhaled breath is higher
than the concentration in the alveolar breath. To avoid this possibility, the operator must not allow the subject to take any
foreign material into his or her mouth for a minimum of fifteen to
twenty minutes prior to the breath test. Likewise, the subject should be observed not to have
belched or regurgitated during this period of time. Mouth alcohol has been shown to dissipate
after fifteen to twenty minutes from its inception.
Independent measurement of duplicate breath samples taken within a few minutes of each
other is another extremely important check of the integrity of the breath test. Acceptable agreement between the two tests
taken minutes apart significantly reduces the possibility of errors arising from the operator, mouth alcohol, instrument
component failures, and spurious electric signals.

Field Sobriety Testing

A police officer who suspects that an individual is under the influence of alcohol usually conducts a series of preliminary
tests before ordering the suspect to submit to an evidential breath or blood test.

These preliminary, or field sobriety, tests are normally performed to ascertain

the degree of the suspect’s physical impairment and whether an evidential test is justified.

Field sobriety tests usually consist of a series of psychophysical tests and a preliminary breath
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test (if such devices are authorized and available for use). This pocket-sized device weighs 5 ounces and uses a fuel cell
to measure the alcohol content of a breath sample. The fuel cell absorbs the alcohol from the breath sample, oxidizes it,
and produces an electrical current proportional to the breath-alcohol content. This instrument can typically perform for
three to five years before the fuel cell needs to be replaced.

Breath-test results obtained with devices must be considered preliminary and non-evidential in nature. They should only
establish probable cause for requiring an individual to submit to a more thorough breath or blood test.

Horizontal-gaze nystagmus, walk and turn, and the one-leg stand constitute a series of
reliable and effective psychophysical tests. Horizontal-gaze nystagmus is an involuntary jerking of the eye as it moves
to the side. A person experiencing nystagmus is usually unaware that the jerking is happening and is unable to stop or
control it. The subject being tested is asked to follow a penlight or some other object with his or her eye as far to the side
as the eye can go. The more intoxicated the person is, the less the eye has to move toward the side before jerking or
nystagmus begins. Usually, when a person’s blood-alcohol concentration is in the range of 0.10 percent, the jerking
begins before the eyeball has moved 45 degrees to the side Higher blood-alcohol concentration causes jerking at smaller
angles. Also, if the suspect has
taken a drug that also causes nystagmus (such as phencyclidine, barbiturates, and other depressants), the nystagmus
onset angle may occur much earlier than would be expected from alcohol alone.

Walk and turn and the one-leg stand are divided-attention tasks, testing the subject’s ability
to comprehend and execute two or more simple instructions at one time. The ability to understand and simultaneously
carry out more than two instructions is significantly affected by increasing blood-alcohol levels. Walk and turn requires the
suspect to maintain balance while
standing heel-to-toe and at the same time listening to and comprehending the test instructions.
During the walking stage, the suspect must walk a straight line, touching heel-to-toe for nine
steps, then turn around on the line and repeat the process. The one-leg stand requires the suspect to maintain balance
while standing with heels together listening to the instructions. During the balancing stage, the suspect must stand on one
foot while holding the other foot several inches off the ground for 30 seconds; simultaneously, the suspect must count out
loud during the 30- second time period.

The Analysis of Blood for Alcohol

Gas chromatography offers the toxicologist the most widely used approach for determining alcohol levels in blood. Under
proper gas chromatographic conditions, alcohol can be separated from other volatiles in the blood. By comparing the
resultant alcohol peak area to ones obtained with known blood-alcohol standards, the investigator can calculate the
alcohol level with a high degree of accuracy.

Another procedure for alcohol analysis involves the oxidation of alcohol to acetaldehyde.
This reaction is carried out in the presence of the enzyme alcohol dehydrogenase and the coenzyme nicotine-amide-
adenine dinucleotide (NAD). As the oxidation proceeds, NAD is converted into another chemical species, NADH. The
extent of this conversion is measured spectrophotometrically and is related to alcohol concentration. This approach to
blood-alcohol testing is normally associated with instruments used in a clinical or hospital setting. On the other hand,
forensic laboratories normally use gas chromatography for determining blood-alcohol content.

Collection and Preservation of Blood

Blood must always be drawn under medically accepted conditions by a qualified individual. It is
important to apply a nonalcoholic disinfectant before the suspect’s skin is penetrated with a sterile needle or lancet, to
negate any argument that an alcoholic disinfectant may have inadvertently contributed to a falsely high blood-alcohol
result. Nonalcoholic disinfectants such as aqueous benzalkonium chloride (Zepiran), aqueous mercuric chloride, or
povidone-iodine (Betadine) are recommended for this purpose.
Once blood is removed from an individual, it is best preserved sealed in an airtight container after adding an anticoagulant
and a preservative. The blood should be stored in a refrigerator until delivery to the toxicology laboratory. The addition of
an anticoagulant, such as EDTA or potassium oxalate, prevents clotting; a preservative, such as sodium fluoride,
inhibits the growth of microorganisms capable of destroying alcohol.

One study performed to determine the stability of alcohol in blood removed from living individuals found that the most
significant factors affecting alcohol’s stability in blood are storage
temperature, the presence of a preservative, and the time of storage.4 Not a single blood specimen examined showed an
increase in alcohol level with time. Failure to keep the blood refrigerated or to add sodium fluoride resulted in a substantial
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decline in alcohol concentration. Longer storage times also reduced blood-alcohol levels. Hence, failure to adhere to any
of the proper preservation requirements for blood works to the benefit of the suspect and to the detriment of society.

Collection of postmortem blood samples for alcohol determination requires added precautions
as compared to collection from living subjects. Ethyl alcohol may be generated in a deceased
individual as a result of bacterial action. Therefore, it is best to collect a number of bloods
samples from different body sites. For example, blood may be removed from the heart and from
the femoral (leg) and cubital (arm) veins. Each sample should be placed in a clean, airtight container containing an
anticoagulant and sodium fluoride preservative and should be refrigerated. Blood-alcohol levels attributed solely to alcohol
consumption should result in nearly similar results for all blood samples collected from the same person. Alternatively,
collection of vitreous humor and urine is recommended. Vitreous humor and urine usually do not suffer from postmortem
ethyl alcohol production to any significant extent.

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 Module Evaluation

The learner’s feedback is vital to us. Taking into account your assessment and impression will help us enrich the
content enhance the quality your learning engagement with us.

From this view, we would appreciate if you could spend some time completing this evaluation by checking the
column you think is appropriate and then providing a qualitative response to the questions raised in this form.

The questionnaire is anonymous and though your participation is voluntary, your utmost cooperation is
encouraged.

Once completed the results of these questionnaires will be analyzed and an overview compiled which will be
reported to the next cohort of students in the module handbook. The overview will also be used to inform discussion at
programme team conference.

INDICATORS Strongly Moderately Slightly Disagree Strongly

Agree Agree Agree Disagree
I. The Module
a. was effectively designed
b. had clear learning outcomes
c. was well organized
d. contained relevant information
e. had clear images
f. had sufficient parts
g. had lessons that are related to
life experiences
II. The Assessment
a. rubrics were clear
b. instructions were comprehensive
c. was sufficiently challenging
d. was aligned to the lessons
e. was done within the prescribed
time
f. had enriched my knowledge
about the lessons
g. were of different types
h. contained critical thinking
questions

What do you like most about the module?

What could have been improved on the module?

What other things you suggest to improve the module?

How satisfied are you with the module?

Thank you.

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A Self-regulated Learning Module 1