SUBJECT FORENSIC SCIENCE

Paper No. and Title Paper No. 4: Instrumental Methods and Analysis

Module No. and Title Module No.15: UV/visible spectroscopy-Forensic application

Module Tag FSC_P4_M15

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 TABLE OF CONTENTS

1. Learning Outcomes
2. Applications of UV/visible spectroscopy in forensic science
2. 1. Analysis of narcotics and drug testing
2. 2. Determination of alcohol in blood
2. 3. Ink analysis
2. 4. Drug overdose study
2. 5. Analysis of carboxyhemoglobin in forensic blood samples
2. 6. Forensic determination of metals by UV/visible spectroscopy
2. 7. Examination of blood stains by UV/Visible spectroscopy
2. 8. Forensic discrimination of dyed textile fibers using UV/Visible
spectroscopy
2.9. Quantitative determination of hemoglobin in tooth and bone sample
using UV/visible spectroscopy
3. Summary

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 1. Learning Outcomes
After studying this module, you shall be able to

 Understand the applications of UV/visible spectroscopy in forensic science.

2. Applications of UV/visible spectroscopy in forensic science

Forensic drug analysis deals with the identification and quantification of illegal drugs.
The testing of drugs is generally carried out in two steps, namely, screening and
confirmation. UV/visible spectroscopy is widely used as a very significant tool for both
qualitative and quantitative drug analysis. Some of the applications of UV/visible
spectroscopy in forensic science as discussed below.

2.1. Analysis of narcotics and drug testing

Analysis of narcotics and related drugs is an important part of forensic science. UV/Vis
molecular absorption is routinely used for the analysis of narcotics and for drug testing.
The presence of these drugs can be confirmed from their absorption maxima i.e. λmax or
by comparing the UV/visible spectra of these drugs with spectra of authentic sample. The
value of λmax (nm) for various abusive drugs is given in Table 1.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 Table 1. Absorption maxima for various abusive drugs
S. No. Drug Absorption maxima (λmax in nm)
1 Cocaine in 0.2 N H2SO4 233
2. Codeine in water 284
3. Ecstasy or MDMA in ethanol 286
4. Morphine in acidic solution 285
5. Phencyclidine or PCP in 0.1 M HCl 262
6. Amphetamine in methanol 258
7. Nicotine in 0.2 N H2SO4 260
8. Caffeine in 0.2 N H2SO4 271
9. Metamphetamine in methanol 259
10. Tetrahydrocannabinol in methanol 276

2.2. Determination of alcohol in blood

UV/visible spectroscopy can be used for quantitative estimation of blood alcohol using
the breathalyzer test. In this test, sample of breath of the person is passed through an
acidic solution of potassium dichromate which is orange in colour. Potassium dichromate
in acidic medium is an oxidizing agent and oxidizes ethanol to acetic acid while
dichromate ions get reduced to green colored Cr3+ ions. The concentration of ethanol in
the breath sample is determined by the decrease in absorbance at 440 nm around which
the dichromate ion absorb. Using this value, by using Lambert-Beers’s law, the
concentration of alcohol can be then measured.

2.3. Ink analysis

Ink analysis is one of the areas in forensic questioned document aims at comparing,
identifying, characterizing and discriminating ink obtained from writing instrument used
to write on a document. Ink becomes important forensic evidence when it is written on a
document suspected to be associated with criminal activities such as threatening letters,
insurance frauds and will frauds.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 The examination of different inks and their classification can be easily done with UV-Vis
spectra of pen inks. This is much simple and quick to perform as compared to other
methods of ink analysis such as chromatographic and spectroscopic techniques.

2.4. Drug overdose study

The drug overdose may lead to death. The cases of drug overdose can be studied by
UV/visible spectroscopy. For example overdose of aspirin can be studied by studying the
UV/visible spectra of blood plasma of a person with overdose of aspirin. Aspirin is
prepared by the acetylation of salicylic acid using acetic anhydride. It is usedas a drug
for many purposes like as an analgesic to reduce pain, anti-inflammatory to reduce
inflammation, antipyretic to reduce temperature, and platelet aggregation inhibitor to thin
the blood and stop clotting. Therapeutic levels taken after a heart attack are typically 150
– 300 mg/L and for post by-pass operations 75 mg/L. The levels of salicylate present in
blood plasma can be analysed using UV-visible spectroscopy to indicate if the subject has
taken a therapeutic dose or an overdose. This method involves measuring the absorbance
of the red-violet complex of ferric and salicylate ions at about 530 nm using a UV/Visible
spectrometer.

2.5. Analysis of carboxyhemoglobin in forensic blood samples

Carbon monoxide (CO) is colorless, tasteless, odorless, and nonirritating and hence its
exposure and intake in human beings is impossible to detect. When inhaled, CO is
instantly absorbed from the lungs into the bloodstream and has highly toxic effect. In the
blood stream, the carbon monoxide slowly forms a reversible complex with hemoglobin
(Hb) known as carboxyhemoglobin (COHb). The presence of COHb in the blood
decreases its oxygen carrying capacity, and hence reduces the availability of oxygen to
body tissues, resulting in tissue hypoxia. It may even lead to death, which can be
explained as follows. The increase in the level of COHb reduces the oxygen delivery. It is
worsened by impaired perfusion resulting from hypoxic cardiac dysfunction. This further
potentially damages the cellular oxidative metabolism and hence lead to death. The
binding of carbon monoxide with heme chromophore effects its absorption in UV/visible
region. That is why, the levels of COHb can be determined by UV/visible spectra of the
blood sample of a person with suspected carbon monoxide poisoning.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 Total hemoglobin can be measured by performing a reaction of the hemoglobin present
with potassium cyanide to form a hemoglobin–cyanide complex. In this method,
hemoglobin is reacted with potassium cyanide to form a hemoglobin–cyanide complex.
This complex has a peak around 546 nm, which can be measured and quantitated.

2.6. Forensic determination of metals by UV/visible spectroscopy

Iron, aluminium, and copper are three metals, which are most widely used for fabricating
various articles of daily need. These elements are found as a part of almost every weapon,
tools, and metal shards in crime scenes. Thus, the forensic investigation of these metal
ions is an important area of study. Weapons often used in robberies to cut metals or in
murders. The investigation of crime scenes often revealed the presence of these metal
ions usually on clothing of suspect. Other metals such as lead, arsenic are sometimes
encountered at industrial work places and many time used as poisons. Arsenic salts are
commonly used as insecticides in houses. In addition to these metal ions are also found in
paints, dyes, explosives such as gunpowder and in glass. The presence and concentration
of these metal ions can be easily measured by UV/visible spectroscopy using various
methods as summarized in Table 2.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 Table 2: UV/visible analysis of various metals

Metal Method λ in nm
Aluminum React with Eriochrome cyanide R dye at pH6 to forms 535
red to pink complex
Arsenic On reaction with Zn arsenic reduces to AsH3 which react 535
with silver to form red color complex
diethyldithiocarbamate
Cadmium On reaction with chloroform solution of dithizone in 518
presence of NaOH gave pink to red complex
Chromium Cr(VI) react with diphenylcarbazide to give red-violet 540
complex
Lead On reaction with chloroform solution of dithizone in 510
presence of with NH3 gave cherry red complex
Mercury On reaction with chloroform solution of dithizone under 492
acidic conditions gave orange complex
Copper React with neocuprine in neutral to slightly acid solution 457
and extract into CHCl3/CH3OH; forms yellow complex

2.7. Examination of blood stains by UV/Visible spectroscopy

At crime scenes, especially related to murder, the most significant of evidence for
forensic investigators are blood stains. The sample of blood stain collected from the
crime scene can be used for DNA-profiling for verifying the suspect’s identity as well as
for pattern analysis in order to reconstruct the crime. UV/visible spectroscopy method
can be used to study blood stains by detecting the presence of enzyme activity in stain.
There is higher activity of enzymes lactate dehydrogenase (LDH), glutamic oxaloacetic
transaminase (GOT) and glutamic pyruvic transaminase (GPT) in bloodstains than in
serum; also there is a comparable activity in completely hemolyzed blood. This forms the
basis for the use of UV/visible spectroscopy method for analysis of blood stains. Using a
combination of LDH/GOT and GOT/GPT ratios, the age estimation up to 12 weeks after
formation of the bloodstain has been estabilished.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 In this method, removal of the bloodstain, followed by extraction with saline is done. The
sample so prepared is then subjected to labeling of the enzymes and is detected at a
wavelength of 340 nm. It is important to note that the GOT/GPT ratio remains stable over
the first weeks, while the LDH/GOT ratio is initially high (>10), and decreases to a ratio
of 5 after eight weeks.

2.8. Forensic discrimination of dyed textile fibers using UV/VIS

spectroscopy

In incidents involving personal contact, such as homicide, assault, and sexual offenses, as
well as hit-and-run accidents and other crimes, fibers are often encountered as trace
evidence. Objects peripherally associated with crimes, such as weapons or cars involved
in armed robberies, etc also most often have fibers associated with them as trace
evidence. From forensic point of view, the knowledge of chemistry of fibres and fibre
dyes is very important for both i.e., as the basis of fiber identification and for the forensic
relevance of various fiber/dye combinations. The UV/visible spectroscopy offers a
significant contribution to differentiate between various type of fibers and dyes. The
UV/visible spectra of the trace evidence as fibre collected from crime scene can be easily
matched with reference spectra of known fibre materials and dyes. For example Figure 1
shows the UV/visible spectrum of various fibers.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 Figure 1. UV-VIS spectrum of yellow fibers mounted in glycerin using quartz slides and
cover slips: (A) cotton; (B) acrylic, (C) nylon; (D) polyester.

2.9. Quantitative Determination of Hemoglobin in Tooth and Bone

sample by UV/visible spectroscopy

In the forensic investigation, the detection and quantification of hemoglobin in the

skeletal or dried remains can be very significant. This can be achieved with the help of
UV/visible spectroscopy using leuco crystal violet dye. E.g., Extractions of hemoglobin
from a femur bone by can be done using 0.5 M hydrochloric acid and from a tooth it can
be done by using 6.0 M urea. A violet product is obtained when these extractions are
treated with leuco crystal violet. Using the measured absorbance at 590 nm of each
extraction, the corresponding amount of hemoglobin in each sample can be determined.
UV/visible spectrum of extract containing hemoglobin is shown in Figure 2.

Figure 2. The UV/Vis absorbance spectrum of the leuco crystal violet dye reaction with
1.0 µM Hb.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application
 3. Summary
 UV/visible spectroscopy provide an important method for both qualitative and
quantitative analysis in forensic investigations.
 UV/visible spectroscopy is most widely used for detection and analysis of drugs of
abuse based upon their absorption maxima.
 It also found application for determination of alcohol in blood.
 Ink analysis which is important part of forensic study can also done with
UV/visible spectroscopy.
 UV/visible spectroscopy is also used for drug overdose study in body.
 UV/visible spectroscopy can also used for study of carbon monoxide poising by
analysis of carboxyhemoglobin in forensic Blood Samples.
 Determination of metals in forensic samples can also done with UV/visible
spectroscopy.
 UV/visible spectroscopy can also used to find the age and for analysis of blood
stains commonly present at crime scenes.
 UV/visible spectroscopy can be used for forensic discrimination of dyed textile
fibers.
 UV/visible spectroscopy can be used for quantitative determination of hemoglobin
in tooth and bone samples.

FORENSIC SCIENCE PAPER No. 4: Instrumental Methods and Analysis

MODULE No. 15: UV/visible spectroscopy-Forensic
application