The toxicology report for rapper Lil Peep, who was found dead in his tour bus in Tucson on Nov. 15. According to the Pima County Media Examiner, Lil Peep died from the toxic effects of fentanyl and...
Chris Cornell toxicology, Wayne County, Detroit, Michigan
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Chemical process safety solution chapter 3 number 3-13, and 3-14 and 3-20Full description
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Directorate of Forensic Science, MHA, Govt. of India
SECTION- 1: INTRODUCTION 1.1
Title:
Forensic Toxicology
1.2
Scope:
Various aspects of toxicological analysis.
1.3
Purpose:
To provide standard toxicological methods of analysis for uniformity.
1.4
Definition:
The word “Toxicology” is derived from the Greek word “Toxicon” which was used as a poisonous substance in arrowheads. Traditionally the toxicology is defined as the science embodying the knowledge source character fatal effect lethal dose analysis of poisons and the remedial measures. ! poison is defined as the substance which is capable of producing in"ury or death when absorbed. !ppropriate dosages can differentiate poison and also the remedial measures. !ll chemicals can produce in"ury or death under certain conditions. #ence a poison can be defined as a substance that is capable of producing detrimental effects on a living organism. !s a result there may be a change in the structure of the substance or functional processes which may produce in"ury or even death. The toxicologist is specially a trained expert to examine the role of such substances and their adverse effects. The variety of potential adverse effects and the diversity of chemicals present in our environment contribute to make toxicology a very broad field of science. Therefore toxicologists are usually specialists to handle various areascategories of toxicology. The professional activities of toxicologists fall into four main i.e. Forensic $ndustrial %linical and &nvironmental Toxicology. Forensic toxicology emerged as the hybrid of analytical %hemistry and toxic principle effects. Forensic toxicologists are also primarily concerned with the medico ' legal aspects of the harmful effects of chemicals on human and animals. The expertise of forensic toxicologistsis primarily utili(ed in establishing the cause of death and elucidating its circumstances in post'mortem investigation. The work of forensic toxicologist is therefore considered as highly complicated as small )uantities of poisons and their metabolites are to be isolated purified and )uantified from a highly complex matrices. 1.
O!"ECTI#E:
This manual is aimed to serve as a didactic text to Forensic &xperts working as Toxicologists. *ue to the preponderance of assorted redundant procedures proclaimed for isolation analysis and estimation of poisons the selection of correct procedures become tedious and obfusca ting. This entails the need for standardised commensurable and pragmatic procedures with emphasis on accuracy and precision. #aving cognisance of these facts it was decide d to orchestrate an adhoc committee of highly experienced and learned experts. This manual assimilates the outcome of the meticulous dialectics of the committee constituting highly experienced and knowledgeable Forensic +cientists from the
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Laboratory Procedure Manual- Forensic Toicolo!y
Forensic +cience /aboratories of the country. The committee has developed this abridged yet comprehensive text which encompasses a thorough revision of erstwhile procedures within the ambit of Toxicology and also introduced some new topics. This text is intended to allay disparity and ambiguity concerning the causes of Toxicology in general with special reference to $ndian context. This text has been prepared with conscientious appraisal of various procedures described in the sundry reference books pertaining to Toxicology. 0e have the propensity to put forward a text that would serve to mitigate the tedious and responsible "ob of Toxicologist. 1y providing a handy and consolidated )uick reference we have scrupulously avoided superfluous details and procedures in this manual. !lbeit it cannot substitute for the reference books and textbooks it should serve as a practical guide to pertinen t Forensic +cientists. This manual does not detail the clinical symptoms or pharmacological effects of poisons but rather focuses on the classification isolation detection and )uantitation of poisons. !s stated above the manual is not directed towards the replacement of accepted procedures2methodologies but incorporate only those which are relevant and practicable. #owever the options are left open for the Toxicologists to adopt any other testing procedure if re)uired. The committee hopes this manual will be received with fervour and meet the ends it has envisaged.
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Directorate of Forensic Science, MHA, Govt. of India
SECTION $2: C%&SSI'IC&TION O' POISONS &ND POISONIN( 2.1
Title:
%lassification of poisons and poisoning.
2.2
Scope:
4oisons encountered in the toxicological analysis
2.3
Purpose:
To classify the poisons and isolate it from the exhibits.
2.4
C%&SSI'IC&TION:
4oisons can be classified in a variety of ways depending upon the interests and needs of classifier e.g. classification may be done on the basis of mode of action of poison toxicity rating in terms of their physical states their labeling re)uirements their chemistry etc. For the purpose of toxicological analysis poison are classified on the basis of their chemical properties and method of isolation from tissues and other biological fluids which are given below5 2.4.1
4oisons are commonly involved in homicidal accidental or suicidal cases. These are also used to destroy the animals and plants. 2..1
&ccient*l poisonin+:
The accidental poisoning commonly takes place as a result of the carelessness2 negligence. The common accidental poisoning cases are5 a7 %oal is allowed to burn in a room unknowingly giving rise to the production of poisonous carbon monoxide gas. b7 0orkers many times unknowingly go into abandoned wells or gutters and die due to the presence of poisonous gases. c7 4oisonous materials like insecticides are carelessly sprayed in the farms and due to accidental inhalation many times may cause the death of farmers2workers. d7 %hildren handle some poisonous articles and develop poisonous symptoms. e7 9verdoses of medicines like barbiturates or fake medicines wrongly prescribed. f7 !llergic conditions. g7 1ites by poisonous insects and snakes. h7 !ccidental cattle or animal poisoning cases.
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Directorate of Forensic Science, MHA, Govt. of India
2..2
5oici*l Poisonin+:
9wing to small fatal doses tasteless and odourless properties miscibility with drink and common availability some poisons are mainly used for homicidal purposes e.g. arsenic salts mercury salts cyanides sodium nitrite methyl alcohol dhatura seeds phosphides etc. Carely cultures of disease germs are also used for homicidal purposes. $n certain parts of the country opium is used as infanticidal poison. 2..3
Suici*l P oisonin+:
+ome poisons are mainly popular for suicidal purposes because of their availability in house or the working place like cyanide in electroplating units insecticidal compounds mostly available with farming communities sodium nitrite in dyeing industries. 1arbiturates are normally used by the educ ated people. The cases of committing suicides by a few doctors by in"ecting anaesthetic agents like thiopental have been reported. 2..4
iscell*neous Poisonin+:
%ases of poisoning other than the above are prevalent all over $ndia which are caused by stupefying agents. The intention behind this is to stupefy the person and commit robbery or other crimes. #owever many a times person dies because of overdoses. These poisons are cigarettes containing dhatura cannabis drugs arsenic etcD sweets containing phenobarbitone or other psychotropic substances like lora(epam etc. +ome times chloral hydrate mixed with drinks is also similarly used. 2./
&CTIONS &ND &DINISTR&TION O' POISONS:
The action of almost all the poisons is the same. They either stop the supply of oxygen to the body tissues or inhibit the en(ymes associated with the respiration mechanism and the person dies due to stoppage of oxygen availability. #owever the mode of stoppage varies e.g. insecticides and pesticides are powerful inhibitors of cholinesterase. The site of action is said to be at the myoneural "unctions and synapses of the ganglions. !t these sites normally acetylcholine is liberated from nerve stimulation. The liberated acetylcholine is hydrolysed into choline and acetic acid by cholinesterase. $n poisoning by insecticides and pesticides the activity of cholinesterase is inhibited acetylcholine therefore accumulates and results in hyper excitation of the voluntary and involuntary muscles. ! drop in the activity of cholinesterase to -@ per cent of normal or lower is associated with toxic arrest. symptoms and leads to death resulting from respiratory failure or circulatory $n carbon monoxide poisoning the gas having greater affinity combines easily with haemoglobin and make them unable to carry oxygen to various tissues of the body. The glycoside poisons affect the heart muscles and the pumping of blood is stopped. 9piates alcohols barbiturates dhatura etc. paralyse the respiratory centers of the brain resulting in respiratory failure.
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Laboratory Procedure Manual- Forensic Toicolo!y
The action of poisons may be local remote local E remote combined or general. The local action means its direct action on the tissues and cause corrosion e.g. strong mineral acids and alkalis irritation and inflammation in cantharides or some nervous effects as in the case of dilation of the pupil by atropine. Cemote action results from the absorption of the poison into the system e.g. alcohol alkaloids or other drugs affect the organs after being absorbed into the system. +ome poisons produce both local and remote actions. $n such cases there is destructive action on the tissue with which they come in contact and there is also a toxic affect after absorption e.g. oxalic acid. General action results when the absorbed poison evokes responses from a wide variety of tissues beyond one or two systems e.g. arsenic mercury insecticidal compounds etc. 8ost of the poison affects the person immediately if not the poison is eliminated from the body by excretions or gets metabolised or detoxified into non'toxic metabolite which is eliminated slowly. #owever some poisons have tendency to get accumulated in the body tissues and when a fatal level is reached the person dies. !rsenic and **T are among such poiso ns which have been repo rtedly used as slow poisons since ages for homicidal purposes. The absorption of **T through skin and accumulation behaviour may result into accidental death. 2.0
'&CTORS &''ECTIN( T5E INTENSIT6 O' POISONIN(:
2.0.1
Dose:
!s a general rule the deleterious effects of a poison depend on its dose. The larger the dose more severe will be the symptoms. 2.0.2
57persensiti,it7:
+ome persons show abnormal response 6idiosyncrasy7 to a drug like morphine )uinine aspirin etc. due to inherent personal hypersensitivity. 2.0.3
&ller+7:
+ome persons are allergic 6ac)uired hypersensitivity7 towards certain drugs like penicillin sulpha etc. 2.0.4
Incop*ti8le co 8in*tions:
$ngestion of certain medications like anti'ulcerous gels with aspirin may lead to fatal effects. 2.0.
Toler*nce
4eople develop a marked tolerance in the case of opium alcohol strychnine tobacco arsenic and some other narcotic drugs by repeated and continued use. The snake charmers develop complete immunity from snakebite by getting snakebites regularly.
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Directorate of Forensic Science, MHA, Govt. of India
2.0./
S7ner+is:
Two poisons e.g. alcohol and barbiturates or cannabis drugs and dhatura in non' toxic doses when taken together may cause severe toxic symptoms due to synergism. 2.0.0
Cuul*ti,e *ction:
The repeated small doses of cumulative poisons like arsenic lead mercury strychnine digitalis etc. may cause death or chronic poisoning by cumulative action. 2.0.
S9oc:
+ome times a large dose of a poison acts differently from a small dose e.g. a large dose of arsenic may cause death by shock while a small dose results in diarrhoea. 2.
'ORS O' POISON:
Gases or vapours act more )uickly than solid and li)uid poisons because they are absorbed immediately. /i)uid poisons act more )uickly than solid s of which fine powders act more )uickly than coarse ones. +ome substances in certain combinations become inert e.g. acids and alkalies strychnine with tannic acid and silver nitrate with hydrochloric acid. +ome substances in certain combinations become poisonous such as lead carbonate and copper arsenite which are insoluble in water but soluble in hydrochloric acid in the stomach and becomes poisons. +imilarly the action of a poison is altered when combined mechanically with inert substances e.g. alkaloids when taken with animal charcoal fail to act. 4oison act slowly when the stomach is full with fatty food. ! poisonous powder does not sink or float when given with a fluid having nearly same specific gravity. For this reason arsenic when administered for homicidal purposes is usually mixed with milk. 2.
ET5ODS O' &DINISTR&TION:
! poison acts more rapidly when inhaled in gaseous form or when in"ected intravenously next when in"ected intramuscularly or subcutaneously and least rapidly when swallowed or applied to skin. +ome poisons act differently when given through different routes. +nake venom is highly poisonous when in"ected but harmless when ingested orally if there is no any internal in"uries. 2..1
Conition of t9e !o7:
The age health and individual allergy towards certain drugs and poisons affect the action of poisons. !s a general rule children old and weaker persons are affected more severely with low doses than young and healthy adults.
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Laboratory Procedure Manual- Forensic Toicolo!y
2..2
'*te of poisons in t9e 8o7:
! poison after being absorbed into the system a part of it is excreted in the urine unchanged. 8ore fre)uently it will be partly or completely metaboli(ed.
DI&(NOSIS O' POISONIN(:
*iagnosis of poisoning is often difficult and has to be made on the available evidences. $n many cases little or no toxicity occurs and the patient parents or relatives are reassured. +ometimes the history may not be available or unreliable. 8any cases of poisoning present vague symptoms and in some fatal acute poisoning symptoms may be delayed for many hours or days even e.g. the symptoms in ethylene glycol may be delayed by 3 hoursD metal vapours' > hoursD methanol' ;> hoursD paracetamol'-3 hoursD para)uate';> hoursD salicylates',: hoursD thallium'; daysD arsine and stibine':; hrs. +ome poisons develop deceptive symptoms e.g. gastro'intestinal type of arsenical poisoning may be mistaken for symptoms of cholera or food poisoning. #ence a working diagnosis has to be made based on clinical features and laboratory investigations. The articles or the containers recovered from the scene of crime or the possession of the victim may provide help in case of suspected poisoning when the diagnosis is not immediately apparent. There are several symptom patterns which are typical for different types of poisoning and can be useful guide to the nature of poison the laboratory tests needed and the treat ment re)uired. The list given below is illustrative and further advice may be re)uired in difficult cases. The examinations should also include a search for signs of trauma and systemic disease because many organic illnesses enter into the differential diagnosis of poisoning. The symptoms clinical examinations andbelow the patterns some commonly used drugs and poisons are given which of arepoisoning essential by in assessing the patient and may help to identify the agent and severity of the problem.
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Directorate of Forensic Science, MHA, Govt. of India
(A) Symptoms
Poisons commonly involved
Vomiting
$rritant poisons like arsenic acids alkalies excess of li)uor and some metallic salts.
*iarrhoea
sually poisons causing vomiting also cause diarrhoea.
%ramps
8etallicpoisonslikearsenic lead antimony mercury etc.
*elirium
*hatura cannabis drugs alcohol atropine hyoscine /+* etc.
/ithium amphetamines lead a rsenic aconite snake venom etc.
%oma
1arbiturates carbon monoxide chloroform trichloro' ethanol opioids and excess of li)uors.
(B) Clinical Findings
Poisons commonly involved
+kin color %herry 4ink
%arbon monoxide
Flushed pink skin
!lcohol cocaine cyanide and anti'cholinergic agents
aundice
#epatotoxic agents like paracetamol
%entral cyanosis
! sign of hypoxia but methaemoglobinaemia also causes similar colour.
+kin changes %utaneous bullae
1arbiturates glutethimide sedative overdoses tricyclic antidepressants and carbon monoxide.
+weating
9rganic conditions likeand hypoglycaemia myocardial infarction pyrexia due to infarctions and poisoning by salicylates organo phosphates or monoamine oxidase inhibitors.
4upils *ilation
4upils dilate in severe hypoxia and in hypothermia. *rugs such as tricyclic antidepressants also cause dilation. Glutethimide and monoamine oxidase inhibitors produce wide dilation.
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Laboratory Procedure Manual- Forensic Toicolo!y
%onstriction
1ody temperature #ypothermia
9pioids typically cause pin point pupils. 9rganophosphate insecticides and trichloroethanol poisoning cause very small pupils. $n barbiturate poisoning the pupils may vary in si(e at times being small at others dilated. %omatose condition for sometime may cause hypothermia. +edative and hypnotic drugs trichloroethanol ethanol and opioids cause hypothermia.
#yperthermia
#yperthermia may be caused by heat stroke and meningitis and poisoning by anti'cholinergic agents tricyclic anti'depressants monoamineoxidase inhibitors carbonmonoxide dhatura phenols and salicylates.
1reath odour
!lcohol a cetone 6 diabetic ketoacidosis a nd starvation7 solvents such as toluene trichloroethane ether turpentine petrol kerosene cyanide and methyl salicylate.
!ppearance of blood urine and vomit
Ced venous blood may carbonmonoxide poisoning.
suggest
cyanide
or
1rown arterial or venous blood may suggest methaemoglobinaemia. Vomit or gastric lavage containing blood may suggest repeated vomiting corrosives para)uat coumarin anticoagulant irritants iron and non'steroidal anti' inflammatory drugs. 8any drugs turn urine black e.g. metronida(ole. rine may be cloudy or red or brown due to haematuria haemoglobinuria or myoglobinuria. 1lood pressure #ypotension
!lmost all sedatives hypnotics dehydration lengthy coma vomiting and sweating may cause hypotension
#ypertension
!mphetamines cocaine phencyclidine sympathometics and anti'cholinergic agents.
%ardiac arrhythmias
%hanges in heart rate or rhythm may be caused by beta blocking drugs organophosphates theophylline tricyclic antidepressants sympathometics barbiturates etc.
Directorate of Forensic Science, MHA, Govt. of India
Chabdomyolitis
4atient lying in coma for a long time on hard surface may develop it due to pressure necrosis of muscle which may lead to renal failure. The agents mostly responsible for the same are barbiturates opioids ethanol and carbon monoxide. Chabdomyolysis can also occur after prolonged and severe muscle spasm due to poisoning by strychnine phencyclidine and monoamine oxidase inhibitors.
(C) Pattern
Poisonscommonlyinvolved
%oma #ypotension Flaccidity.
1arbiturates ben(odia(epines glutethimide trichloro' ethanol ethanol opiods etc.
! peculiar smell on opening the body5 The substance detectable by their smell are alcohol cyanide carbolic acid petroleum products camphor nicotine opium paraldehyde phosphorus insecticides pesticides etc.
•
4resence of any foreign material in the form of powder capsules tablets leaves or seeds in the stomach.
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Laboratory Procedure Manual- Forensic Toicolo!y
•
$rritation ulceration and perforation or discoloration and change in color or softening of the mucous membrane of the stomach.
•
/aryngeal oedema commonly in the death is due to alcohol and barbiturates.
•
!cute lung congestion and oedema.
•
!cute swelling of brain with or without a pressure cone.
•
*istended urinary bladder.
•
$ntravascular sickling.
•
•
1ody is highly decomposed.
•
*ilation or contraction of pupils.
2.12
&N&(EENT &ND EDICO%E(&% &S PECTS O' POISONIN( C&SES:
The principle of management of poisoned patients is careful attention and treatment to prevent cerebral anoxia cardiovascular neurological and other complications. The main aim respiratory is to help the patient to stay alive. 8ost patients need only supportive care. The initial management should always be active until the contributions of drug induced damage and pre'existing organic disease are established. The main ob"ects of treatment of poisoning are ; Cemoval of unabsorbed poison from the system ; !dministration of antidotes ; &limination of absorbed poison
H +ymptomatic treatment H 8aintenance of the patientIs general condition. $t should always be remembered that over'treatment of the patient with large doses of antidotes sedatives or stimulants does far more damage than the poison itself. The "udicious use of drugs and necessary therapeutic measures are re)uired.
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Directorate of Forensic Science, MHA, Govt. of India
2.13
REO#&% O' UN&!SOR!ED POISON:
2.13.1 In9*le Po isons:
$n the cases of poisoning by inhalation the patient should immediately be removed to fresh air. ! clear airway should be ensured and artificial respiration commenced at once. $n some cases the respiratory centre may be stimulated by a mixture of ?J of oxygen and J of carbon dioxide. 2.13.2 In
The use of tourni)uet above the site of in"ection may slow the absorption. The tourni)uet should be loosened every ,@ min. for : min. suck the poison by making incisions at the site and use chemical antidotes cold packing and vasoconstrictors e.g. in"ection of epinephrine to prevent absorption. The common examples of in"ected poisons are hypnotics insulin snake or other insects bites etc. 2.13.3 Cont*ct Poisons:
$f the poison is applied to eyes skin wound or inserted the poison is removed with specific antidotes. 2.13.4 In+este Pois ons:
Gastric lavage or emesis may be induced before gastric emptying time usually ; to 3 hours after ingestion. 2.13. Eetic:
0hen the poison has been ingested early vomiting 6emesis7 is of greater value in avoiding absorption than the most energetic gastric lavage carried out after some delay. &metics are the substances which produce vomiting. 8ost poisons are themselves emetics and may cause vomiting. #owever it is advisable to give an emetic to ensure a more thorough emptying of the stomach. &mesis is easier than gastric lavage and less traumatic for the patient if the patient is conscious and co' operative and vomiting is not contra'indicated 6like corrosives strychnine petroleum distillates and coma7. &mesis should be avoided in corrosive poisoning for fear of damage of oesophagus and stomach. $t must also not be used when there is danger of aspiration into lungs either from volatile poisons like petroleum distillates or frombeinhalation gastric impaired consciousness. &mesis should induced of either by contents tickling due the to fauces or by emetics. The household emetics are5 •
%opious lukewarm water.
•
, gm of mustard powder in :@@ ml of water.
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Laboratory Procedure Manual- Forensic Toicolo!y
•
!bout -@ gms of common salt in :@@ ml of water .
•
Binc sulphate5 ,': gms in :@@ ml of water.
•
!pomorphine has a remarkable selective action on vomiting centre. $t is used in a dose of 3 mg by sub'cutaneous in"ection for )uick emesis followed by levallorphan ,': mg if necessary to or counteract preferably its naloxone ',@ mg intramuscular or intravenous narcotichydrochloride effects.
2.13./ (*stric &spir*tion *n %*,*+e:
Gastric aspiration and lavage is the only suitable method of emptying the stomach of an unconscious patient. $n unconsciousness protect airway with cuffed endotracheal tube. 4lace the head of the patient over the end or side of the bed so that mouth and throat are below the larynx and trachea. se a wide bore tube lubricated with vaseline or glycerin 6ac)ues gauge @cm in adults @ cms will reach the stomach7. &nsure the tube is not in the trachea. !spirate and save the first sample for analysis. se -@@'3@@ ml. of water at body temperature for washing. $f the tube becomes blocked gentle suction can be applied. %ontinue stomach wash with water or saturated lime or starch water or ,5@@@ potassium permanganate or ;J tannic acid etc. till a clear odourless fluid comes out. /eave some amount of antidote in the stomach for neutrali(ing left over poison. $t is best method if undertaken early i.e. within about ;'3 hours after ingestion of poison. !fter then it may not be useful. %ertain extra precautions are necessary for gastric lavage in cases of coma petroleum distillates and strychnine poisoning. Following the ingestion of corrosive passage of stomach tube may lead to perforation. /avage is dangerous in cases of petroleum distillates which may be inhaled rapidly may cause fatal results unless the glottis is sealed. $n coma there is a serious risk of aspiration pneumonia due to depression of cough reflex unless the airway is sealed by cuffed intubations by an anesthetist. $n the cases of strychnine ingestion a convulsion may be induced by this method unless the patient was first anaestheti(ed. 2.13.0 Use o f &ntiotes:
!ntidotes are remedies to counteract the effects of poison. They are used because the poison may not have been completely removed by emesis or lavage or the poison is already absorbed or it has been administered by other route than ingestion. !ccording to their mode of action they are divided into the following classes5 2.13.0.1 ec9*nic*l or P97sic*l:
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Directorate of Forensic Science, MHA, Govt. of India
These are the substances which tend to impede the absorption of poisons by their presenceD they are5 &cti,*te C9*rco*l:
$t is recommended as a general'purpose oral adsorbent. !ctivated means the brand of charcoal which meets certain adsorbance standards. To be fully effective a ratio of about ,@ parts of charcoal to , part of poison is needed. $t is more beneficial for adsorbance of substances which are toxic in small amounts such as tricyclic antidepressants and alkaloids like strychnineD but less effective when large amount of poison has been ingested e.g. in aspirin or paracetamol poisoning. The treatment with charcoal is most effective during the first four to six hours after ingestion. #owever some poisons are also adsorbed by oral use of activated charcoal many hours after ingestion and even after intravenous administration presumably by back diffusion or interruption of enterohepatic circulation. !spiration of charcoal into the lungs is a risk that should be guarded against. gms. Deulcents *n !ul7 'oos:
These are the substances which prevent the absorption of the poison by forming a coating on the mucous membrane of the stomach. $n doing so they act both in corrosive and irritant poisoning except in phosphorus poisoning as phosphorus is soluble in them thereby increasing the absorption. The common demulcents are fats oils milk and egg album in. 1ulky food like banana acts as a mechanical antidote to glass by imprisoning its particles and thus preventing its action. 2.13.0.2 C9eic*l:
These are the substances which act chemically to form a non toxic compound by forming insoluble compound or by oxidi(ing the poison to non toxic constituents e.g. dilute acetic acid neutrali(es alkalis. 8agnesium oxide or calcium oxide will neutrali(e acids. +imilarly lime can be used for oxalic acid magnesium sulphate for carbolic acid copper sulphate for phosphorus sulphates of alkalis for lead and freshly precipitated iron oxide for arsenic. Tannin produces insoluble compounds with most alkaloids glucosides and metals. 4otassium permanganate being oxidi(ing agent reacts with organic substances a ,5,@@@ strength a)ueous solution of potassium permanganate is effective against all oxidi(able poisons phosphorus cyanides etc. like alkaloids amidopyrin antipyrin barbiturates 2.13.0.3 P97siolo+ic*l or P9*r*colo+ic*l:
These chemicals do not enter into any chemical combination but produce opposite effects to that of the poison. #ence they are known as physiol ogical antagonists e.g. atropine for pilocarpine chloroform for strychnine caffeine for morphine atropine and oximes for organophosphorous compounds. #owever ,;?
Laboratory Procedure Manual- Forensic Toicolo!y
the antagonism is usually not complete and the remedy may itself produce most undesirable results.
$t is used when the iden tity of poison is not known or when a com bination of poisons is suspected. $t consists of a mixture of the following substances5 $
4owdered animal charcoal 6or burnt toast 7
: parts
!dsorbs alkaloids
$$ 8agnesiumoxide
,part
$$$ Tannic acid 6or strong tea 7
, part
4recipitates alkaloids certain glucosides E many metals.
The mixture is given in doses of a tablespoonful stirred up in : ltrs. of water. This dose may be repeated once or twice if necessary. 1esides some household products may be used as safe antidotes which are as follows.
2.14
•
+trong li)uid tea precipitates many alkaloids and metal poisons.
•
8ilk and raw egg white being protein rich precipitates mercury arsenic and other heavy metals. $n addition they have excellent demulcent properties.
•
8ashed potatoes in water are also good adsorbents and can be used in place of charcoal when it is not available.
•
8ilk of magnesia or soap solution may be used in poisoning due to acids.
•
Tinned "uice or vinegar can be used in poisoning due to alkalis.
E%IIN&TION O' &!SOR!ED PO ISONS:
!fter a considerable absorption of poison into the blood stream has occurredD procedures must then be employed to accelerate the excretion of the toxic agent mainly through urine. &limination by catharsis when not contra'indicated and by sweating by means of hot packs may be enco uraged. Fluid administration to maintain ade)uate renal function with periods of dialysis will be beneficial. $n cases of aspirin and barbiturate poisoning forced diuresis using intravenous
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Directorate of Forensic Science, MHA, Govt. of India
chlorothia(ide and2or mannitol infusion have been proved valuable. 4eritoneal dialysis has been recommended for salicylate poisoning in children. #aemodialysis has been used for eliminating barbiturates bromides glutethimide methanol salicylates and thiocyanates for the blood. &xchange transfusion is only feasible with small children and has been applied to poisoning by salicylates barbiturates iron salts carbon monoxide etc. !ll toxic substances are removed by this techni)ue. 2.1
TRE&TENT O' (ENER&% S6PTOS:
The treatment should be applied as indic ated by the symptoms. $n unknown poisoning cases the symptoms are the clues to the treatment of the case. +trong analgesic should be given for pain and oxygen for artificial respiration in respiratory failure. !ntibiotics should not be given routinely but only if infection is suggested by purulent sputum pyrexia and leucocytosis treatment is re)uired. %ardiac stimulants in circulatory failure and anaesthetic for convulsions should be given. The symptomatic effect of the poison should be treated by general means. +aline infusion is useful in counteracting dehydration and encouraging diuresis. #owever careful attention must be paid to water metabo lism as over hydration may lead to pulmonary oedema and circulatory impairment. The addition of sodium bicarbonate to the infusion may be of value when the alkali reserve is diminished. !dministration of glucose will combat depletion of liver glycogen and the restoration of potassium ' sodium imbalance may be necessary. 2.1/
&INTEN&NCE O' T5E P&TIENT=S (ENER&% CONDITION:
The patient should be kept warm and comf ortable. !fter coming out of the effects of poison one of the main dangers is the subse)uent development of upper respiratory tract infection. this is a special ha(ard in elderly people who had a respiration infection before and who inhaled vomitus. #ence if infection is suggested antibiotic should be given. Good environment and nursing care should be provided to the patient 6especially if unconscious7. !ll patients who have attempted suicide should not be allowed to leave the hospital without being interviewed by psychiatrist who can give further necessary supportive psychotherapy. References:
,.
!lbert !./. +elective Toxicity %hapman and #all /ondon ,?=?.
+perling F. Muantitation of Toxicology' The *ose N Cesponse Celationship in +perly F 6&d7 Toxicology5 4rinciples and 4ractices Vol.: 0iley <. Kork ,?>;.
,;.
Cowland 8. To(ev T.<. %linical 4harmaco Ainetics %oncepts E !pplications :nd &dn. /ea E Feliger.
,.
%larke &.G.% $solation and $dentification of *rugs : nd &dn. the 4harmaceutical 4ress /ondon ,?>3.
,:
nd
&dn. Caven 4ress <.
&dn. Von
rd
&dn. %harlesL %. th
Directorate of Forensic Science, MHA, Govt. of India
SECTION - 3: ISO%&TION &ND PURI'IC&TION O' POISONS 3.1
Title:
$solation and purification of poisons.
3.2
Scope5
General procedures for isolation and purification of poisons.
3.3
Purpose:
To know the methods adopted for isolation or extraction of poisons from different matrices.
3.4
Responsi8ilities: Ga(etted officers and other associated scientific staff.
3.
!&SIC ST EPS IN & N&%6TIC&% TO>ICO%O(6:
3./
,.
&xtraction of active constituent i.e poison in matrices of interest.
:.
+tripping or purification of active constituent thus separated.
-.
Capid +creening and $dentification
;.
Muantitation
.
$nterpretation 2 %onclusion.
(%OSS&R6 O' TER RE%&TED TO E>TR&CTION: *tri): !ny material substance in the universe wherein the active constituent
may be dispersed accumulated left absorbed or chemically bound. &cti,e Constituent: The toxic chemical of interest i.e. poison. Strippin+: 4urification. 3.0
For the purpose of chemical analysis poisons are grouped according to the methods used for isolation of the substance from matrices. These are given below.
There are various classica l and modern methods of extraction. The selection of proper method of extraction depends on various controlling factors vi(. nature of poison matrix or matrices and also )uantity of samples available or forwarded for analysis. The active constituent should be extracted from sample in minimum steps to avoid loss during processing. The extracted material also re)uire proper stripping or purification to avoid interferences of matrices as far as possible. The efficiency of extraction and stripping determines the lower limit of detection precision and accuracy in the determination. The analyst plays a significant role in the selection of proper methodology on the basis of different parameters vi(. case history amount physical state of matrices analytical re)uirements and also infrastructural facilities available. $t will be befitting if the methods are presented schematically depending on the classification of poisons. Cl*ssofpoison
Gases Volatile $norganic
Volatile 9rganic
Cl*ssic*l et9o
8icro'diffusion! dsorption' desorption Gut(eit 8ethod 8arsh' 1er(elius 8ethod 8icro' diffusion *igestion with specific reagents 2 under specific conditions of 4# *istillation +team *istillation *iffusion *ry and wet ashing Group analysis &lectro'dialysis *igestion under appropriate analytical conditions 4aper and Thin layer chromatography +olvent &xtraction +tas' 9tto *igestion with ammonium sulphate sodium tungstate or other modified methods of the above
,;
oernet9o
+ensor 1ased Gas !naly(er Gas %hromatography. 8icrowave oven techni)ue for digestion followed by $on %hromatography +pectroscopy68ass7 etc %hromatographic methods 8icrowave oven techni)ue for digestion followed by $on %hromatography using $on'exchange resins #4/% 4aired ion extraction %hromatography #4T/% +upercritical fluid chromatography +olid phase extraction 8icellar extraction !ffinity chromatography 8icrowave
Directorate of Forensic Science, MHA, Govt. of India
!nion
3..1
*ialysis %hemical *igestion 4aper and Thin /ayer %hromatography
The various unit processes 2 operations related to extraction are as given below5 3..1.1 Sol,ent E)tr*ction:
! system of two immiscible li)uid is re)uired for the separation of material by solvent extraction. The active constituent should be unevenly soluble in the system thereby facilitating extraction of the constituent from one phase to the other. The efficiency of extraction is determined by distribution co'efficient 6*7. *O
Total wt 6gms.7 of solute in the organic phase '''''''''''''''''''''''''''''''''''''''''''''''''''''' Total wt 6gms.7 of solute in the a)ueous phase
$f one of The the two li)uids contains solute this and method found to settle. be more+ome suitable. system in this case isa first shaken thenisallowed of the solute is transferred to the other li)uid. &ach of the li)uid in a mixture of two immiscible li)uids of this kind is referred to as a phase. Thus some of the solutes is transferred from one phase to the another phase. The amount transferred depends on the relative affinity of the solute for each of the two solvents 6relative solubility7. $t is determined by *. Greater the value of * greater is the efficiency of extraction. The immiscible system may involve two organic solvents. The extraction for this system may be impaired due to formation of emulsion. +olvent extraction is a common techni)ue in forensic toxicology related to biological matrices. +olvent extraction method has now been upgraded and made automatic vi(. accelerated solvent extraction. $n case of solid non'biological matrices continuous extraction by a soxhlet may be employed i.e. continuous extraction.
3..1.2 Distill*tion:
The process involves heating a sample of li)uid to convert it into vapour which is then allowed to flow in another location where it is cooled condensing it back into a li)uid. Various modifications of the basic distillation process are used for ,
Laboratory Procedure Manual- Forensic Toicolo!y
specific purpose vi(. steam distillation fractional distillation distillation under reduced pressure sweep co'distillation etc. 3..1.2.1 Ste* Distill*tion:
+team volatile substances can be separated or isolated from blood urine and properly minced viscera by steam distillation. +team is passed into the sample and the a)ueous distillate is collected by condensation. Toxicants from acidic distillation process include ethanol methanol phenol halogenated hydrocarbons cyanides etc. 9n the other hand toxicants from basic distillation process include basic drugs such as amphetamine methadone and also aniline pyridine nicotine etc. 3..1.2.2 'r*ction*l Distill*tion:
This is a type of distillation which enables separation of a mixture of volatile li)uid differing marginally in boiling point. ! mixture of kerosene oil or mineral turpentine oil in an oil'water emulsion may be separated by this method. 3..1.2.3 Distill*tion Uner #*cuu:
This is another type of distillation method which provides separation of thermally labile volatile compounds at a low temperature without decomposition. 3..1.2.4 S@eep Co-istill*tion:
This is a special type of distillation based on the preferential volatili(ation of organic compounds specially pesticides from oil lipids or plant extracts using a stream of inert gas and subse)uent isolation of volatiles on cold traps or solid adsorbent. $t is a purge and trap techni)ue involving dispersion of the sample in thin films on deactivated glass beads or florisil or alumina or silica gel or tenax as trapping media at elevated temperatures. niversal Trace Cesidue &xtractor 6<$TC&P7 and !ccelerated +olvent &xtractor 6!+&7 i.e. the highly automatic extraction system for rapid extractions of multiple samples work on this principle. 3..1.3 icro-iffusion:
8icro'diffusion is a convenient and popular method that facilitates toxicants 6gaseous and volatiles7 in blood urine and gastric aspirates to be isolated detected and determined. This is done by %onway 8icro'diffusion dish 6elaborated separately for convenience in section ;.=.-7. 3..1.4 Di*l7sis:
$t involves separation of a crystalloid from a colloid by filtering through a semi' permeable membrane. This separation method may be employed for the separation of toxic cations and anions in a colloidal solution or dispersion or colloidal matrices especially biolo gical materials. The separation process may be accelerated by applying e.m.f. i.e. electro'dialysis.
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Directorate of Forensic Science, MHA, Govt. of India
3..1. Su8li*tion:
This is similar to distillation except the sample is a solid to begin with and is converted directly into vapour and then back into solid. +ublimation is applicable to isolate a toxicant in solid matrices vi(. naphthalene anthracene which sublimes. 3..1./ Di+estion or C9eic*l Tre*tent:
+ometimes active constituents 6toxicant7 are separated on treatment with acid or alkali or digestion on a water bath or muffle furnace vi(. biological matrices are digested on a water bath for , hour or above or digested in muffle furnace with acid 2 alkali 2 or chemicals to isolate inorganic metals. Volatile inorganic poisons vi(. phosphine arsine and hydrogen sulphide are isolated from their salts on treatment with dilute acids. 3..1.0 icro@*,e Di+estion:
8atrices are digested with acids 2 alkalis in microwave oven to facilitate isolation of inorganic poison in organic matrices under a specific analytical condition of operation vi(. operation of oven at a specific microwave for sometime. The interaction of microwave with matrices results in production of heat with rise of temperature for which digestion occurs. 3..1. &8sorption:
$t is a slow process 6compared to adsorption at the surface7 involving diffusion of one substance into the interior of absorbent material. Toxic gases and volatiles in oil are entrapped and enriched by the process using a tube containing diverse absorbing materials specific for a particular toxicant and on' line detection and determination is facilitated. 3..1. C9ro*to+r*p97:
%hromatography involves the separation of substances based on their relative affinity for two phases vi(. stationary and mobile. +ubstances which have higher affinity for the mobile phases are moved or carried along with it and are thus separated from those with higher affinities for the stationary phase. Thus the toxicants in molecular mixtures may be separated conveniently under different chromatographic methods and operating conditions in a particular chromatography. There are different controlling parameters vi(. nature of toxicant mobile and stationary phase and temperature. +alient aspects of different chromatographic methods are given below. 3..1..1 Colun C9ro*to+r*p97:
+eparation of active constituent is achieved here by preferential absorption of active constituents on the adsorbent or stationary phase. $n this method a vertical glass tube is filled with a granular adsorbent. This adsorbent acts as the stationary phase. The sample is then added to the top of the column in the form of a solution in a suitable solvent or mixture of solvents. The system of solvent is then added as a mobile phase which is to be selected beforehand. !s the solvent system flows ,=
Laboratory Procedure Manual- Forensic Toicolo!y
through the column under the influence of gravity or pressure various components of the sample mixture will migrate at different rates and then arrive at the lower end of the column at different rates i.e. time. Fractions collected at various intervals will thus contain the different components separated at different intervals. The efficiency of separation in the column is dependent on the adsorbent material selection of solvent system nature of active constituent as well as mobile phase. These controlling factors have been explored to develop different method of chromatography vi(. #4/% #4T/% affinity gel permeation ion'exchange and ion chromatography solid phase extraction and super critical fluid chromatography. These have been discussed separately. The analytical conditions re)uired for separation of toxicants are either available in standard texts or references or may be developed conveniently by standardi(ation depending on nature of toxicant and matrices. 3..1..2 P*per C9ro*to+r*p97:
The separation of active constituent occurs on cellulose paper as the stationary phase. This is the primitive method in chromatography and used for separation of organic dyes pigments inks cations and anions etc. 3..1..3 T9in %*7er C9ro*to+r*p97:
The separation takes place on a thin layer of adsorbent material such as alumina silica gel G or cellulose coated on to an inert support material such as glass plate plastic or aluminium sheet. There are different methods that are popular in T/% vi(. ascending descending two dimensional etc. Muantitative separation is achieved by optimi(ing analytical conditions. 3..1..4 5i+9 Perfor*nce T9in %*7er C9ro*to+r*p97:
! type of thin layer chromatography wherein the stationary phase is designed to offer enhanced separation and resolution properties. The enhanced separation is due to special structural feature of adsorbent attained by special processing for which optimum resolution of molecular mixtures 6for separation by chromatography7 occur on plate coated with specially prepared adsorbent. The system 6#4T/%7 is now fully automatic and multiple samples may be handled )uickly precisely and conveniently.
3..1.. 5i+9 Perfor*nce %iAui C9ro*to+r*p97:
$t is based on the different attractions of non'volatile analytes vi(. drugs pesticide explosive organics etc. between a li)uid phase 6 pumped through a column7 and a solid phase 6 packed within the column'stationary phase 7. The operating parameters includes composition of mobile phase adsorbent nature of analyte etc. for optimi(ations of resolution. 3..1../ Ion C9ro*to+r*p97:
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Directorate of Forensic Science, MHA, Govt. of India
$t works on the same principle as in #4T/%. The composition of stationary phase and also the mobile phase is so selected that cations and anions can be separated conveniently. The active constituents in the matrix material are chemically bonded to molecules which have a fixed charge. $n suitable form ion exchange resins can be packed into columns and used for separation of molecular mixtures which have the opposite charges 6because unlike charges attract 7 vi(. cations and anions. $n some cases the net charge on the column material or sample molecules or both is dependent on p# giving rise to greater analytical flexibility. 3..1..0 (*s C9ro*to+r*p97:
$t is a procedure whereby volatile components of a mixture may be separated by partition between a solid or li)uid stationary phase and a gaseous mobile phase. The efficiency in the separation is achieved by controlling several factors vi(. column type 6capillary or wide bore7 column length column diameter nature of li)uid phase carrier gas flow rate and temperature. This techni)ue has now been hyphenated with other techni)ues vi(. +F%'G% G%'8+. 3..2
oern et9os of E)tr*ction 6> ?7:
The methods include various methods in chromatography as well as special extraction methodologies of the present generation vi(. ion'pair formation solid phase extraction solid phase micro extraction micellar extraction. +pecial types of digestion as a means of isolation of toxicant through chemical processing i.e. microwave digestion and digestion by plasma source for inorganic samples are also emerging for their consideration in routine toxicological analysis. Thus there are various methods of extraction under the head “ 8odern 8ethod” which are either underutili(ed or unexploited in the $ndian perspective due to lack of infrastructural facilities and analytical expertise. !s it has become very difficult to cope up with much inflow of exhibits modern methods are replacing traditional methods of extraction for speedy analysis. 3..2.1 5e* Sp*ce Tec9niAue 6,37:
The headspace method is especially suitable for the very fast separation of volatile components 6alcohols acetone aldehydes7 in complex biological matrices specially blood in mass'li)uor and prohibition law related cases. This method has the advantage of avoiding the risk of contamination of non'volatile components which may be eliminated due to on'line analysis by gas chromatography. The principle underlying the headspace analysis is that in a sealed vial at constant temperature is established volatile a li)uid samplee)uilibrium and the gas phase above it between 6the head the space7. !ftercomponents allowing theoftime for e)uilibrium 6normally , minutes or so for @ or more samples in a single run7 a portion of the headspace may be withdrawn one by one from vials using a gas' tight syringe and in"ected to G% for on'line analysis. 3..2.2 D7n*ic 5e*sp*ceB Pur+e *n Tr*p Tec9niAue:
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Laboratory Procedure Manual- Forensic Toicolo!y
These techni)ues are basically modified or higher version of #eadspace method to optimi(e the separation of volatiles for on'line analysis by G%. $n the purge and trap method volatile compou nds 6toxicants7 are liberated from the sample by bubbling with an inert carrier gas and subse)uently either condensed in a receiver cooled usually with solid carbon'dioxide or li)uid nitrogen adsorbed on a cartridge filled with solid adsorbent material such as Tenax. $t is polymer based diphenyl'p'phenylene oxide and available in various forms. Tenax T! is a highly purified form of the polymer and stable upto-= o% and gives insignificant bleeds of organics. ! material suitable for the recovery of low molecular weight compounds is Tenax GC which contains :-J graphite. This adsorbent is suitable for efficient trapping of compounds of low to medium polarity and recovering them )uantitatively by either solvent extraction or thermal desorption. !lternatively cartridges filled with activated charcoal can be used to trap the volatiles which are then extracted into a small volume of carbon disulphide prior to the analysis. This techni)ue is widely used in the analysis of volatile compounds in water samples till date mainly because of the difficulty in interpreting the results at concentration below those which can be measured using ordinary headspace method. 4urge and trap techni)ue are similar to dynamic headspace sampling except that the gas is passed through the sample. %learly any apparatus used for dynamic headspace sampling can also be used for purge sampling by using on appropriate sampling vessel. 1oth dynamic and purge methods are available in a variety of automatic systems to enable separation of constituent of concentration in the ppb N ppt range. 3..2.3 Soli P9*se E)tr*ction SPE:
The old observation in the extraction of drugs by adsorption on solid materials vi(. Florisil 6! synthetic magnesium silicate7 or activated charcoal and selective elution by solvent thereafter has given rise to solid phase extraction method. $n this method siliceous materials with relatively close si(e distribution 6, to ,@@µ 7 and various silica bonded phases vi(. n'octadecyl 6%,> 9*+7 n'octyl 6%>7 n'hexyl 6%37 ethyl 6%:7 methyl 6%,7 cyanopropyl 6nitrile %<7 aminopropyl 6amino !4+ etc.7 have been employed as adsorbent with much greater efficiency of separation. The method is based on the use of small tubes or cartridges filled with ,@@'@@ mg. of an appropriate adsorbent as stated above. There lies however a recently developed type of +4& cartridge in which very small particles of the adsorbent are enriched in a well of 4TF& microfibrils having similar efficiency as in a packed column but re)uire less pressure drop. The sample is applied to the +4& cartridge either from a syringe or sampling manifold by applied pressure or suction at the lower end. %omponents are subse)uently recovered by solvent flushing. The method is attractive because of the small amounts of the sample and materials are necessary and also much greater speed of the procedure compared to classical adsorption method. !nalyte concentration may often be achieved more easily with +4& than with li)uid'li)uid extraction as use of +4& column to concentrate an analyte from solvent extract may provide a )uicker and possibly safer alternative to solvent ,3@
Directorate of Forensic Science, MHA, Govt. of India
evaporation. ! ma"or advantage of +4& is that batch processing can be simplified. Further feature when screening for unknown is that a range of analyte can be extracted simultaneously although this may create problem if analyte of a single component is re)uired. 8oreover +4& columns are expensive and it may not be possible to retain very water'soluble analyte. The +4& protocol is now available. +olid 4hase &xtraction 6+4&7 cartridges are used primarily to clean up samples for analysis and2or concentrate samples to improve detection limits. The lack of sufficient sample preparation will result in poor detection limits identification and )uantitation errors contamination problems and rapid deterioration of G% or #4/% column performance. +4& techni)ues usually provide better sample cleanup and recoveries than li)uid'li)uid extraction techni)ues. +4& uses small volumes of common solvents re)uires very simple laboratory skills does not re)uire the use of highly speciali(ed laboratory e)uipments and allows rapid sample throughput. ! li)uid sample or solid sample dissolved in a solvent is poured into the conditioned +4& cartridge. Vacuum or pressure is used to force the sample through the sorbent in the cartridge. $n +4& vacuum manifold is normally used to simultaneously process multiple cartridges. sually +4& methods are designed to retain the analytes of interestD other sample components similar to the analytes also will be retained. The analytes of interest are then eluted from the sorbent using another solvent. This solvent is collected for analysis for additional processing. SPE C*rtri+es
!n +4& cartridge is composed of three basic parts5 ,7 %artridge or tube body :7 Frits -7 4hase or sorbent C*rtri+e or Tu8e !o7
The cartridge body usually is a syringe like barrel made of serological grade polypropylene. 'rits
The frits are used to hold the sorbent in the barrel and to act as a particulate filter. P9*se or Sor8ent
The most common +4& phases are bonded silica'based materials. $rregular shaped Qm silica particles withfunctional 3@R poresgroups are used starting material. Various ;@ silanes are used to attach to as thethe accessible areas of the silica particle. $n addition several non'silica based phases are commonly used. +olvent reservoirs can be used to increase the volume of barrel above the phase. /arge amounts of sample or solvent 6up to = ml7 can be added directly to +4& cartridges in one volume instead of in small increments. %oupling fittings are used to attach the reservoirs to the +4& cartridges. P9*ses.
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Laboratory Procedure Manual- Forensic Toicolo!y
There are three types of phases5 normal reverse and ion' exchange. Nor*l P9*se
Table : lists common normal phase sorbents. !ll of these phases are polar and are used to retain 6extract7 polar analytes. For normal phase sorbents solvent strength increases as the solvent becomes more polar. For example a retained analyte will completely elute from a normal phase sorbent in a smaller volume of methanol than chloroform. !ll of the solvents in Table , are commonly used with normal phase sorbents. 8ixtures of two solvents often are used to refine the solvent strength for optimal sample cleanup and analyte recovery. Re,erse P9*se
!ll of these phases are non'polar and will be used to retain 6extract7 non'polar analytes. For reverse phase sorbents the solvent strength relationship is the opposite from normal phase sorbents 6Table :7. For reverse phase sorbents solvent strength increases as the solvent becomes more non'polar. For example a retained analyte completely elutes from the sorbent in a smaller volume of acetonitrile than water. $n most cases the solvents used with reverse phase sorbents are limited to water methanol isopropanol and acetonitrile as mentioned in Table , 6Ceverse 4hase7. 9n occasion acetone or dichloromethane may be used as an elution solvent for highly retained analytes. Ion E)c9*n+e P9*se
$on exchange phases are more dependent on p# ionic strength and counter ion strength than solvent strength. $on exchange phases depend on ionic interactions as the primary retention mechanism. $onic interactions occur between an analyte molecule carrying a positive or negative charge and a sorbent carrying an opposite charge. There are two groups of ion exchange phases. The cation exchange phases retain positively charged or “cationic” compounds. !mines and carboxylic acid are not charged species. They can be charged by varying p#. The anion exchange phases retain negatively charged or “anionic” compounds. Table - lists the classifications and characteristics for several common ion exchange phases charge7. T*8le 1: Sol,ent stren+t9s
For each category the highest selectivity counter ions were normali(ed to ,@D thus the values are relative.
,3-
Laboratory Procedure Manual- Forensic Toicolo!y
3..2.4 Soli P9*se icro E)tr*ction SPE:
+olid phase micro extraction 6+48&7 is an extraction techni)ue for organic compounds in a)ueous samples in which analytes are adsorbed directly from the sample onto a fused silica fiber that is coated with an appropriate stationary phase. 0hen the fiber is inserted in the sample the analyte portion from the sample matrix enters the stationary phase until e)uilibrium is reached. The fiber in then inserted into the in"ection port of a gas chromatograph 6G%7 where it is heated and the analytes are rapidly desorbed thermally into a capillary G% column for analysis. 3..2. Super Critic*l 'lui E)tr*ction S'C:
Gases above their critical pressure and temperature are in a supercritical state intermediate between that of a gas and li)uid. +upercritical fluids have strong extraction properties because the solubility of compounds in fluid is close to that of a true solvent and much lower viscosity allows it to percolate through packed bed of sample. Thus not only there is an efficient contact between the extracting fluid and the sample but the fluid is easily removed when it is released from its supercritical state. %arbon dioxide is nearly always the chosen gas for +F% in view of its innocuous nature and mild critical condition namely critical pressure of = bar and a critical temperature of -,@% which are relatively easy to achieve at present. The sample holder is composed of a number of small stainless steel cartridges which are filled with the sample in a particular state. +olid sample such as soil or sediment are packed into the cartridges without any pretreatment and a)ueous samples can be flashed through the cartridges filled with an appropriate adsorbent to concentrate all of the contaminates. The cartridges are subse)uently fed into the extraction oven and the carrier gas line which at this stage consists of supercritical carbon dioxide. The extracted compounds are carried to the cold trap and condensed after the heated constriction which restore carbon dioxide to its true gaseous state. !fter the appropriate extraction period the circulation of coolant ceases and trap is rapidly heated to vapori(e the components. !t the same time the column temperature is programmed according to the re)uired condition. The ad"ustable split partitions the sample si(e to avoid the possibility of overloading effect. +F% is suitable for extraction of pesticide traces in solid and a)ueo us samples. 3..2./ icell*r E)tr*ction:
8icellar extraction is a special type of extraction procedure that appears to be uni)ue in the separation of drugs plant poisons and pesticides in biological matrices 6viscera7. $n the extraction of active constituents as above micellar environment of surfactant of different classes is employed. +urfactant or surface active agents at a particular concentration in solution known as critical micellar concentration 6%8%7 form micelle or association colloid. !t this concentration or ,3;
Directorate of Forensic Science, MHA, Govt. of India
above marked changes in the properties vi(. viscosity conductance electrical conductance are exhibited. +urfactant in solution also acts at the interface of two phase system of oil and water or organic solvent and water resulting solubilisation of one phase into the other. !n emulsion or micro'emulsion is formed by the process. The emulsion may be destabili(ed by increasing the ionic concentration of additives including surfactant. 1iological matrices 6Viscera7 contains fats degraded protein and colouring matter etc. makes the extraction of active constituent difficult. $n the solvent extraction process if surfactant is added to the extractant 6organic solvent7 deprotei ni(ation and also solubilisation with the formation of emulsion will occur due to micellar interaction. The emulsion thus formed is due to solubilisation of fat in biological matrices in the added solvent 6containing traces of water7. +imultaneous formation of emulsion occurs due to micellar interactions. The emulsion thus formed due to solubilisation is destabili(ed on increasing the concentration of surfactant in the system. !s a result fats are separated as semisolid material due to lowering of (eta potential between the electrical double layers of the colloidal system. !s protein and fats are separated out the supernatant li)uid containing active constituent may be extracted for poison by organic solvents. The detailed analytical conditions have also been presented at appropriate places in this manual. 3..2.0 icro@*,e &cceler*te Re*ction S7ste:
The method of extraction is used for isolating pesticides in biological materials especially in liver and kidneys. $n this process the sample is sub"ected to rapid heating with organic solvent by microwaves at elevated pressure resulting isolation of active constituent. The biological material 6N ,@ gms.7 is placed inside a microwave transparent vessel with a polar solvent or ionic solution 6usually an acid7 and is sub"ected to rapid heating by microwave in a microwave accelerated reaction system 6digester7. The analytical conditions 6temp. time of digestion pressure7 may vary depending on active constituent and nature of sample vi(. monocrotophos and phosphamidon are successfully extracted within ,':@ minutes from viscera using dichloromethane as a solvent at >@',@@ @% and ,@@ 4si. #owever optimi(ation of analytical conditions to covers different classes of pesticides are re)uired for a rapid extraction by this method. The method finds application in the digestion of biological materials for isolation of some toxic metals 6%u !s 4b etc.7 . 3..2. Uni,ers*l Tr*ce Resiue E)tr*ction:
$tIs a system that has been developed for the recovery of pesticides and organic residue from a wide range of samples including biological materials. $t is based on the principle sweep co'distillation thatfrom reliesbiological on preferential volatili(ation of pesticides or ofother organic chemicals materials lipids plant extracts using a stream of inert gas and subse)uent isolation of volatiles on cold traps of solid adsorbents. $t is a purge and trap techni)ue involving dispersion of the sample in thin films on deactivated glass beads at elevated temperatures. The extractor system is specifically designed to recover volatile thermally stable organochloro and organophosphorous pesticides from lipids meat butter viscera etc. !t present the distillation tube does not contain glass beads or glass wool as it ,3
Laboratory Procedure Manual- Forensic Toicolo!y
renders less recovery. Florisil in con"unction with sodium sulphate has been found satisfactory for trapping many different classes of volatile organic compounds !lumina +ilica gel and Tenax are materials that have potential for use as trapping media with advantages over Florisil in specific applications. The method is expected to fail for thermally labile pesticides. The consumption of solvent is minimum. The method re)uires optimi(ation of analytical conditions before its application to biological samples 6viscera7 in forensic cases. 3..2. &cceler*te Sol,ent E)tr*ction:
The name of method signifies multiple sample handling in a very short time by a very updated extraction system which also works on the same principle as niversal Trace Cesidue &xtractor i.e. +weep %o'distillation. $n this method a commonly used solvent is pumped into an extraction cell containing the sample which is then brought to an elevated temperature and pressure. 8inutes later the extract is transferred from the heated cell to a standard collection vial for clean up analysis. The entire extraction process is fully automated and performed in minutes for fast and easy extraction of multiple samples with a very minimum solvent consumption. The standard or optimum analytical conditions are to be arrived for its application to biological matrices in forensic toxicological work covering a broad spectrum of pesticides. #owever the method has been found to be effective for soil samples. 3..2.1 Sie E)clusion C9ro*to+r*p97:
The techni)ue can be used to advantage as a preparation techni)ue for the prior fractionation of oils fats environmental samples etc. into discrete molecular weight fractions and as a way of removing very high molecular weight material from complex sample. 3..2.11 Ion-P*ir E)tr*ction:
This method is applicable for the extraction of highly water'soluble organic compound. These compounds cannot be extracted from samples by direct solvent extraction. The difficulty has been overcome by forming ion pair with a suitable reagent vi(. )uaternary ammonium salts as ion'pairing agent. The ion'pair formation is an attraction between a positive and a negative charge. 9nce this pair is formed it will act exactly like an organic molecule and not like as ionic compound at all. The extraction may be carried out thereafter by direct solvent extraction preferably in presence of a buffer. The method is also applicable for extraction of drugs in the form of )uaternary salts in urine or blood. The biological are deproteini(ed and treated with a dye 6selective7 in presence of buffer tomatrices form drug'dye complex which is extracted by organic solvent. The dye is destroyed by the action of acid or alkali and the drug is isolated for analysis. 3..2.12 57p9en*te Tec9niAues 5P%C-(C &ND S'C-(C6,@7:
The hyphenated techni)ues vi(. #4/%'G% and +F%'G% allow complex samples to be pre'fractionated rapidly according to molecular weight or chemical ,33
Directorate of Forensic Science, MHA, Govt. of India
classification on a suitable #4/% pre'column or +F% and appropriate fractions then separated by on line capillary G%. The coupling of two techni)ues has been made by a bypass valve fitted with a sample loop of appropriate volume. +elected fractions are passed to the capillary column via a large retention gap which utili(es the process of concurrent solvent evaporation to retain the fraction in the retention gap thus avoiding any preliminary contact with column. 3..3
E)tr*ction of #ol*tile Poisons 87 Distill*tion:
The method is applicable for volatile organic or inorganic poison under different conditions of p# i.e. acidic and alkaline. 3..3.1 Neutr*l *n &ci Distill*tion: Proceure:
@',@@ gms. of viscera 6properly minced7 stomach contents vomit or other materials are to be examined separately. They should be brought to the consistency of a thin gruel by adding -' times of distilled water and acidified with tartaric or sulphuric acid and submitted to steam distillation. The condenser and the receiving flask should be well cooled with ice especially during the hot season the outlet of the condenser being dipped in a little water or
!fter the completion of the acid distillation the flask is allowed to cool and its contents are rendered alkaline by adding
E)tr*ction of To)ic et*ls in *trices:
3..4.1 Non-!iolo+ic*l *trices:
The non'biological matrices such as tablets powders may be sub"ected to chemical analysis by preparing solution of samples and their systematic group separation. ,3=
Laboratory Procedure Manual- Forensic Toicolo!y
3..4.2 !iolo+ic*l *trices:
The extraction of metals in biological matrices may be carried out by the following methods. *ry !shing 8ethodD 0et *igestion or !cid *igestion 8ethodD Fresenius and 1abo 8ethodD +elective %hemical Treatment. The organic matters which constitute the bulk portion are destroyed by chemical means to get the active constituents 6metal ions7 free completely for )ualitative and )uantitative analysis. 3..4.2.1 Dr7 &s9in+ et9o:
!bout ,@'@ gm.of tissue or other biological materials is taken in a silica crucible and heated in a 1unsen burner for removing the moisture and partially destroying the organic material. Then the crucible is kept in a muffle furnace. The temperature of the furnace is raised up to @ °% and at this temperature the incineration of the organic matter is performed by keeping the silica crucible for one residue hour. !fter complete the crucible is taken colour of the is toincineration be noted asinwhen hot because in presence of out. (inc The the residue assumes yellow colour while in presence of copper the colour of the residue is somewhat bluish green. The residue in the silica basin is boiled with ,@ ml. of ;< hydrochloric acid and then filtered. The clear acidic solution is tested for metallic poisons such as copper bismuth (inc barium etc. by performing general group analysis using semi'micro methods chromatographic and instrumental techni)ues. 3..4.2.2 Fet Di+estion et9o: Proceure :
!bout @gms. of biological material or ,@ ml. of blood is take n into a large A"eldahl flask and :@ to ;@ ml. of %onc. #<9 - is added to cover the material and flask is gently heated in a small flame when the mass begins to li)uefy. The heating is continued until the li)uefication of the material is complete and that must be done in the presence of copious brown fumes of nitrogen dioxide in the flask. !t this stage about :@ N-@ ml. of %onc. # :+9; is added and the flask is heated strongly over a wire gauge and %onc. #<9 - is added in drops 6by using dropping funnel7 to the contents of the flask at the rate of about ,@ drops per minute so that the atmosphere in the flask must at no times be free from brown fumes. #eating is continued until all organic matter is destroyed and the li)uid becomes clear and colourless or straw coloured.
,3>
Directorate of Forensic Science, MHA, Govt. of India
To find out if the oxidation is complete the flask is heated without adding any #<9-. $f there is any un'burnt organic matter the li)uid begins to darken and if the digestion is complete no darkening takes place and the white fumes of +9 - are given off. $n the former case the addition of #<9 - and heating are continued further till the organic matter is completely oxidi(ed. #eating is continued for , minutes more to expel the nitric acid completely. Then after cooling : ml. of saturated ammonium oxalate solution is added. The li)uid is boiled until +9 fumes appear. This ensures complete removal of #<9 -. $t is then cooled diluted with an e)ual volume of water and carefully transferred to a beaker. The beaker is heated on a hot plate or sand bath to expel the excess # :+9;. The solution is cooled and diluted with water in such a way that the strength of acid is about,@J. !t this stage a precipitate may be formed which contains the insoluble salts of lead bismuth tin barium strontium or silver etc. The precipitate is filtered off and tested for the metals mentioned above. The filtrate will now contain all other metals except mercury. $t is sub"ected to systematic group analysis and )uantitative determination thereafter as and re)uired. 3..4.2.3 'reseniu *n !*8o et9o for ercur7:
The nitric'sulphuric acid 6wet digestion7 method of destruction of organic matter is not at all suitable for mercury which is almost completely lost by volatili(ation. This method is considered most suitable for liberation of mercury although there is a possibility of some loss of mercury by vaporisation.
Proceure :
! definite amount of biological material vi(. :@': gms. of viscera or ',@ ml. of blood is taken in a flask fitted with a reflux condenser. $n the case of viscera or other solid material sufficient water is added to make gruel like consistency. 9ne third of its volume of chemically pure hydrochlo ric acid and a few gms. ofolid s A%,9 - are added. The content are mixed by shaking. The mixture is heated over a wire gauge on a burner flame or on a boiling water bath. +mall amount of A%,9- is added time to time and the flask is shaken. %hlorine gas evolves. The heating is continued until the contents of the flask becomes a uniform straw coloured li)uid free from organic matter except some fatty substances in suspension which can not be oxidi(ed. $f heating for an hour after the last addition of A%,9- produces no darkening of the mixture the oxidation of organic matter may be taken as completed. $t takes ;'3 hours to attain the stage. $t is filtered and washed with water. The filtrate and washings are collected. +ufficient sodium sulphite or bisulphite is added to reduce the excess of ischlorine hydrochloric acid. The li)uid is warmed on water bath and a current of air passed into to expel the excess +9 :. The solution is now ready for analysis. 3..4.2.4 Selecti,e C9eic*l Tre*tent:
! few toxic metals vi(. arsenic and antimony in their specific oxidation state 6S7 may be sub"ected to the reduction process by nascent hydrogen 6by the reaction between (inc and dil sulphuric acid7 for isolation of metals in matrices vi(. burnt ,3?
Laboratory Procedure Manual- Forensic Toicolo!y
bones nail hair and non'biological matrices like food preparation drinks tea coffee etc. in the form of their volatile hydrides 6!s# - and +b# -7. The process may be carried out by Gut(eit or 8arsh'1er(elius method. $n case of presence of !s and +b in their higher oxidation state 6S7 reduction to S- state is to be carried out prior to chemical treatment. The method and analyticals have been presented separately. The analyst should not be biased by positive findings as there are chances of interference due to the presence of phosphorous sulphide !s or +b in the matrix itself. ! blank test should invariably be carried out as !s or +b may be present in matrices or the reagent. 3..
E)tr*ction of To)ic &nions in 'orensic *trices:
The extractions of toxic anions in non'biological matrices re)uire very minor processing and clean up. $nconveniences is felt in case of biological matrices vi(. viscera stomach and gastric contents stomach wash urine and blood. The extraction procedures include the following method. 4rotein 4recipitation. *ialysis. +elective %hemical Treatment. 8icro *iffusion. $on %hromatography 3...1 Protein Precipit*tion:
! slurry of the sample 6minced viscera ,@':@ gm. of stomach contents or gastric lavage etc. is prepared. The protein in the sample is coagulated by adding @.',.@ gm. of ammonium sulphate. $t is filtered through a : cm. layer of cotton wool held in the barrel of the syringe. The excess of ammonium sulphate remaining in the filtrate is precipitated by methanol. The supernatant li)uid is collected and evaporated to a small volume on a water bath. The concentrated li)uid is ready for analysis of anions. 3...2 Di*l7sis:
,@ gms of the tissue is cut into small pieces and placed in a cellophane membrane made the shapewater of a bag. The bag slowlymotor rotated a beaker containing ,@@ mlinto of distilled by means of is anthen electrical or in mechanical device. *ialysis occurs rapidly. !fter one hour the water in the beaker is replaced by fresh water and the bag is rotated for further half an hour. The water is then taken out mixed with the previous fraction and evaporated to a small volume on water bath. This is filtered if necessary and tested for toxic anions. 3...3 Selecti,e C9eic*l Tre*tent:
,=@
Directorate of Forensic Science, MHA, Govt. of India
4hosphides and sulphides present in biological matrices vi(. Viscera stomach content and gastric lavage is sub"ected to treatment with dilute acid on hot plate or water bath. The toxicants that are liberated in the form 4# - or #:+ are sub"ected to chemical analysis. This has been covered separately in the monograph. 3...4 icro-Diffusion:
The unit operation may be employed for biological matrices vi(. blood urine and stomach wash and also non'biological matrices vi(. water drinks tea coffee containing traces of toxic anions 6cyanide phosphide sulphide7 by using selective sealing liberating and detection reagent in %onway 8icro'*iffusion assembly 6as and when re)uired in case of volatile poisons. Vi(. ethanol methanol acetaldehyde chloroform toxic gases vi(. carbon monoxide phosphine ammonia hydrogen sulphide and toxic anions cyanide nitrate etc.7. Con@*7 icro-Diffusion &sse8l7:
The assembly consists of brink type polypropylene cells with clear polystyrene covers. The cells have an outermost annular sealing well 6
The preferential exchange of ions on ion'exchange resins packed in the column of ion chromatograph renders separation of anions by using mobile phases usually buffers of diverse p#. 3../
E)tr*ction of No n-,ol*tile Or+ *nic Poisons:
The group includes pesticides drugs 6acidic basic neutral and amphoteric7 and plant poisons 6specially alkaloids glycosides etc.7. 9ut of the above classes of poisons pesticides account for more than >@J of fatal cases of poisoning in $ndia thereby re)uiring special attention to it..
,=,
Laboratory Procedure Manual- Forensic Toicolo!y
3../.1 E)tr*ction of Pesticies in *trices:
The extraction of pesticides in biological materials vi(. viscera stomach contents gastric lavage and blood is difficult due to the interferences of fat degraded protein and colouring matter in the matrices. The extracts re)uire proper cleanup except for 8icellar method. The extraction in case of non'biological matrices is less cumbersome and re)uires either minor cleanup or no cleanup. The methods described hereunder are based on solvent extraction cum stripping under diverse conditions vi(. nature and condition of matrices use of organic solvent etc. 3../.1.1 et9o-I:
1iological materials 6viscera stomach content or gastric lavage etc.7 are macerated into fine slurry by mixing with e)ual amount of anhydrous sodium sulphate and transferred into a conical flask with an air condenser. @ ml of n' hexane is added to the flask and heated on a hot water bath for one hour. The contents are cooled and filtered. The residual slurry is extracted twice with : ml portion of n'hexane. The filtered n'hexane fractions are combined and taken into a separating funnel. This hexane layer is vigorously shaken with , ml ,@ ml and again ,@ ml portion of acetonitrile which are previously saturated with n'hexane. The acetonitrile layers are mixed and taken into another clean separating funnel and diluted ,@ times with distill ed water. : ml of saturated sodium sulphate solution is added to it and extracted thrice with : ml portion of n'hexane. The n' hexane layers are combined concentrated to ml by evaporating on water bath and gms of anhydrous sodium sulphate is added. The extract is evaporated as and when re)uired for analysis. 3../.1.2 et9o-II:
@ gms of macerated tissues or biological materials are mixed with e)ual amount of anhydrous sodium sulphate and ,@@ ml of acetone in a conical flask and then refluxed on hot water bath for one hour. !fter cooling the acetone extract is filtered. The residue is extracted twice with further @ ml portion of acetone. The acetone fractions are combined and concentrated by evaporation up to @ ml for further processing 6cleanup7. The above acetone extract 6@ ml7 is taken into a separating funnel and diluted with ,@ ml of water. !dd :@ ml of saturated solution of sodium sulphate to the same. The contents are extracted thrice with : ml portions of chloroform with gentle shaking. The chloroform extracts are combined washed with water'acetone mixture 6, :,7 and finally with @ ml of water. The washed chloroform layer is passed through anhydrous sodium sulphate and then evaporated to dryness by passing air. 3../.1.3 et9o $ III: E)tr*ction of Pesticies in sto*c9-@*s9B urine *n ,oit:
The sample 6:@ ml of stomach wash or urine or,@' :@ gms of vomit7 is taken in a conical flask. @ ml of n'hexane is added. $t is refluxed on a water bath for half' an'hour. !fter cooling the li)uid is filtered mixed with :@ ml of n'hexane and taken in a separating funnel. The n'hexane layer is separatedD passed through ,=:
Directorate of Forensic Science, MHA, Govt. of India
anhydrous sodium sulphate and evaporated to dryness by passing a current of dry air through it. 3../.1.4 et9o-I#: E)tr*ction of Pesticies in !loo:
:@ ml of blood is mixed with ,@ ml of ,@J sodium tungstate solution and , ml of ,< sulphuric acid shaken for two minutes and then filtered. The filtrate is kept reserved. The residue is washed with two , ml portions of @.,< sulphuric acid. The washings are collected mixed with filtrate 6kept reserved7 transferred into a separating funnel and extracted thrice with :@ ml portion of n'hexane. The hexane layers are combined passed through anhydrous sodium sulphate and the solvent is removed by passing a stream of air as stated in the previous methods.
3../.1. et9o-#: Direct Sol,ent E)tr*ction:
The biological materials 6@ gms of viscera7 are mixed with gms of ammonium sulphate and homogeni(ed. !fter addition of ,@@ ml of diethyl ether the mixture is shaken at intervals and kept overnight. $t is filtered and concentrated as before. The concentrated cleaned up by passing through chromatographic column 6diameter extract :.cm7 iscontaining three successive layersa of different lengths vi(. cm layer of alumina 6top layer7 :.cm of activated charcoal 6middle7 and :.cm layer of anhydrous sodium sulphate 6bottom7 previously washed with ether. The elute is evaporated to dryness as before. 3../.1./ et9o-#I: E)tr*ction 87 Ste* Distill*tion cu Sol,ent E)tr*ction:
0hen the biological materials are clean and purified i.e. less degraded and contain very little fat and colouring matter the following method may be adopted. Proceure:
@ gms of biological material is treated with a few drops of phosphoric acid and steam distilled for , minutes. The distillate 6,@@ ml7 is collected and sub"ected to solvent extraction with ,@@ ml of diethyl ether in :@ ml portion. The ethereal layers are collected during extractions combined and sub"ected to clean up by passing through chromatographic column as before 6method V7. 3../.1.0 et9o-#II Isol*tion of Pesticies in Non-8iolo+ic*l *teri*ls:
,=-
Laboratory Procedure Manual- Forensic Toicolo!y
8atrices5 :@': gms of rice or more if available ,@@ ml of drinking water or tea or coffee or milk wearing apparel 6:@': round pieces cut out from fabric each of :.cm diameter7 :@': gms of soil sand grains or cereals. Proceure:
For the above materials direct solvent extraction is carried out with @',@@ ml of diethyl ether without adding ammonium sulphate. The ethereal extract is concentrated to :@ ml and cleaned up by column chromatography as stated above. The ethereal extract is collected evaporated to dryness by passing stream of air.
3../.1. icell*r E)tr*ction in !iolo+ic*l *trices:
@ gms of biological material 6viscera7 is mixed with gms of ammonium sulphate and homogeni(ed. !fter addition of ,@@ ml of diethyl ether the mixture is shaken at intervals kept overnight and filtered. The ethereal extract is taken into a separating funnel. ,@ mg of sodium lauryl sulphate is added and stirred gently. 9n settling fat in li)uid and semi'solid form is separated and taken off from the system. The addition of surfactant is continued till all the fatty materials and proteins are separated and settled at the bottom. The end'point is indicated by a change of dark colour of ethereal layer to colourless. The ethereal extract is shaken with : ml portion of water twice gently. The ethereal layer is collected. $n case of emulsion formation ethereal layer is collected by breaking the emulsion with excess ether and gentle stirring. The collected ethereal layer in dried over anhydrous sodium sulphate to remove traces of water. The ethereal layer is decanted and evaporated to dryness as before. 3../.1. E)tr*ction of Pesticies in 'ruitsB #e+et*8lesB !utter '*t 87 Uni,ers*l Tr*ce Resiue E)tr*ctor: Proceure:
The sample is extracted by direct solvent extraction with dichloromethane. The extract is dried over granular anhydrous sodium sulphate in a column. The dried extract thus obtained is concentrated by a stream of nitrogen. The concentrated extract is then sub"ected to sweep co'distillation in the niversal Trace Cesidue &xtractor at :-@o% for -@ minutes by passing nitrogen 6:-@ ml 2 min7 and using sodium sulphate ' ,@J florisil 6, :,7. The &lution is made by ml of acetone hexane or ml of dichloromethane. extract is collected for,@J analysis. Thein applications of this method to different pesticides for their analysis in biological matrices re)uire further standardi(ation to set up optimum analytical conditions to cover different classes of pesticides. 3../.1.1 E)tr*ction of Pesticies in SeientB SoilB Dr7 F*ste *n Tissue 87 &cceler*te Sol,ent E)tr*ction &SE:
,=;
Directorate of Forensic Science, MHA, Govt. of India
Proceure:
The sample is mixed thoroughly or passed through a , mm sieve. +ufficient sample is introduced into the grinding apparatus to yield at least ,@':@ gm after grinding. The sample is air dried at room temperature for ;> hours in a glass tray or on hexane cleaned aluminium foil. The drying may also be made by mixing with anhydrous sodium sulphate until a free flowing powder is obtained. 6!ir drying is not recommended for volatile pesticides.7 Gummy fibrous or oily materials not amenable to grinding should be cut shredded or otherwise separated to allow mixing. These may be grinded after mixing with anhydrous sodium sulphate ,:cell. , proportion. ! cellulose is placed at in the,,outlet extraction !pproximately ,@ gmdisk of each sample ml orand :@ end gm of the each sample in :: ml extraction cell. 6+urrogate spikes and matrix spikes may be added to the appropriate sample cell7. The extraction cells were placed into the auto' sample tray and the collection trays are loaded in appropriate number 6up to :;7. The tray is loaded with ;@ ml pre'cleaned clapped vials with septa. The conditions for extraction in !+& are set for extraction of pesticides by using acetone 5 hexane 6, 5 , v2v7 as the solvent. The operating conditions include oven temp. of ,@@% pressure at ,@@ psi oven heat time and static time each of minutes and flush volume in the proportion of 3@J of extraction cell volume. The extracts are collected for analysis. The method has been validated for analysis of pesticides in soil sediment dry wastes and fish tissues. #owever further standardi(ation is re)uired for application of !+& to biological matrices in forensic toxicological work. 6,7 3../.2 E)tr*ction of Dru+s *n Poisons of Pl*nt Ori+in in !iolo+ic*l *trices:
This group comprises alkaloids glycosides barbiturates phenothia(ines salicylates sulphonal groups of different classes and plant poisons. The extraction of these poisons depends on their solubility at different p# i.e. at low or high acidic or alkaline condition and also differential solubility in organic solvents. The methods include +tas'9tto or *ragendorff or their different modifications ammonium sulphate method and modern method especially solid phase extraction micellar and en(yme digestion for the above poisons in biological materials. The main difficulty in all the methods for extraction of poisons in biological materials is to get rid of fats degraded protein and pigments which interfere with their isolation in pure form and subse)uent identification and )uantitation. The problem is that )uantities of these poisons present in biological matrices are small and multiple steps in the extraction and purification may incur loss of poison giving a minus error. $f not sufficiently purified a positive error arises. This is the reason why the result of )uantitative determination especially the alkaloids are not dependable and the negative findings in many cases do not represent the actual state of affairs. The )uantitative result actually represents the recovery not the exact )uantity present in the tissues. #owever the percentage of recovery is improving excellently by using modifications of the existing methods and also modern methods. The methods are described hereunder with special reference to biological materials. 3../.2.1 oifie St*s-Otto et9o:
,=
Laboratory Procedure Manual- Forensic Toicolo!y
Proceure:
!.
@ gms of biological material is minced mixed with plenty of rectified spirit 6about :'- times the weight of material7 in a flask and acidified with tartaric acid. The mixture is heated on the steam bath for ,': hours with thorough shakings at fre)uent intervals. The extraction is then allowed to proceed for about :; hours with steam off. $t is then filtered through a flutted filter paper. The filtrate is evaporated and the residue is again extracted with acidulated alcohol in the same way filtered and washed several times with hot rectified spirit. The combined filtrates are evaporated in a porcelain basin on the steam bath to a syrupy consistency.
1.
To the syrupy residue about ,@@ ml of rectified spirit is added very slowly with constant stirring so that insoluble matter may be granular and not gummy. $f the alcohol is added rapidly or all at a time the insoluble matter will be gummy causing much loss of alkaloids by enclosing them in the sticky mass. $t is warmed with occasional stirring for about half an hour and filtered. This process is repeated once more and the combined alcoholic extracts are evaporated almost to dryness.
%.
The residue is now dissolved in about @ ml of water acidulated with dilute sulphuric acid and filtered after about an hour. The poisons are thus dissolved out by the a)ueous solution which is transferred to a separating funnel and extracted with a suitable solvent such as ether chloroform etc. in portions of about : ml. The solv ent would take up from the acid solution colouring matters toxic oils and resins salicylic acid and its derivatives 6aspirin salol etc.7 barbiturates sulphonal acetanilide narcotine and alkaloids of ergot certain glycosides such as thevetin which has escaped initial treatments for purification.
*.
The acid a)ueous solution is then rendered alkaline with a solution of sodium carbonate or ammonia which would liberate the free base from its salt. The alkaline solution is now extracted with :ml portions of chloroform in the same way as in the previous stage. $t will take up all the alkaloids except morphine 6only a trace being extracted7 and those feebly basic substances which are partially extracted from the acid solution. The extraction is repeated : or - times more.
&.
$f morphine is suspected it may be extracted at this stage by amyl alcohol or chloroform N ether 6-5,7 mixture or chloroform N alcohol 6?5,7. 9f these amyl alcohol is the best. 1ut as it is prone to form annoying emulsions ether mixture isextraction used by many. morphine is likely to be the the chloroform only poisonN the chloroform 6stage$f*7 described above may be omitted altog ether. The combined chloroform or amyl alcohol extracts are evaporated to dryness and the residue is now ready for further purification and analysis.
F.
The evaporated chloroform extract is purified by dissolving it in about :@ ml of water acidulated with sulphuric acid and filtering through a small filter. The filtrate is extracted with chloroform first in acid and then in ,=3
Directorate of Forensic Science, MHA, Govt. of India
alkaline medium as in the initial stages of extraction. These extracts are evaporated to dryness for analysis. 3../.2.2 'urt9er oific*tion of St*s-Otto et9o:
!s there are too many steps in the extraction of non'volatile organic poison by +tas'9tto method and also chances of loss of poison in each of the steps there may be even a complete failure to detect it in case of considerable loss of poisons. The ob"ectives of modifications are to minimi(e the steps as far as practicable under special circumstances. The following modifications of the techni)ue are sometimes necessary. i7
The alcoholic extraction of biological materials is to be carried out at room temperature 6not exceeding ;@ o%7 i.e. without using steam bath and preferably with absolute alcohol 6to prevent hydrolysis7 in place of rectified spirit for suspected poisoning by aconite belladonna datura or cocaine. The evaporation of alcoholic extract should be done under reduced pressure.
ii7
The extraction with rectified spirit is to be done for ;> hours if biological materials are preserved in saturated saline solution.
iii7
For stomach contents comprise off too much of fluid the extraction should be done with absolute alcohol for - or ; times.
iv7
For stomach wash as th e sample extraction with double the )uantity of absolute alcohol acidulated by tartaric acid should be done and then allowed to evaporate on a steam bath.
v7
For filtration 1uchner funnel is preferred.
vi7
To prevent loss due to emulsion formation agitation with organic solvents 6ether or chloroform or amyl alcohol7 should be done gently in the beginning and steadily in an violent manner thereafter 6: N - time in the beginning and not exceeding ,: times at the end7. $f emulsion persists it is to be evaporated on a steam bath and the residue taken up in fresh solvent.
3../.2.3 &oniu Sulp9*te et9o:
This method is most useful for preliminary analysis 6screening7 of barbiturates alkaloids and tran)uili(ing drugs etc. and also identification and semi' )uantitation. Proceure:
The visceral materials 6about @' ,@@ gms.7 are cut into small pieces macerated mixed with,@@ ml of percent acetic acid and taken into a 3@@ ml beaker. +olid ammonium sulphate is then added to it by fre)uent shaking to make a saturated solution. !bout :@ gms. of solid ammonium chloride are added in excess. The
,==
Laboratory Procedure Manual- Forensic Toicolo!y
mixture is then heated in a boiling water bath for three hours 6for suspected poisoning by aconite the temperature should not exceed 3@°%7. The mixture is cooled slightly and filtered through the filter paper. The residue on the funnel is again extracted with two portions of ,@@ ml of percent acetic acid and filtered as before. The filtrates are combined and taken into a @@ ml separating funnel. The residue slurry on filter paper is leached with ,@@ ml of diethyl ether and the same is received in a cold container. The ether fraction is added to the a)ueous acidic extract in the separating funnel and shaken for minutes and separated. ,@@ ml of ether is again added to the acidic layer shaken for minutes and separated. The ether layers are combined. The acidic ether extract is tested for salicylic acid aspirin barbiturates meprobamates lysergides ben(odia(epines etc. The a)ueous solution remaining in the separating funnel after separation of acidic drugs is made alkaline by addition of ammonium hydroxide and extracted three time with ,@@ ml portions of chloroform ether mixture 6, 5 -7. The a)ueous layer is retained for extraction of opium alkaloids. The organic layers after separation are combined and washed with @ ml of water. $t is extracted three times with : ml portions of ,@J sulphuric acid. The sulphuric acid fractions are combined and taken into another separating funnel. 69rganic layer is discarded7 @ ml mixture of chloroform N ether 6, 5 -7 is added to it. *ilute ammonium hydroxide solution is added to make the solution alkaline shaken for minutes. The organic layer is separated. The extraction is repeated thrice. The organic layer after separation are combined washed with l@ ml of water and then dried by passing through anhydrousdatura sodiumand sulphate and evaporated dryness. The extract is tested for opium aconite alkaloids to amphetamines meprobamate and metha)ualone etc. T9e *ci $ et9er e)tr*ct *7 8e furt9er 8e sep*r*te into t9ree fr*ctions.
i7
The acid N ether fraction is shaken with : ml of J sodium bicarbonate solution. The a)ueous layer is removed and taken into another separating funnel. $t is acidified with dilute sulphuric acid and re'extracted with : ml of ether. This ether fraction is passed through on hydrous sodium sulphate and then dried "ust to dryness. The residue fraction , contains salicylates.
ii7
The ether layer 6of acid ether fraction7 after washing with sodium bicarbonate is extracted twice with : ml portions of <'.sodium hydroxide solution. The a)ueous layer are separated from ether layer combined and taken into another separating funnel. $t is made acidic with dilute sulphuric acid and extracted twice : ml portions of ether. This ether fraction is washed with : ml of water and then dried by passing through anhydrous sodium sulphate and then evaporated to dryness. The residue fraction : contains barbiturates in relatively purified form.
iii7
The ether layer after extracting with
,=>
Directorate of Forensic Science, MHA, Govt. of India
3../.3 E)tr*ction of Dru+s in Urine:
+ufficient phosphoric acid or tartaric acid is added to ,@ ml of urine to ad"ust the 4# to -. $t is then extracted with two -@ ml portions of ether. The extracts are combined and washed with ml of water. The washings are added to the sample. The a)ueous solution is retained for possible presence salicylates 6Fraction !7. The ethereal solution is extracted with ml of @. 8 sodium hydroxide and the extract is retained for examination of barbiturates and weakly acidic substances 60eak !cid Fraction 1 N 1arbiturates Glutethimide 4aracetamol 4henytoin 4henylbuta(one etc.7. The ethereal solution is washed with water. The washing is discarded. The ethereal solution is then dried over anhydrous sodium sulphate and evaporated to dryness. The residue may contain neutral drugs 6 . $t is extracted with two ,@ ml portions of chloroform. The combined ether extracts are washed with water filtered. ! little tartaric acid is added to prevent the loss of volatile bases. $t is evaporated to dryness. The residue may contain basic drugs 61asic Fraction * N !mphetamine !mitriptylene %affeine 4henothia(ines &rgot !lkaloids 8orphine 8etha)ualone Flura(epam /ora(epam etc.7. The p# of the a)ueous solution obtained after extraction of Fraction * is ad"usted to p# - by the addition of hydrochloric acid. $t is heated at ,@@ °% for -@ minutes cooled and extracted with two ,@ ml portions of ether. The a)ueous solution is kept reserved. The combined ether extracts are washed with ml of , 8. sodium hydroxide and evaporated to dryness. The residue may contain ben(odia(epines as ben(ophenones 6Fraction &7. The p# of the a)ueous solution is ad"usted to p# ? and cooled. $t is extracted with a mixture of ethyl acetate and isopropyl alcohol 6?5 ,7. The solvent layer is separated and evaporated to dryness. The residue may contain opiates 6Fraction F7. 3../.4 E)tr*ction of Dru+s in !loo:
!s sample'volume in case of blood or serum or plasma is small and only a limited number of drugs may easily be detected and identified in them the extraction procedure is slightly different from that for urine and stomach contents. *ifferent fractions of extraction vi(. ! 1 % * bear the same meaning as stated in -.>.3.-. i.e. extraction of drugs in urine. The initial extraction is carried out at p# =.; as many basic drugs are recovered by chloroform extraction at this p#. !s a result the substance looked for is most likely to be found in either fraction 1 or % and preparation of fraction * is only necessary either to ensure that nothing has been missed or where no drug has been found in fractions 1 and %.
,=?
Laboratory Procedure Manual- Forensic Toicolo!y
4rocedure5 : ml of phosphate buffer 6p# O =.;7 and ;@ ml of chloroform are added to ; ml of the sample and shaken vigorously. : gms of anhydrous sodium sulphate is added and shaken again to produce a solid cake. The decanted chloroform is passed through a filter paper and the cake is extracted with a further :@ ml of chloroform. The chloroform extracts are combined. The chloroform layer is extracted with sodium carbonate to remove salicylate 6+trong !cid Fraction !7 if detected in the preliminary tests. To the chloroform layer > ml of @. < sodium hydroxide solutions is added. The mixture is shaken and centrifuged. The sodium hydroxide may contain barbiturates and other weakly acid substances. 60eak !cid Fraction 17. The chloroform layer is washed with a little water. The washing is discarded. The chloroform layer is dried over anhydrous sodium sulphate filtered and evaporated to dryness. The residue may contain 6caffeine carbromal ben(odia(epines meprobamate phena(one etc.7 neutral drugs together with a number of bases 6
ml of urine is added to : ml of phosphate buffer 6@., mol.2/ 4# 3.@7 in a glass test tube and the 4# is ad"usted to 4# . N 3. using @., mol.2l a)ueous sodium hydroxide or , mol.2/ a)ueous acetic acid 6depending on the nature of the drug7. +4& column is inserted into vacuum manifold and washed with , ml of methanol and , mltoofthe phosphate 6@., mol.2l 3.@7. !nis>added ml fitted is is attached top of thebuffer extraction column4# and urine to it.reservoir The column dried under vacuum and washed with , ml of phosphate buffer 6@., mol.2/ 4# 3.@7 followed by @. ml of a)ueous acetic acid 6, mol.2l7. The column is dried under vacuum and washed with , ml of hexane. The elution for acidic and neutral drugs is made with ; x , ml portions of dichloromethane. The eluate is evaporated to dryness under a stream of nitrogen at -@ N ;@ o%. The residue is kept reserved for analysis of acidic and neutral drugs. The column is then washed with methanol 6, ml7 and elution for basic drugs is made with : ml of methanolic ammonium ,>@
Directorate of Forensic Science, MHA, Govt. of India
hydroxide 6:J V2V7. - ml of de'ionised water is added and elution is made with @.: ml of chloroform. The chloroform layer is collected and evaporated to dryness under a stream of nitrogen as above. The residue is kept reserved for analysis of basic drugs. 3.././ Tissue Di+estion usin+ Proteol7tic En7e:
The digestion with proteolytic en(ymes gives much improved recovery and has the advantage that once the digest has been prepared analogous methodology to those used with plasma can be employed. $t is obviously important to ensure that use of the en(yme preparation does not introduce interferences. ! further potential problem is that con"ugates and other metabolites may not survive. The procedure has been described below. ! solution 6: gms2/7 of lyophili(ed subtilisin in sodium dihydrogen orthophosphate 2 disodium hydrogen orthophosphate buffer 6= mol.2/ 4# =.;7 ,@@ mg portions of tissue are dissected and the excess of fluid removed by filter paper. The tissue is added to ,@ ml of tapered glass tubes and exact weights are recorded. , ml of subtilisin solution is added. The tubes are sealed with ground glass stroppers and incubated in a water bath at @ °% for ,3 hours. The tubes are cooled. The contents are mixed on a vortex mixer. Thereafter extraction is done with @.: ml portions as for plasma or serum. 3../.0 icell*r E)tr*ction of Dru+s in !iolo+ic*l *teri*ls:
@',@@ gms of biological materials are finely minced in a mincing machine. !n excess of rectified spirit 6about :@@ ml7 is added to it in a flask mixed thoroughly and made acidic with glacial acetic acid by drop wise addition and stirring. The mixture is then heated on a steam bath for , hour with through shaking at intervals and the flask containing the mixture is kept for :; hours at room temperature. $t is then filtered through a 1uchner funnel having a bed of sand [email protected] cm7 on the filter paper. The filtrate is collected in a separating funnel. !fter adding of @ ml of solvent ether cloudiness is observed. Fine particles of fat separate on standing which is drained off. The contents of separating funnel is shaken with , mg portions of sodium lauryl sulphate when semi'solid fat separates. The separation of fat is accelerated by addition of a few drops of water. Ceddish brown oily mass of fat settles at the bottom of the separating funnel. This is drained off. The process is repeated till the settling of fat ceased 6indicated by a clear transparent extract in the separating funnel7. The contents of the separating funnel is evaporated to dryness. The residue is extracted gently with :@ ml portions of water five times. The a)ueous extract is made distinctly acidic by drop wise addition of acetic acid and extracted with >@ ml of diethyl ether in :@ ml portion . The a)ueous part is kept separately. The combined ether extract as above 6acid' ether part for acidic and neutral drugs7 is washed with water till free from acid. The washings are mixed up with the a)ueous part. The combined ether extract is dried over anhydrous sodium sulphate. The dried ether extract is decanted off. $t is evaporated to dryness. The residue is kept ready for analysis.
,>,
Laboratory Procedure Manual- Forensic Toicolo!y
The combined a)ueous part is made distinctly basic with drop wise addition of ammonia solution. $t is extracted with >@ ml of chloroform in :@ ml portion. The combined chloroform extract is washed with water till free from alkali and dried over anhydrous sodium sulphate. $t is decanted and evaporated to dryness. The residue is kept ready for analysis 6basic drugs7.
3..0
5e*sp*ce proceure for Isol*tion of #ol*tiles in !iolo+ ic*l *teri*ls:
The principle underlying headspace analysis is that in a sealed vial at constant temperature e)uilibrium is established between volatile components of a li)uid sample in the vial and the gas phase above it 6the headspace7. !fter allowing due time for e)uilibrium 6normally , minutes or so7 a portion of the headspace may be withdrawn using a gas'tight syringe and in"ected into the G% column. Proceure:
The internal standard solution 6: mg 2 l ethyl ben(ene and ,@ mg 2 U l , : trichloroethane is added to a :@@µl of a mixture of expired blood and deionised water 6,5:; v2v7 in a =ml glass septum vial using a semi'automatic pipette. The vial is sealed using a crimped on 4TF&' lined silicone disc. The vial is incubated at 3 o% in a heating block and a portion 6,@@'-@@l7 of headspace is withdrawn using a warmed gas'tight glass syringe for onward analysis in gas chromatograph. 6:7 3.1
C%E&N-UP P ROCEDURES:
$n the extraction techni)ues described above it is likely that the extract will contain interfering substances which will create problems in the analysis in various ways. The occurrence is particularly so in the extracts from samples of effluent soil sediment and tissues or organic matrices which contain fats oils and other naturally occurring substan ces. The long established procedure for the removal of these substances is to pass the extract through an alumina column and then to separate the target compounds into different batches by passing the cleanup extract through a silica column. The supply of materials by preparative thin layer chromatography is also followed if the active components are identified by preliminary screening. The purification in the same way may also be achieved by G% or #4/%. For volatile toxicants stripping may also be done by G%'#ead +pace method 64urge or Trap techni)ue7. There are different analytical conditions for the purpose which have been specified in literature. $n different hyphenated techni)ues vi(. G%'8+ /%'8+ #4/%'$C the first techni)ue is applied for separation of components in the pure state and their subse)uent identification by the second techni)ue.
,>:
Directorate of Forensic Science, MHA, Govt. of India
Cecently the cleanup has been streamlined by the use of commercially available solid'phase extraction cartridges or discs 6described earlier7. *ifferent methods of stripping are described below in short.
3.1.1 Cle*nup usin+ &luin* *n Silic * Colun:
This techni)ue removes interfering compounds by passing the extract through basic and acidic column and then separating active constituents specially pesticides on a silica column. Prep*r*tion of Soli &sor8ents:
The preparation of solid adsorbents N basic alumina acidic alumina and silica gel is as follows. i7
1asic !lumina5 !bout ,@@ gms of alumina is placed in a silica dish heated in a muffle furnace for ; hrs at >@@ o% cooled to about :@@ o% and then to room temperature in a dessicator. 0ater 6;J w2w7 is added to the weighed portion in a stoppered flask which is shaken well sealed and then stored.
ii7
!cidic !lumina5 ! portion of the alumina is washed with ,< #%l by making slurry in a beaker. $t is filtered through a sintered funnel and dried in a silica dish at ,@o% for ; hours and cooled in a desiccator. 0ater 6;J w2w7 is added to a weighed portion in a stoppered flask. $t is mixed thoroughly and stored. These alumina preparations get slowly deactivated on exposure to air and should be discarded after two weeks.
iii7
+ilica Gel5 !bout ,@@ gms of silica gel is heated in a silica dish in a muffle furnace for : hours at @@ °% cooled and then placed in a desiccator. ! portion is weighed into a stoppered glass container and water e)uivalent to -J of the silica is added. The silica gel deactivates more rapidly than the alumina and should preferably be prepared daily. Proceure:
The column is pre'washed with acetone followed by hexane and allowed to dry. The acid 2 base alumina column is prepared by first adding : gms. of acidic alumina then , gm of basic alumina to the column. The column is tapped to settle. ,>-
Laboratory Procedure Manual- Forensic Toicolo!y
The silica column is filled by adding :. gms of silica gel pre pared as stated above and tapped to settle. ,@ ml of hexane is passed through the column 6to wet the column7 and run off in excess till the hexane meniscus is at level with the column material. The organic layer 6obtained after extraction of matrices for pesticides or drugs7 is then transferred on to the column. The active constituents 6pesticide or drugs7 are then eluted from the column by hexane or diethyl ether or suitable organic solvent. The elute is collected. $t is then passed through the silica column. The elute is collected and dried over anhydrous sodium sulphate. $t is evaporated to dryness. 3.1.2 oifie et9o for Cle*n-Up *n sep*r*tion usin+ &luin* ? Silic* (el: Prep*r*tion of Soli &sor8ent:
6i7
!lumina N !bout ,@@ gms of alumina is heated in a silica dish at @@ o% for ; hours and then cooled. To a weighed portion in a stoppered glass container deioni(ed water e)uivalent to =J 6w2w7 weight of alumina. is added and the sample is agitated to mix thoroughly. The alumina is kept in a sealed container. This adsorbent is stable for only about a week once re' exposed to atmosphere.
6ii7
!lumina 2 +ilver
The chromatographic column is plugged at the base with hexane washed glass wool or cotton wool . , ml of hexane is added. , gm of alumina 2 silver nitrate is poured and allowed to settle. Then : gms of alumina is added and again allowed to settle. This is then charged with a little anhydrous sodium sulphate. #exane is run off in excess until the li)uid level is at the top of the column. The concentrated organic layer 6containing pesticides or drugs as the active constituents7 is added with rinsing the topsolvent of the 6as colum -@ may ml ofbe7hexane or diethyl or any suitabletoorganic then.case is passed throughether the column and the elute is collected. The elute is concentrated further to ,@ ml. ! silica column is then prepared by adding : gms of silica to a plugged chromatographic column with a layer of anhydrous sodium sulphate at the top. The concentrated elute from the alumina 2 silver nitrate column is added with rinsing to the silica gel column and allowed to be adsorbed. ,@ ml of hexane or diethyl ether or any suitable organic solvent or a mixture of solvents is added to the top of the column and the elute is collected. ,>;
Directorate of Forensic Science, MHA, Govt. of India
This is dried over anhydrous sodium sulphate and evaporated to dryness for further analysis. 3.1.3 Cle*nup 87 * Siple Colun C9ro*to+r*p9ic et9o:
The method can be profitably employed to purify the material after extraction from biological materials for pesticides or drugs. The concentrated extract 6organic layer7 is cleaned up by passing through a chromatographic column 6dia.:.cm7 containing from top alumina layer of cm activated charcoal layer of :.cm 6middle layer7 and a anhydrous sodium sulphate layer of :.cm 6bottom layer7 previously arranged after plugging the chromatographic column and washing by hexane or diethyl ether or any suitable solvent or mixture of solvents 6as the case may be7. The elution is made with appropriate organic solvent. The elute is dried over anhydrous sodium sulphate and evaporated to dryness. 3.1.4 Cle*nup usin+ Soli P9*se E)tr*ction SPE C*rtri+es:
There are a variety of cartridges available vi(. 1ond &lute from Analytic "#e$ International. The cartridge is filled with a chemically modified silica adsorbent and the appropriate one is selected according to the nature of the material 6toxicant7 vi(. aminopropyl for organo'chloro compounds. Proceure:
! selective cartridge is taken with an adapter fitted on top. ! ,@ ml glass syringe is fitted onto the adaptor. ml of methanol is put into the syringe. $t is passed through the tube . The tube is then washed with ml of hexane. The syringe is detached. The concentrated organic layer containing active constituent is added to the top of the tube and allowed to pass. The clean extract is collected. The tube with the syringe is washed with the same solvent. This is added to the clean extract as above. The combined layer is dried over anhydrous sodium sulphate. $t is then evaporated to dryness. 3.1. Cle*nup 87 Prep*r*ti,e T%C et9o 6,;7:
This is applicable if the identity of toxicant is to be known. Thereafter by screening of the T/% plate developed by applying similar conditions i.e. developing solvent system adsorbent temperature etc. The spots containing the active components separated at different Cf values on the developed plates are scrapped off. The scrapped materials at different (ones are eluted with selective organic solvent. This is collected dried over anhydrous sodium sulphate and evaporated to dryness. 3.1./ Cle*nup 87 5P%C et9o:
$n this method also the identity of toxicant is to be known by noting retention time under specific chromatographic conditions vi(. solvent system flow rate column etc. !gain the same #4/% method is employed and the separated components at various retention times 6as established earlier7 are collected for further analysis. ,>
Laboratory Procedure Manual- Forensic Toicolo!y
*ifferent methods of extraction 2 isolation of toxicants in diverse matrices and their stripping methods have been described in the previous paragraphs. The methods are to be employed depending on the nature of toxicant matrices and availability of infrastructure facilities. $n the forthcoming chapters the analysis of different classes of toxicants will be described. References:
,. !pplicable
1ough 4 Gas %hromatography N ! practical approach p',=; , edition 9xford niversity 4ress
st
!nalysis of gaseous and volatile poisons. +ystematic analysis of gaseous and volatile poisons in various type of formulations biological materials and non'biological matrices. To identify and estimate gaseous and volatile poisons .
4. 4 Responsi8ilities: Ga(etted officers and associated scientific staff. 4.
#O%&TI%E POISONS:
+ome of the volatile poisons with their characteristics are given below5'. T!1/& N ;.,
!cetone
!niline
1en(ene
+ynonym E +ource !cetic aldehyde ethanal &thyl aldehyde. +ource5 1y'product in alcoholic fermentation.
9ther %haracteristics 8etaboli(ed through oxidation to acid and eventually to carbondioxide and water. 8ore toxic than ethanol or acetic acid. %etona *imethyl ! clear colourless /arge amounts are Aetone volatile inflammable excreted unchanged :'4ropanone. li)uid 1.4.3oc but small doses may +ource5 ! product 8iscible with water be oxidi(ed to carbon of destructive ethanol chloroform dioxide. !cetone is distillation of wood and ether. the main metabolite a by'product in the of isopropyl alcohol fermentation of occurs naturally in starch. blood and urine of diabetics abused in glue sniffing. !minoben(ene /i)uid colorless but on $ntoxication occurs 4henylamine keeping turns to brown by inhalation due to formation of ingestion or
4henyl hydride
4hysical 4roperties %olourless inflammable li)uid 1.4. :@.:o% miscible with water ethanol and ether.
@ nitroso . cutaneous absorption. 1.4.,>;compound %. 8iscible Toxic symptoms with alcohol ben(ene include cyanosis chloroform and other methemoglobinamea organic solvents. vertigo headache and $nsoluble in water. confusion. Forms salts with mineral acids. ! clear colourless 1en(ene is absorbed
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Directorate of Forensic Science, MHA, Govt. of India
+ource5 $n light oil fraction obtained by distillation of coal tar.
inflammable li)uid characteristic aromatic smell 1.4.' >@o% practically immiscible with water miscible with dehydrated alcohol acetone ether and glacial acetic acid.
%arbon tetrachloride
%arboneum tetrachloratum medicinale +ource5 +ynthesis by chlorination of hydrocarbon etc.
! heavy clear colourless li)uid with chloroform like odour volatile. !lmost insoluble in water miscible with dehydrated alcohol chloroform and ether.
%hloral hydrate
!)uachloral %hloradom %hlo' ralex *ormel &lix'
%olourless or white crystals it li)uefies between @o% and boils at ?>o% soluble in ether chloroform and partially soluble in water. &thyl alcohol' solubilityW
from the gastro' intestinal tract thro' ugh skin and lungs. @J of inhaled ben(ene may be eliminated via the lungsD small amount is excreted unchanged in the urine mainly as phenol catechol and )uinol in the con"ugated form. 4henylcarbinol ! colourless li)uid with se5 4henylmethanol a faint aromatic odour /ocal anaesthetic +ource5 !s ester of 1.4.' *isinfectant. cinnamic acid E :@-o%. +oluble in ben(oic acid in water methanol 1alsam of Tolu and chloroform and ether. 4eru. %arbon disulphide ! colourless li)uid with sed in the synthesis %arbonic anhydride a faint aromatic odour of different +ource5 %oal tar. 1.4.' :@-':@>o% dithiocarbamates. soluble in water miscible with ethanol chloroform and ether.
Ceadily absorbed by oral administration readily metabolised by reduction to trichloroethanol and finally to trichloroacetic acid by steps. sed as hypnotic as well as sedative. ! colourless volatile !bsorbed by oral li)uid 1.4.' about 3,o% administration and insoluble in water inhalation. $t is miscible with exhaled unchanged ,>?
Laboratory Procedure Manual- Forensic Toicolo!y
dehydrated alcohol and between :@J to =@J. ether. General anaesthetic. %resols +ynonym 5 !n almost colourless to !bsorbed after 6! mixture of o' %resylic !cid pale brownish yellow ingestion and through m' E p'cresols7 Tricresol li)uid becoming darker skin and mucous 4roprietary
Formic aldehyde 8ethylene oxide 9xamethane
! colourless gas soluble in water slightly soluble in ethanol and ether.
#ydrocyanic acid
#ydrogen cyanide prussic acid +ource5
+mell like bitter almonds. +alts are highly soluble in water and alkaline in nature.
$sopropyl alcohol
! clear colourless volatile li)uid 1.4.'>, N >-o% miscible with water ethanol chloroform and ether.
,?@
$nflammable irritant to mucous membrane inhalation through respiratory tract may cause bronchitis and pneumonia rapidly metabolised in the body tissue to formic acid and methanol. #aemoglobin is converted to cyano haemoglobin. %onstriction of throat di((iness vertigo.
$nflammable readily absorbed by oral administration slowly metaboli(ed than ethanol largely
Directorate of Forensic Science, MHA, Govt. of India
+ource5 Fermentation.
Aerosene
8ethyl alcohol
converted to acetone then to acetate and formate. %oal oil Varsol. 9ily li)uid of $nflammable +ource5 1y characteristic odour consumption or fractionation of boiling range5 exposure causes petroleum. ,@'-@@o%. irritation to eyes headache and blurred vision. 8ethanol %arbinol ! colourless li)uid &xtremely poisonous wood spirit. 1.4. 3;.=o% mixes with E causes blindness +ource5 The li)uid water and organic and even death. fraction pyro solvents peculiar odour 8etabolised by ligneous acid in the and a burning taste. oxidation to destructive formaldehyde formic distillation of wood acid E finally contains methanol formate. as a ma"or constituent. %an be synthesi(ed commercially. 8iddle oil fraction %olourless transparent 1urns with a smoky of coal tar scales 8.4.' >@o% flame used as distillation. 1.4.' :@>o% practically insecticide and in the insoluble in water preparation of dyes
4araldehyde
4aracetaldehyde +ource5 4olymeri(ation of acetaldehyde.
4henol
%arbolicacid Fenol 4henyl hydrate. +ource5 /ight oil fraction of coal tar
soluble in ethanol %hloroform highlyand soluble in ether. ! pale yellow oily li)uid practically insoluble in water miscible with ether and methanol 1.4.' :@3o% characteristic odour of bitter almonds 6shoe' polish7. ! clear colourless or pale yellow li)uid 1.4. ,:-',:3o% soluble in water miscible with ethanol chloroform and ether.
%olourless or faintly pink deli)uescent crystals or crystalline mass which becomes pink on storage ,?,
such as indigo a(o' dyes. sed for preparation of aniline. 4oisonous.
sed as hypnotic readily absorbed on administration metabolised to accetaldehyde by depolymerisation E then oxidi(ed to acetic acid and finally carbon dioxide. 4henol denatures and precipitates cellular protein which may rapidly cause poisoning. Capidly
Laboratory Procedure Manual- Forensic Toicolo!y
Turpentine
4./
distillation.
1.4.,>,o% partially soluble in water soluble in ethanol chloroform and ether.
9il of Turpentine 9il of 4ine +ource5 4ine tree.
%olourless oil with peculiar odour immiscible with water but miscible with alcohol ether and chloroform.
absorbed through G.$. tract or penetrate skin metabolised to phenyl glucuronide and phenyl sulphate and small amounts are oxidised to catechol E )uinol 6con"ugated form7. !bsorbed through skin or by inhalation.
ET5ODO%O(6:
The distillation methods for isolation of volatile poison have already been described 6section'-7. The distillates in two fractions acid steam distillate and alkaline steam distillate are kept to undertake systematic screening and other detailed analysis as mentioned hereunder. 4./.1
Preliin*r7 screenin+ of #ol*tile Poisons:
The screening is done in three steps vi( a7 +mell b7 %olour tests c7 V'spectra ! small portion of the distillate may also be sub"ected to G%2G% #eadspace examination and the retention time of the peaks can be compared with the standard references available in literature and confirmed by the in"ection of a control. For this purpose normal Flame $oni(ation *etector is used and the columns used normally are %arbowax or #ellcomid. X max 6nm7 values of some commonly known volatile in water are given below5' T&!%E $ 4.2
Xmax 6nm7
!niline 1en(ene %amphor %resol Aerosene
:>@ ::>::-:->:;::;>:;:33. :>: :=:=@ 6may vary depending on inter batch variations7 ,?:
Directorate of Forensic Science, MHA, Govt. of India
$n biological materials 6viscera7 stomach contents urine isolation by acid' distillation as described earlier. The distillate is sub"ected to the following tests5 4./.2.1 Tests for *cetone:
,.
Ioofor Test:
!.
Fit9 I2 G N*O55
To , ml. of the distillate a few drops of ,@J
Fit9 I2 G N54O55
4ositive only in case of acetone by using <# ;9# in place of
%e+*l=s Test :
To , ml. of the distillate : drops of saturated solution of sodium nitroprusside 6freshly prepared7 and , drop of ,@J sodium hydroxide is added. ! red or yellowish red colour is produced. ! few drops of glacial acetic acid is then added to acidify the solution which changes the colour to carmine or purplish red. +ubse)uently the solution is heated which produces violet colour. 4./.2.2 Test for *cet*le97e:
$t can be isolated from tissue and other biological material by acid'distillation. The distillate may be sub"ected to the following tests. ,.
Ioofor Test:
0ith $: S
Sc9iff=s re*+ent Test:
To , ml. of the distillate : ml. of +chiffIs reagent 6decolourised basic fuchsine7 is added and kept for -@ minutes. ! purple colour is obtained 6formaldehyde also gives purple colour7. To the developed purple colour @. ml. of conc. sulphuric acid is added. The purple colour disappears if acetaldehyde is present.
,?;
Directorate of Forensic Science, MHA, Govt. of India
-.
To , ml. of the distillate a few drops of freshly prepared sodium nitroprusside solution and piperidine are added. The formation of blue colour indicates the presence of acetaldehyde.
4./.2.3 Tests for for*le97e:
Formaldehyde is isolated from biological materials by acid'distillation. The distillate is sub"ected to the following tests. ,.
Sc9iff=s re*+ent Test:
To , ml. of the distillate : ml. of +chiffIs reagent is added and kept aside for -@ minutes. 4urple colour is formed. $f the colour persists even after addition of @. ml. of conc. sulphuric acid the presence of formaldehyde is confirmed. :.
C9rootropic *ci Test:
To @. ml. of the distillate ml. of %hromotropic acid solution 6prepared by dissolving @. mg. of sodium salt of %hromotropic acid in ,@@ ml. of %onc. sulphuric acid7 is added. The contents are heated on a hot water bath at 3@o% for -@ minutes and then cooled. Violet colour is produced 6Xmax of the coloured complex O=@ mQ7. 4./.2.4 Test for et97l *lco9ol:
&thyl alcohol is isolated from biological materials by acid distillation. The distillate is sub"ected to the following tests. ,.
Ioofor Test:
0ith $: S
Sulp9ool78ic &ci N 6, gm of molybdic acid in : ml. of %onc.
sulphuric acid75 To : ml. of the distillate : ml. of the hot reagent is added. ! deep blue ring appears at once at the "unction of two li)uids. 9n shaking the whole mixture becomes deep blue. $t indicates the presence of ethyl alcohol 6This test is very sensitive and is negative with acetone acetaldehyde and dilute solution methyl alcohol. +trong solution of methyl alcohol gives only a light blueofcolour after several minutes7. -.
Et97l !eno*te Test:
: drops of 1en(oyl %hloride are added to : ml. of the distillate. The mixture is made alkaline by adding ,@J solution of sodium hydroxide drop by drop. 9n warming irritating smell of 1en(oyl %hloride disappears
,?
Laboratory Procedure Manual- Forensic Toicolo!y
and the sweet fruity odour of ethyl ben(oate appears 6methyl alcohol also gives this test but it does not give the iodoform test7. 4./.2. Tests for isoprop7l *lco9ol:
The acid'distillation is used to isolate isopropyl alcohol from tissue and other biological material. The distillate is sub"ected to the following tests. ,.
Ioofor Test:
0ith iodine S ml. of water7 in one of the test tubes. This is allowed to stand for minutes. The colour if any left after oxidation is decolourised with a pinch of sodium bisulphite. , ml of ,@J
,.
Sc9iff=s re*+ent Test:
To ; ' ml. of the distillate @. ml. of ethyl alcohol : ml. of -J A8n9 ; solution and @.: ml. 9f phosphoric acid are added and kept aside for ,@ minutes. The , ml. of ,@J oxalic acid is added followed by , ml. of conc. sulphuric acid and the contents are cooled to room temperature. ml. of +chiffIs reagent is added to it and the colour is observed after -@ minutes. 4urple colour appears if methanol is present 6confirms the presence of methanol in presence of ethanol7. The blank run is also made side by side. The )uantitation may also be made by matching the intensity of colour developed with different known standards of methanol. :.
C9rootropic &ci Test:
To @. ml. of the distillate in a test tube @.: ml. of J potassium permanganate solution is added. !fter minutes saturated solution of sodium bisulphite is added drop by drop until the permanganate colour is discharged. $f a brown colour still persists a drop of phosphoric acid is again added to it a drop of sodium bisulphite solution. To this colourless solution @. ml. of freshly prepared chromotropic acid solution 6prepared by dissolving mg. of sodium salt of chromotropic acid in ,@ ml. of concentrated sulphuric acid and heating on a water bath at 3@ o% for -@ minutes and cooling thereafter. ! violet colour is observed. ,?3
Directorate of Forensic Science, MHA, Govt. of India
4./.2.0 Test for c9lorofor:
%hloroform is isolated from biological materials by acid'distillation. The distillate is sub"ected to the following tests5 ,.
'u
To , ml. of the distillate , ml. of pyridine and : ml. of :@J sodium hydroxide solution are added in a test tube and this is heated on a water bath for , minute. ! pink to red colour indicates the presence of chloroform 6chloroform and polyhalogenated compounds respond to this test7. :.
To , ml. of the distillate , ml. of alcoholic solution of potassium hydroxide 6,@J7 is added followed by , drop of aniline. The mixture is heated. ! disagreeable odour of phenyl isocyanide evolves.
-.
To , ml. of the distillate , ml . of < esslerIs reagent is a dded.
;.
To , ml. of the distillate , ml. of strongly alkaline solution of β'naphthol is added and then heated. ! blue colour turning to green and finally brown is observed.
4./.2. Test for turpentine:
Turpentine is isolated by acid distillation. The distillate is collected in an ice cooled receiver and extracted four times with ml. portion of diethyl ether. This extract is dried by passing through anhydrous sodium sulphate and kept reserved for the following tests. ,.
: ml. of ether extract is evaporated to dryness on a spotting tile. , drop of conc. sulphuric acid is added. ! deep reddish brown colour is produced.
:.
: ml. of ether extract is evaporated to dryness. To this residue a few drops of 8ar)uis reagent is added and warmed. ! pink'red colour is observed.
-.
: ml. of e ther extract is evaporated to dryness. To this residue : ml. of ethanol followed by , ml. of ,J Vanillin in conc. #%l is added. The mixture is heated. Green colouration turning to blue is observed.
4./.2. Test for p*r*le97e:
4araldehyde is isolated from biological materials by acid'distillation. The distillate is sub"ected to the following tests. ,.
@. ml. of distillate is added to ml. of chilled :J sodium bisulphite solution in an ice'bath. To , ml. of the chilled mixture , drop of freshly ,?=
Laboratory Procedure Manual- Forensic Toicolo!y
prepared p'hydroxydiphenyl solution is added 6, gm in : ml. of hot :<.
%arbon disulphide is isolated by acid'distillation. The distillation occurs slowly 6due to presence of carbon disulphide7. $t is therefore better to collect :@ ml. in two fractions 6latter portion contains much higher proportion of carbon disulphide if present7. The distillate is sub"ected to the following tests. ,.
%e* *cet*te Test:
: drops of lead acetate is added to the solution to be tested. !n excess of potassium hydroxide solution is added and boiled. ! black precipitate is obtained if carbon disulphide is present. :.
T9ioc7*n*te Test:
To a portion of the distillate , ml. of ammonium hydroxide and , ml. of ethyl alcohol are added. $t is then boiled for minutes concentrated to , ml. and acidified with dilute hydrochloric acid. , drop of ferric chlori de solution is added. The formation of red colour indicates the presence of carbon disulphide. -.
C*sti+lionis Test:
To , ml. of distillate , ml. of alcoholic solution is added. The formation of yellow precipitate indicates the presence of carbon disulphide. 4./.2.11 Test for c*r8on tetr* c9lorie:
%arbon tetra chloride is isolated by acid distillation. The distillate is sub"ected to the following tests5 ,.
P7riine Test:
ml. of -@J
P9en7l Isoc7*nie Test:
To ,@ ml. of the distillate , ml. of distilled or purified aniline and : ml. of :@J sodium hydroxide are added. The mixture is heated for a few minutes. The evolution of foul smell of phenyl isocyanide indicates ,?>
Directorate of Forensic Science, MHA, Govt. of India
presence of carbon tetra chloride 6chloral hydrate and chloroform also respond7. 4./.2.12 Tests for c9lor*l 97r*te:
%hloral hydrate is isolated by acid'distillation. The distillate is sub"ected to the following tests. ,.
'u
!s stated in case of test for chloroform. :.
Nessler=s re*+ent Test:
! few drops of the reagent are added to a few drops of the distillate. ! yellow or reddish brown precipitate changing to grey or black is formed 6negative in case of chloroform7. -.
Resorcinol $ Pot*ssiu 8roie Test:
To : drops of reagent 6@.: gm of resorcinol and , gm of potassium bromide in ,@ ml of water7 : ml of conc. sulphuric acid is added carefully followed by : ml of distillate. This is then heated on a water bath. %hange of colour is observed as yellow to pink and finally to violet at ?@ @%. !t ,@@@% blue colour appears which turns to orange'red by adding : ml of water. 9n adding strong A9# solution the colour again changes to violet and finally to a reddish tint 6not given by chloroform7. 4./.2.13 Tests for 8enene:
The acid distillation is used to isolate ben(ene from tissue and other biological materials. The distillate is extracted with diethyl ether and the ethereal extract is dried over anhydrous sodium sulphate. ,.
: ml. of nitrating mixture 6, ml. of %onc. nitric acid S , ml. of conc. sulphuric acid7 is cooled in an ice bath and then added to residue obtained after evaporation of : ml. of ethereal extract from the distillate. The contents are transferred to a hard glass test tube and then heated by keeping it in a hot water bath. !fter that the contents are cooled and diluted with water. *ue to the formation of nitroben(ene bitter almond odour is obtained.
:.
The procedure as above is to f orm nitroben(ene by nitration. The following test may be carried out to confirm the presence of nitroben(ene i.e. the presence of ben(ene in distillate. The mixture obtained after nitration is reduced on a boiling water bath by using tin and hydrochloric acid. !fter the reaction is ceased 6usually after minutes7 , ml. of aniline and , ml. of alcoholic potassium hydroxide
,??
Laboratory Procedure Manual- Forensic Toicolo!y
solution are added. The mixture is heated. 9bnoxious smell of phenyl isocyanide indicates the presence of ben(ene. Gas chromatography5 The retention index and retention time of some common volatile poisons are given below5 T&!%E $ 4.4
%olumn N :.J +& -@ on >@ N ,@@ mesh %hromosorb G 6acid washed and dimethyl dichlorosilane treated7 :m x ; mm $.*. glass column. The support should be fully deactivated. %olumn Temperature 5 ,@@ N -@@o% %arrier Gas 5
%olumn N @.-J %arbowax :@ 8 on >@ N,@@ mesh %arbopak % :m x : mm $.*. glass column. %olumn Temperature5 -°% per : minute and then programmed at °% per minute to ,=°% and hold for at least > minutes. Gas %arrier
5
4./.2.14 Tests for 57roc7*nic *ci:
#ydrocyanic acid may be detected by screening tests with test papers by placing in air tight conditions inside the "ar containing biological materials. 6,7 Guaiacum N %opper sulphate test paper turns blue after -@ minutes 6test paper is prepared by dipping strips of
:@@
Directorate of Forensic Science, MHA, Govt. of India
6:7 4icric acid test pape r turns from yello w to tan or brown within minutes 6picric acid test paper is prepared by dipping strips of
Prussi*n 8lue Test:
To : ml. of concentrated alkaline distillate 6concentrated by heating on water bath7 a few drops of freshly prepared J solution of ferrous sulphate and , drop of ferric chloride solution are added. The mixture is heated "ust to boiling followed by the addition of dilute sulphuric acid dropwise to make the solution "ust acidic and warmed. ! 4russian blue colour formation indicates the presence of hydrocyanic acid. 9n keeping the content over night a precipitate may be seen. :.
Nitroprussie Test #ort*n=s:
To ml. of the alkaline distillate a few drops of a freshly prepared a)ueous potassium nitrite solution is added followed by the addition of four drops of ferric chloride solution. Then dilute sulphuric acid is added while shaking till the mixture assumes a bright yellow colour. The mixture is then heated to boiling. +ubse)uently it is cooled and dilute ammonium hydroxide solution is added to precipitate out excess of iron and then filtered. To the clear filtrate a few drops of dilute solution of ammonium sulphide are added. ! violet colour is obtained which changes gradually to blue green and finally to yellow 6in presence of very small amount of hydrocyanic acid a bluish green colour is observed instead of violet colour7. -.
Sulp9oc7*n*te Test:
To : ml. of concentrated alkaline distillate ,ml. of yellow ammonium sulphide is added in a porcelain basin and evaporated to dryness on a boiling water bath. Then the residue is dissolved in , ml. of acidulated water 6containing : drops of hydrochloric acid7. $t is warmed and filtered. To the clear filtrate a few drops of very dilute ferric chloride solution is added. ! blood red colour is obtained in presence of hydrocyanic acid. 9n adding a few drops of mercuric chloride solution the red colour disappears. 4./.2.1 Test for P9enol:
4henol is isolated in biological materials by acid distillation 64referably with ,5 ; sulphuric acid7. The distillate is collected in ml. of ,<
:@,
Laboratory Procedure Manual- Forensic Toicolo!y
,.
'erric c9lorie Test:
To about : ml. of distillate : drops of a very dilute solution of ferric chloride solution are added. ! violet or bluish violet colouration is produced. 6cresol other phenols and salicylic acid gives this test with the shades of colour N violet to bluish violet being different in each case7. !ntipyrine pyramidon and similar non'phenolic compounds also give this test. $n case of phenol the colour produced disappears on adding alcohol. :.
%i8er*nn=s Test:
ml. of alkaline distillate is evaporated to dryness over a small flame in a porcelain dish. $t is cooled. ! pinch of sodium nitrite is added. Thereafter conc. sulphuric acid is added. 9n mixing carefully blue or green colouration is observed. $t turns to red on adding water to it which turns green if made alkaline by sodium hydroxide or ammonia solution. 6%resol and other phenols also respond to this test7. -.
F*re=s nitrite $ nitr*te Test:
To ml of the alkaline distillate in a mortar ,@ ml. of conc. hydrochloric acid and @. gms. of nitrite'nitrate mixture 6sodium nitrite', part sodium nitrate or potassium nitrate', part and anhydrous sodium sulphate : parts7 are added . Then it is mixed thoroughly to dissolve the contents and thereafter allowed to stand for :' minutes. $n the presence of carbolic acid a rich crimson or blood red colour appears. , ml. of this blood N red or crimson coloured mixture is added drop by drop to ,@J ammonia solution. ! deep emerald green colour is produced. !lternatively : drops of formalin are added to : ml. of the blood red mixture as above. The colour changes to purple. This mixture is added drop by drop in to ,@J ammonia solution. Greenish blue or deep blue colour is produced. 6$n presence of cresols no blood red colour is produced. 9nly brown or reddish brown colour appears.
%resols are isolated by acid distillation. The distillate is collected in ml. of , <. sodium hydroxide solution in ice'cold condition. The distillate thus collected is sub"ected to the following tests5 ,.
'erric c9lorie Test:
!s described in the test for phenol. :.
%i8er*nn=s Test:
!s described in the test for phenol :@:
Directorate of Forensic Science, MHA, Govt. of India
-.
F*re=s nitrite $ nitr*te Test
!s described in the detection of phenol brown or reddish brown colour appears 6phenol gives blood red colour7 which does not change on adding a few drops of formalin or ,@J ammonia solution 6distinction from phenol7. 4./.2.10 Tests for &niline:
!s aniline is basic it is isolated in biological materials by alkaline steam distillation. The distillate is collected in ice cold condition in ml. of , < sodium hydroxide solution. The alkaline distillate is extracted with three ,@ ml. portions of diethyl ether. The ether is dried by passing through anhydrous sodium sulphate and is used for following tests. ,.
Dic9ro*te Test:
! portion of ether extract is evaporated to dryness in a porcelain basin. drops of conc. sulphuric acid and , drop of potassium dichromate solution are added. $n a few minutes the edge of the mixture begins to show a blue colour which becomes uniformly blue on adding a few drops of water. :.
P9en7l isoc7*nie Test:
!s stated in a case of chloroform obnoxious smell of phenyl isocyanide evolves. -.
57poc9lorite Test:
! portion of ether extract is evaporated to dryness. The residue is taken up in water. To it a few drops of sodium hypochlorite or freshly prepared solution of bleaching powder is added. ! purple or violet'blue colour appears which changes to reddish brown or dirty red. 9n adding a few drops of a dilute a)ueous solution of phenol containing some ammonia the violet or dirty red solution changes to blue 6indophenol7. 4./.2.1 Test f or Nitro8enene:
! portion of ether extract is evaporated to dryness. To the dried residue ml. of concentrated hydrochloric acid and metallic tin are added. This is warmed on a hot water bath for minutes and filtered. To the filtrate : ml of chloroform and : ml of alcoholic caustic potash solution are added. The mixture is heated strongly. The evolution of obnoxious smell of phenyl isocyanate indicates the presence of nitroben(ene. :@-
Laboratory Procedure Manual- Forensic Toicolo!y
:.
! portion of ether extract is evaporated to dryness. To the dried residue in a test tube ml. of ammonium chloride solution and (inc dust are added. The mixture is heated on a water bath for minutes. To the mixture , ml. of ammoniacal silver nitrate solution is added and heated on water bath for minutes. The formation of mirror on walls of test tube or formation of black precipitate indicates the presence of nitroben(ene.
4./.2.1 Test for N*p9t9*lene:
Test @it9 picric &ci:
! portion of ether extract is evaporated to dryness. To the dried mass a few drops of picric acid solution is added. Kellow crystals of naphthalene picrate are formed which show characteristic shape under microscope. :.
U# Spectru:
! portion of ether extract is evaporated to dryness. The residue is dissolved in s'ectra 'ure ethanol. $t shows maximas at :33 nm and :>> nm in V +pectra. 4./.2.2 Test for Herosene oil:
Aerosene oil in biological materials is isolated by steam distillation. The distillate is collected in ice cold condition in :@ ml saturated solution of sodium chloride. The top layer 6, cm.7 of the distillate is taken out after some time and extracted four times with ,@ ml. portions of diethyl ether. The combined ethereal extracts are dried by passing through anhydrous sodium sulphate evaporated at room temperature and sub"ected to the following tests. ,.
U# Spectr*:
! portion of ethereal extract is evaporated. To the residue water is added. 9n scanning in V +pectrophotometer maximas are observed at ::- N ::> m and also at -33 nm 6weak band7. 4./.2.21 Tests for Nicotine:
Directorate of Forensic Science, MHA, Govt. of India
,.
Roussin=s Test:
To the ethereal extract , ml. of ether solution of iodine is added. ! brownish red amorphous precipitate is formed which turns to crystalline ruby red needle shape crystals after sometime. :.
Sc9ineleister Test:
! portion of ethereal extract is evaporated to dryness. To the , drop of -@J formaldehyde solution 6free from formic acid7 and a drop of concentrated sulphuric acid are added. The mixture becomes rose red. 4./.2.22 Et97lene Di8roie: S7non7s: ,:'*ibromoethaneD &thylene bromide &*1
&thylene dibromide is a pesticide used as fumigant for storage of food grains. $t is a heavy li)uid having the odour like chloroform soluble in about :@ parts of water and miscible in alcohol ether. &thylenedibromide is potentially a cause of a variety of acute health effects including damage to liver stomach and adrenal cortex along with significant reproductive system toxicity particularly in testes. (*s C9ro*to+r*p9ic et9o:
These are homogenous solutions of coal tar acids or similar acids derived from petroleum with or without hydrocarbons or other phenolic compounds including substituted phenolic compounds or a mixture of these and a suitable emulsifier. $t is black in colour and on dilution with water gives translucent off'white turbidity 6-7. 4henyl is used as disinfectant in houses hospitals and laboratories for sanitary purposes. The tests prescribed for phenols and cresols should be applied. 4./.3
ET5ODS O' U&NTIT&TION !IO%O(IC&% &TERI&%S:
O'
SOE
4./.3.1 Deterin*tion of Et97l &lco9ol in 8loo *n urine.
:@
#O%&TI%ES
IN
Laboratory Procedure Manual- Forensic Toicolo!y
,.
oifie Hoel* $ 5ine et9o:
The apparatus used in this method consists of four hard glass tubes 6length :@ cm dia :. cm7 connected with each other by means of )uick fit "oints. $n the first tube ml of :J potassium dichromate in concentrated sulphuric acid is taken through which air is bubbled to remove the moisture and other organic volatiles. $n the second tube : ml. of blood sample or ,@ ml. of urine sample is taken. To this sample : ml. of ,@J sodium tungstate and @. ml. of : < sulphuric acids are added to deproteini(e the blood 6deproteini(ation is not re)uired for urine sample7. $n the third tube ml. of saturated mercuric chloride and ml. of saturated sodium hydroxide solution are added and mixed uniformly when a yellow precipitate of hydrated mercuric oxide is obtained 6used to trap acetone acetaldehyde etc.7. This tube is interposed between the tube of blood or urine samples and the fourth tube. To the fourth tube ,@ ml. of @.,< potassium dichromate solution and ,@ ml. of concentrated sulphuric acid are added. !ll the four tubes are arranged in such a manner so that the first tube remains out of hot water while the rest of the tubes are dipped into the hot water bath. The air is sucked with the help of an aspirator 6at the rate of : ml. 2 min.7 through the tube containing :J dichromate'sulphuric acid mixtureD then successively through the tube of blood sample the tube containing alkaline mercuric chloride and finally the tube containing @.,< potassium dichromate'sulphuric acid mixture. !fter one hour the dichromate solution is taken out of the tube i.e. fourth tube and made up to ,@@ ml. with distilled water. This solution is titrated iodometrically with @., < sodium thiosulphate solution. ! blank experiment is also performed side by side by taking all the reagents except the blood sample
%alculation5'
%onsidering redox reaction
,.@ ml. of @., A:%r:9= solution O ,., mg. of ethanol. Therefore blood alcohol J O ,., x 6P N K7 x ,@@ blood. V
mg per ,@@ ml. of
0here VO
Volume 6 ml.7 of blood taken for the experiment.
PO
Volume 6ml.7 of @.,< sodiu m thiosulphate re)uired in blank experiment.
KO
Volume 6 ml.7 of @.,< sodium thiosulphate solution re)uired in the experimentation with the blood sample. :@3
Directorate of Forensic Science, MHA, Govt. of India
:.
Deterin*tion of *lco9ol in 8loo 87 9e* sp*ce +*s c9ro*to+r*p9ic et9o:
,.
@. ml. of blood and @. ml. of internal standard 6@.:J w2v solution of n'propanol in distilled water7 is taken into the glass vial and placed into the turntable head space sampler thermostat at =@o% for ,@ minutes. The head space sample is withdrawn and sub"ected to gas chromatography.
:.
+imilar sets of experiment are made with different standards of alcohol for calibration purpose as per the following chromatographic conditions.
C9ro*to+r*p9ic conitions
,.
%olumn
5 :m x ,2>” 9.*. +tainless steel column. 420 -.>J #alcomid 8'> on %hromosorb 06mesh5,@@',:@7.
:.
*etector
5 F.$.*.
-.
%arried Gas
5
;.
Temperature 5 Transfer $n"ectiontemperature temperature %olumn temperature F .$.*. temperature
+odium %hloride. !ll the reagents should be of !.C. Grade.
Oper*tion:
The li)uid sample 6:@ l to ,ml.7 is placed into a glass septum vial together with sufficient sodium chloride to assure saturation of the li)uid 6and after dilution in fixed proportion with the internal standard solution when that procedural modification is used7 Ceference samples are treated identically and all sealed vials are inserted into the thermo stated water bath. !fter a ; minute e)uilibration period a fixed )uantity of the head space vapour of each septum vial is se)uentially sampled with a gas tight syringe and in"ected into the preset gas chromatograph. The analysis proceeds for a fixed time interval and the response of detector is recorded as a function of time. (*s C9ro*to+r*p9ic Conitions:
%olumn
5 %arbowax ,@@ 6@.;J7 on 3@ 2 >@ mesh.
%arbopack % ,.> 8 x -.: mm 9.*. 63 ft. x ,2> in.7 stainless steel column. %arrier Gas
5 # elium
5 $nlet pressure
Flow rate
5 ; ml.2 min.
Temperature 5 %olumn 9ven
5 psig.
5 >@ °%
$n"ection port 5 ,= °% *etector
5 :: °%
F.$.*.
5 #ydrogen
Flow rate
5 ; ml.2 min.
!ir
5 $nletpressure
Flow rate
5 - -@ ml.2min.
5 $nletpressure
5 ,psig.
Temperature 4rogramme
5 $sothermal.
%hart+peed
5 mm.2min. :@>
5 :@psig
Directorate of Forensic Science, MHA, Govt. of India
Proceure :
!.
,.
! well mixed ali)uot of the blood specimen is mixed an identical volume of acetonitrile internal standard solution using fully )uantitation techni)ue.
:.
&thanol or mi xed volatiles reference solutions 6usually low and high calibrators7 are treated identically.
-.
!fter the minimum e)uilibration period a fix ed identical volume of the headspace vapour is withdrawn using the gas'tight syringe ad"usted to fixed volume 6usually :@ l7 with the chaney adapter or otherwise the syringe having been preheated to ->o% in the incubator or similar device. Cepeated rapid pumping of the syringe is employed to obtain a fully representative ali)uot of the headspace vapour which is then immediately in"ected into the gas chromatograph immediately following the headspace sample withdrawal each septum vial is vented with a : gauge needle to reestablish atmospheric pressure. The syringe and needle are thoroughly purged by rapid repetitive intake and expulsion of room air and the syringe is returned to the incubator until its meet use.
;.
The analyte is allowed to proceed for approximately l minutes 6or - minutes7 if ethanol is the only compound.
.
The same gas tight syringe with volume calibration intact is used for the entire unknown and reference sample. &ach specimen is analysed by this procedure in at least independent duplicate as in each calibrator after allowing each septum vial to re'e)uilibrate for at least :@ minutes after the prior headspace sample withdrawal.
3.
The analysis record is a ppropriately identified for computation of result as follows.
Results Coput*tion 87 Pe* 5ei+9t e*sureent.
C, 1lood !lcohol concentration 61!%7 O ''''''' P %onc. &thanol %alibrator C: 0here 4# O peak height of the indicated detector response recording 6e.g. 4# i.s. O 4eak height of the acetonitrile internal standard response7.
:@?
Laboratory Procedure Manual- Forensic Toicolo!y
1.
4# &thanol 4# $.+
O C, For unknown sample 'internal standard mixture
4# &thanol mixture 4# $.+
O C: For the %alibrator sample 'internal standard
Result Coput*tion 87 Pe* &re* e*sureent.
C, 1!% unknown O '''''' P %onc. &thanol %alibrator. C: 0here 4! O 4eak area of the indicated detector responserecording 6e.g. 4! i.s. N 4eak area of the acetonitrile internal standard response7. 4! &thanol C, O
for the unknown ' sample internal standard mixture. 4! i.s. 4! &thanol
C: O
4.!. i.s.
for the %alibrator N internal standard mixture.
C. ResultB coput*tion *7 *lso 8e *e for ot9er ,ol*tiles *ccorin+l7.
4. et9o 'or T9e Deterin*tion Of &lco9ol In !loo !7 (C-5S
! volume of @. ml of blood and @. ml of internal standard 6@.: w2 v solution of n'propanol in distilled water7 was taken into the glass vial and placed in to the turntable of head space sampler thermo stated at =@ c for :@ minutes vapors were in"ected into the column and analysed. The conditions for analysis are as below. Gas N %hromatographic %ondition5 $nstrument5 ' 4erkin ' &lmer %larius Gas %hromatograph coupled with ,,@ #ead +pace sampler and analytical data +tation. 4erkin'&lmer >;,@>3@@ G% coupled with #+' ,@, is also used for this purpose. %olumn5 ' ,2>”o.d. s.s. column : meter p2w -.> Jhalcomid m',> on chromosorb w 6hp7 mess range ,@@',:@ *etector5 ' Flame ioni(ation detector. %arrier Gas: -
Directorate of Forensic Science, MHA, Govt. of India
Temperature5 ' $n"ection temp. O @>@ c Transfer temp. O @>= c %olumn temp. O @3= c F$* temp. O ,@ c +ample temp. O @3 c 4rogramme5 ' Thermostat time O :@ min. $n"ection time O @.@ min. 4ressuri(ation time O @., min. %ycle time O ;.@ min. %alculation5 ' cubic S srcin fit type multilevel using peak area at different levels. This ensures the linearity fit curve to ;@@ mg J of ethanol check standard are used to verify the initial calibration
4./.3.2 (*s C9ro*to+r*p9ic eterin*tion of Et9*nol *n ot9er #ol*tiles in !loo?Urine?,iscer*l istill*te. Isol*tion of ,ol*tiles in 8loo 5 , ml. of blood is diluted with ; ml. of water. $t is
acidified with a few drops of J tartaric acid solution and then distilled. ml of distillate is collected in ice cold condition and an ali)uot 6,@ l7 of it is in"ected into the gas chromatograph as per conditions stated below. Isol*tion of ,ol*tiles in Urine 5 , ml. of urine is taken into a micro centrifuge and
centrifuged for , minutes. l of supernatant li)uid is in"ected into the gas chromatograph as per conditions stated below. %olumn N 4orapak Y N 4olymer bead >@ N ,@@ mesh I P ; mm. 6id.7 glass column. %olumn Temperature N ,3@°% %arrier Gas
'
Gas Flow
'
*etector
' -@@ ml.2min.
' F.$.*.
T&!%E : 4. :Retention Tie of Different #ol*tile Su8st*nces Rel*ti,e to nProp*nol.
The presence of a volatile 2 volatiles may be made from the above study. u*ntit*tion :
The )uantitative estimation of volatile substance is made by determining the area of the corresponding peak and calculating the )uantity of the sample from the following relation ship. %a %b ''''' O '''''' !a !b 0here %a O %oncentration of volatile substance in exhibit. !a O !rea of peak of volatile substance present in the exhibit. %b O %oncentration of the standard. !b O 4eak area of the standard. %b is once determined by in "ecting ,@ l of su pernatant prepared by standard blood sample having ,@ mg !b of volatile substance per ,@@ ml. of blood 6the ratio remains constant7. 4./.3.3 Deterin*tion of c7*nie:
$.
!7 &r+entoetric Titr*tion
!ll silver salts except the sulphide are readily soluble in excess of a solution of alkali cyanide. #ence chloride bromide and iodide do not interfere. The only difficulty in obtaining a sharp end point lies in the fact that silver cyanide is often precipitated in curdy forms which do not readily re'dissolve and moreover the end point is not easy to detect with accuracy. There are two methods for overcoming these disadvantages. $n the first the precipitation of silver argentocyanide at the end point can be :,:
Directorate of Forensic Science, MHA, Govt. of India
avoided by the addition of ammonia solution in which it is readily soluble. $f a little potassium iodide solution is added before the titration is commenced the sparingly soluble silver iodide which is insoluble in ammonia solution will be precipitated at the end point. !g Z!g 6%<7:[ S ;<#- O : Z!g6<# -7:[ S S:%< − Z!g 6<#-7:[S S $− O !g$ S :<#!t the end point silver iodide in precipitated. Viewing against a black background best sees the precipitation. $n the second method diphenyl carba(ide is employed as an adsorption indicator. The end point is marked by the pink colour becoming pale violet 6almost colourless7 on the colloidal precipitate in dilute solution before the opalescence is visible. The colour change is observed on the precipitated particles of silver argentocyanide. Prep*r*tion of solutions: .1N Sil,er Nitr*te solution5>.;?; gms. of !.C. grade silver nitrate is
weighed accurately dissolved in distilled water and volume is made up to @@ ml. volumetric flask. This gives @.,< silver nitrate solution. !lternatively about >. gms. of pure dry silver nitrate is weighed out accurately and dissolved in @@ ml. of water in a graduated flask. The molar concentration is calculated from the weight of silver nitrate employed. *iphenyl %arba(ide +olution5 $t is prepared by dissolving @., gm of the *4%6solid7 in ,@@ ml. of ethanol. 9n the above basis cyanide is determined as follows in salts as well as in biological materials. ,7
Deterin*tion of C7*nie in suspecte s*lt: Proceure:
The sample is weighed accurately and dissolved in :@ ml. of water in a volumetric flask. $t is shaken well. From the stock solution thus prepared cyanide may be determined by any of the two methods. et9o I:
: ml. of the stock solution is transferred to a :@ ml. conical flask by means of a burette 6not by a pipette due to toxicity of cyanide7. = ml. of water '3 ml. of 3 8 ammonia solution and : ml. of ,@J potassium iodide solution are added. The flask is placed on sheet of black paper and the content of the flask is titrated against @.,< silver nitrate solutions. The silver nitrate solution is added drop :,-
Laboratory Procedure Manual- Forensic Toicolo!y
wise till sign of persistence of yellow colour of silver iodide is observed. 0hen a further drop of silver nitrate produces a permanent turbidity the end point is noted. et9o II:
: ml. of the stock solution of sample is transferred to a :@ ml. conical flask as above. = ml. of water '3 ml. of 3 8 ammonia solution and : ml. of ,@J potassium iodide solution are added. The content of the flask is titrated against @., < silver nitrate solution using :'- drops of diphenyl carba(ide as an indicator. The end point is recorded when a permanent violet colour is produced by drop wise addition of silver nitrate solution. C*lcul*tions:
The amount of cyanide or potassium cyanide may be calculated as follows5 , ml. of , 8 !g<9- ≡ @.@:@; g of %<− ≡ @.,-@; g of A%<. 2
Deterin*tion of C7*nie in !iolo+ic*l *teri*ls: Proceure:
,@@ gms. of visceral material is placed in a distilling flask of the standard fit distillation unit and acidified with tartaric acid and distilled. The distillate is collected in ice cold condition in a graduated conical flask containing ,@ ml. of 38 ammonia solution. ,@@ ml. of distillate is collected. 0hile collecting the distillate in the receiver tube should be dipped into the ammonia solution. : ml. of ,@J potassium iodide solution is added . $t is then placed on a sheet of black paper and titrated against @., 8 silver nitrate solution by drop wise addition and shaking till the persistence of yellow colour of silver iodide is observed. The addition of drops of silver nitrate solution is done carefully. 0hen a drop produces a permanent turbidity the end point is reached and noted. The relation calculates the amount of cyanide as follows. , ml. of , 8 !g<9- ≡ @.@:@; g of %<− ≡ @.,-@; g of A%<. The above titration can also be carried out by using diphenyl carba(ide solution as indicator.
:,;
Directorate of Forensic Science, MHA, Govt. of India
