The document discusses methods for analyzing toxic substances and poisons. It describes: - LD50 testing which measures the lethal dose (LD) of a substance required to kill 50% of test subjects. This allows comparison of relative toxicities. - Extraction methods used to isolate acidic, basic, volatile, and non-volatile poisons from tissues through use of solvents and acid-base reactions. - Various analytical techniques like TLC, GC, AAS that can identify specific poisons isolated from samples based on their chemical properties. - Examples of tests used to detect common poisons like ethanol, barbiturates, DDT, and methods to analyze heavy metals like mercury and arsenic

1. Forensic Analysis
Dr. S. H. Burungale

2. Forensic Analysis
Any substance harmful to living organism
According to German Swiss Physician Paracelsus
“All substances are poisons, there is none which
is not a poison. The right dose differentiate a
poison from a remedy." Thus, it is a substance
(solid, liquid or gas), introduced or brought into
contact with any body part, will produce ill-
health or death.

3. LD stands for "Lethal Dose”, it figures a
substance's acute toxicity. Median Lethal
Dose is quantity of the chemical that is
estimated to be fatal to 50% of the organisms.
Initially introduced by J.W. Trevan in 1927.
It is an index determination of medicine and
toxin’s virulence.

4. Importance
Different chemicals cause different toxic effects, comparing
the toxicity of one with another is hard. To compare the
toxic potency or intensity of different chemicals, researchers
must measure the same effect. One way is to carry out
lethality testing (the LD50 tests) by measuring how much of a
chemical is required to cause death.
Significance
As an aid in developing emergency procedures in case of a
major spill or accident. To help develop guidelines for the
use of appropriate safety clothing and equipment. For the
development of transportation regulations. As an aid in
establishing occupational exposure limits. As a part of the
information in Material Safety Data Sheets

5. Significance of LD-50 and LC-50.
Definition
LD stands for "Lethal Dose”, it figures a
substance's acute toxicity. ...
To compare the toxic potency or intensity of
different chemicals, researchers must measure the
same effect.
One way is to carry out lethality testing
(the LD50 tests) by measuring how much of a
chemical is required to cause death.

6. CLASSIFICATION ON THE BASIS OF MODE OF ACTION OF
POISON
Corrosive SystemicIrritant
Strong Acid
HCl,HNO3,
H2SO4
Strong Base
NaOH,KOH
Organic
Animal
Snakes
Plants
Inorganic
Poisoins
Metals and
Nonmetals

8. Classification
ON
THE BASIS OF THEIR CHEMICAL PROPERTIES AND METHOD OF
ISOLATION FROM TISSUES AND OTHER BIOLOGICAL FLUIDS
Poison Volatile Alcohol Chloroform Ether Acetone
Nicotine Pyridine
Non Volatile: All drugs like: Sleeping pills Pain killers
Anti- depressants
Pesticides: DDT, Eldrin Malathion
Miscellaneous
Toxic Anions : Cyanide, Phosphate Clorate Chloride
Toxic Cations: Mercury Arsenic Lead Zinc
Animal :Snake Venom, Insect bite
Vegetable Dhatura, Cannabis, Kuchila
Food Poisoning Due to Micro organism

9. Extraction of Poisons
An acid is any substance that releases hydrogen ions (H+) when
dissolved in water. A base is any substance that accepts hydrogen
ions (H+) when dissolved in water. Most drugs and poisons are
either acids or bases. For example, most barbiturates have a pH
below 7; therefore, they are acidic. Most amphetamines have a pH
above 7 and, therefore, are basic. During an acid-base extraction
procedure, body fluids, tissues, or organs are placed in an acidic
solution and/or a basic solution. Acidic drugs or poisons are easily
extracted from an acidic solution; basic drugs or poisons are easily
extracted from a basic solution. After these acid-base procedures
are completed, the drug or poison is identified as an isolated
sample. The isolated sample(s) then goes through a screening test
and, finally, through a confirmation test.

10. Isolation of Poisons
Analysis of Poisons Toxicological analysis of
biological tissues involves, Separation of drug
from the biological tissues. Purification.
Analytical detection. This is done by any of the
analytical techniques like TLC, HPTLC, IR, Raman,
FTIR, FT, FT Raman, LC, LCMS, GC, GCMS,
HPLC, GC MS/MS, AAS, AES, AES, SEM, TEM,
PCR, UV Spectrophotometry, ICPMS, EXDRF.

14. Tests: Ex.1 - 𝑪𝑯 𝟑 𝑪𝑶𝑪𝑯 𝟑:
1. Iodoform Test: In 1 ml of distillate, add 2 drops of
1% NaOH. This is followed by drop wise addition of 𝐼
2 till the solution becomes brown. The solution is
allowed to warm in low flame. The ppt observed
under the microscope, crystals are often found standing,
characteristic Hexagonal crystal of iodoform is seen. 2.
Legal’s Test: In 1 ml distillate add a few drops of
KOH and add 2-3 drops of saturated solution of freshly
prepared Sodium nitroprusside. First red color is
produced, then Yellow color occurs, then add few drops
of glacial acetic acid are trickled down the side of the
test tube so at the line of junction of the two fluids is
formed a carmine or purple ring.

15. Ex.2 - 𝑪𝑯 𝟑 𝑪𝑯 𝟐 𝑶𝑯:
1. Iodoform Test:
CH3CH2OH + 𝐼2 + 𝑁𝑎𝑂𝐻 + 𝑣𝑒 𝑅𝐸𝑆𝑈𝐿𝑇
2. Sulfomolybdic acid Test:
In 2 ml of distillate known reagent is added.
Reagent – 1 gm of molybdic acid in 25ml
of conc. 𝐻2 𝑆𝑂4.
If a deep blue ring appearance at the junction
of the 2 liquids is found – Ethanol present

16. Analysis of Acidic Drugs / Poisons (Non- Volatile) Ex.
- Barbiturates
They are drugs which are associated with criminal
case poisoning. (Homicidal & Suicidal) Analysis of
Barbiturates:
Extraction:
The viscera material about 50-100gm is cut into
small pieces. Mix with 100ml of 𝐶𝐻3 𝐶𝑂𝑂𝐻 and
add solid (𝑁𝐻4)2 𝑆𝑂4. The mixture is then heated
in water bath. It is cooled and filtered; the filtrates
were taken in separating funnel. Add 100ml of 𝐶2 𝐻
5 − 𝑂 −𝐶2 𝐻5 into it. Shake it & than take ether layer.
The acidic extract is tested for arbiturates, salicylate
etc

17. Test for Barbiturates:
1. Dille – Koppayani Test:
2. A small amount of extract material is
placed in spot plate.
3. 3-4 drops of acetate solution and iso-
propyl amine solution are added.
4. Purple or blue violet color indicates
presence of barbiturates. 2. By U.V:
Compound 𝝀 𝒎𝒂𝒙 Phenobarbitone 230nm
Allobarbitol 241nm

18. Analysis of Basic Drugs / Poisons Basic Drugs: Drugs
which are alkaline in nature are called “Basic Drugs”.
Mainly contains N, S and O in the ring.
Narcotics, Cardial Poison Analysis of Basic Drugs:
Extraction:
The aqueous solution remaining in the funnel after
the separation of acidic drugs is made alkaline by
addition of 𝑁𝐻4 𝑂𝐻 & 100ml 𝑪𝑯𝑪𝒍 𝟑:Ether (1:3)
solution.
The aqueous solution layer is retained for opium
alkaloid.
Take 50ml of 𝐶𝐻𝐶𝑙3–Ether mixture and add to it.
This is done for 3 times. The extract which is best is
taken for alkaloids & other basic drugs like opium.

19. Organo Chloro:
These are extensively used in agriculture and
domestic application.
Ex.: DDT, BHC, Anarine, Lindane. A.
Extraction:
The sample is taken in a conical flask.
50ml of n-hexane is added to it.
It is refluxed on water bath for half an hour.
After cooling, the liquid is filtered and
evaporated to dryness. Use it for analysis.

20. By TLC:
Plate: Silver
Solvent SVS: Hexane: Acetone (9:1)
Spray Reagent: Zinc chloride diphenyl
Color of spot: Bluish Green C. Color test of DDT:
5 ml of chloro benzene is added to the extract +
warm it & filter through AlCl3. D. By GC:
Column: SE30
Column Temp.: 190° C
Carrier Gas: N2 at 95ml/min.

21. Mercury
Most volatile of all metals
Highly toxic in vapor form
Liquid mercury itself is not highly toxic, and most
of that ingested is excreted
Health effects
Skin burns
Irritation of nose and skin
Rashes
Excessive perspiration Damage to the kidneys
Damage to vision Minamata disease
Dysfunctions of the central nervous system Loss of
hearing and muscle coordination Severe brain
damage Death

22. Arsenic
Arsenic oxides were the common poisons
used for murder and suicide from roman times
through to the middle ages
Arsenic compounds were used widely as
pesticides before the organic chemicals era
Arsenic is very much similar to phosphorous

23. Sources of Arsenic
Pesticides
Mining, smelting of gold, lead, copper and
nickel
Production of iron and steel
Combustion of coal
Leachate from abandoned gold mines
Used as a wood preservative
Herbicides
Tobacco smoke
Wallpaper paste and pigments in wallpaper

24. Methods for measurement of trace metals
Most common method of collecting
particulate matter is through filters
Identification and concentration of individual
trace metals like lead,cadmium, arsenic,
mercury and chromium is determined by
1: Atomic absorption spectrophotometer
2: X-ray fluorescence

25. Atomic absorption spectrophotometry is a
destructive method and requires at least 1 to 2
ml of solution
X-ray fluorescence is a nondestructive
method and works independent of the chemical
state of the sample.

26. Mercury (Hg)
Determination Mercury is determined by AAS
(Atomic Absorption Spectroscopy) after dissolution of
the sample in nitric acid. Note: This analysis is only
performed on food grade calcium chloride.
Arsenic Determination Heavy metals (Ag, As, Bi,
Cd, Cu, Hg, Pb, Sb and Sn) are precipitated as their
corresponding sulphides using a saturated H2S
solution. The turbidity of the sample is compared to
a standard containing only lead that is treated in the
same way as the sample.

27. THANK YOU