SECTI

ON-1:
INTRODUCTI
ON
1.
1 Ti
tl
e: For
ensi
cToxi
col
ogy

1.
2 Scope: Var
iousaspect
soft
oxi
col
ogi
cal
anal
ysi
s.

1.
3 Pur
pose: Toprov
idest
andar
dtox
icol
ogi
calmet
hodsofanal
ysi
s
f
oruni
for
mity.

1.
4 Def
ini
ti
on:

Thewor d“ Toxi col ogy ”isder ivedf rom t heGr eekwor d“ Toxi con”whi ch
wasusedasapoi sonoussubst ancei nar rowheads.Tr adi tional l
y ,the
toxi
col ogyi sdef inedast hesci enceembody ingt heknowl edge,sour ce,
charact er,f atalef fect,lethaldose,anal ysisofpoi sonsandt her emedi al
measur es.A poi soni sdef inedast hesubst ance,whi chi scapabl eof
produci ng i njur y ordeat h when absor bed.Appr opriate dosages can
diff
er entiatepoi sonandal sot her emedi almeasur es.Al lchemi calscan
producei njuryordeat hundercer taincondi ti
ons.Hence,apoi soncanbe
definedasasubst ancet hati scapabl eofpr oduci ngdet riment aleffect son
alivingor gani sm.Asa r esul t
,t heremaybeachangei nt hest r
uct ureof
thesubst anceorf unct ionalpr ocesses,whi chmaypr oducei njuryorev en
deat h.Thet oxi col ogisti sspeci all
yat r
ainedexper tt oexami net her ol eof
such subst ances and t heiradv erse ef fect s.The v ariety ofpot ential
adver seef f
ect sandt hedi versityofchemi calspr esenti nourenv i
ronment
cont ri
butet omaket oxicologyav erybr oadf iel
dofsci ence.Ther ef ore,
toxi
col ogi stsar eusual lyspeci al i
ststohandl ev ar i
ousar easoft oxi cology .
Thepr ofessi onalact ivi
tiesoft oxicologistsf al lintof ourmai ncat egor i
es
i
.e.For ensic,I ndust rial,ClinicalandEnv ir
onment alToxi cology .For ensic
toxi
col ogy emer ged as t he hy brid ofanal y ti
calChemi st ry and t oxic
pri
nci pleef fect s.For ensict oxi cologistsar eal sopr imar i
lyconcer nedwi t
h
themedi co-l egalaspect soft hehar mfulef f
ect sofchemi calsonhuman
andani mal s.Theexper ti
seoff orensict oxicol ogi stsispr imar i
lyut il
izedi n
establ i
shingt hecauseofdeat handel ucidat i
ngi tsci rcumst ancesi npost -
mor tem i nvest igat ion.Thewor kof f or ensi ct oxi cologisti st her efore
consi deredashi ghl ycompl icatedassmal lquant itiesofpoi sonsandt heir
met abol i
tes ar et o be i solated,pur ifi
ed and quant if
ied f rom a hi ghl y
compl exmat rices.

1.
5 OBJECTI
VE:

Thismanuali sai medt oser veasadi dacti

ct exttoForensicExpert
s
wor ki
ng as Toxicologi
sts.Due t ot he preponder ance of assort
ed
redundantproceduresproclai
medf ori
solati
on,anal ysi
sandest i
mati
onof
poisons,the selecti
on of cor r
ect procedures become t edi
ous and
obfuscati
ng.Thisentail
st heneedf orstandardisedcommensur abl
eand
pragmaticprocedureswithemphasi sonaccur acyandpr eci
sion.Havi
ng
cogni
sance oft hese f acts,i twas deci
ded t o or
chestr
ate an adhoc
commi tt
ee ofhi ghly experienced and lear
ned experts.This manual
assi
milatestheout comeoft hemeticul
ousdi al
ecti
csoft hecommi t
tee
const
ituti
nghighlyexper iencedandknowl edgeableForensi
cScienti
sts
fr
om the
Forensi c Sci ence Labor ator
ies of t he count ry.The commi ttee has
dev el
opedt hisabr idgedy etcompr ehensi vet ext ,whi chencompassesa
thoroughr ev isionofer stwhi l
epr ocedur eswi thi nt heambi tofToxi col ogy
and al so i nt r
oduced somenew t opi cs.Thi st exti si ntended t o al lay
dispar i
tyandambi guityconcer ningt hecausesofToxi cologyi ngener al
withspeci alr efer encet oI ndiancont ext .Thist exthasbeenpr eparedwi th
consci ent i
ousappr aisalofv ariouspr ocedur esdescr ibedi nt hesundr y
referencebooksper t
ainingt oToxi col ogy .Wehav et hepr opensi tyt oput
forwar dat extt hatwoul dser vetomi ti
gat et het ediousandr esponsi bl ejob
ofToxi cologi st.Bypr ovidingahandyandconsol idatedqui ckr efer ence,
wehav escr upul ousl yav oidedsuper fluousdet ai l
sandpr ocedur esi nt his
manual .Albei t,itcannotsubst it
utef ort her efer encebooksandt extbooks,
i
tshoul dser veasapr act i
calguidet oper t
inentFor ensicSci entists.Thi s
manualdoesnotdet ai
lt heclini
calsy mpt omsorphar macol ogicalef fect s
ofpoi sonsbutr at herf ocusesont hecl assifi
cat ion,i solati
on,det ection
andquant itationofpoi sons.Asst at edabov e,t hemanuali snotdi r ected
towar ds t he r eplacementofaccept ed pr ocedur es/ methodol ogies,but
i
ncor por ateonl yt hose,whi char erel ev antandpr acti
cable.Howev er ,the
optionsar el ef topenf ort heToxi col ogistst oadoptanyot hert esting
procedur e,ifr equi red.Thecommi tteehopest hismanualwi llber ecei ved
withf ervourandmeett heendsi thasenv i
saged.
 SECTI
ON–2:
CLASSI
FICATI
ONOFPOI
SONSANDPOI
SONI
NG

2.
1 Ti
tl
e: Cl
assi
fi
cat
ionofpoi
sonsandpoi
soni
ng.

2.
2 Scope: Poi
sonsencount
eredi
nthet
oxi
col
ogi
cal
anal
ysi
s

2.
3 Pur
pose: Tocl
assi
fyt
hepoi
sonsandi
sol
atei
tfr
om t
heexhi
bit
s.

2.
4 CLASSI
FICATI
ON:

Poi sons can be cl assif

ied inav arietyofway s,depending upon the
i
nt er estsandneedsofcl assi
fi
ere. g.classi
ficati
onmaybedoneont he
basi sofmodeofact i
onofpoi son, t
oxicit
yrati
ng, i
ntermsoft heirphy
sical
stat es,theirlabel
i
ngr equirements,theirchemi st
ryet
c.Forthepur poseof
toxicol ogicalanal
ysis,poisonar eclassifi
edont hebasisoft
hei rchemical
proper t
iesandmet hodofi solat
ionfrom t i
ssuesandot herbi
ologicalf
lui
ds
whi char egivenbelow:

2.
4.1 Noxi
ousGases:

(
1) Car bon Monoxi de (CO) , (2) Carbon di oxi
de ( CO2), (3)
Hydrogensulphi
de(H2S),(
4)Sulphurdi
oxide(SO2),(5)Chlori
ne(Cl2)
,(6)
Ni
trousoxi de(N2O)
,(7)Methane(CH4),(8)Methyli
socyanide(CH3NCO),
(
9)Wargases, (
10)Ammonia,(11)Teargas(chl
oracetophenone).

2.
4.2 Vol
ati
l
eInor
gani
c:

(1)Cy ani
de (2)Phosphine (
3)Arsi
ne (4)Phosgene,(
5)St
il
bine (
6)
Carbonylchl
ori
de(8)Fl
urocar
bon(
9)Isocy
anide.

2.
4.3 Vol
ati
l
eOr
gani
c:

(1)
Met hanol(2)Ethanol(3)Formal
dehyde(4)
Acetal
dehyde( 5)Chl
oral
hydrat
e( 6)Py
ridi
ne(7)Ket
ones(8)Hy
drocar
bons(9)Chl
orofor
m

2.
4.4 Non–v
olat
il
eInor
gani
c:

2.
4.4.
1Ani
ons:

(1)Hali
des(2)Seleni
de( 3)Dichromate(4)Chl
orat
e(5)Azide(6)Nit
ri
te(
7)
Sulphi
de(8)Sulphate(9)Nitr
tate(10)Phosphi
de(11)Cyanideetc.

2.
4.4.
2Cat
ions:

(
1)Mercur
y(2)Ar
senic(
3)Bar
ium (
4)Thall
ium (
5)Lead(
6)Ant
imony
(
7)Bi
smuth(8)Copper(
9)Al
umini
um (10)Zincetc.
2.
4.5 Non-
vol
ati
l
eOr
gani
cNeut
ral
Compounds:

(1) Pest
ici
des (Organophosphor
ous,Organochl
oro,Car
bamat
es and
Pyret
hroi
ds)(2)Neutr
alCompounds.
2.
4.6 Non-vol
ati
leOrganicAcidi
cCompounds:

Aci
dic Drugs li
ke bar
bit
urat
es, sul
pha and phenol
i
c compounds,
sal
i
cyl
atesetc.

2.
4.7 Non-
Vol
ati
l
eOr
gani
cAl
kal
i
neCompounds:

Al
lbasi
cdr
ugsl
i
kebenzodi
azepi
nes,
alkal
oidset
c.

2.
4.8 Pl
antPoi
sons:

Dhat
ura,
Aconi
te,
Oleander
,Nuxv
omi
ca,
Abr
usPr
ecat
ori
uset
c.

2.
4.9 Mi
scel
l
aneousPoi
sons:

(
A)Mechanical:
Glasspowder,Di
amonddust
,choppedhai
ret
c.
(
B)Foodpoisons/my cot
oxins.
(
C)Animal
/insectpoi
sons.
(
D)Watersolubl
ecompounds.

2.
5 POI
SONI
NG:

Poisonsar
ecommonlyi
nvol
vedinhomici
dal
,acci
dentalorsui
cidalcases.
Theseareal
sousedt
odestr
oytheani
malsandplant
s.

2.
5.1 Acci
dent
alpoi
soni
ng:

The accident
alpoisoni
ng commonl
ytakes pl
ace as a r
esul
toft he
car
elessness/negl
i
gence.Thecommonacci
dent
alpoi
soni
ngcasesar
e:

a)Coalisall
owed to bur
ni n ar
oom unknowi
ngl
ygi
vi
ng r
iset
othe
pr
oduct
ionofpoi
sonouscarbonmonoxi
degas.

b)Worker
smanyti
mesunknowi ngl
ygointoabandonedwel
l
sorgut
ter
s
anddi
eduetot
hepresenceofpoi
sonousgases.

c)Poisonousmater
ialsli
keinsect
ici
desarecarel
essl
yspr
ayedint he
far
msandduet oaccident
ali
nhalat
ion,manyt
imesmaycausethedeath
offar
mers/wor
ker
s.

d)Chi
l
dren handl
e some poi
sonous ar
ti
cles and dev
elop poi
sonous
 sy
mpt
oms.

e)Overdosesofmedi
cinesl
i
kebar
bit
urat
esorf
akemedi
cineswr
ongl
y
pr
escri
bed.

f
)Al
l
ergi
ccondi
ti
ons.

g)Bi
tesbypoi
sonousi
nsect
sandsnakes.

h)Acci
dent
alcat
tl
eorani
mal
poi
soni
ngcases.

2.
5.2 Homi
cidal
Poi
soni
ng:

Owi ngt osmallfataldoses,tast

elessandodour lessproperti
es,misci
bil
i
ty
withdr inkandcommonav ail
abili
ty,somepoi sonsar emai nlyusedfor
homi cidalpurposese. g.arsenicsal t
s,mercurysalts,cyanides,sodi
um
nitr
ite,methylalcohol,dhat
ur aseeds,phosphidesetc.Rar el
y,cult
uresof
diseaseger msar eal sousedf orhomi ci
dalpurposes.Incer tai
npartsof
thecount ry
,opium isusedasi nfanti
cidal
poison.

2.
5.3 Sui
cidal
Poi
soni
ng:

Somepoi sonsaremai nl
ypopularf orsuicidalpurposesbecauseoft heir
avai
labil
it
yi nhouseort hewor kingpl acel ikecy anidei nelectr
oplati
ng
unit
s,insecti
cidalcompoundsmost lyavailablewithf armingcommuni t
ies,
sodium nitri
teindyeingi
ndustr
ies.Barbituratesarenor mallyusedbyt he
educatedpeopl e.Thecasesofcommi tti
ngsui ci
desbyaf ew doctorsby
i
njecti
nganaest heti
cagentsl
ikethiopentalhav ebeenr eported.

2.
5.4 Mi
scel
l
aneousPoi
soni
ng:

Casesofpoi soningotherthantheabov eareprev al

entalloverIndia
whichar ecausedbyst upefy
ingagent s.Thei nt
enti
onbehi ndt hisist o
stupef
yt heper sonandcommi trobberyorothercrimes.Howev er
,manya
ti
mesper sondi esbecauseofov erdoses.Thesepoi sonsar ecigarettes
contai
ning dhat ura,cannabis,dr ugs,arsenic etc;sweet s containi
ng
phenobarbitone orot herpsy chotr
opicsubstancesl ike lor
azepam et c.
Somet i
mes, chlor
alhydratemixedwi t
hdr i
nksisalsosimilarl
yused.

2.
6 ACTI
ONSANDADMI
NISTRATI
ONOFPOI
SONS:

Theactionofalmostallthepoisonsisthesame.Theyei t
herstopthe
supplyofoxygent ot
hebodyt i
ssuesorinhibi
ttheenzymesassociat
ed
wit
ht herespi
rat
ionmechani
sm andt hepersondiesduetostoppageof
oxygen avail
abi
lit
y. However
,t he mode of st oppage var
ies e.g.
 i
nsectici
desandpest i
cidesar epower fulinhibitorsofchol i
nesterase.The
sit
eofact ioni
ssaidt obeatt hemy oneur aljunctionsandsy napsesoft he
gangli
ons.Att hesesites,nor mally,acet y
lcholineisl i
ber at
edf rom ner ve
sti
mulation.Thel i
beratedacet y
lcholinei shy dr
olysedi nt
o chol i
neand
aceti
caci dbycholi
nester ase.I npoisoni ngbyi nsecticidesandpest i
cides,
the activi
ty of chol i
nest erase i s i nhibi
ted, acet ylcholi
ne t herefore
accumul at
es and r esultsi n hy per exci t
ation of t he v oluntary and
i
nv ol
untarymuscles.Adr opi nt heact i
v i
tyofchol inesteraset o30percent
ofnormalorl owerisassoci atedwi thtox i
csy mpt omsandl eadst odeat h
resul
ti
ngf rom r
espir
atoryf ailureorcirculatoryar r
est.

Incar bonmonoxidepoi soni

ng,thegashav i
nggr eateraff
init
ycombi nes
easil
ywi t
hhaemogl obi
nandmaket hem unabletocar ryoxygent ov ari
ous
ti
ssuesoft hebody .Theglycosi
depoi sonsaffectthehear tmuscl esand
thepumpi ngofbloodi sstopped.Opiates,al
cohols,barbit
urates,dhatura
etc.par al
ysether espi
ratorycenter
soft hebrai
nr esult
inginr espirat
ory
fail
ure.

Theact ionofpoi sonsmaybel ocal,r emot e,local&r emot ecombi nedor

general.Thelocalact i
onmeansi tsdi r
ectact i
onont het issuesandcause
corrosi
one. g.strongmi neralacidsandal kalis,irr
it
ationandi nflammat i
on
i
ncant haridesorsomener vousef fect sasi nt hecaseofdi lationoft he
pupilbyatropine.Remot eactionr esultsfrom t heabsor pti
onoft hepoison
i
ntot hesy stem e.g.al cohol,alkaloidsorot herdr ugsaf fecttheor gans
aft
erbei ngabsor bedi ntothesy stem.Somepoi sonspr oducebot hlocal
andr emot eact i
ons.I nsuchcases,t herei sdest ruct i
veact i
onont he
ti
ssuewi t
hwhi cht heycomei ncont actandt herei salsoat oxicaffect
aft
erabsor pti
one. g.oxalicacid.Gener alactionr esultswhent heabsor bed
poisonev okesr esponsesf r
om awi dev ari
et yoft issuesbey ondoneor
twosy stemse. g.arsenic,mercur y,i
nsect ici
dal compoundset c.

Mostoft hepoisonaf f
ectstheper soni mmedi atel
y,ifnot,t
hepoisoni s
eli
minat edfrom thebodybyexcr eti
onsorget smet abol
isedordetoxi
fi
ed
i
nto non- toxic metaboli
te which is eli
mi nated slowly.Howev er
,some
poisonshav etendencytogetaccumul atedi nthebodyt i
ssuesandwhena
fat
all evelisreached,theper sondies.Ar senicandDDTar eamongsuch
poisons,whi chhav ebeenr eport
edlyusedassl owpoi sonssi
nceagesf or
homi cidalpurposes.Theabsor pt
ionofDDTt hroughskinandaccumulati
on
behav iourmayr esulti
ntoaccidentaldeath.

2.
7 FACTORSAFFECTI
NGTHEI
NTENSI
TYOFPOI
SONI
NG:

2.
7.1 Dose:

Asageneralr
ule,t
hedelet
eri
ouseff
ect
sofapoi
sondependoni
tsdose.
Thel
argert
hedose,moreseverewi
l
lbethesy
mpt
oms.
2.
7.2 Hy
per
sensi
ti
vi
ty:

Somepersonsshow abnormalresponse(idi
osyncrasy
)to adrug l
ike
morphi
ne,
quini
ne,
aspi
ri
netc.duetoinher
entpersonalhy
per
sensi
ti
vi
ty.

2.
7.3 Al
l
ergy
:

Some per
sons are al
l
ergi
c( acqui
red hy
per
sensi
ti
vi
ty)t
owar
ds cer
tai
n
drugsl
i
kepeni
ci
lli
n,sul
phaetc.

2.
7.4 I
ncompat
ibl
ecombi
nat
ions:

I
ngest
ionofcertai
nmedi
cat
ionsl
i
keant
i-
ulcer
ousgel
swi
thaspi
ri
nmay
l
eadtofat
alef
fects.

2.
7.5 Tol
erance

People develop a marked tol

erance i
nt he case ofopium,alcohol
,
str
ychni
ne,t obacco,ar
senicandsomeot hernarcot
icdrugsbyrepeated
andcontinueduse.Thesnakechar mersdevel
opcompl et
eimmunit
yf r
om
snakebi
tebyget ti
ngsnakebit
esregul
arl
y.

2.
7.6 Sy
ner
gism:

Twopoisonse.g.al
coholandbarbi
tur
atesorcannabi
sdrugsanddhatur
a
i
n non-t
oxic doses,when taken toget
her,may cause sever
et oxi
c
sympt
omsduet osyner
gism.

2.
7.7 Cumul
ati
veact
ion:

The repeated smalldoses ofcumulati

ve poi
sons l
i
ke arsenic,lead,
mercury,st
rychnine,
digi
tal
i
setc.maycausedeat
horchroni
cpoisoningby
cumulati
veaction.

2.
7.8 Shock:

Somet i
mes,alargedoseofapoisonact
sdif
fer
ent
lyf
rom asmal
ldose
e.g.alargedoseofarseni
cmaycausedeat
hbyshockwhi
leasmal
ldose
resul
tsindiar
rhoea.

2.
8 FORMSOFPOI
SON:

Gasesorv apoursactmorequickl
ythansol
i
dandl iquidpoisonsbecause
theyar eabsorbedimmediatel
y.Liqui
dpoisonsactmor equickl
yt han
soli
ds,ofwhichf i
nepowdersactmor equi
cklythancoarseones.Some
substancesincert
aincombinati
onsbecomeiner
te. g.aci
dsandal kal
ies,
 str
ychninewithtannicaci dandsi l
v erni
trat ewithhy drochlori
caci
d.Some
substancesin certai
n combi nati
onsbecomepoi sonous,such asl ead
carbonateandcopperar senitewhichar ei nsolubleinwat erbutsolublein
hydrochlor
icacidi nthest omachandbecomespoi sons.Simil
arly,the
acti
onofapoi soni sal tered whencombi ned mechani cal
lywit
hi nert
substancese.g.alkaloidswhent akenwi thani malchar coalfai
lt oact .
Poisonactslowl ywhent hest omachi sf ullwithf att
yf ood.Apoisonous
powderdoesnotsi nkorf loatwhengi venwi thaf lui
dhav i
ngnearl
ysame
specif
icgravi
ty.Forthisr eason,arsenicwhenadmi ni
steredforhomi cidal
purposes,i
susual l
ymi x
edwi thmilk.

2.
9 METHODSOFADMI
NISTRATI
ON:

Apoisonact smor erapi

dlywheninhal
edi
ngaseousf or
m orwheni nject
ed
i
ntravenousl
y ,nextwheni nj
ect
edintr
amuscul
arl
yorsubcut aneouslyand
l
eastr apidl
ywhen swal l
owed orappli
ed to ski
n.Some poi sons act
di
fferent
lywhengi venthroughdif
fer
entrout
es.Snakev enom i shighl
y
poi
sonouswheni nj
ect
edbutharmlesswheningest
edor al
l
y,ifthereisno
anyinter
nalinjur
ies.

2.
9.1 Condi
ti
onoft
heBody
:

Theage,healthandindiv
idualal
l
ergytowardscertai
ndr ugsandpoisons
aff
ecttheactionofpoisons.Asageneralrule,chil
dren,oldandweaker
persons ar
e aff
ect
ed mor e sev
erel
ywi t
hl ow doses t han y
oung and
healt
hyadult
s.

2.
9.2 Fat
eofpoi
sonsi
nthebody
:

Apoi sonaf t
erbei ngabsorbedi nt othesystem,apar tofi tisexcretedi n
the urine unchanged.Mor ef requentl
y ,i
twi llbe par tlyorcompl etely
met aboli
zed.Nor mall
yt hepoi soni snotcompl etel
ydest r
oyed byt he
met aboli
cpr ocessesandi tmaybedet ect
edi ntheor iginalform orinthe
for
m ofi ntermediateproductsi nt heti
ssueort heexcr eta.Thel i
veristhe
guardianoft hebodyagai nstpoi sons.Therefore,thechancesofget ti
ng
maxi mum amountofpoi sonsori tsmet aboli
tesar einliver.From l
iver,t
he
poisonpassesi ngeneralcirculati
onandexer t
sitsact i
onei theroncel l
sof
apar ti
cularorganortissuesf orwhi chithasanaf f
ini
ty,oront hecel l
ular
ti
ssuesi ngener al
.

Themainrout
esofexcret
ionofpoisonori t
sendpr oductsistheur
inar
y
tr
act,
butot
herchannel
saretheint
esti
ne,thebil
educts,t
hesweatglands,
sal
iv
a,mucousorserousoutfl
ows,breastti
ssueduri
nglactati
onandthe
 l
ungs.Theexoandendoskel et
onsuchasepi dermis,nai
ls,hai
randbony
skel
etonret
aininorganicmetalsforl
ongperiodsevenafterthesamehave
beeneli
minatedfrom restoft
hebodyt i
ssues.

2.
10 DI
AGNOSI
SOFPOI
SONI
NG:

Diagnosi sofpoi soni ng i sof tendi fficultand hast o bemadeont he

av ailabl eev i
dences.I nmanycases,l it
tleornot oxici
tyoccur sandt he
pat ient ,parent sorrelat i
vesar er eassur ed.Somet imes, thehist orymaynot
be av ailable orunr eliable.Many cases ofpoi soning pr esentv ague
sympt omsandi nsomef atalacut epoi soni ng,sy mpt omsmaybedel ay ed
formanyhour sorday sev ene. g.t hesy mpt omsi net hylenegl ycolmaybe
del ay ed by 6 hour s;met alv apour s-8 hour s;met hanol -48 hour s;
par acet amol -
36hour s;par aquat e-48hour s;sal i
cy l
ates-12hour s;thal
li
um-
4 day s;ar sine and st i
bine-24 hr s.Some poi sons dev el
op decept ive
sympt oms e. g.gast ro-i
ntest inalt ype ofar senicalpoi soning may be
mi st akenf orsy mpt omsofchol eraorf oodpoi soning.Hence,awor ki
ng
diagnosi s has t o be made based on cl i
nicalf eatures and l aborator y
i
nv est i
gat i
ons.Thear ti
clesort hecont ainersr ecov eredf rom t hesceneof
crime ort he possessi on oft he v ictim maypr ovi
de hel pi n case of
suspect edpoi soni
ngwhent hedi agnosi si snoti mmedi atelyappar ent .
Ther ear esev eralsy mpt om pat ternswhi char et ypicalf ordifferenttypes
ofpoi soni ng and can be usef ulgui de t ot he nat ure ofpoi son,t he
l
abor atoryt estsneededandt het reat mentr equired.Thel istgivenbel owi s
i
llust rat i
veand f urtheradv i
cemayber equired i n di
f f
icultcases.The
exami nat i
ons shoul d al so i ncl ude a sear ch f orsi gns oft rauma and
syst emi c di sease because many or gani ci l
lnesses ent er into t he
differ ent i
aldi agnosisofpoi soni ng.Thesy mpt oms,cl i
nicalexami nati
ons
andt hepat ternsofpoi soningbysomecommonl yuseddr ugsandpoi sons
aregi v enbel owwhi char eessent i
alinassessi ngt hepat i
entandmayhel p
toi dent i
fytheagentandsev erityoft hepr oblem.

(
A)Sy
mpt
oms Poi
sonscommonl
yinv
olv
ed

Vomi
ti
ng Ir
ri
tantpoi
sonsli
kearseni
caci
ds,al
kal
i
es
excessofl
iquorandsomemetall
i
csalt
s.

Di
arr
hoea Usuall
ypoisonscausi
ngv
omi
ti
ngal
socause
di
arrhoea.

Cr
amps Met
all
i
cpoisonsl
i
kear
seni
c,l
ead,
ant
imony
,
mer
curyet
c.
Del
i
rium Dhatur
a,cannabi
sdrugs,
alcohol
,at
ropi
ne,
hyosci
ne,LSDetc.

Conv
ulsi
ons Strychni
ne,ni
coti
ne, cy
ani des,tri
cycli
c
anti
depressant
s,phenot hiazines,
carbonmonoxide,ethyl
enegl ycol,opi
oids,
organophosphateinsecticides,sali
cyl
ates.

Par
aly
sis Li
thium,amphet
amines,
lead,
arseni
c,aconi
te,
snakevenom et
c.

Coma Barbi
turates, carbon monoxi de, chlor
oform,
tr
ichl
oro-ethanol
,opi
oidsandexcessofl
iquors.

(
B)Cl
i
nical
Findi
ngs Poi
sonscommonl
yinv
olv
ed

Ski
ncolor
Cher
ryPink Car
bonmonoxi
de

Fl
ushedpi
nkski
n Alcohol
,cocai
ne,
cyani
deandant
i-
chol
i
ner
gic
agents

Jaundi
ce Hepat
otoxi
cagent
sli
kepar
acet
amol

Cent
ral
cyanosi
s Asignofhypoxi
abutmethaemogl
obi
naemi
a
al
socausessimil
arcol
our.
Ski
nchanges
Cut
aneousbul
lae Barbit
urates,gl utethimide, sedative
overdoses, t
ricy cl
icantidepr essantsandcar bon
monoxi de.
Sweat
ing Organiccondi tionsl i
kehy poglycaemi a,
my ocardi
al i nfarction and py rexia due t o
i
nfarcti
onsandpoi soningbysal i
cylates,organo
phosphat es,ormonoami neoxi daseinhibit
ors.

Pupi
ls Pupilsdi
l
ateinseverehypoxiaandi nhypothermi
a.
Di
lat
ion Drugssuchast r
icy
cli
cant i
depressantsalso
causedil
ati
on.Glut
ethi
mi deandmonoami ne
oxidasei
nhibi
tor
sproducewi dedilati
on.

Const
ri
cti
on Opioi
ds t ypi
cal
ly cause pi n poi nt pupi l
s.
Organophosphate i
nsecti
cides and
tr
ichl
oroethanol poisoni
ng cause v ery small
pupil
s.In barbit
urat
e poisoni
ng the pupi l
s may
varyi
nsi zeatti
mesbei ngsmall
,atot hersdi
lat
ed.
Bodyt
emper
atur
e Comat ose condit
ion forsometime may cause
Hypot
her
mia hypothermia. Sedative and hy pnotic drugs,
 tr
ichl
oroet
hanol
, et
hanol and opi
oids cause
hypot
hermia.

Hy
per
ther
mia Hyperthermiamaybecausedbyheatst r
okeand
meningiti
s and poi soning by ant i
-choli
nergi
c
agents, tr
icy
cli
c ant
i-
depressants,
monoami neoxi
dase,inhi
bit
ors,carbonmonoxi de,
dhatura,phenol
sandsal i
cyl
ates.

Br
eat
hodour Alcohol, acetone (diabeti
c ket oaci
dosis and
starv
ation) sol vents such as t ol
uene,
tr
ichl
oroethane,et
her,turpenti
ne,petr
ol,ker
osene,
cyanideandmet hylsal
icylat
e.

Appear
anceof Red venous bl
ood may suggest cy
ani
de or
bl
ood,uri
neandv
omi
t car
bonmonoxi
depoisoni
ng.

Br
own arter
ialorv enous bl
ood may suggest
methaemogl
obinaemi
a.

Vomitorgast ri
cl av
age cont ai
ning bl
ood may
suggestrepeatedv omiti
ng,cor r
osiv
es,paraquat,
coumar i
n,anticoagul
ant,ir
ri
tants,iron and non-
ster
oidalant
i-
inflammatorydrugs.

Manydr
ugst
urnur
inebl
acke.
g.met
roni
dazol
e.

Uri
ne maybe cloudyorr ed orbrown due to
haemat
uri
a,haemoglobi
nur
iaormyoglobi
nur
ia.

Bl
oodpressur
e Al
mostall sedat
ives,
hypnot
ics,
dehydr
ati
on,
Hypot
ensi
on l
engthycoma, vomiti
ngandsweati
ngmaycause
hypot
ension
Hy
per
tensi
on
Amphet
amines,cocai
ne,
phencycl
idi
ne,
sy
mpathometi
csandanti-
chol
iner
gicagent
s.

Car
diacar
rhy
thmi
as Changesi nhear trat
eorrhythm maybecausedby
betablockingdr ugs,or
ganophosphates,
theophyll
ine,t
ricycli
canti
depressant
s,
sympat hometics,barbi
tur
ates
etc.

Pul
monar
yoedema Petroleum products, organophosphat
es,
ethylenegly
col,i r
ri
tant gases (metal vapour)
,
sali
cy l
ates,opi
oidsetc.
Rhabdomy
oli
ti
s Patientlyi
ngincomaf oralongt imeonhar d
surfacemaydev elopi tduetopr essurenecrosisof
muscl e,whichmayl eadt orenal f
ail
ure.The
agentsmost l
yresponsi bleforthesamear e
barbitur
ates,opioids,ethanolandcar bon
monoxi de.Rhabdomy olysiscanal sooccurafter
prolongedandsev eremuscl espasm, dueto
poisoningbyst r
y chnine,phency cl
idi
neand
monoami neoxidasei nhibit
ors.

(
C)Pat
ter
n Poi
sonscommonl
yinv
olv
ed

Coma,Hypot
ensi
on, Barbi
turat
es,benzodi
azepi
nes,gl
utethi
mide,
Fl
acci
dit
y. tr
ichl
oro-ethanol
,et
hanol,
opiodsetc.

Coma, hyper
tensi
on, Tri
cycl
i
cantidepr
essant
s,ant
i-
chol
i
ner
gicagent
s,
techy
cardia,
dil
atedpupi
l
s. phenot
hiazi
nes.

Malaise,
rest
lessness, Car
bon monoxide,sol
vent
s,i
nsect
ici
des,l
ead,
nausea,weakness. mercur
y,ar
seni
c.

Restl
essness,hy
pertoni
a, Monoamine oxidase inhibi
tors,ant
i-
choli
nergi
c
hyper
refl
exia,
pyrexi
a. agent
s,st
rychni
ne,phencycli
dine,
amphetamines.

Behaviour
al Psychotropic drugs, anti
chol
iner
gic drugs,
Dist
urbances cor
ticosteroi
des, sol
vent abuse, psi
locy
bin-
mushr ooms.

Bur
nsi nmout h, Cor
rosi
ves,
caust
ics,
par
aquat
.
di
sphagia,abdominal
pai
n,
di
stension.

Renal
fai
l
ure Paracetamol,mer
cur
ialcompound,
acids
(phosphori
c,oxal
i
c,f
ormic),
phenol
s,arsi
ne,
sti
bine,l
ead.

Jaundice,
hepat
ic Paracet
amol ,
carbontetrachl
ori
de,
fai
lur
e. phosphorous,or
ganiclead.

2.
11 POSTMORTEM FI
NDI
NGSI
NPOI
SONI
NG:

· Apeculi
arsmellonopeningthebody:Thesubstancedet ect
ablebythei
r
smel
larealcohol,cy
anide,carbol
icacid,pet
rol
eum pr oducts,camphor
,
ni
cot
ine,
opium,paral
dehyde,phosphor
usinsecti
cides,
pestici
desetc.

· Pr
esenceofanyf
orei
gnmat
eri
ali
nthef
orm ofpowder
,capsul
es,
tabl
ets,
 l
eav
esorseedsi
nthest
omach.

· Ir
rit
ati
on,ul
cer
ati
onandper
for
ati
onordi
scol
orat
ionandchangei
ncol
or
orsoft
eningoft
hemucousmembraneoft
hestomach.

· Lary
ngealoedemacommonl
yint
hedeat
hisduet
oal
coholand
bar
bit
urates.

· Acut
elungcongest
ionandoedema.

· Acut
eswel
l
ingofbr
ainwi
thorwi
thoutapr
essur
econe.

· Di
stendedur
inar
ybl
adder
.

· I
ntr
avascul
arsi
ckl
i
ng.

· Negat
iveevidencesuchasnotraumaornosi
gnofdi
seasei
nanyor
gan
t
oaccountforthecauseofdeat
h.

· Bodyi
shi
ghl
ydecomposed.

· Di
l
ati
onorcont
ract
ionofpupi
l
s.

2.
12 MANAGEMENTANDMEDI
COLEGALASPECTSOFPOI
SONI
NGCASES:

Thepr inci
pleofmanagementofpoi sonedpatientsiscarefulatt
entionand
tr
eatment t o prevent cerebral anoxia, respir
atory
, car diovascular,
neurologicalandothercomplicati
ons.Themai naimi st
ohel pt hepatient
to stay al i
ve.Mostpat i
ents need only suppor ti
ve care.The i nitial
managementshoul d alwaysbe act i
ve unti
lt he cont
ributi
onsofdr ug
i
nduceddamageandpr e-exi
sti
ngorganicdiseaseareestabl i
shed.

Themai
nobj
ect
soft
reat
mentofpoi
soni
ngar
e

*Remov
alofunabsor
bedpoi
sonf
rom t
hesy
stem

*Admi
nist
rat
ionofant
idot
es

*El
i
minat
ionofabsor
bedpoi
son

*Sy
mpt
omat
ict
reat
ment

*Mai
ntenanceoft
hepat
ient
’sgener
alcondi
ti
on.

I
tshouldal
waysber emember edthatover
-t
reat
mentofthepati
entwi
th
l
argedosesofant
idotes,sedat
ivesorst
imulant
sdoesfarmoredamage
 t
han t
he poi
son it
sel
f.The j
udici
ous use ofdr
ugs and necessar
y
t
her
apeut
icmeasur
esarer
equi
red.

2.
13 REMOVALOFUNABSORBEDPOI
SON:

2.
13.
1Inhal
edPoi
sons:

I
nt hecasesofpoisoni
ngbyinhal
ati
on,
thepati
entshoul
di mmediatel
ybe
removed tofreshair.A cl
earair
wayshould beensured and art
if
ici
al
respi
rat
ioncommencedatonce.Insomecasestherespi
rator
ycentremay
bestimulat
edbyami xtur
eof95%ofoxygenand5%ofcar bondi
oxide.

2.
13.
2Inj
ect
edPoi
sons:

Theuseoft ourni
quetabovet hesit
eofi nj
ectionmayslowtheabsorpti
on.
Thet ourni
quetshouldbel oosenedev er
y10mi n.for2mi n.suckt he
poisonbymaki nginci
sionsatt hesiteandusechemi calant
idot
es,cold
packing and v
asoconstri
ctors e.
g.injecti
on ofepinephri
ne to pr
event
absorpti
on.Thecommonexampl esofi njectedpoi
sonsar ehypnoti
cs,
i
nsulin,
snakeorotherinsectsbit
esetc.

2.
13.
3Cont
actPoi
sons:

I
fthepoi
sonisappli
edtoeyes,ski
n,woundori
nser
ted,t
hepoi
soni
s
r
emovedwit
hspeci
fi
cant
idot
es.

2.
13.
4Ingest
edPoi
sons:

Gastr
iclav
ageoremesismaybei nducedbef
oregast
ri
cempt
yingt
ime
usual
ly4to6hour
saf
teringest
ion.

2.
13.
5 Emet
ic:

Whent hepoi sonhasbeeni ngested,earlyvomi ti

ng( emesi s)isofgr eater
val
ueinav oi
dingabsorpti
ont hant hemostener geti
cgast riclavagecar r
ied
outaftersomedel ay.Emeticsar ethesubstances, whi chpr oducev omiting.
Mostpoi sonsar ethemselvesemet i
csandmaycausev omi ti
ng.Howev er,
i
tisadv i
sabletogiveanemet i
ct oensureamor et horoughempt y i
ngof
thestomach.Emesi siseasiert hangast r
iclavageandl esst r
aumat icfor
thepatient,i
fthepatienti
sconsci ousandco- operati
veandv omi ti
ngisnot
contr
a-indicat
ed (li
ke corrosives,strychnine,pet roleum di sti
ll
ates and
coma).Emesi s should be av oided i
n cor rosive poi soning forfearof
damageof oesophagusandst omach.Itmustal sonotbeusedwhent here
 i
s dangerofaspi rat
ion i
ntolungs,eit
herf r
om volat
il
e poi
sons l
i
ke
petrol
eum dist
il
lates orfrom inhal
ati
on ofgastri
c content
s due t
o
i
mpai r
edconsciousness.Emesisshouldbeinducedei
therbyti
ckl
ingt
he
faucesorbyemet i
cs.Thehouseholdemeti
csare:

· Copi
ousl
ukewar
m wat
er.

· 15gm ofmust
ardpowderi
n200ml
ofwat
er.

· About30gmsofcommonsal tin200mlofwat er.Normall

ythi
s
emeticagentshoul
d beav oi
ded becausei ti
sapooremet i
cand
excessi
vedosesareeasil
ygiven,resul
tinginhypernatr
aemia.Ther
e
aremanydocumenteddeathsf
rom thiscauseeveninrecentyear
s.

· Zi
ncsul
phat
e:1-
2gmsi
n200ml
ofwat
er.

· Apomor phinehasar emarkabl

esel ecti
veactiononv omiti
ngcentr
e.It
i
susedi nadoseof6mgbysub- cutaneousi njecti
onf orqui
ckemesis
fol
lowedbyl evall
orphan1-2mg,i fnecessar yorpr efer
ablynal
oxone
hydrochl
ori
de5- 10mgi ntr
amuscul arori nt
ravenoust ocounter
actit
s
narcoti
ceffects.Now-a-day s,i
tiswi del
yaccept ed.

2.
13.
6 Gast
ri
cAspi
rat
ionandLav
age:

Gast r
icaspirat
ionandlav agei st heonl ysuitabl
emet hodofempt yingthe
stomachofanunconsci ouspat ient.Inunconsci ousness,pr otectairway
wi t
hcuf fedendotr
achealt ube.Pl acet heheadoft hepat ientov ertheend
orsi deoft hebedsot hatmout handt hroatarebel ow t hel arynxand
trachea.Use a wi de bor et ube l ubri
cated wi thv asel i
ne orgl yceri
n
(Jacquesgauge50cm i nadul t
s,50cmswi llr
eacht hest omach) .Ensure
thet ubei snoti nthet rachea.Aspi rateandsav ethef i
rstsampl efor
analysis.Use300-600ml .ofwat eratbodyt emper at
uref orwashi ng.Ifthe
tubebecomesbl ocked,gent lesuct i
oncanbeappl i
ed.Cont inuest omach
washwi thwaterorsat uratedl i
meorst archwat eror1: 5000pot assi
um
permanganat eor4%t annicaci det c.til
laclearodour lessf luidcomesout .
Leav esomeamountofant i
dot ei nthest omachf orneut ralizingleftover
poison.I ti
sbestmet hod,i funder t
akenear l
yi.e.withinabout4- 6hour s
afteringesti
onofpoison.Af terthen, i
tmaynotbeusef ul.

Cer
t ainextr
apr ecauti
onsar enecessar yf orgastri
clav ageincasesof
coma,pet rol
eum di sti
ll
ates and st r
ychnine poisoni
ng.Fol lowing t he
i
ngest i
onofcorrosive,passageofst omacht ubemayl eadtoper f
or ati
on.
Lavagei sdanger ousi n casesofpet roleum disti
ll
ateswhi ch maybe
i
nhal edrapi
dly
,maycausef atalr
esult
sunl essthegl otti
sisseal ed.In
coma,t her
eisaser i
ousr i
skofaspi r
ationpneumoni a,duet odepr ession
ofcoughr efl
ex,unlesst heai r
wayisseal edbycuf fedintubati
onsbyan
 anest
het
ist
.Inthecasesofst
rychni
neingest
ion,aconv ul
sionmaybe
i
nducedbythi
smethodunl
esst
hepati
entwasfirstanaest
het
ized.

2.
13.
7 UseofAnt
idot
es:

Antidot
esareremediestocounteracttheef
fectsofpoi
son.Theyareused
becausethepoisonmaynothav ebeencompl et
elyr
emovedbyemesi sor
l
av ageorthepoisonisalreadyabsorbedorithasbeenadmi nist
eredby
otherrout
et haningest
ion.Accordingtotheirmodeofact i
on,theyare
di
v i
dedint
othefoll
owingclasses:

2.
13.
7.1Mechani
cal
orPhy
sical
:

These ar
et he subst
ances,whi
ch t
end t
oimpede t
he absor
pti
on of
poi
sonsbythei
rpresence;t
heyar
e:

Act
ivat
edChar
coal
:

Iti
srecommendedasagener al
-purposeor aladsorbent.Acti
vatedmeans
thebrandofchar coalwhichmeet scer t
ainadsor bancestandards.Tobe
ful
lyeff
ectiv
e,ar at
ioofabout10par tsofchar coalto1par tofpoisonis
needed.Iti
smor ebenefi
cialforadsor banceofsubst anceswhicharet oxi
c
i
nsmal lamountssuchast ri
cycl
icant i
depressantsand alkaloi
dsl i
ke
str
ychnine;butlessef f
ecti
vewhenl argeamountofpoi sonhasbeen
i
ngestede.g.inaspir
inorparacetamol poisoni
ng.

Thet r
eatmentwi t
hchar coalismostef f
ecti
vedur i
ngt hef i
rstfourt osi x
hoursaf t
eringest
ion.Howev er,somepoisonsar ealsoadsor bedbyor al
useofact i
vated charcoalmanyhour saf t
eri ngesti
on and ev en af
ter
i
ntravenousadmi nist
rati
on,presumablybybackdi f
fusionori nterrupt
ion
ofenterohepati
ccircul
ation.Aspir
ati
onofcharcoalintot hel
ungsi sarisk
thatshouldbeguar dedagainst.Normal
lycharcoalisgiveninadoseof4-
8gms.

Demul
cent
sandBul
kyFoods:

Thesear ethesubst anceswhichpreventtheabsor ptionofthepoisonby

formingacoat i
ngont hemucousmembr aneoft hestomach.Indoingso,
theyactbot hincor rosiv
eandi rr
itantpoi soningexceptinphosphorus
poisoning as phosphor us i
s solublei nt hem t hereby i
ncr
easing t
he
absor pt
ion.Thecommondemul centsaref ats,oil
s,milkandeggalbumin.
Bulky food like banana acts as a mechani calant i
doteto gl
ass by
i
mpr isoningit
spar t
iclesandthuspreventi
ngi tsaction.

2.
13.
7.2Chemi
cal
:
 Thesear et hesubst anceswhi ch actchemi cal
lytof orm a non t oxic
compoundbyf or
mingi nsolublecompoundorbyoxi dizingthepoi sonto
non t oxic const i
tuents e. g. di
lute acet i
c acid neut ral
izes alkal
is.
Magnesi um oxideorcal ci
um oxi dewillneutrali
zeaci
ds.Si mil
arly
,li
mecan
beusedf oroxalicacid,magnesi um sul phateforcarbolicacid,copper
sulphatef orphosphor us,sul phates ofal kal
isforl ead and f r
eshly
precipi
tatedi r
onoxidef orarsenic.Tanninpr oducesinsolublecompounds
withmostal kal
oidsglucosidesandmet als.

Potassium permanganate being oxidi

zing agentreact
s with organic
substances, a 1:1000 st r
ength aqueous sol uti
on of pot assium
permanganateisef f
ecti
veagai nstalloxidi
zabl
epoisonslikealkaloi
ds,
amidopy r
in,
ant
ipyr
in,bar
bit
urates,phosphorus,
cyani
desetc.

2.
13.
7.3Phy
siol
ogi
cal
orPhar
macol
ogi
cal
:

Thesechemi cal
sdonotent eri
ntoanychemi calcombinati
on,butproduce
opposite ef f
ectst ot hatoft he poi son.Hence,t hey are known as
physiologicalantagonists e.
g.atropine forpi l
ocar
pine,chlorofor
m f or
stry
chni ne, caffei
ne f or mor phine, at ropine and oxi mes f or
organophosphor ouscompounds.Howev er
,theantagonism i
susual l
ynot
compl eteandt her emedymayi tsel
fpr oducemostundesi rableresult
s.
Nalorphinehaspr ovideditsel
fasav eryv al
uablespecifi
cantidoteagainst
mor phineandi t
sgroup.

Certainchelati
ngagent sarewi delyusedasspeci fi
canti
dotesagainst
someheav ymet al
s.Thesesubst ancesproduceafir
m non-i
onizedcycli
c
compl ex (
chelate)with cati
ons.Such compounds can f orm stable,
solutbl
enon-toxiccomplexeswithcalci
um andcert
ainheavymet al
s.They
areBAL( Dimercaprol
),EDTA(Ethy l
enediaminet
etr
aacetat
e),penci
lamine
anddesf er
ri
oxami newhichareusedi nheavymetalpoi
soni
ng.

2.
13.
7.4Uni
ver
sal
Ant
idot
es:

Iti
susedwhentheidenti
tyofpoi
sonisnotknownorwhenacombinat
ion
ofpoisons i
s suspect
ed.Itconsist
s ofa mi xt
ure oft
he f
oll
owing
subst
ances:

I Powderedanimalchar
coal 2par
ts Adsor
bsal
kal
oids
(orbur
nttoast)

I
I Magnesi
um oxi
de 1par
t Neut
ral
i
zesaci
ds

I
IITanni
caci
d(orst
rongt
ea) 1par
t Preci
pit
atesalkal
oids,
cert
ainglucosi
des&
manymet al
s.
 Themi xt
urei
sgivenindosesofat abl
espoonfulsti
rr
edupi n2l t
rs.of
water
.Thisdosemayber epeat
edonceortwice,ifnecessar
y.Besides,
somehouseholdproduct
smaybeusedassaf eant i
doteswhichareas
fol
l
ows.

· St
rongl
i
qui
dteapr
eci
pit
atesmanyal
kal
oidsandmet
alpoi
sons.

· Mi
lkandraw eggwhit
e,bei
ngprot
einri
ch,preci
pit
atesmer cur
y,
arseni
c and ot
herheavy met
als.I
n addi
ti
on,they have excell
ent
demulcentpr
opert
ies.

· Mashedpotat
oesinwat
erareal
sogoodadsor
bent
sandcanbeused
i
nplaceofchar
coal
wheniti
snotavai
l
abl
e.

· Mil
kofmagnesi
aorsoapsol
uti
onmaybeusedi
npoi
soni
ngduet
o
aci
ds.

· Ti
nnedj
uiceorv
inegarcanbeusedi
npoi
soni
ngduet
oal
kal
i
s.

2.
14 ELI
MINATI
ONOFABSORBEDPOI
SONS:

Afteraconsi der ableabsor pti

onofpoi son i ntot hebl ood st ream has
occur red;procedur esmustt henbeempl oyedt oaccel eratetheexcr etion
ofthet oxi
cagentmai nlythroughur i
ne.Eliminationbycat harsiswhennot
cont r
a- i
ndi
cated and by sweat ing by means ofhotpacks may be
encour aged.Fl uidadmi ni
str
ationt omai ntainadequat er enalf unctionwi th
periodsofdi alysiswi llbebenef icial.I
ncasesofaspi ri
nandbar bit
ur ate
poisoni ng, f orced di ur
esis usi ng i nt r
avenous chl orothi
azi de
and/ ormanni t
oli nf usi
onhav ebeenpr ovedv al
uabl e.Per i
tonealdi alysis
has been r ecommended f or sal i
cy l
ate poi soni ng i n chi ldren.
Haemodi alysi
s has been used f orel iminating bar biturates,br omi des,
gl
ut ethimide,met hanol,sal i
cylates and t hiocyanat es f or t he blood.
Exchanget ransf usioni sonlyf easiblewi thsmal lchildr enandhasbeen
applied t o poi soni ng by salicylates,bar biturat
es,i ron sal ts,car bon
monoxi deetc.Al ltoxicsubstancesar eremov edbyt hist echnique.

2.
15 TREATMENTOFGENERALSYMPTOMS:

The t reatmentshoul d be appl i

ed as indicated byt he symptoms.I n
unknownpoi soningcases, thesymptomsar et hecluest othetreat
mentof
the case.St rong anal gesi
cshoul d be given f orpain and oxygen for
arti
fici
alr espirat
ioni nrespirat
oryfail
ure.Antibioti
csshoul dnotbegiv en
routinely,butonl yi finfect
ionissuggest edbypur ulentsputum,pyrexia
andl eucocy tosist r
eatmenti srequir
ed.Car diacst i
mul ant
si ncir
cul
atory
fai
lureandanaest heti
cf orconvul
sionsshoul dbegi ven.Thesy mptomat i
c
effectoft hepoi sonshoul dbet r
eatedbygener almeans.Sal ineinf
usi
oni s
 usefulincount eracti
ngdehydrati
onandencour agi
ngdi ur
esis.Howev er
,
carefulat
tenti
onmustbepai dtowat ermetaboli
sm asov erhydrati
onmay
l
eadt opulmonar yoedemaandci rculat
oryimpairment.Theaddi t
ionof
sodium bicarbonat etothei nfusi
on maybeofv al
uewhen t healkal
i
reserveisdiminished.Administ
rati
onofgl ucosewillcombatdepl et
ionof
l
iverglycogen,andt herest
orati
onofpot assium -sodium imbalancemay
benecessar y
.

2.
16 MAI
NTENANCEOFTHEPATI
ENT’
SGENERALCONDI
TION:

Thepat i
entshoul dbekeptwar m andcomf ortable.Aftercomingoutoft he
eff
ectsofpoi son, oneoft hemai ndangersi sthesubsequentdev elopment
ofupperr espi r
ator ytractinfect
ion.,thi
si saspeci alhazardi nel derl
y
people,whohadar espir
ati
oni nfecti
onbef ore,andwhoi nhaledv omit
us.
Hence,i fi nfecti
on i s suggested,ant ibioti
c shoul d be gi v
en.Good
envi
ronmentandnur singcareshoul dbepr ovidedt othepatient(especiall
y,
i
funconsci ous).Al lpatient
swhohav eat tempt edsui ci
deshoul dnotbe
al
lowedt ol eav et hehospi t
alwi thoutbeingi nter
viewedbypsy chiat
ri
st
whocangi vef urthernecessarysuppor ti
vepsy chotherapy.

Ref
erences:

1. Al
ber
t,A.
L.,
Sel
ect
iveToxi
cit
y,ChapmanandHal
l
,London,
1979.

2. Lu,F.C.
,BasicToxi
col
ogy
,Hemi
spher
ePubl
i
shi
ngCor
por
ati
on,
Washi
ngton,D.
C.,
1985.

3. Subrahmanyam,B.V(Ed)
,Modi ’
sMedi calJur
ispr
udenceand
Toxi
col
ogy,12t
hEdn.
,But
ter
worths,
Indi
a,1999.

4. Curr
y,A.S.
,Adv
ancesi
nFor
ensi
candCl
i
nicalToxi
col
ogy
,CRCPr
ess,
Cl
evel
and,1991.

5. Hayes,A.W.,Pri
nci
plesandMet
hodsofToxi
col
ogy
,3r
dEdn.
,Rav
en
Pr
ess,N.York,1991.

6. Hodgson,E.
,Mailman,R.
B.andChamber
s,J.E.
,Dict
ionar
yof
Toxi
col
ogy,VanNost
randRei
nhol
dCompany
,N.Yor
k,1988.

7. Ott
obani
,M.
A.,TheDoseMakest
hePoi
son,2ndEdn.
,VonNast
rand
Rei
nhol
dCompany,N.Yor
k,1991.

8. Sul
l
ivan,
J.B.andKrei
gev,G.
R( Eds)
,Hazar
dousMat
eri
alsToxi
col
ogy
,
Wi
l
li
amsandWi l
ki
nson,
Balti
more,1992.

9. Di
Mai
oDJ,
DiMai
o.VJM,
For
ensi
cPat
hol
ogy
,CRCPr
ess,
BocaRat
on,
 1989.

10. Spi
tz,
WU( Ed)
,Medi
col
egal
Inv
est
igat
ionofDeat
h,3r
dEdn.
,Char
les\C.
Thomas,N.York,
193.

11. Basel
t,R.
C.,Cr
avey,R.
H.,Di
sposi
ti
onofToxi
cDr ugsandChemi
cal
sin
Man,4t
hEdn,Chemical
Technol
ogyInst
it
ute,
CA,1995.

12. Gol
dfrank,LewisR.
,Toxi
col
ogi
calEmer
genci
es,5t
hEdn.
,Appl
eton&Lange,
Connect
icut,1994.

13. Sper
li
ng,F.,Quanti
tat
ionofToxicol
ogy-TheDose–ResponseRelat
ionshi
pin
Sperl
y,F(Ed)Toxi
cology:Pr
inci
plesandPr
acti
ces,
Vol
.2,
Wil
ey,N.Yor
k,1984.

14. Rowl
and,M.,Tozev,T.
N.,Cl
i
nicalPhar
macoKi
net
icsConcept
s&Appl
i
cat
ions,
2ndEdn.
,Lea&Fel
iger
.

15. Clarke,E.
G.C,I
sol
ati
onandI
dent
if
icat
ionofDr
ugs,2ndEdn.
,thePhar
maceut
ical
Pr
ess,London,1986.
 SECTI
ON-3:
ISOLATI
ONANDPURI
FICATI
ONOFPOI
SONS

3.
1 Ti
tl
e: I
sol
ati
onandpur
if
icat
ionofpoi
sons.

3.
2 Scope: Gener
alpr
ocedur
esf
ori
sol
ati
onandpur
if
icat
ionofpoi
sons.

3 Pur
pose: Toknow t
hemet
hodsadopt
edf
ori
sol
ati
onorex
tract
ionofpoi
sonsf
rom di
ff
erent
mat
ri
ces.

3.
4 Responsi
bil
i
ties:
Gazet
tedof
fi
cer
sandot
herassoci
atedsci
ent
if
icst
aff
.

3.
5 BASI
CSTEPSI
NANALYTI
CALTOXI
COLOGY:

1. Ext
ract
ionofact
iveconst
it
uenti
.e,
poi
soni
nmat
ri
cesofi
nter
est
.

2. St
ri
ppi
ngorpur
if
icat
ionofact
iveconst
it
uentt
hussepar
ated.

3. Rapi
dScr
eeni
ngandI
dent
if
icat
ion

4. Quant
it
ati
on

5. I
nter
pret
ati
on/Concl
usi
on.

3.
6 GLOSSARYOFTERM RELATEDTOEXTRACTI
ON:

Matr
ix:Anymat er
ialsubst
anceintheuni v
ersewherei
ntheact
iveconst
it
uentmaybe
di
spersed,
accumul
ated,l
eft
,absor
bedorchemical
l
ybound.

Act
iveConst
it
uent
:Thet
oxi
cchemi
cal
ofi
nter
esti
.
e.poi
son.

St
ri
ppi
ng:
Pur
if
icat
ion.

3.
7 CLASSI
FICATI
ONOFMATRI
CES:

A. Biol
ogical:Viscera,bl
ood,uri
ne,sal
iv
a,st
omachcontents,i
ntest
inal
contents,gastriclavage,vomit
,brai
nmat t
er,st
ool
,faecalmat t
er,
bone,nails,hair
,skin.

B. NonBi ologicalmat r
ices:Water
,remnantsortr
acesofpoi sonin
smallcont ai
ner,foodandf oodproduct
s,mil
kandmi l
kproducts,
fr
uit
s,v egetables,tea,coff
ee,cooked mat
eri
als,dr
inks,cereal
s,
pul
ses,wi nes,etc.

C. Vi
scera:Internalorgansviz.l
iver
,kidney
,st
omach,i
ntest
ine,gal
l
bl
adder,uterus,hear
t,l
ungs,br
ainetc.

3.
8 DI
FFERENTCLASSESOFPOI
SON:

Forthepurposeofchemi
calanal
ysi
s,poi
sonsaregroupedaccordingt
o
themethodsusedfori
sol
ati
onoft
hesubstancef
rom matri
ces.Thesear
e
 gi
venbel
ow.
· Noxiousgases
· Volati
l
epoi sons(organi
candinor
gani
c).
· Non-volati
lepoi
sons( or
gani
candinor
ganic)
.
· Pl
antpoi sons
· Miscell
aneouspoi sons.

3.
9 DI
FFERENTMETHODSOFEXTRACTI
ON(
1,2,
3,4,
5,6,
7):

There ar ev ari
ous cl assicaland moder n met hods ofext r
act i
on.The
selecti
onofpr opermet hodofext ractiondependsonv ariouscont rol
li
ng
factorsv i
z.natureofpoi son,mat ri
xormat ri
cesandal soquant ityof
sampl esav ail
ableorf orwardedforanal ysis.Theact i
veconst it
uentshoul d
be ex tracted fr
om sampl ei n minimum st eps t o av oidl oss dur i
ng
processi ng.The ext r
acted mat eri
alal so r equi r
e pr operst r
ipping or
purifi
cationt oav oidi nt
erfer
encesofmat ri
cesasf araspossi bl
e.The
effi
ciency ofext racti
on and st ri
pping det ermi nes t he l owerl i
mi tof
detection,preci
sionandaccur acyinthedet ermination.Theanal ystplaysa
signif
icantr ol
ei nt hesel ect
ionofpr opermet hodol ogyont hebasi sof
diff
erentpar ameter sviz.casehistory,amount ,physicalst ateofmat ri
ces,
analyti
calr equi
rement sandal soinfrastructuralfacili
ti
esav ai
lable.Itwill
bebef itt
ingifthemet hodsar epresent edschemat icall
ydependi ngont he
classif
icationofpoi sons.
Classofpoi
son Classical Met hod Moder nMet hod
Gases Micr o-diffusion, Adsor ption- SensorBasedGasAnal yzer,
desor pt i
on GasChr omat ography .
Vol
ati
l
eInor
gani
c Gutzei tMet hod,Mar sh- Berzelius Mi cr owav eov ent echniquef or
Met hod, Mi cro-diffusion, di gest i
onf oll
owedby
Digest i
onwi t
hspeci ficr eagent s I on Chr omat ography,
/underspeci fi
c condi tions of Spect roscopy (Mass)et c
PH,
Vol
ati
l
eOr
gani
c Distill
ation, St eam Di stil
lati
on, Chr omat ographi cmet hods
Diffusion
Non-Vol
ati
l
e Dry and wet ashi ng, Gr oup Mi cr owav eov ent echniquef or
I
norgani
c analy sis, Elect ro-dialysis, digest i
on f ollowed by I on
Digest i
on under appr opriate Chr omat ographyusi ng
analy ti
calcondi ti
ons,Paperand I on-exchanger esins
Thinl ay erchr omat ogr aphy
Non-Vol
ati
l
e Solv entExt raction,St as-Ot to, HPLC, Pai r
ed i on ext r
action
Organi
c Digest i
on wi t
h ammoni um Chr omat ography , HPTLC,
sulphat e,sodi um t ungst at e or Super crit
ical f
luid
othermodi fiedmet hodsoft he chr omat ography ,Sol id phase
abov e extr action,Mi cellarext ract
ion,
Affini t
y chromat ography,
Mi cr owav e Assi sted React ion
Syst em, Accel erated sol vent
Ext raction, Sweep Co-
Dist il
lati
on Uni versal Tr ace
Resi dueExt raction.
 Ani
on Dial
ysi
s, Chemical Di
gesti
on, I
on-Chromat
ography by I
on-
Paper and Thi
n Layer Exchanger
esins.
Chromatogr
aphy

3.
9.1 Uni
tpr
ocesses/
oper
ati
oni
next
ract
ionmet
hods:

Thevar
iousuni
tpr
ocesses/oper
ati
onsr
elat
edt
oext
ract
ionar
easgi
ven
bel
ow:

3.
9.1.
1 Sol
ventExt
ract
ion:

A system oftwo immisci

bleliqui
di sr equir
ed f orthe separ
ation of
materi
albysol
ventextr
acti
on.Theact iveconstit
uentshouldbeunev enly
sol
ubleinthesystem therebyfaci
l
itati
ngext racti
onoft heconst it
uent
fr
om onephasetotheother.Theeff
iciencyofext r
acti
onisdeterminedby
di
str
ibuti
onco-
eff
ici
ent(D).

Totalwt(gms.)ofsol
uteint
heorgani
cphase
D= -
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
--
-
Totalwt(gms.)ofsol
uteint
heaqueousphase

Ifoneoft hetwoliqui
dscont ainsasol ute,thismet hodi sfoundt obemor e
suitabl
e.Thesystem, i
nt hi
scasei sfi
rstshakenandt henallowedt osettle.
Someoft hesol
ut eist
ransf err
edt otheot herliqui
d.Eachoft heli
quidina
mixt ur
eoft woimmi scibleliquidsoft hiskindi sr eferr
edt oasaphase.
Thus,someoft hesolut esist ransf
erredf rom onephaset otheanot her
phase.Theamountt ransferreddependsont her elat
iveaf fi
nit
yoft he
soluteforeachoft hetwosol vents(r
elati
vesol ubili
ty).Iti
sdet erminedby
D.Gr eatert
hevalueofD, greateristheef f
iciencyofext r
acti
on.
Theimmi sciblesystem mayinv
olvet woorgani
csolvents.Theext r
act
ionfort hissy stem maybe
i
mpai r
edduet of ormati
onofemul sion.Sol
ventextracti
onisacommont echni quei nfor
ensi
c
toxi
cologyrelatedtobiol
ogi
calmatrices.Sol
ventextr
actionmet hodhasnowbeenupgr adedand
madeaut omat i
cv i
z.accel
erat
edsol ventextr
acti
on.I ncaseofsol idnon- biologicalmatri
ces,
conti
nuousext ract
ionbyasoxhletmaybeempl oyedi.
e.continuousextract
ion.

3.
9.1.
2 Di
sti
l
lat
ion:

Theprocessi
nv olvesheati
ngasampl eofliquidtoconvertiti
ntov apour,whichisthenal
lowedt o
fl
owinanotherlocation,
whereitiscooled,condensi
ngitbacki ntoaliquid.Vari
ousmodificat
ions
ofthebasicdistil
l
ationprocessareusedf orspeci
fi
cpur posev iz.steam dist
il
lati
on,f
racti
onal
di
sti
ll
ati
on,di
sti
llat
ionunderreducedpressure,sweepco-disti
l
lati
onetc.

3.
9.1.
2.1St
eam Di
sti
l
lat
ion:

St
eam v
olat
il
esubst
ancescanbesepar
atedori
sol
atedf
rom bl
ood,ur
ineandpr
oper
lymi
nced
vi
scerabyst eam disti
l
lati
on.Steam i
spassedi nt
ot hesampleandt heaqueousdist
il
lateis
col
lectedbycondensat ion.Toxi
cant
sfrom aci
dicdist
il
lat
ionpr
ocessi
ncludeethanol
,methanol,
phenol,halogenated hydrocar
bons,cyani
des,etc.On the ot
herhand,toxi
cantsfrom basic
di
stil
lati
on processincludebasicdrugssuch asamphet ami
ne,met
hadoneand also anil
ine,
pyr
idine,ni
cotineetc.

3.
9.1.
2.2Fr
act
ional
Dist
il
lat
ion:

Thi
si sat y
peofdisti
l
lati
on,whi
chenabl essepar
ati
onofamixtur
eofv ol
ati
l
eliqui
ddi
ff
eri
ng
marginal
lyi
nboil
ingpoint
.A mixtur
eofker oseneoilormi
ner
altur
pent
ineoili
nanoil
-wat
er
emulsionmaybesepar
atedbythi
smet hod.

3.
9.1.
2.3Di
sti
l
lat
ionUnderVacuum:

Thi
sisanothert
ypeofdi
sti
ll
ati
onmethodwhichprovi
dessepar
ati
onoft
her
mal
l
ylabi
l
evol
ati
l
e
compoundsatal
owtemperat
urewi
thoutdecomposi
ti
on.

3.
9.1.
2.4SweepCo-
dist
il
lat
ion:

Thisisaspecialty peofdist
il
lati
onbasedont hepr ef
erenti
alv ol
at i
l
izat
ionofor ganiccompounds
speciall
ypesti
cidesf r
om oil
,li
pidsorpl antext
racts,usingast ream ofi ner tgasandsubsequent
i
solationofvolati
lesoncol dtrapsorsol idadsorbent.Itisapur geandt raptechniqueinvol
vi
ng
dispersi
onofthesampl ei
nthinfilmsondeact i
v at
edglassbeadsorf l
ori
si loraluminaorsil
i
cagel
ortenaxastrappingmedi aatelevatedtemperatures.UniversalTraceResi dueExt r
actor(UNI
TREX)
andAccel er
atedSol ventExtractor(ASE)i .
e.thehi ghl
yaut omat i
cext ractionsystem forrapi
d
extracti
onsofmul ti
plesampleswor kont hi
sprinci
ple.

3.
9.1.
3 Mi
cro-
dif
fusi
on:

Micr
o-dif
fusi
on isa conveni
entand popularmethod thatfacil
it
atestoxi
cant
s( gaseousand
vol
ati
les)inblood,uri
neandgast
ricaspir
atestobeisolated,detect
edanddetermined.Thisi
s
donebyConwayMi cr
o-di
ff
usi
ondi
sh(elabor
atedsepar
atelyforconveni
encei
nsecti
on4. 7.
3).

3.
9.1.
4 Di
aly
sis:

Iti
nvol
vesseparati
on ofa cryst
alloi
df r
om a colloi
d byfil
ter
ing thr
ough a semi-permeabl
e
membrane.Thi
ssepar ati
onmethodmaybeempl oyedforthesepar at
ionoft oxi
ccationsand
ani
onsinacoll
oidalsoluti
onordispersi
onorcoll
oidalmatri
ces,especi
allybiol
ogi
calmat er
ial
s.
Thesepar
ati
onprocessmaybeaccel er
atedbyappl
y i
nge.m.f
.i.
e.elect
ro-
dialy
sis.

3.
9.1.
5 Subl
i
mat
ion:

Thi
sissi mil
artodist
il
lat
ionexceptthesampl ei
sasoli
dtobegi
nwit
handi sconvert
eddi
rect
ly
i
ntov apourandt henbacki ntosolid.Subl
imat
ioni
sappli
cabl
etoisol
ateat oxi
canti
nsoli
d
matr
icesv i
z.napht
hal
ene, ant
hracenewhichsubl
i
mes.

3.
9.1.
6 Di
gest
ionorChemi
cal
Treat
ment
:

Somet imesactiv
econst i
tuents( t
oxicant)are separated on t
reatmentwith aci
d oralkalior
digesti
ononawat erbathormuf f
lefurnaceviz.
,biol
ogicalmatri
cesaredigest
edonawat erbath
for1hourorabov eordi gestedinmuf f
lefurnacewi t
haci d/al kal
i/orchemi cal
st oisol
ate
i
nor ganicmetal
s.Vol at
il
ei norgani
cpoi sonsv iz.phosphine,ar
sineandhy drogensulphi
dear e
i
solatedf r
om t
heirsal
tsont reatmentwi t
hdil
uteacids.
3.
9.1.
7 Mi
crowav
eDi
gest
ion:

Mat r
icesar
edigestedwi thacids/alkal
isinmicrowav eovent ofaci
li
tat
eisolat
ionofinorgani
c
poisoninorgani
cmat r
ices,underaspecif
icanalyti
calcondit
ionofoper at
ionviz.
,operat
ionof
ovenataspecifi
cmi cr
owav eforsometime.Theinter
actionofmicrowavewithmat r
icesr
esult
sin
producti
onofheatwithriseoftemper
atureforwhichdigesti
onoccurs.

3.
9.1.
8 Absor
pti
on:

I
tisasl owpr ocess(comparedtoadsorpti
onatt hesur
face)i
nvol
v i
ngdiff
usi
onofonesubstance
i
ntothei nter
iorofabsor
bentmateri
al.Toxicgasesandv ol
ati
l
esinoilareentr
appedandenr
iched
bythepr ocessusingat ubecontaini
ngdi verseabsorbi
ngmat eri
alsspeci
ficforapart
icul
ar
toxi
cantandon-l i
nedetect
ionanddeterminati
onisfaci
li
tat
ed.

3.
9.1.
9 Chr
omat
ogr
aphy
:

Chromatographyi nv olvesthesepar ationofsubst ancesbasedont hei

rr elati
veaf f
init
yf ortwo
phasesv i
z.st ati
onaryandmobi l
e.Subst anceswhi chhav ehi gheraffi
nit
yf ort hemobi lephases
aremov edorcar riedal ongwithitandar et hussepar at
edf r
om t hosewithhi gheraf fi
niti
esf ort
he
stati
onaryphase.Thus,t hetoxicantsinmol ecularmi xt
uresmaybesepar atedconv enientl
yunder
diff
erentchromat ogr aphicmet hodsandoper ati
ngcondi t
ionsi napar ticularchr omat ography.
Therearedifferentcont rol
li
ngpar ametersv iz.natureoft oxi
cant, mobil
eandst ationaryphaseand
temperatur
e.Sal i
entaspect sofdi ff
erentchr omat ographicmet hodsaregiv enbel ow.

3.
9.1.
9.1Col
umnChr
omat
ogr
aphy
:

Separat i
onofact iveconsti
tuenti sachi
ev edher ebypr eferent i
alabsorpti
onofact iv
econst i
tuents
ontheadsor bentorstationaryphase.Int hismet hodav erticalglasstubeisf i
ll
edwi t
hagr anular
adsorbent .Thi
sadsor bentact sast hest ati
onar yphase.Thesampl eist henaddedt ot hetopof
thecol umni nt heform ofasol uti
oninasui tablesol ventormi xtur
eofsol vents.Thesy st
em of
solventi sthenaddedasamobi l
ephase,whi chi st obesel ectedbeforehand.Ast hesol vent
system f l
owst hroughthecolumnundert hei nfluenceofgr av i
tyorpressure,vari
ouscomponent s
ofthesampl emi xt
urewillmigrat eatdi
ff
er entr atesandt henar ri
veatthelowerendoft hecolumn
atdifferentratesi.e.t
ime.Fr actionscoll
ect edatv ar
iousint erval
swi l
lthuscont ai
nthedi f
ferent
component sseparatedatdifferentint
ervals.

Theef f
iciencyofsepar at
ionint hecol umni sdependentont headsor bentmat eri
al,selectionof
solventsy st
em,nat ureofacti
v econst it
uentaswel lasmobi lephase.Thesecont r
ollingfactor s
havebeenexpl oredt odevel
opdi ff
erentmet hodofchr omat ographyv i
z.HPLC, HPTLC, affi
nity,gel
permeat i
on,ion-exchangeandi onchromat ography,solidphaseext racti
onandsupercr i
ti
calfluid
chromat ography.Thesehav ebeendi scussedsepar atel
y .Theanaly t
icalcondit
ionsr equiredf or
separ at
ionoft oxicantsareeitheravailableinstandardt extsorreferencesormaybedev eloped
conv enientlybyst andardi
zati
ondependi ngonnatureoft oxicantandmat r
ices.

3.
9.1.
9.2PaperChr
omat
ogr
aphy
:

Theseparat
ionofact
iveconsti
tuentoccur
soncel
l
ulosepaperast
hestat
ionar
yphase.Thi
sisthe
pri
miti
vemet hodinchromatographyandusedforseparat
ionofor
ganicdyes,pi
gments,inks,
cat
ionsandanionset
c.

3.
9.1.
9.3Thi
nLay
erChr
omat
ogr
aphy
:

Thesepar
ati
ont
akespl
aceonat
hinl
ayerofadsor
bentmat
eri
alsuchasal
umi
na,si
l
icagelGor
cell
ulosecoatedont oani ner
tsupportmater
ialsuchasglasspl at
e,plast
icoral
umi
nium sheet.
Therearedi f
fer
entmet hodsthatarepopul
arinTLCv i
z.ascending,descendi
ng,t
wodi
mensi onal
etc.Quanti
tati
veseparati
onisachievedbyopt
imizi
nganaly
tical
conditi
ons.

3.
9.1.
9.4Hi
ghPer
for
manceThi
nLay
erChr
omat
ogr
aphy
:

At y
peoft hi
nlayerchromatogr aphywher ei
nt hestati
onaryphaseisdesignedt oofferenhanced
separat
ionandr esoluti
onproper t
ies.Theenhancedsepar ati
onisduet ospecialst
ructuralfeature
ofadsorbentat t
ainedbyspeci alprocessi
ngf orwhichopt i
mum resoluti
onofmol ecularmi xtures
(f
orsepar ati
onbychr omatography )occuronpl at
ecoatedwi t
hspeciall
ypreparedadsor bent.The
system (HPTLC)i snow full
yaut omat i
candmul ti
plesamplesmaybehandl edquickly,precisely
andconv enient
ly.

3.
9.1.
9.5Hi
ghPer
for
manceLi
qui
dChr
omat
ogr
aphy
:

I
ti sbasedont hedi ff
erentat
tract
ionsofnon- v
olatil
eanalytesv i
z.drugs,pest
ici
de,expl
osi
ve,
organi
cset c.betweenal i
qui
dphase(pumpedt hroughacol umn)andasol idphase(packed
withi
nthecol umn- st
ati
onaryphase) .Theoperat
ingpar ametersincl
udescomposi t
ionofmobil
e
phase,adsorbent,natur
eofanalyteetc.f
oropti
mizationsofresolut
ion.

3.
9.1.
9.6I
onChr
omat
ogr
aphy
:

Itworksont hesamepr inci

pleasi nHPTLC.Thecomposi t
ionofst ati
onaryphaseandal sothe
mobilephaseissosel ectedthatcat i
onsandani onscanbesepar atedconveni
ent l
y.Theact iv
e
consti
tuent
sinthematrixmat er
ialarechemi call
ybondedt omol ecul
es,whichhaveaf i
xedchar ge.
Insuit
ableform,ionexchanger esi
nscanbepackedi nt
ocol umnsandusedf orseparationof
molecularmixt
ureswhichhav etheopposi t
echar ges(becauseunl i
kechargesatt
ract)viz.cati
ons
andanions.Insomecases, thenetchar geont hecolumnmat er
ialorsamplemol ecul
esorbot his
dependentonpHgi v
ingrisetogreateranalyti
calflexi
bil
i
ty.

3.
9.1.
9.7GasChr
omat
ogr
aphy
:

Iti
saprocedurewherebyvolati
lecomponent sofami xtur
emaybesepar atedbypar t
it
ionbetween
asoli
dorliqui
dstati
onaryphaseandagaseousmobi l
ephase.Theef f
iciencyintheseparati
onis
achi
evedbycontroll
i
ngsev eralfact
orsv i
z.,columnt ype( capi
ll
aryorwidebor e),col
umnl ength,
col
umndi ameter
,natureofl i
quidphase,car r
iergas,f l
ow rateandtemper at
ure.Thistechni
que
hasnowbeenhy phenatedwithothertechni
quesv iz.SFC-GC, GC-MS.

3.
9.2 Moder
nMet
hodsofExt
ract
ion(
8,9)
:

The met hods i nclude vari

ous met hods i n chr omat ography as wel las speci alext raction
methodologiesoft hepr esentgener ationv i
z.,ion-pairf ormat i
on,solidphaseext racti
on,sol i
d
phasemi croext racti
on,micell
arext ract i
on.Speci altypesofdi gesti
onasameansofi solationof
toxi
cantthroughchemi calprocessingi .e.microwav edi gestionanddi gesti
onbypl asmasour cef or
i
norganicsampl esar ealsoemer gingf orthei
rconsi derationi nrouti
netoxicological analy
sis.Thus,
ther
ear ev ariousmet hodsofext ract i
onundert hehead“Moder nMet hod”whi char eei ther
underuti
li
zedorunexpl oit
edi ntheI ndi anper spectiv
eduet olackofi nfrastructuralfacil
it
iesand
anal
yticalexpertise.Asi thasbecomev er
ydi ff
icultt ocopeupwi t
hmuchi nflow ofexhi bits,
moder nmet hodsar ereplaci
ngt r
aditional met hodsofext racti
onf orspeedyanal ysis.

3.
9.2.
1 HeadSpaceTechni
que(
16)
:

Theheadspacemethodisespeci
all
ysui
tabl
eforthever
yfastsepar
ationofvol
ati
l
ecomponents
(al
cohol
s,acet
one,al
dehydes)i
ncomplexbiol
ogi
calmatri
cesspeci
all
ybloodinmass-
li
quorand
prohibi
ti
on law related cases.This met hod has the adv antage ofav oidi
ng the ri
sk of
contaminati
onofnon- vol
ati
lecomponents,whichmaybeel i
mi natedduetoon- l
i
neanalysisbygas
chromatography.Thepr inci
pleunderl
yingt heheadspaceanal ysisist hati naseal edv ialat
constanttemperature,equil
ibr
ium i
sest abli
shedbetweent hev ol
ati
l
ecomponent sofal i
quid
sampl eandt hegasphaseabov eit(t
heheadspace) .Af
teral lowi
ngt het imef orequil
ibri
um
(normall
y15mi nutesorsof or50ormor esamplesinasingl erun)apor t
ionoft heheadspace
maybewi t
hdrawnonebyonef rom vi
alsusingagas-ti
ghtsyr i
ngeandi njectedtoGCf oron-l
ine
analysi
s.

3.
9.2.
2 Dy
nami
cHeadspace,
Pur
geandTr
apTechni
que:

Thesetechni
quesarebasical
l
ymodi f
iedorhi
gherv
ersi
onofHeadspacemet
hodt
oopt
imi
zet
he
separ
ati
onofv ol
ati
l
esforon-l
ineanal
ysi
sbyGC.

Int hepur geandt r

apmet hod,v ol
ati
lecompounds( toxicants)ar eliberatedfrom t hesampl eby
bubbl ingwit hani nertcar r
iergasandsubsequent lyeithercondensedi nar eceivercooledusual ly
wi t
hsol i
dcar bon-dioxideorl iquidnitrogenadsor bedonacar t r
idgef i
ll
edwi thsol i
dadsor bent
mat er i
alsuchasTenax .Iti spol ymerbaseddi phenyl-p-phenyl
eneoxi deandav ail
ableinv arious
forms.TenaxTA i sahi ghl ypur ifi
edf orm oft hepol ymerandst ableupt o375oC andgi ves
i
nsi gnifi
cantbl eedsofor gani cs.A mat erialsuitablef orther ecov eryofl ow mol ecularwei ght
compoundsi sTenaxGRwhi chcont ains23% gr aphite.Thi sadsor bentissui tableforef fi
cient
trappingofcompoundsofl ow t omedi um pol ar
ityandr ecoveringt hem quant itati
velybyei ther
solv entextract i
onort hermal desor pt
ion.Al ter
nativel
y,car t
ri
dgesf i
ll
edwi t
hactivatedchar coal can
beusedt ot rapt hev olati
l
eswhi char et henext r
actedi nt
oasmal lv olumeofcar bondi sulphide
priort otheanal ysis.Thi stechni quei swi delyusedi nt heanal y
sisofv olati
lecompoundsi nwat er
sampl estil
ldat emai nlybecauseoft hedi ffi
cultyininterpreti
ngt her esultsatconcent rati
onbel ow
thosewhi chcanbemeasur edusi ngor dinaryheadspacemet hod.

Purgeandt r
apt echni
quearesimil
artodynamicheadspacesampl i
ngexceptt hatthegasi s
passedthrought hesampl
e.Cl
earl
yanyappar
atususedf ordy
namicheadspacesamplingcanalso
beusedf orpur gesampli
ngbyusingonappr opr
iatesampli
ngvessel
.Bothdy namicandpur ge
methodsar eav ail
abl
einavari
etyofaut
omaticsy st
ems,toenabl
eseparationofconsti
tuentof
concent
rationintheppb–pptrange.

3.
9.2.
3 Sol
i
dPhaseExt
ract
ion(
SPE)
:

Theol dobser vati

oni nt heext ractionofdr ugsbyadsor pt i
ononsol i
dmat er
ialsv iz.Florisi
l( A
syntheticmagnesi um si li
cate)oract ivatedchar coalandsel ecti
veelutionbysol ventt hereafterhas
givenr iset osol idphaseext ractionmet hod.I nt hismet hod,sili
ceousmat er
ialswi threl
at i
vely
closesi zedist r
ibuti
on( 15t o100)andv arioussi li
cabondedphasesv iz.n-oct
adecy l( C18,ODS) ,
n-octyl( C8),n-hexyl( C6) ,ethyl( C2) ,met hyl( C1),cy anopr opyl(nitr
ile,CN)ami nopr opyl(ami no,
APSet c.)hav ebeenempl oyedasadsor bentwi thmuchgr eateref fi
ciencyofsepar ati
on.The
met hodi sbasedont heuseofsmal ltubesorcar t
ridgesf il
ledwi t
h100- 500mg.ofanappr opriate
adsor bentasst at
edabov e.Ther el ies,howev er,ar ecent l
ydev elopedt ypeofSPEcar t
ri
dgei n
whichv erysmal lparticlesoft headsor bentar eenr i
chedi nawel lofPTFEmi cr
of ibri
l
shav ing
similaref f
ici
encyasi napackedcol umnbutr equirel esspr essuredr op.Thesampl ei sappliedt o
theSPEcar t
ridgeeitherf rom asy ringeorsampl ingmani foldbyappl iedpr essur
eorsuct i
onatt he
l
owerend.Component sar esubsequent lyr ecov eredbysol v entfl
ushing.Themet hodi sat t
ractive
becauseoft hesmal lamount soft hesampl eandmat er
ialsar enecessar yandal somuchgr eater
speedoft hepr ocedurecompar edt ocl assical adsor ptionmet hod.

Analy
te concentr
ati
on mayof ten be achi
eved mor e easi
lywith SPE than wi
thliqui
d-l
iquid
ext
racti
onasuseofSPEcol umnt oconcentrateananal yt
ef r
om solv
entextr
actmaypr ovi
dea
qui
ckerandpossi bl
ysaferal
ternati
vetosolventevaporati
on.Amaj oradvant
ageofSPEi st hat
bat
chpr ocessi
ngcanbesimplif
ied.Fur
therf
eaturewhenscr eeni
ngforunknownisthatarangeof
anal
ytecanbeext r
actedsi
multaneousl
yalthought
hismaycreat
eprobl
em i
fanal
yteofasingl
e
componentisrequi
red.Moreov
er,SPEcolumnsareexpensi
veandi
tmaynotbepossi
blet
oretai
n
ver
ywater-
solubl
eanalyt
e.TheSPEpr ot
ocolisnowavai
labl
e.

SolidPhaseExt raction( SPE)cartri

dgesar eusedpr i
mar i
lyt ocl eanupsampl esf or
analysisand/ orconcent r
atesampl est oi mpr ovedet ectionl imits.Thel ackofsuf f
icient
sampl epr eparationwi l
lresultinpoordet ecti
onl imits,i dent ifi
cationandquant itation
errors,cont aminat i
on pr oblems and r api d deterioration of GC orHPLC col umn
performance.SPEt echniquesusual l
ypr ovidebet t
ersampl ecl eanupandr ecover
iest han
l
iquid-li
quid extraction techniques.SPE uses smal lv olumes ofcommon sol vent s,
requiresv erysimpl el aboratoryskil
ls,doesnotr equiret heuseofhi ghlyspeci alized
l
abor atoryequipment sandal lowsr apidsampl et hroughput .A l i
quidsampl eorsol i
d
sampl edissolvedi nasol ventispour edi ntot hecondi tionedSPEcar t
ri
dge.Vacuum or
pressureisusedt of orcethesampl et hrought hesor benti nthecar t
ridge.InSPE, vacuum
mani fol
di snor mal l
yusedt osi multaneousl yprocessmul tiplecar tri
dges.Usual l
y,SPE
met hodsar edesi gned t oretaintheanal y t
esofi nter est ;ot hersampl ecomponent s
similartotheanal y
tesal sowi l
lberetained.Theanal y t
esofi nterestaret henelutedf rom
thesor bentusi nganot hersolvent.Thissol venti
scol lect edf oranal ysisf oradditional
processing.

SPECar
tri
dges
AnSPEcar tr
idgeiscomposedoft
hreebasi
cpar
ts:
1)Cartri
dgeortubebody
2)Frit
s
3)Phaseorsor bent

Car
tri
dgeorTubeBody
The cart
ri
dge body usual
l
yis a sy
ringe l
i
ke bar
relmade of ser
ologi
calgr
ade
pol
ypropy
lene.

Fr
it
s
Thef ri
tsar eusedtohol dthesor bentinthebarrelandtoactasapar t
iculatefil
ter
.
PhaseorSor bent
ThemostcommonSPEphasesar ebondedsi l
ica-
basedmat er
ials.Irr
egul arshaped,40
µm si l
icapar t
icl
eswi t
h60Åpor esar eusedast hestar
tingmateri
al.
Varioussi lanesareusedt oattachf uncti
onalgroupstot heaccessibl
ear easoft hesili
ca
parti
cle.I n additi
on,sev eralnon- sil
i
ca based phases are commonl yused.Sol v
ent
reservoirscanbeusedt oincr
easet hev ol
umeofbar r
elabovethephase.Lar geamount s
ofsampl eorsolvent( upto75ml )canbeaddeddi r
ectlytoSPEcar tr
idgesi nonev ol
ume
i
nsteadofi nsmallincrements.Coupl i
ngfitt
ingsareusedt oattacht her eservoi
rstothe
SPEcar t
ridges.

Phases.

Therearet hreetypesofphases:normal
, r
everseandi
on-exchange.
Nor
mal
Phase
Table2listscommonnor malphasesor bents.Al
loft
hesephasesar epol
arandareused
toretai
n( extract
)polaranal
ytes.Fornormalphasesorbent
s,solventst
rengt
hincr
eases
asthesolv entbecomesmor epolar.

Forexampl e,aretai
nedanal yt
ewi l
lcompl etel
yelut
ef r
om anor malphasesorbenti
na
smal l
ervolumeofmet hanolthanchloroform.Alloft
hesolventsinTabl
e1ar ecommonl y
usedwi thnormalphasesor bents.Mixturesoftwosolventsoftenareusedtorefi
nethe
solventstr
engthforopti
mal samplecleanupandanal yt
erecovery.
Rev
ersePhase
Allofthesephasesar enon-polarandwi llbeusedtoretai
n( ext
ract
)non-pol
aranal
ytes.
 Forr eversephasesor bent s,thesol ventst r
engthr elationshipist heopposi tefrom nor mal
phasesor bents( Table2) .Forr ev er
sephasesor bent s,solventst rengthincreasesast he
solventbecomesmor enon- polar.Forexampl e,aret ainedanal ytecompl etelyelut
esf rom
thesor benti nasmal lerv olumeofacet onit
ri
lethanwat er.Inmostcases,t hesol v ents
used wi t
hr everse phase sor bentsar el i
mited to wat er,met hanol,isopropanoland
acetonit r
il
e as ment ioned i n Tabl e1( Reverse Phase) .On occasi on,acet one or
dichloromet hanemaybeusedasanel utionsolventf orhighl yr
et ainedanal yt
es.
I
onExchangePhase
Ionexchangephasesar emor edependentonpH, ionicst rengthandcount erionst rengt h
thansol v
entst r
engt h.Ionexchangephasesdependoni oni cinteracti
onsast hepr imar y
retenti
onmechani sm.I onici nteractionsoccurbet weenananal ytemol eculecarryinga
positiv
eornegat iv
echar geandasor bentcar r
yinganopposi t
echar ge.Ther earet wo
groupsofi onexchangephases.Thecat i
onexchangephasesr etainposi t
ivel
ychar gedor
“cati
oni c”compounds.Ami nesandcar boxyli
caci dar enotchar gedspeci es.Theycanbe
chargedbyv ar
yingpH.Theani onexchangephasesr et ai
nnegat ivelychargedor“ ani onic”
compounds.Tabl e3l istst hecl assi
ficationsandchar acteri
sti
csf orseveralcommoni on
exchangephaseschar ge).

Tabl
e1:
Solventst rengths
NORMALPHASE Weak REVERSEPHASE
Hexane Wat er
Isooct ane Met hanol
Toluene I
sopr opy l
alcohol
Chlor ofor
m Acet onitr
il
e
Met hy l
enechlori
de Acet one
Tetrahy drofur
an Ethyl acetate
Ethylether Ethylether
Ethyl acetate Tetrahy drofur
an
Acet one Met hy l
enechlori
de
Acet onitr
il
e Chlor ofor
m
Isopropy l
alcohol Toluene
Methanol STRONG I
sooct ane
Wat er Hexane

Tabl
e2:
Sor
bent
s
NORMALPHASE
Cy ano( CN)*
Diol (
DIOL)
Sil
ica( SI)
Ami no( NH3)+1**
REVERSEPHASEOct adecy
l(C18ORODS)
Oct yl(
C8)
Met hyl(C3)
Pheny l(Ph)
I
ON EXCHANGE PHASE BENZENE SULFONYLPROPYL
(scx)
Quat ernaryamine(SAX)

*
maybeusedasarev
ersephaseal
so
*
*maybeusedasani
onexchangeal
so
 Rel
ati
vecount
eri
onexchange.

Table3:I
onExchangephases
CATI
ONS ANIONS
Li+1H+1 0.
5 OH- 1,F-1,Propionat
e 0.
1
Na+1 1.
5 Acet ate,
For mate 0.
2
(NH4)
+1 2.
0 (HPO42- )
,(HCO3–1) 0.
4
Mn+2,K+1,
Mg+2,Fe+2,
+ 2. 5 CI–1, (
NO2) -1 1.
0
3
Zn+2,
Co+2,Cu+1,
Cd+2 3.
0 (HSO3) -
1,CN-1 1.
5
Ca+2 4.
5 (NO2) -1 4.
0
Cu+2 6.
0 (CIO3) –1 4.
5
pb+1,
Ag+1 8.
5 (HSO4) -
1 5.
0
Ba+1 10.0 Cit
rat e 9.
5
Benzenesul fonate 10.
0

Foreachcategor
y,thehi
ghestsel
ect
ivi
tycount
eri
onswer
enor
mal
i
zedt
o10;t
hus,t
he
val
uesarerel
ati
ve.

3.
9.2.
4Sol
i
dPhaseMi
croExt
ract
ion(
SPME)
:

Soli
d phase micro extraction (SPME)i s an ext
racti
on techni
que f oror gani
c compounds i n
aqueoussamplesi nwhi chanalytesar eadsorbeddirect
lyfr
om thesampl eontoaf usedsi
li
caf i
ber
thati
scoatedwi t
hanappr opri
atest ati
onaryphase.Whent hef i
berisi nsert
edint hesample,the
analy
teport
ionfrom t hesampl emat r
ixentersthestati
onaryphaseunt ilequi
li
bri
um isreached.
Thef i
beri
ntheni nsertedint othei nj
ecti
onpor tofagaschr omat ograph( GC)wher eiti
sheat ed
andtheanalyt
esar erapidlydesor bedthermall
yintoacapill
aryGCcol umnf oranal
ysis.

3.
9.2.
5 SuperCr
it
ical
Flui
dExt
ract
ion(
SFC)
:

Gasesabov etheircr
it
icalpressureandt emper at
ureareinasuper cri
ti
calst ate,int
ermedi
ate
betweent hatofagasandl i
quid.Super
cri
ti
calflui
dshavestrongextract
ionpr opert
iesbecausethe
sol
ubili
tyofcompoundsi nflui
disclosetothatofat r
uesolventandmuchl owerv i
scosit
yall
owsit
topercolatethroughpackedbedofsampl e.Thus,notonlythereisanef f
icientcontactbetween
theextracti
ngf l
uidandthesampl ebutthefluidiseasi
lyremov edwheni tisreleasedfrom i
ts
supercr
iti
calstate.

Carbondioxi
deisnearl
yal
way sthechosengasf
orSFCi
nviewofitsi
nnocuousnat
ureandmil
d
cri
ti
calcondi
ti
onnamelycri
ti
calpressur
eof75barandacr
iti
calt
emperat
ureof310Cwhichar
e
rel
ati
vel
yeasytoachiev
eatpresent
.

Thesampl ehol deriscomposedofanumberofsmal lst ai

nlesssteelcartri
dges,whi charef i
lled
withthesampl ei napar t
icularstate.Solidsampl esuchassoi lorsedimentar epackedi ntot he
cart
ridgeswi t
houtanypr etreatmentandaqueoussampl escanbef l
ashedt hrought hecartri
dges
fi
ll
edwi thanappr opri
ateadsor bentt oconcent ratealloft hecont aminates.Thecar t
ri
dgesar e
subsequent l
yf edi ntotheext racti
onov enandt hecar r
iergasl i
newhi chatthisstageconsi stsof
supercri
ticalcar bondi oxide.Theext r
actedcompoundsar ecarri
edtot hecoldtrapandcondensed
aft
ert heheat edconst ri
ctionwhi chr estorecar bondioxi det oitstruegaseousst ate.Aftert he
appropriateext racti
onper i
od,t heci r
culati
onofcool antceasesandt rapi srapidlyheatedt o
vaporizet hecomponent s.Att hesamet imet hecol umnt emper atur
ei sprogrammedaccor dingt o
therequi redcondi ti
on.Theadj ustablesplitparti
tionsthesampl esizet oav oi
dt hepossibili
tyof
overl
oadi ngef fect.SFCi ssui tabl
ef orextr
actionofpest icidetracesinsoli
dandaqueoussampl es.
3.
9.2.
6 Mi
cel
l
arExt
ract
ion:

Micel l
arext ract i
oni saspeci alty peofext ractionpr ocedur et hatappear st obeuni quei nt he
separ ationofdr ugs, plantpoi sonsandpest i
cidesi nbi ologicalmat rices( viscer a).Int heext raction
ofact ive const ituents as abov e,mi cell
arenv ironmentofsur f
act antofdi f
fer entcl asses i s
empl oy ed.Sur fact antorsur faceact iveagent satapar ti
cularconcent r
ationi nsol utionknownas
cri
ti
calmi cell
arconcent rati
on( CMC)f orm mi celleorassoci ationcol loid.Att hisconcent rationor
abov e,mar kedchangesi nt hepr oper t
iesv iz.viscosi ty,conduct ance,el ect ri
calconduct ancear e
exhibited.Sur fact antinsol utional soact satt hei nter faceoft wophasesy st em ofoi landwat eror
organi csol ventandwat err esul tingsol ubil
isationofonephasei ntot heot her .Anemul si onor
micr o-emul sioni sf ormedbyt hepr ocess.Theemul sionmaybedest abilizedbyi ncreasi ngt he
i
onicconcent rationofaddi tiv esi ncl udingsur fact ant.Bi ol
ogi calmat ri
ces( Viscer a)cont ainsf ats,
degr adedpr otei nandcol our ingmat t
eret c.makest heext ract i
onofact iv
econst ituentdi fficul t
.In
the sol ventext ract i
on pr ocess,i fsur factanti s added t ot he ext ract ant( organic sol vent),
depr oteinizationandal sosol ubi l
isat ionwi tht hef ormat ionofemul sionwi lloccurduet omi cell
ar
i
nter action.Theemul siont husf ormedi sduet osol ubi l
isati
onoff ati nbiol ogicalmat ricesi nt he
addedsol vent( cont ainingt racesofwat er )
.Si mul taneousf ormat i
onofemul si
onoccur sduet o
micel l
ari nteract ions.Theemul si
ont husf ormedduet osol ubili
sat i
oni sdest abi l
i
zedoni ncr easi ng
theconcent r
at ionofsur factanti nt hesy stem.Asar esul t,fatsar esepar atedassemi solidmat erial
duet olower ingofzet apot ent ialbet weent heel ect ricaldoubl elay ersoft hecol l
oi dalsy st em.As
proteinandf at sar esepar atedout ,thesuper nat antl i
quidcont ainingact iveconst i
tuentmaybe
extracted f orpoi sonbyor gani csol v
ent s.Thedet ailed anal yticalcondi tionshav eal so been
present edatappr opriatepl acesi nt hismanual .

3.
9.2.
7 Mi
crowav
eAccel
erat
edReact
ionSy
stem:

Themet hodofext r
actionisusedf orisolatingpest icidesinbi ol
ogicalmat er
ialsespeci all
yinl i
ver
andki dney s.Int hispr ocess,t hesampl eissubj ect edt or apidheat i
ngwi t
hor gani csol ventby
microwav esatel evatedpr essur er esulti
ngi sol
ationofact iveconst i
tuent.Thebi ologi calmat eri
al
(5–10gms. )isplacedi nsideami crowav etranspar entv esselwithapol arsolventori onicsoluti
on
(usuall
yanaci d)andi ssubj ectedt or apidheat ingbymi crowav ei nami crowav eaccel erat
ed
reacti
onsy st em (digester).Theanal yti
calcondi t
ions( temp.,ti
meofdi gesti
on, pressur e)mayv ary
dependingonact iveconstituentandnat ureofsampl ev i
z.monocr otophosandphosphami donar e
successf ullyext ractedwi thi
n15- 20mi nutesf r
om v iscerausingdi chloromethaneasasol ventat
80-1000Cand100Psi .Howev er,opt i
mi zati
onofanal yti
calconditi
onst ocov ersdi fferentclasses
ofpest i
cidesar erequiredf orar apidext racti
onbyt hismet hod.Themet hodf indsappl icati
oni n
thedigest ionofbi ol
ogical mater i
al sforisolati
onofsomet oxicmet al
s( Cu,As,Pbet c.).

3.
9.2.
8 Uni
ver
sal
TraceResi
dueExt
ract
ion:

I
t’sasy stem t
hathasbeendev el
opedf ortherecoveryofpest ici
desandor gani
cr esi
duef r
om a
wider angeofsampl esi ncludingbiologicalmateri
als.I
tisbasedont heprinci
pleofsweepco-
disti
ll
ationthatrel
iesonpr eferenti
alv ol
atil
i
zati
onofpest ici
desorot heror ganicchemicalsfrom
biologicalmateri
als,li
pids,plantextractsusingastream ofi nertgasandsubsequenti solati
onof
volati
lesoncol dtrapsofsol idadsorbent s.I
tisapurgeandt raptechniqueinvolvi
ngdispersi
onof
thesampl eint
hinf i
l
msondeact iv
atedgl assbeadsatelevatedtemper atures.

Theext ractorsy st
em isspecif
icallydesignedt or ecov ervol
atil
e,thermallystabl
eor ganochl oro
and or ganophosphor ous pesticides from l ipi
ds,meat ,butter,v i
scera etc.Atpr esent ,t he
dist
il
lati
ont ubedoesnotcont ai
ngl assbeadsorgl asswoolasi trenderslessrecovery.Flor i
silin
conjunctionwi thsodium sul
phat ehasbeenf oundsat isfact
oryfortrappi
ngmanydi f
ferentcl asses
ofvolatil
eor ganiccompoundsAl umi na,Sil
icagelandTenaxar emat eri
alsthathavepot ent ialfor
useast r appingmedi awit
hadv antagesov erFlorisilinspecifi
cappl i
cations.Themet hodi s
expectedt of ailforther
mall
yl abilepesticides.Theconsumpt i
onofsol ventismi nimum.The
methodrequir
esopti
mizat
ionofanal
yti
calcondi
ti
onsbef
orei
tsappl
i
cat
iont
obi
ologi
calsampl
es
(
viscer
a)infor
ensi
ccases.

3.
9.2.
9 Accel
erat
edSol
ventExt
ract
ion:

Thenameofmet hodsigni f
iesmul ti
plesampl ehandl i
ngi nav eryshor ttimebyav eryupdat ed
extr
act i
onsy stem whichal sowor ksont hesamepr i
ncipleasUni versalTraceResi dueExt ractori.
e.
SweepCo- di
still
ati
on.Int hismet hod,acommonl yusedsol v
enti spumpedi ntoanext ractioncell
containingthesampl ewhi chi st henbr oughtt oanel evatedtemper at
ureandpr essure.Mi nut
es
l
ater,theext ractist r
ansf erredf rom theheat edcel ltoast andar dcollectionv ialforcl eanup
analysis.Theent ir
eextract i
onpr ocessi sf ull
yautomat edandper formedi nmi nutesf orf astand
easyext ract
ionofmul t
iplesampl eswi thav er
ymi ni
mum sol ventconsumpt i
on.Thest andar dor
opti
mum anal yti
calconditionsar et obear r
ivedforit
sappl i
cationtobi ological matricesinf orensi
c
toxi
cologicalwor kcoveringabr oadspect rum ofpesticides.Howev er,t
hemet hodhasbeenf ound
tobeef f
ecti
vef orsoi
lsampl es.

3.
9.2.
10Si
zeExcl
usi
onChr
omat
ogr
aphy
:

Thetechni
quecanbeusedt oadvantageasapr epar
ati
ont echni
quef orthepri
orfr
act
ionat
ionof
oil
s,f
ats,envi
ronment
alsamplesetc.intodiscr
etemol ecularweightfract
ionsandasawayof
removi
ngv er
yhighmolecul
arwei
ghtmat eri
alfr
om compl exsample.

3.
9.2.
11I
on-
Pai
rExt
ract
ion:

Thismet hodisappl i
cablef ort heextracti
onofhi ghlywat er-
sol ubleor ganiccompound.These
compoundscannotbeext r
act edfrom sampl esbydi rectsolv entext raction.Thedi f
fi
cult
yhas
beenov ercomebyf ormi ngionpai rwithasuitabler eagentv iz.quat ernaryammoni um sal t
sasi on-
pair
ingagent .Thei on-pairf
or mationisanat tr
actionbet weenaposi t
iveandanegat i
vechar ge.
Oncet hi
spai ri
sf or
medi twillactexactl
ylikeanor ganicmol ecul eandnotl ikeasi oni ccompound
atall.Theext racti
onmaybecar ri
edoutt hereaft erbydi rectsol v entext ract
ionpr efer
ablyi n
presenceofabuf fer.Themet hodisal soappl i
cabl ef orext racti
onofdr ugsi nt hef orm of
quaternarysalt
sinur i
neorbl ood.Thebi ologicalmat ri
cesaredepr oteinizedandt reat edwi t
hady e
(sel
ective)inpresenceofbuf fertoform drug-dyecompl exwhi chi sext ractedbyor ganicsolvent.
Thedy eisdestroyedbyt heact i
onofacidoral kaliandt hedr ugi sisolatedf oranaly si
s.

3.
9.2.
12Hy
phenat
edTechni
ques(
HPLC-
GCANDSFC-
GC)(
10)
:

The hy phenated techniques v i

z.HPLC- GC and SFC-GC al low compl ex sampl es to be pr e-
fracti
onatedrapidlyaccordingt omol ecularweightorchemi calclassi
fi
cationonasui t
ableHPLC
pre-col
umnorSFCandappr opri
atefractionsthenseparatedbyonl i
necapi l
l
ar yGC.Thecoupl ing
oft wot echniqueshasbeenmadebyaby passv al
vef i
ttedwi t
hasampl eloopofappr opr
iat e
volume.Sel ectedfract
ionsar epassedt othecapi l
l
arycol umnv iaal arger etent
iongapwhi ch
util
izesthepr ocessofconcur r
entsol ventevaporati
ontor etainthefracti
oni nt heret
enti
ongap
thusav oidi
nganypr el
iminarycont actwi t
hcolumn.

3.
9.3 Ext
ract
ionofVol
ati
l
ePoi
sonsbyDi
sti
l
lat
ion:

Themet hodisappli
cabl
eforv
olat
il
eor
gani
cori
nor
gani
cpoi
sonunderdi
ff
erentcondi
ti
onsofpH
i
.e.aci
dicandalkal
i
ne.

3.
9.3.
1 Neut
ral
andAci
dDi
sti
l
lat
ion:

Pr
ocedur
e:

50-
100gms.ofv
iscer
a(pr
oper
lymi
nced)
,st
omachcont
ent
s,v
omi
torot
hermat
eri
alsar
e
tobeexami nedsepar ately.Theyshoul dbebr oughtt otheconsi stencyofat hi
ngr uelbyaddi ng3- 5
ti
mesofdi sti
ll
edwat erandaci dif
iedwi t
ht ar t
ari
corsul phur i
caci dandsubmi ttedt ost eam
disti
ll
ati
on.Thecondenserandt hereceiv i
ngf laskshouldbewel lcooledwi thiceespeci all
ydur ing
thehotseason,t heout l
etoft hecondenserbei ngdippedi nal ittl
ewat erorNaOHsol ut
ionorany
otherreagentasnecessar y
.Af ew piecesofpumi cest onemaybet akeni nt heflaskt opr event
bumpi ng.Itisbettert ocol l
ectthedi sti
ll
atein4or5f r
act i
ons,ofwhi cht hef i
rstoneshoul dnot
exceed20ml .andt her emaini
ngf r
act i
onsshoul dbe50ml .each.Thef l
askcont aini
ngt he
mat er
ialshoul d preferablybe heat ed on t he wat erbat h.I fphosphor ous i s suspect ed,t he
disti
ll
ati
onshoul dbecar r
iedoutinadar kr oom andabl ackscr eenispl acedbet weent hebur ner
andcondensersot hatphosphor escencemaybeseencl early.Thedi still
ateasst atedabov e
containsalcohols,par aldehyde,otheral dehydes,acet one,car bolicacidandphenol icderivati
ves,
carbondi sulphi
de,thy mol ,camphor ,turpentine,nitr
oglycerine,benzene,ot herv olati
leacidset c.
Forcy anidesandcar boli
caci d,thedi sti
ll
atei scoll
ectedi n10ml .of0. 1N sodi um hy droxide
soluti
on.

3.
9.3.
2 Al
kal
i
neDi
sti
l
lat
ion:

Afterthecompl eti
onoft heaciddisti
ll
ati
on,thef l
askisall
owedt ocoolandi tscont
ent
sar e
renderedal
kali
nebyaddi ngNaOHsol ut
ion.Thealkali
nemixt
ureist hendisti
ll
edagai
nint
hesame
wayasbef oreandt hedisti
l
lat
ecoll
ectedintwof racti
ons–thefirstfracti
onofabout20ml.and
thesecondf r
actionofabout50ml .Thedi sti
ll
atefrom t
healkal
inemi xt
uremaycontai
nanil
ine,
pyridi
ne,ni
cot
ine,coni
ine,ammoniaandv olat
il
ebases.

3.
9.4 Ext
ract
ionofToxi
cMet
alsi
nMat
ri
ces:

3.
9.4.
1 Non-
Biol
ogi
cal
Mat
ri
ces:

Thenon-biol
ogi
calmatr
icessuchastabl
ets,powder
smaybesubj ect
edt
ochemi
calanal
ysi
sby
prepar
ingsol
uti
onofsamplesandthei
rsy
stematicgr
oupsepar
ati
on.

3.
9.4.
2 Bi
ologi
cal
Mat
ri
ces:

Theext
ract
ionofmet
alsi
nbi
ologi
cal
mat
ri
cesmaybecar
ri
edoutbyt
hef
oll
owi
ngmet
hods.

Dr
yAshi
ngMet
hod;

WetDi
gest
ionorAci
dDi
gest
ionMet
hod;

Fr
eseni
usandBaboMet
hod;

Sel
ect
iveChemi
cal
Treat
ment
.

Theorgani
cmatterswhi
chconst
it
utethebul
kporti
onar
edestroy
edbychemicalmeanst
ogett
he
act
iveconst
it
uents(met
ali
ons)f
reecomplet
elyf
orqual
i
tat
iveandquant
it
ati
veanal
ysi
s.

3.
9.4.
2.1Dr
yAshi
ngMet
hod:

About10- 50gm.oftissueorot herbiologicalmat eri

alsistakeni nasi l
icacrucibleandheat edina
Bunsenbur nerforremov ingthemoi stureandpar ti
all
ydest royi
ngt heorganicmat eri
al.Then,t he
cruci
bleiskeptinamuf flefurnace.Thet emper atureofthef ur
nacei srai
sedupt o550 Candat
thi
st emperatur
e,theincinerat
ionoft heor ganicmat t
erisper formedbykeepi ngt hesi l
icacr ucibl
e
foronehour .Aft
erincinerat
ionincompl ete,t
hecr ucibl
eist akenout .Thecol ouroft heresi dueis
tobenot edaswhenhotbecausei npresenceofzi ncther esidueassumesy ellowcol ourwhi l
ein
presenceofcoppert hecol ouroft heresiduei ssomewhatbl ui
shgr een.Ther esidueint hesi li
ca
basinisboiledwith10ml .of4Nhy drochloricacidandt henf i
lt
ered.Thecl earaci di
csol utionis
test
edformetal
li
cpoisonssuchascopper
,bi
smuth,
zinc,
bari
um et
c.byperforminggener
algr
oup
anal
ysi
susingsemi-
mi cr
omet hods,
chr
omatogr
aphi
candinst
rumental
techniques.

3.
9.4.
2.2WetDi
gest
ionMet
hod:

Procedur e:
About50gms.ofbi ologi
calmateri
alor10ml .ofbl oodi stakenintoal argeKjeldahlflask
and20t o40ml .ofConc.HNO3i saddedt ocovert
hemat er
ialandf l
askisgent l
yheatedi nasmal l
fl
amewhent hemassbegi nst oli
quefy.Theheat i
ngi scont inuedunt i
lthel i
quefi
cat i
onoft he
mat eri
aliscompl eteandt hatmustbedonei nthepr esenceofcopi ousbrownf umesofni tr
ogen
dioxideint hef lask.Att hi
sst ageabout20–30ml .ofConc.H2SO4i saddedandt hef l
aski s
heatedst ronglyov erawi r
egaugeandConc.HNO3i saddedi ndrops( byusingdroppingf unnel)to
thecont entsoft heflaskatt herat
eofabout10dr opspermi nutesot hattheat mospher eint he
fl
askmustatnot i
mesbef reefr
om brownf umes.Heat ingiscont i
nuedunt ilal
lorganicmat teris
destroyedandt heliquidbecomescl earandcolour
lessorst rawcol oured.

Tof indouti ft heoxidationiscompl ete,thef laskisheat edwi thoutaddi nganyHNO3.I fthereis

anyun- bur
ntor ganicmat ter
,theliquidbegi nst odar kenandi ft hedigest i
oni scompl et e,no
darkeningtakespl aceandt hewhitefumesofSO3ar egivenof f.Int heformercase, theadditionof
HNO3andheat i
ngar econt i
nuedfurthertilltheor gani cmat teriscompl etelyoxi di
zed.Heat ingis
continuedf or15mi nutesmor etoexpelt heni tri
caci dcompl etely.Then,af tercool ing,25ml .of
saturatedammoni um oxalatesol
utioni sadded.Thel i
quidisboi l
edunt ilSO3f umesappear .This
ensur escompl eteremov alofHNO3.I tist hencool ed,dilut
edwi thanequalv ol umeofwat erand
carefull
yt r
ansf erredtoabeaker .Thebeakeri sheat edonahotpl at
eorsandbat ht oexpelt he
excessH2SO4.Thesol uti
oniscooledanddi lutedwi thwat eri nsuchawayt hatt hest r
engt hof
acidi sabout 10%.Att hisstageapr ecipi
tatemaybef ormedwhi chcont ainst hei nsolublesaltsof
l
ead,bi smut h,tin,bari
um,st r
onti
um orsi lveret c.Thepr eci
pitatei sf i
lt
eredof fandt est
edf orthe
met alsment ionedabov e.Thefil
tratewi llnow cont ai
nal lot hermet alsexceptmer cury.Itis
subjectedtosy stematicgroupanalysisandquant i
tativedetermi nationther eafterasandr equired.

3.
9.4.
2.3Fr
eseni
um andBaboMet
hod(
forMer
cur
y):

Thenitri
c-sul
phur i
caci d(wetdi
gesti
on)methodofdestr
uct
ionofor gani
cmatt
erisnotatall
suit
abl
ef ormer cury,whichi
salmostcomplet
elyl
ostbyv
olat
il
izat
ion.Thi
smet
hodisconsi
dered
mostsuitableforliberat
ionofmer
curyalt
houghther
eisapossibi
l
ityofsomel
ossofmercur
yby
vapori
sati
on.

Pr
ocedur
e:

Adef i
nit
eamountofbi ologi
calmat eri
alv i
z.20-
25gms.ofv i
scer aor5- 10ml .ofbl oodist akenina
fl
askfit
tedwi thar efluxcondenser.Inthecaseofv isceraorot hersolidmat erialsuffi
cientwat eris
addedt omakegr uell i
keconsist
ency .Onet hi
rdofi tsvolumeofchemi cal
lypur ehy drochloricacid
andaf ew gms.ofsol idKC1O3ar eadded.Thecont entar emi xedbyshaki ng.Themi xtur
ei s
heatedov erawi r
egaugeonabur nerf l
ameoronaboi l
ingwat erbath.Smal lamountofKC1O3i s
addedt i
met oti
meandt hefl
aski sshaken.Chl orinegasev olv
es.Theheat i
ngi scont inuedunt i
l
thecont entsoft hef l
askbecomesauni for
m,st raw colour edliquidf r
eef rom or ganicmat ter
exceptsomef att
ysubst ancesinsuspensi onwhi chcannotbeoxi dized.Ifheat i
ngf oranhour
aft
erthel astaddit i
onofKC1O3pr oducesnodar keningoft hemi xture,theoxi dat i
onofor ganic
mattermaybet akenascompl et
ed.Ittakes4- 6hour stoat t
aint hestage.Itisf il
teredandwashed
withwat er.Thef i
ltrateandwashi ngsar ecollected.Suf f
icientsodium sul phi t
eorbi sulphit
ei s
addedt oreducetheexcessofchl ori
nei ntohydrochloricacid.Thel i
qui di
swar medonwat erbath
andacur rentofairispassedt oexpel t
heexcessSO2.Thesol ut
ionisnowr eadyf oranal ysis.
3.
9.4.
2.4Sel
ect
iveChemi
cal
Treat
ment
:

Af ew t oxi
cmet alsv i
z.arsenicand anti
monyi ntheirspeci
fi
coxi dati
onst at
e( +5)maybe
subjectedtot her educti
onpr ocessbynascenthydr
ogen( bythereactionbetweenzi ncanddi l
sulphuricacid)forisolati
onofmet alsinmatri
cesviz.bur
ntbones,nai l
,hairandnon- bi
ological
mat r
icesl i
kef ood preparati
on,drinks,t
ea,coff
eeet c.i
nt heform oft heirvolat
il
ehy drides
(AsH3andSbH3) .Thepr ocessmaybecar ri
edoutbyGutzei
torMarsh-Berzel
iusmethod.

Incaseofpr esenceofAsandSbi nthei

rhi
gheroxidationstate(+5),r
educti
ont o+3stateistobe
car r
ied outpriorto chemicaltreat
ment.The met hod and anal yt
ical
s have been presented
separ at
ely.Theanal y
stshould notbebi ased byposi ti
vef i
ndingsast her
ear echancesof
i
nt erfer
enceduet othepresenceofphosphorous,sulphi
de,AsorSbi nthemat r
ixit
self
.Abl ank
testshouldinvari
abl
ybecar r
iedoutasAsorSbmaybepr esentinmat ri
cesorthereagent.

3.
9.5 Ext
ract
ionofToxi
cAni
onsi
nFor
ensi
cMat
ri
ces:

Theext r
acti
onsoftoxi
canionsinnon-bi
ologicalmatr
icesrequir
ev eryminorpr
ocessingandcl
ean
up.Inconveni
encesisf
elti
ncaseofbiologicalmatr
icesv i
z.vi
scera,stomachandgastri
ccont
ents,
stomachwash, uri
neandblood.Theextracti
onproceduresincl
udet hefol
lowi
ngmet hod.

Pr
otei
nPr
eci
pit
ati
on.

Di
aly
sis.

Sel
ect
iveChemi
cal
Treat
ment
.

Mi
croDi
ff
usi
on.

I
onChr
omat
ogr
aphy

3.
9.5.
1 Pr
otei
nPr
eci
pit
ati
on:

Asl urr
yoft hesampl e(mincedviscera10- 20gm.ofst omachcont ent
sorgast ricl
av ageetc.is
prepared.Thepr otei
nint hesamplei scoagulat
edbyaddi ng0.5-1.0gm.ofammoni um sulphate.I
t
i
sf il
teredthrougha2cm.l ayerofcot tonwoolheldint hebar relofthesy ri
nge.Theexcessof
ammoni um sulphateremaini
ngint hef i
l
trat
eispreci
pit
atedbymet hanol.Thesupernatantli
quidis
coll
ectedandev aporatedtoasmal lvol
umeonawat erbat h.Theconcent rat
edliqui
disr eadyfor
analysi
sofani ons.

3.
9.5.
2 Di
aly
sis:

10gmsoft het
issueiscutintosmallpiecesandpl acedinacellophanemembr anemadei nt
othe
shapeofabag.Thebagi sthenslowlyrotatedi nabeakercontaining100mlofdi sti
l
ledwaterby
meansofanel ect
ri
calmot orormechani caldev i
ce.Dial
ysi
soccur srapi
dly
.Afteronehour ,t
he
waterint
hebeakeri srepl
acedbyf reshwat erandt hebagisrotatedforfurt
herhalfanhour.The
wateristhentakenout ,mi
xedwi ththepr eviousfract
ionandev apor
atedtoasmal lv
olumeon
waterbat
h.Thisisfi
lt
ered,i
fnecessaryandt estedfortoxi
canions.

3.
9.5.
3 Sel
ect
iveChemi
cal
Treat
ment
:

Phosphidesandsul
phi
despresenti
nbiologi
calmat
ri
cesv
iz.Vi
scer
a,stomachcontentandgastri
c
l
avagei ssubj
ect
edtotr
eatmentwit
hdiluteaci
donhotpl
ateorwaterbath.Thet
oxicant
sthatare
l
iberatedinthefor
m PH3orH2Sar esubject
edtochemicalanal
ysis.Thishasbeencov ered
separatel
yint
hemonograph.

3.
9.5.
4 Mi
cro-
Dif
fusi
on:
Theuni toperationmaybeempl oyedf orbi
ologicalmatricesv iz.bl
ood,ur i
neandst omachwash
andal sonon- biol
ogicalmatricesv iz.water
,drinks,tea,cof f
eecont ainingtracesoft oxicanions
(cyanide,phosphi de,sulphi
de,)byusi ngselectivesealing,liberat
inganddet ect
ionr eagentin
ConwayMi cro-Diff
usionassembl y( asandwhenr equir
edi ncaseofv olati
lepoisons.Viz.ethanol,
met hanol,acetaldehyde,chloroform,t oxi
cgasesv iz.car bonmonoxi de,phosphi ne,ammoni a,
hydrogensulphi deandt oxi
cani onscy ani
de,nit
rateetc.)
.

ConwayMi
cro-
Dif
fusi
onAssembl
y:

Theassemblyconsi
stsofbri
nktype,pol
ypr
opylenecell
swithcl
earpoly
styr
enecovers.Thecell
s
haveanoutermostannul
arseal
i
ngwel l(
No.
1),aninter
mediateannul
arwell
(No.
2)forthesample
andtheliber
ati
ngagentandacent erwel
l(No. 3)forthereagentwhichisusedt otrapthe
di
ff
usinggasorvapour.

The seal ing agent( usual l

y2 ml .
)i si ntr
oduced i ntot he out ermostseal ing wel l
(No. 1) .An
approximat eamount( 1ml .)ofl iberatingagent( usuallysameasseal i
ngagent )v i
z.10%H2SO4as
sealingandl i
beratingagentf orcar bonmonoxi deandsat urat
edsodi um car bonat esol utionf or
met hanolandet hanolet c.isplacedasapooli nonehal foft heintermedi atewel li
.e.int hesampl e
well( No.2).Trappingagent2mlsayPdCl 2i
ncaseofcar bonmonoxi det urnst obl ack,aci di
fi
ed
potassium di chromat et urnst ogr eeni ncaseofmet hanoloret hanolet ci spl acedi nt hecent re
well(No.3).Sampl e(1ml .
)isi ntroducedi ntotheot herhal fofsampl ewel l
(No. 2)t akingcar et hatit
doesnotmi xwi ththel iberati
ngagent .Thecov erispl
acedandcel lisr
ot atedt oef fectai rti
ghtseal .
Theassembl yisfittedback&f or t
ht omi xthesampl eandl i
beratingagent .Thecel lispl acedon
tabl
eorwat erbath,i fneededf orr eacti
on.Thecont r olsampl ecel li
sal sopr epar edusi ngsame
reagentbutwi thoutsampl ei .
e.1ml .ofwat eri nplaceofsampl e.Thecol orchangei ncent ral
compar tmentcont ainingtrappi ngagent( No.1)i snoted.

3.
9.5.
5 I
onChr
omat
ogr
aphy
:

The pr
efer
enti
alexchange ofions on i
on-
exchange r
esins packed i
nthe col
umn ofion
chr
omatographrender
ssepar
ati
onofani
onsbyusingmobil
ephasesusuall
ybuf
fer
sofdi
versepH.

3.
9.6 Ext
ract
ionofNon-
vol
ati
l
eOr
gani
cPoi
sons:

Thegr oupi
ncl
udespest i
cides,drugs( aci
dic,basi
c,neutralandamphoter
ic)andpl antpoisons
(speci
all
yal
kal
oids,gl
ycosi
deset c.).Outoft heaboveclassesofpoi
sons,pesti
cidesaccountfor
mor et
han80%off at
alcasesofpoi soningi
nI ndi
atherebyrequi
ri
ngspeci
alatt
enti
ont oit
..

3.
9.6.
1 Ext
ract
ionofPest
ici
desi
nMat
ri
ces:

Theextracti
onofpest i
cidesinbiologicalmat er
ial
sv iz.v i
scera,st omachcont ents,gastr
iclavage
andbloodisdi ff
icultduetot heinter
ferencesoff at,degr adedpr otei
nandcol ouringmat terinthe
matri
ces.Theext r
actsrequirepropercl eanupexceptf orMi cell
armet hod.Theext racti
onincase
ofnon-biol
ogicalmat ri
cesisl esscumber someandr equi r
esei thermi norcleanupornocl eanup.
Themet hodsdescr i
bedher eunderarebasedonsol v entext ractioncum st ri
ppingunderdi verse
condi
ti
onsv iz.natureandcondi ti
onofmat ri
ces,useofor ganicsol ventetc.

3.
9.6.
1.1Met
hod-
I:

Biol
ogicalmateri
als(vi
scera,st
omachcont entorgast ri
clavageet c.
)ar emacer atedintofine
sl
urrybymi xi
ngwi t
hequalamountofanhy droussodium sulphateandt r
ansferr
edi ntoaconical
fl
askwi thanaircondenser
.50mlofn- hexaneisaddedt otheflaskandheat edonahotwat er
bathforonehour.Thecontentsarecooledandf i
lt
ered.Ther esi
dualslurr
yisextractedtwicewith
25 mlpor ti
on ofn-hexane.The fil
ter
ed n-hexane fracti
onsar e combined and t aken i
ntoa
separati
ngfunnel
.Thishexanel ayerisv i
gorousl
yshakenwi th15ml ,10mlandagai n10ml
por ti
onofacet onit
ri
le,whicharepreviousl
ysaturatedwithn-hexane.Theacet oni
tr
il
el ay
ersare
mi xedandt akenintoanothercleanseparat
ingfunnelanddil
uted10t i
meswi t
hdisti
l
ledwat er
.25
ml ofsat
uratedsodium sulphat
esoluti
onisaddedt oitandext
ractedthri
cewi t
h25ml port
ionofn
-hexane.Then- hexanel ay
ersar ecombined,concentrat
edto5mlbyev aporati
ngonwat erbath
and5gmsofanhy dr
oussodi um sul
phateisadded.Theext ractisev aporat
edasandwhen
requiredforanalysi
s.

3.
9.6.
1.2Met
hod-
II
:

50gmsofmacer at
edt i
ssuesorbi ologicalmat erialsaremixedwi t
hequalamountofanhy drous
sodium sulphateand100mlofacet onei naconi calf l
askandt henref
luxedonhotwat erbat hf or
onehour .Aftercooli
ng,theacetoneext ractisf i
ltered.Theresidueisextractedtwicewi thfurther
50mlpor t
ionofacet one.Theacet onef r
acti
onsar ecombinedandconcent rat
edbyev aporation
upto50mlf orf
urtherprocessi
ng( cleanup).Theabov eacetoneextract(50ml )i
stakeni nt
oa
separati
ngf unnelanddi l
utedwi t
h150mlofwat er.Add20mlofsat uratedsol ut
ionofsodi um
sul
phat etot hesame.Thecont entsar eext r
actedt hri
cewith25mlpor t
ionsofchl oroform wi th
gentleshaking.Thechl or
oform extractsar ecombi ned,washedwi t
hwat er-acetonemi xtur
e( 1:1)
andf i
nall
ywi th50mlofwat er
.Thewashedchl orof ormlay
erispassedt hr
oughanhy dr
oussodi um
sul
phat eandt henevaporatedtodrynessbypassi ngai r.

3.
9.6.
1.3Met
hod–I
II
:

Ext
ract
ionofPest
ici
desi
nst
omach-
wash,
uri
neandv
omi
t:

Thesampl e( 20mlofstomachwashorur ineor10-20gmsofv omi t

)i stakeninaconi calf
lask.50
mlofn- hexaneisadded.Itisr
efluxedonawat erbathforhal
f-
an-hour .Aftercool
ing,theli
quidis
fi
lt
ered,mi xedwith20mlofn- hexaneandt akeninaseparatingf unnel.Then- hexanelayeris
separat
ed;passedt hroughanhydroussodium sul
phateandev aporatedt odrynessbypassi nga
curr
entofdr yairt
hroughit
.

3.
9.6.
1.4Met
hod-
IV:

Ext
ract
ionofPest
ici
desi
nBl
ood:

20mlofbl oodismixedwi t
h10mlof10%sodi um tungstat
esol uti
onand15mlof1Nsul phuri
c
acid,shakenfortwomi nutesandthenf i
ltered.Thefilt
rat
eiskeptr eserved.Theresi
dueiswashed
witht wo15mlpor t
ionsof0. 1N sulphuricacid.Thewashi ngsar ecol l
ected,mixedwithfil
tr
ate
(keptreserved)
,tr
ansferredint
oasepar ati
ngf unnelandextractedt hr
icewi t
h20mlpor ti
onofn-
hexane.Thehexanel ayersarecombi ned,passedt hroughanhy droussodi um sul
phat
eandt he
solventisremovedbypassi ngastream ofai rasstatedinthepreviousmet hods.

3.
9.6.
1.5Met
hod-
V:

Di
rectSol
ventExt
ract
ion:

Thebi ologicalmaterials(50gmsofv iscera)ar emixedwi th5gmsofammoni um sulphat

eand
homogeni zed.Af
teraddi t
ionof100mlofdi ethylether,
themi xt
ureisshakenatinter
valsandkept
overnight.Itisfi
lter
edandconcent r
atedasbef ore.Theconcent r
atedextractiscleanedupby
passingt hroughachr omatographiccolumn( diameter2.5cm)containingt
hreesuccessivelay
ers
ofdifferentlengt
hsv iz.5cm layerofal
umi na(topl ay
er),2.
5cm ofactiv
atedcharcoal(middl
e)and
2.
5cm l ayerofanhy droussodium sulphate( bottom)previousl
ywashedwi thet her
.Theeluteis
evapor atedtodrynessasbef ore.
 3.
9.6.
1.6Met
hod-
VI:

Ext
ract
ionbySt
eam Di
sti
l
lat
ioncum Sol
ventExt
ract
ion:

Whenthebiol
ogi
calmat er
ial
sarecl
eanandpur
if
iedi
.e.lessdegr
adedandcont
ainv
eryl
i
ttl
efat
andcol
our
ingmatter
,thefoll
owi
ngmethodmaybeadopted.

Pr
ocedur
e:

50gmsofbi ol
ogicalmater
ialistr
eatedwithaf ewdr opsofphosphor
icacidandst eam di
sti
ll
ed
for15mi nutes.Thedisti
l
lat
e( 100ml)iscoll
ectedandsubj ect
edtosol
ventextr
act
ionwi t
h100ml
ofdiet
hylet herin20mlpor t
ion.Theethereallayer
sar ecoll
ect
edduri
ngext r
act
ions,combined
andsubjectedt ocleanupbypassingthroughchr omatogr
aphiccol
umnasbef or
e(met hodV).

3.
9.6.
1.7Met
hod-
VII

I
sol
ati
onofPest
ici
desi
nNon-
biol
ogi
cal
Mat
eri
als:

Matri
ces:20-25gmsofr i
ceormore,i
favai
labl
e,100mlofdr i
nki
ngwat
erorteaorcoff
eeormi
l
k,
weari
ngappar el(
20-25roundpi
ecescutoutfr
om fabr
ic,eachof2.
5cm di
ameter
),20-
25gmsof
soi
l,
sand,grainsorcer
eals.

Pr
ocedur
e:

Fortheabovemateri
als,di
rectsolventextracti
oniscar r
iedoutwit
h50-100mlofdi et
hylether
wit
houtaddi
ngammoni um sulphate.Theetherealext
racti
sconcentr
atedt
o20mlandcl eanedup
bycolumnchromatographyasst atedabov e.Theet her
ealext
ractiscol
lect
ed,ev
aporatedt o
dry
nessbypassi
ngstream ofair.

3.
9.6.
1.8Mi
cel
l
arExt
ract
ioni
nBi
ologi
cal
Mat
ri
ces:

50 gms ofbi ologicalmat er i

al( viscera)i s mi xed wi th 5 gms ofammoni um sulphat e and
homogeni zed.Afteraddi ti
onof100mlofdi ethy lether,themi xtureisshakenati ntervals,kept
overnightandf i
ltered.Theet herealext ractist akeni ntoasepar ati
ngf unnel.10mgofsodi um
l
aury lsul phateisadded and st ir
red gent ly.Onset tl
i
ng,f ati nliqui
d and semi -
soli
df orm is
separ atedandt akenof ffr
om t hesy stem.Theaddi t
ionofsur f
actantiscont i
nuedtil
lallthef atty
mat erialsandpr oteinsaresepar atedandset t
ledatt hebot t
om.Theend- poi
ntisindicatedbya
changeofdar kcolourofet hereallay ertocol our l
ess.Theet herealextr
actisshakenwi th25ml
portionofwat ert wicegent l
y.Theet hereallay eriscol lected.I ncaseofemul si
onf ormat ion,
ethereall ayeriscol l
ectedbybr eaki ngt heemul sionwi thexcesset herandgent lestir
ring.The
coll
ect edet her
eallay erindr i
edov eranhy droussodi um sul phatet oremov etracesofwat er.The
ethereal l
ay eri
sdecant edandev apor atedt odrynessasbef ore.

3.
9.6.
1.9Ext
ract
ionofPest
ici
desi
nFr
uit
s,Veget
abl
es,
But
terFatbyUni
ver
sal
TraceResi
dueExt
ract
or:

Pr
ocedur
e:

Thesampl eisextr
actedbydi rectsolventext
racti
onwi thdi
chlor
omet hane.Theextracti
sdr i
ed
overgranularanhydrous sodium sulphatein a column.The dr i
ed extractthus obt
ained i
s
concent
ratedbyast ream ofni t
rogen.Theconcentratedext
ractisthensubj ect
edtosweepco-
di
sti
ll
ati
oni ntheUniv
er salTr
aceResi dueExtr
actorat230oCf or30mi nutesbypassingnitr
ogen
 (230ml/mi n)andusi ngsodi
um sulphat
e-10% f lor
isil(1:
1).Eluti
onismadeby5mlof10%
acetone in hexane or5 mlofdi chl
oromethane.The ext ractis col
l
ected f
oranalysis.The
appli
cationsoft hismethodtodi
ff
erentpesti
cidesf
ort heiranalysi
sinbi
ologi
calmatri
cesr equi
re
furt
herst andardizat
iontosetupopt imum analyt
icalconditionstocov erdi
ff
erentclassesof
pesti
cides.

3.
9.6.
1.10Ext r
acti
onofPest
ici
des i
n Sedi
ment
,Soi
l
,Dr
yWast
eandTi
ssueby Accel
erat
edSol
vent
Ext
ract
ion(ASE)
:

Pr
ocedur
e:

Thesampl ei smi xedt hor oughl yorpassedt hrougha1mm si ev e.Suffi

cientsampl ei si ntroduced
i
nt othegr indi ngappar at ust oy i
eldatl east10- 20gm af tergr indi
ng.Thesampl eisai rdr iedat
room t emper aturef or48hour si nagl asst rayoronhex anecl eanedal umi nium foil.Thedr yi
ng
mayal sobemadebymi xingwi thanhy dr oussodi um sul phat eunt i
laf reef l
owi ngpowderi s
obtained.( Ai rdr y
ingi snotr ecommendedf orv olatilepesti
cides. )Gummy ,fibrousoroi lymat erials
notamenabl et ogr indingshoul dbecut ,shr eddedorot herwi sesepar atedt oal l
owmi xing.These
maybegr indedaf t
ermi xingwi thanhy droussodi um sulphat e1: 1propor tion.Acel lulosedi ski s
placedatt heout letandendoft heext r
actioncel l.Approximat ely10gm ofeachsampl ein11ml
or20gm ofeachsampl ei n22mlext ractioncel l.(Surr
ogat espi kesandmat r
ixspi kesmaybe
addedt otheappr opriatesampl ecell).Theext ract i
oncel l
swer eplacedi ntot heaut o-sampl et ray
andt hecol lectiont raysar el oadedinappr opr iatenumber( upt o24) .Thet rayi sl
oadedwi th40ml
pre-cleaned, clappedv ial
swi thsepta.Thecondi tionsf orextract ioninASEar esetforext ract ionof
pesticidesbyusi ngacet one:hexane( 1:1,v / v)ast hesolv ent .Theoper atingcondi ti
onsi nclude
ovent emp.of100º C,pr essur eat1500psi ,ov enheatt i
meandst ati
ct i
meeachof5mi nut esand
fl
ushv olumei nt hepr opor tionof60% ofext ract ioncellv olume.Theext ractsarecol lectedf or
analysi s.Themet hodhasbeenv al
idatedf oranal y si
sofpest icidesinsoi l
,sediment ,dr ywast es
andf isht i
ssues.Howev er ,fur t
herst andardizat i
oni srequiredf orapplicationofASEt obi ol ogical
mat ri
cesi nf orensi ctoxicol ogical work.(1)

3.
9.6.
2 Ext
ract
ionofDr
ugsandPoi
sonsofPl
antOr
igi
ninBi
ologi
cal
Mat
ri
ces:

Thisgr oupcompr i
sesalkal
oids,gly
cosides,barbi t
urat
es,phenothiazi
nes,sali
cyl
ates,sulphonal
groupsofdi f
ferentclassesandplantpoisons.Theext ract
ionofthesepoi sonsdependsont hei r
solubi
lit
yatdi fferentpH i.
e.atl ow orhighaci di
coral kalineconditi
onandal sodi f
ferential
solubi
lit
yinor ganicsolvents.Themet hodsi ncludeSt as-
Ot t
oorDr agendorffortheirdiff
erent
modi f
icati
ons,ammoni um sulphat
emet hodandmoder nmet hodespecial
lysoli
dphaseextraction
micell
arandenzy medigesti
onfortheabov epoisonsi nbiol
ogicalmater
ials.

Themai ndif
ficult
yi nallthemet hodsforext racti
onofpoi sonsi nbiol
ogi calmat eri
alsistogetr i
d
off ats,degr adedpr oteinandpi gment s,whi chinterferewi ththei
risol ati
oni npur efor m and
subsequenti dentifi
cat i
on and quant it
at i
on.Thepr oblem i st hatquant i
ti
esoft hesepoi sons
presenti nbiologicalmat r
icesar esmal landmul t
iplest epsi ntheextract i
onandpur i
fi
cationmay
i
ncurl ossofpoi songi vingami nuserror.Ifnotsuf fi
cientlypur i
fi
ed,aposi tiveerrorari
ses.Thi sis
thereasonwhyt her esultofquant it
ati
vedet erminat i
onespeci al
lythealkaloidsarenotdependabl e
and t henegat i
vef i
ndingsi n manycasesdo notr epr esentt heact ualst ateofaf fairs.The
quant itat
iveresultact uall
yr epresentsther ecoverynott heexactquant itypresentinthet issues.
Howev er
,theper centageofr ecoveryi simpr oving excel l
entlybyusi ng modi fi
cati
onsoft he
existingmet hodsandal somoder nmet hods.Themet hodsar edescri
bedher eunderwi t
hspeci al
referencet obiological material
s.

3.
9.6.
2.1Modi
fi
edSt
as-
Ott
oMet
hod:

Pr
ocedur
e:

A. 50gmsofbi
ologi
calmat
eri
ali
smi
nced,
mixedwi
thpl
ent
yofr
ect
if
iedspi
ri
t(about2-
3ti
mes
 thewei ghtofmat eri
al)inaf l
askandaci di
fi
edwi t
htartaricacid.Themi xtureisheatedont he
steam bathf or1-2hour swi t
hthoroughshakingsatf requentintervals.Theext r
acti
oni sthen
all
owedt opr oceedf orabout24hour swithsteam off
.I tisthenf i
l
teredt hroughaf l
uttedfi
lt
er
paper.Thef i
lt
rateisev apor
atedandtheresidueisagai
next r
actedwi thacidulat
edalcoholinthe
sameway ,fi
lt
eredandwashedsev eralt
imeswi thhotrectif
iedspiri
t.Thecombi nedfi
ltr
atesare
evaporatedinapor celai
nbasinonthesteam bathtoasy rupyconsistency .

B. Tot hesy r
upyr esi
dueabout100mlofrecti
fi
edspiri
tisaddedv er
yslowlywi t
hconstant
sti
rri
ngsot hatinsol
ublematt
ermaybegr
anularandnotgummy .I
fthealcoholi
saddedr api
dlyor
allatat i
me,t heinsol
ublematt
erwil
lbegummycausi ngmuchl ossofal kal
oidsbyenclosing
them int hestickymass.Iti
swarmedwit
hoccasionalst
ir
ri
ngforabouthalfanhourandf il
ter
ed.
Thispr ocessisr epeat
edoncemoreandthecombinedalcohol
i
cext r
actsareevaporat
edalmost
todr y
ness.

C. Ther esidueisnowdi ssolvedinabout50mlofwat eracidulat

edwi thdil
utesulphuri
caci dand
fi
lteredaf teraboutanhour .Thepoi sonsar ethusdissolvedoutbyt heaqueoussol uti
onwhi chis
tr
ansf erredt oasepar ati
ngf unnelandext ractedwi
thasui t
ablesol ventsuchaset her,chloroform
etc.i npor tionsofabout25ml .Thesol v entwouldt akeupf r
om t heacidsolution,colour i
ng
mat ters,toxi coi
lsandr esins,salicyl
icacidandi tsder i
vativ
es( aspirin,sal
oletc.)
,bar bi
turates,
sulphonal ,acetanil
ide,narcoti
neandal kaloidsofergot,certai
ngly cosidessuchast hev eti
nwhi ch
hasescapedi nit
ial
t r
eatment sforpurif
icat
ion.

D. Theacidaqueoussolutionisthenrender
edal kal
inewi t
hasol uti
onofsodi um carbonateor
ammoni a,
whichwouldl i
beratethefr
eebasef r
om i t
ssal t.Thealkal i
nesol ut
ioni
snowext r
acted
wit
h25mlpor ti
onsofchloroforminthesamewayasi nthepr evi
ousst age.Itwi
l
ltakeupal lthe
al
kaloi
dsexceptmor phine( onl
yat racebeingext racted)andt hosef eebl
ybasicsubstances,
whicharepar
tial
lyext
ractedfrom t
heacidsolut
ion.Theext r
actioni srepeated2or3t i
mesmor e.

E. Ifmorphineissuspected,itmaybeext r
actedatthisstagebyamy lalcoholorchlorof
orm–
ether(3:1)mi xt
ureorchlorof
or m –al cohol( 9:
1).Ofthese,amy lalcoholist hebest.Butasi tis
pronet oform annoyi
ngemul sions,thechl oroform –ethermi xturei susedbymany .I
fmor phine
i
sl ikel
yt obet heonl
ypoi son,t hechl oroform ext
racti
on( stageD)descr ibedabov emaybe
omi tt
edaltogether
.Thecombi nedchl oroform oramy lal
coholext r
actsar eev apor
atedtodry ness
andt heresidueisnowreadyf orfurt
herpur if
icati
onandanal ysis.

F. Theev aporat
edchlorof
orm extractispurifi
edbydissolv
ingi ti
nabout20mlofwat er
aci
dulatedwithsulphur
icaci
dandf il
ter
ingthroughasmal lf
il
ter.Thef i
lt
rat
eisextr
actedwith
chl
oroform,fi
rsti
nacidandt heninalkali
nemedi um asi
nthei ni
ti
alstagesofext
ract
ion.These
ext
ractsareevaporat
edtodrynessforanalysi
s.

3.
9.6.
2.2Fur
therModi
fi
cat
ionofSt
as-
Ott
oMet
hod:

Ast herearetoomanyst epsint heextr

acti
onofnon- vol
atil
eorgani
cpoisonbyStas-Ot
tomet hod
andal sochancesoflossofpoi sonineachoft hesteps,theremaybeev enacompletefai
lur
et o
detectiti
ncaseofconsider abl
el ossofpoi
sons.Theobj ecti
vesofmodifi
cat
ionsaretominimize
thestepsasf araspracticableunderspecialci
rcumstances.Thefoll
owingmodifi
cati
onsoft he
techniquearesometi
mesnecessar y.

i
) Thealcoholi
cextract
ionofbiol
ogicalmater
ialsi
stobecarri
edoutatr oom temperat
ure(not
exceeding40oC)i .e.wit
houtusingsteam bathandpr ef
erabl
ywithabsolutealcohol(
topr ev
ent
hydrolysi
s)inplaceofr ecti
fi
edspiri
tforsuspectedpoisoni
ngbyaconite,bel
ladonna,daturaor
cocaine.Theev aporat
ionofalcohol
i
cext r
actshouldbedoneunderreducedpressure.

i
i
) Theextr
act
ionwit
hrect
if
iedspir
iti
stobedonef
or48hour
sifbi
ologi
calmat
eri
alsar
e
pr
eser
vedinsat
urat
edsal
i
nesolut
ion.
i
i
i) Forst
omachcontent
scompriseof
ftoomuchoff
lui
d,t
heext
ract
ionshoul
dbedonewi
th
absolut
ealcohol
for3or4ti
mes.

i
v) Forstomachwashasthesampl
e,ext
ract
ionwit
hdoubl
ethequanti
tyofabsol
utealcohol
aci
dulat
edbytar
tar
icaci
dshoul
dbedoneandthenal
l
owedtoevapor
ateonasteam bat
h.

v
) Forf
il
tr
ati
on,
Buchnerf
unnel
ispr
efer
red.

v
i) Topr ev
entlossduet oemulsi
onf or
mation,agi
tat
ionwithorganicsolvents,(
etheror
chl
orof
orm oramy lal
cohol
)shouldbedonegent lyi
nt hebegi
nni
ngandst eadilyi
nanv iol
ent
mannerthereaft
er( 2– 3t i
meint hebeginningandnotexceeding12t i
mesatt heend).If
emulsi
onper si
sts,iti
stobeev aporat
edonast eam bathandther esi
duet akenupi nfresh
sol
vent.

3.
9.6.
2.3Ammoni
um Sul
phat
eMet
hod:

Thismethodi
smostusefulf
orpr el
i
minaryanal
ysi
s(screening)ofbar
bit
urat
es,al
kal
oidsand
tr
anqui
l
izi
ngdr
ugset
c.andal
soidenti
fi
cat
ionandsemi-
quanti
tation.

Pr
ocedur
e:

Theviscer
almat eri
als(about50-100gms. )arecuti ntosmallpi
eces,macer
ated,mixedwith100
mlof5per centacet i
caci dandtakeni nt
oa600mlbeaker .Soli
dammoni um sulphat
ei sthen
addedtoitbyfrequentshakingtomakeasat ur atedsol
uti
on.About20gms.ofsolidammoni um
chl
ori
deareaddedi nexcess.Themi xtureisthenheat edinaboili
ngwaterbathforthreehours
(f
orsuspectedpoisoningbyaconit
e,thetemper atureshoul
dnotexceed60C)
.

Themi xtureiscooledsli
ghtlyandf i
lt
eredt hr
ought hef i
lt
erpaper .Ther esi
dueont hef unneli s
againextractedwithtwopor t
ionsof100mlof5per centacet i
cacidandf i
l
ter
edasbef ore.The
fi
lt
ratesarecombi nedandtakenintoa500mlsepar atingfunnel.Ther esi
dueslurr
yonf il
terpaper
i
sl eachedwi th100mlofdi ethyletherandt hesamei sreceivedi nacol dcontai
ner.Theet her
fr
actionisaddedt otheaqueousacidicext r
actinthesepar ati
ngf unnelandshakenfor5mi nutes
andsepar ated.100mlofet herisagai naddedt ot heaci diclayer,shakenfor5mi nutesand
separated.Theet herl
ayersar ecombi ned.Theaci di
cet herext r
actist est
edforsalicyli
caci d,
aspiri
n,barbi
turat
es,meprobamates,lysergi
des,benzodiazepinesetc.

Theaqueoussol uti
onr emainingi nthesepar at i
ngf unnelaf t
ersepar ati
onofaci dicdr ugsi smade
alkal
inebyaddi tionofammoni um hy droxideandext r
act edt hr
eet imewi th100mlpor ti
onsof
chlorofor
m et hermi xture(1:3) .Theaqueousl ayerisr etainedf orextracti
onofopi um al kaloids.
Theor ganiclayer saftersepar ationarecombi nedandwashedwi th50mlofwat er.Itisext r
acted
threetimeswi t
h25mlpor t
ionsof10%sul phur icaci d.Thesul phuri
caci dfracti
onsar ecombi ned
andt akenintoanot hersepar atingf unnel.(Organi clay erisdiscarded)50mlmi xtureofchl oroform
–et her(1:3)i saddedt oi t
.Di luteammoni um hy droxi desol uti
onisaddedt omaket hesol uti
on
alkal
ineshakenf or5mi nutes.Theor ganicl ayerissepar at
ed.Theext ract
ionisr epeatedt hrice.
Theor ganicl ayeraftersepar ationar ecombi ned,washedwi thl50mlofwat erandt hendr i
edby
passingt hroughanhy dr
oussodi um sul phateandev apor atedt odryness.Theext r
acti stestedf or
opium, daturaandaconi t
ealkal oids,amphet ami nes, mepr obamat eandmet haqualoneet c.

Theaci
d–et
herext
ractmaybef
urt
herbesepar
atedi
ntot
hreef
ract
ions.

i
) Theacid–et herfracti
onisshakenwith25mlof5% sodium bicarbonatesolut
ion.The
aqueousl ayerisremov edandt akenint
oanothersepar
ati
ngfunnel
.I tisacidi
fiedwithdilute
sulphuri
caci dandre-extract
edwi t
h25mlofet her.Thi
setherfr
actionispassedt hroughon
hydrous sodium sulphate and t
hen dri
ed j
ustto dr
yness.The r
esidue fract
ion 1 contains
sali
cylat
es.
i
i
) Theet herl
ayer(ofaci detherfracti
on)af t
erwashi ngwi thsodium bi carbonat
eisextracted
twicewith25mlpor t
ionsofN- .sodi
um hy dr
oxidesolution.Theaqueousl ay eraresepar
atedfrom
etherlayer,combinedandt akeni ntoanot herseparatingf unnel
.I ti
smadeaci di
cwithdi l
ute
sulphuri
cacidandext ractedtwi ce25mlpor t
ionsofet her.Thisetherfract i
oniswashedwi th25
mlofwat erandthendr i
edbypassi ngthroughanhy droussodi um sulphat eandt henevaporated
todryness.Theresiduefracti
on2cont ai
nsbar bit
urat
esi nrelati
vel
ypur i
fiedf orm.

i
i
i) Theet herl
ayeraf
terext
ract
ingwi
thNaOHi swashedwi t
hwat erandthenev apor
atedto
dr
yness.Theresi
duefr
acti
on3contai
nsmeprobamat
e,othercarbamatesandotherneutr
aldr
ugs.

3.
9.6.
3 Ext
ract
ionofDr
ugsi
nUr
ine:

Suffici
entphosphori
caci
dortartar
icaci
disaddedto10ml ofur
inetoadj
ustt
hePHt o3.I
tisthen
extractedwit
ht wo30mlpor t
ionsofether.Theext
ract
sarecombinedandwashedwi t
h5mlof
water .Thewashingsareaddedt ot hesample.Theaqueoussoluti
onisret
ainedforpossibl
e
presencesali
cylat
es(Fr
acti
onA).

Theetherealsoluti
onisextr
act edwit
h5mlof0. 5M sodium hydroxi
deandtheextr
actisret
ained
forexami nat
ion ofbar bi
turates and weakl
y aci
dic substances (Weak Aci
d Fract
ion B –
Barbi
tur
ates,Glutet
himide,
Par acet
amol,Pheny
toi
n,Phenylbut
azoneetc.)
.

Theet her
ealsoluti
oni swashedwi t
hwat er.Thewashingi sdiscar
ded.Theet herealsolut
ionis
thendriedoveranhy dr
oussodium sulphat
eandev aporatedtodryness.Ther esiduemaycont ai
n
neutraldrugs (Neut r
alFracti
on C – Caf fei
ne,Car bromal,Chlordi
azepoxi de,Flurazepam,
Lorazepam,Mepr obamate,
Met haqual
one,Methypry
lone,Nitr
azepam,Paracetamol ).

Tot heaqueoussol ut
ionretainedaf terthef i
rstextr
acti
onsuffici
entdi
luteammoni asoluti
onis
addedt oadj ustthePHt o8.I tisextract
edwi thtwo10mlpor t
ionsofchlorofor
m.Thecombi ned
etherextractsar ewashedwi thwat er
,fil
tered.Alit
tl
et ar
tar
icacidisaddedt opreventt
hel ossof
volati
lebases.Itisevaporatedt odryness.Ther esi
duemaycont ainbasicdrugs(BasicFracti
onD
– Amphet ami ne, Amitri
ptylene, Caf fei
ne, Phenot hi
azi
nes, Ergot Al kal
oids, Mor phine,
Met haqualone,Flur
azepam, Lorazepam etc.).

ThepHoft heaqueoussol ut
ionobtainedaf t
erextr
actionofFr acti
onDi sadj
ustedt opH3byt he
addit
ionofhy drochl
ori
cacid.Iti
sheat edat100 Cfor30mi nutes,cooledandext ract
edwi t
htwo
10mlpor t
ionsofether.Theaqueoussol ut
ioni
skeptr eser
ved.Thecombi nedet herext
ractsare
washedwi th5mlof1M.sodi um hydroxideandev aporatedtodr yness.Theresiduemaycont ain
benzodiazepinesasbenzophenones( Fracti
onE).

ThepHoft heaqueoussol
uti
oni sadj
ustedt opH9andcooled.Iti
sext
ract
edwit
hami xtur
eof
et
hylacetat
eandi sopr
opylalcohol(
9:1) .Thesolventl
ayerissepar
atedandevapor
atedto
dry
ness.Theresi
duemaycontainopi
ates(Fract
ionF)
.

3.
9.6.
4 Ext
ract
ionofDr
ugsi
nBl
ood:

Assampl e-v
olumei ncaseofbl oodorser um orpl asmai ssmal landonl yal imitednumberof
drugsmayeasi l
ybedet ectedandi dent i
fiedint hem,t heext r
act
ionprocedurei sslight
lydi
ffer
ent
from thatforuri
neandst omachcont ents.Differentfracti
onsofextracti
onv i
z.A,B,C,Dbeart he
samemeani ngasst at
edi n3.8.
6.3.i.e.ext r
actionofdr ugsi nuri
ne.Thei ni
ti
alextracti
oniscarr
ied
outatpH7. 4asmanybasi cdrugsar er ecov eredbychl or
oform ext
racti
onatt hi
spH.Asar esult
,
thesubst ancelookedfori smostl ikelyt obef oundi neitherfr
acti
onBorCandpr epar
ati
onof
fract
ionDi sonlynecessar yeit
hertoensur et hatnot hinghasbeenmi ssedorwher enodrughas
beenf oundinfract
ionsBandC.

Procedur
e:2mlofphosphatebuf
fer(
pH=7.4)and40mlofchl
orof
orm ar
eaddedt
o4mloft he
sampleandshakenvi
gorousl
y.2gmsofanhydr
oussodi
um sul
phat
eisaddedandshakenagai
n
 toproduceasol
i
dcake.Thedecant
edchlor
ofor
mi spassedthr
oughafi
lt
erpaperandt
hecakei
s
ext
ractedwi
thafur
ther20mlofchl
orof
orm.Thechl
orofor
m ext
ract
sar
ecombined.

Thechl or
oform layerisextract
ed wi thsodi
um car bonat
et or emovesali
cyl
ate(St
rong Aci
d
Fracti
onA) ,ifdetectedinthepreli
mi naryt
ests.Tot hechlor
oformlayer,8mlof0.5N sodium
hydroxidesoluti
onsi sadded.Themi xturei
sshakenandcent ri
fuged.Thesodi
um hy
droxidemay
containbarbit
uratesandotherweaklyacidsubstances.(WeakAcidFracti
onB).

Thechl or
oformlayeri
swashedwi t
halit
tlewater.Thewashingisdi
scar
ded.Thechlor
oformlayer
i
sdr iedov eranhydr
oussodium sul
phate,f
il
teredandev aporat
edtodryness.Theresi
duemay
contain(caffei
ne,carbr
omal,benzodi
azepi
nes,mepr obamate,phenazone et
c.)
,neutraldr
ugs
togetherwithanumberofbases(Neutr
alandBasi cFr
acti
onC) .

Ifsuff
icientoforigi
nalsampleisav ai
labl
e,afurt
herporti
onofi tismadealkali
newi t
hdi l
ute
ammoni asoluti
onandext r
act
edwi thtwo10mlpor t
ionsofchlor
oform.Thechl
orof
orm extr
actis
dri
edov eranhydroussodi
um sulphateandevapor
atedtodryness.Theresi
duemaycontainbasic
drugs(BasicFracti
onD-asstatedintheprevi
oussecti
on)
.

Ifthereisnotsuffi
cientoft heor igi
nalsampl ef ort
heextract
ionofbasi cfracti
onF,t hefoll
owi ng
procedure may be car r
ied out .Af terfraction C has been chemi cal
ly exami ned by UV or
Chr omatogr
aphicmet hods,t her emai ni
ngr esidue,i
fanyisdissolvedi nchlorof
orm andext racted
with0.5M sul phuri
caci d.Thisext ractedpor tionisaddedt othesodi um sulphatecaker etained
afterthefir
stext r
acti
on( f
orFr acti
onA) .Itismadeal kali
newi t
hdi luteammoni asoluti
onand
extract
edwi thtwo10mlpor t
ionsofchl orofor m.Thechloroforml ayersarecollected,dr
iedov er
anhy dr
oussodi um sulphateandev aporatedt odryness.Ther esiduemaycont ainbasi
cdr ugs
(BasicFract
ionD-asst atedint hepr evi
oussect i
on).

3.
9.6.
5 Sol
i
dPhaseExt
ract
ionofDr
ugsf
rom Ur
ine:

5mlofur i
neisaddedt o2mlofphosphat ebuf fer( 0.1mol .
/L,PH6. 0)inagl asst esttubeandt he
PHi sadj ustedt oPH5. 5–6. 5usi ng0. 1mol .
/laqueoussodi um hy droxi
deor1mol .
/Laqueous
acet i
caci d(dependi ngont henat ureoft hedr ug) .SPEcol umni sinser t
edint ov acuum mani f
old
andwashedwi th1ml ofmet hanol and1ml ofphosphat ebuf f
er(0.1mol ./l
,PH6. 0) .An8ml f
it
ted
reser voirisat t
achedt ot het opoft heext racti
oncol umnandur i
nei saddedt oit.Thecol umni s
driedunderv acuum andwashedwi th1mlofphosphat ebuf fer(0.
1mol ./
L,PH6. 0)fol
lowedby
0.5mlofaqueousacet icaci d(1mol .
/l)
.Thecol umni sdriedunderv acuum andwashedwi th1ml
ofhexane.The el uti
on f oraci dic and neut r aldr ugs is made wi t
h 4 x 1 mlpor ti
ons of
dichloromet hane.Theel uat eisev apor atedt odr y
nessunderast r
eam ofni trogenat30–40oC.
Ther esiduei skeptr eservedf oranal ysisofaci dicandneut raldrugs.Thecol umni st henwashed
withmet hanol(1ml )andel uti
onf orbasi cdr ugsi smadewi th2mlofmet hanolicammoni um
hydr oxide( 2%,V/ V).3mlofde- i
onisedwat eri saddedandel utioni smadewi th0.2mlof
chlor oform.Thechl oroforml ayeri scol lectedandev aporatedt odr ynessunderast ream of
nitrogenasabov e.Ther esidueiskeptr eser vedf oranalysi
sofbasi cdr ugs.

3.
9.6.
6 Ti
ssueDi
gest
ionusi
ngPr
oteol
yti
cEnzy
me:

Thedigest i
onwi t
hproteol
yti
cenzy mesgivesmuchi mprovedrecoveryandhast headvant
agethat
oncet hedi gesthasbeenpr epared,anal
ogousmet hodol
ogyt othoseusedwi thpl
asmacanbe
employ ed.Itisobv i
ouslyimpor t
antt oensuret hatuseoft heenzy mepr eparat
iondoesnot
i
ntroducei nter
fer
ences.Afurtherpotenti
alprobl
em isthatconjugatesandothermet abol
i
tesmay
notsurv i
ve.Theprocedurehasbeendescr ibedbel
ow.

Asoluti
on(2gms/ L)oflyophil
i
zedsubti
li
si
ninsodium di
hydr
ogenorthophosphate/di sodi
um
hydr
ogenorthophosphat
ebuffer(7mol.
/L,PH7.
4)100mgpor tionsofti
ssuear edissect
edand
theexcessoffl
uidremovedbyf i
lt
erpaper
.Thet
issueisaddedto10mloft aperedglasstubes
 andexactwei ghtsarerecor
ded.1mlofsubti
li
sinsolut
ionisadded.Thetubesar eseal
edwith
groundglassstroppersandi
ncubat
edinawaterbathat50Cfor16hours.Thetubesarecool
ed.
Thecontentsaremi xedonavort
exmixer
.Ther
eaft
erextr
acti
onisdonewith0.2ml por
ti
onsasfor
pl
asmaorser um.

3.
9.6.
7 Mi
cel
l
arExt
ract
ionofDr
ugsi
nBi
ologi
cal
Mat
eri
als:

50-100gmsofbi ologi calmat erialsarefinelymi ncedi nami ncingmachi ne.Anexcessofr ect i

fied
spiri
t( about200ml )isaddedt oitinaf lask, mixedt hor oughl yandmadeaci dicwi thgl acialacet i
c
acidbydr opwi seaddi tionandst i
rri
ng.Themi xtureist henheat edonast eam bathf or1hourwi th
throughshaki ngati nt ervalsandt heflaskcont ai
ningt hemi xtur
ei skeptf or24hour satr oom
temper ature.Itist henf i
lteredt hroughaBuchnerf unnelhav ingabedofsand( 0.3cm)ont hef il
ter
paper .Thef i
l
tratei scol lectedi nasepar at i
ngf unnel .Af teraddingof50mlofsol ventet her ,
cloudi nessi sobser ved.Fi nepar ti
clesoff atsepar ateonst andingwhi chi sdr ainedof f
.The
cont entsofsepar atingf unneli sshakenwi th1mgpor tionsofsodi um l aurylsulphat ewhensemi -
solidf atsepar ates.Thesepar ationoff ati saccel er atedbyaddi tionofaf ew dr opsofwat er
.
Reddi shbr ownoi lymassoff atset tl
esatt hebot tom oft hesepar atingf unnel.Thisi sdr ainedof f.
Thepr ocessi sr epeat edt il
ltheset tl
ingoff atceased( indicatedbyacl eartranspar entext racti n
thesepar ati
ngf unnel ).Thecont entsoft hesepar atingf unneli sev aporatedt odr yness.The
residuei sextract edgent l
ywi t
h20mlpor t
ionsofwat erf i
vet i
mes.Theaqueousext r
acti smade
disti
nct lyacidicbydr opwi seaddi tionofacet i
caci dandext ractedwi th80mlofdi ethylet heri n20
mlpor tion.Theaqueouspar tiskeptsepar ately.Thecombi nedetherext r
actasabov e(aci d-ether
partf oraci di
candneut ral drugs)i swashedwi thwat ert il
lfreef r
om aci d.

Thewashingsaremi xedupwi t
htheaqueouspart.Thecombi
nedetherext
ractisdr
iedov er
anhy
droussodium sulphat
e.Thedriedet
herext
racti
sdecant
edoff
.Iti
sevaporat
edtodryness.
Theresi
dueiskeptreadyforanal
ysi
s.

Thecombi nedaqueousparti
smadedist
inct
lybasicwithdropwi seaddit
ionofammoni asol
uti
on.
Iti
sextractedwith80mlofchl or
ofor
mi n20mlpor t
ion.Thecombi nedchl or
ofor
m extr
actis
washedwi thwaterti
l
lfr
eef r
om al
kal
ianddriedov eranhydroussodium sulphat
e.Iti
sdecanted
andevaporatedtodry
ness.Ther
esi
dueiskeptreadyforanalysis(basi
cdrugs).

3.
9.7 Headspacepr
ocedur
eforI
sol
ati
onofVol
ati
l
esi
nBi
ologi
cal
Mat
eri
als:

Theprinci
pleunderl
yingheadspaceanal y
sisisthati
naseal edv i
alatconstanttemper at
ure
equi
li
bri
um isestabl
i
shedbetweenv olatil
ecomponentsofaliquidsampleint
hev ialandthegas
phaseaboveit(t
heheadspace)
.Afterallowingdueti
meforequil
ibri
um (nor
mall
y15mi nut
esorso)
aporti
onoft heheadspacemaybewi thdrawnusingagas-t
ightsyri
ngeandinjectedintotheGC
col
umn.

Pr
ocedur
e:

Thei nt
ernalstandardsol
ution(25mg/let hylbenzeneand10mg/ι l
,1,2t ri
chloroethaneis
addedt oa200lofami xtureofexpi
redbloodanddei oni
sedwater(1:
24,v/ v
)ina7mlgl ass
septum vialusingasemi-automati
cpipett
e.Thev iali
ssealedusi
ngacr i
mpedonPTFE-l i
ned
si
li
conedi sc.Thev i
ali
si ncubat
edat65oC i naheat i
ngblockandapor t
ion( 100-300μl)of
headspacei swi thdr
awnusi ngawar medgas- t
ightglasssyri
ngeforonwar danal ysisingas
chromatograph.(2)

3.
10 CLEAN-
UPPROCEDURES:

I
ntheext
ract
iont
echni
quesdescr
ibedabov
e,i
tisl
i
kel
ythatt
heext
ractwi
l
lcont
aini
nter
fer
ing
 substances which willcreate problems int he analysisi nv ari
ous way s.The occur r
ence is
parti
cular
lysoi nt heextractsf r
om sampl esofef fluent,soi l
,sedimentandt issuesoror ganic
mat ri
ceswhichcont ai
nf ats,oil
sandot hernat ur
all
yoccur ringsubst ances.Thelongest abl
ished
procedureforther emovaloft hesesubstancesi stopasst heext r
actthroughanal uminacolumn
andt hentosepar atet
het argetcompoundsi ntodifferentbat chesbypassi ngthecleanupext r
act
throughasili
cacol umn.Thesuppl yofmat er
ialsbypr eparativethinl
ay erchr
omat ographyisalso
fol
lowedi ft
heact i
vecomponent sareidenti
fi
edbypr eli
minar yscreening.Thepur i
fi
cati
onint he
samewaymayal sobeachi evedbyGCorHPLC.

Forv olat
il
etoxi
cant
s,str
ippingmayal sobedonebyGC-HeadSpacemethod(Pur
georTr ap
technique)
.Ther
earedif
ferentanal
yti
calcondi
ti
onsf
ort
hepur
posewhi
chhavebeenspeci
fi
edin
l
iterat
ure.

Indiff
erenthy
phenat
edtechni
quesvi
z.GC-MS,LC-
MS,HPLC-I
R,thef
ir
sttechni
quei
sappli
edfor
separati
onofcomponentsinthepurestat
eandthei
rsubsequenti
dent
ifi
cati
onbythesecond
techni
que.

Recent
ly,t
hecleanuphasbeenstreamli
nedbytheuseofcommer
cial
l
yav
ail
abl
esol
i
d-phase
ext
ract
ioncar
tri
dgesordi
scs(
descr
ibedearl
i
er)
.

Di
ff
erentmet
hodsofst
ri
ppi
ngar
edescr
ibedbel
owi
nshor
t.

3.
10.
1 Cl
eanupusi
ngAl
umi
naandSi
l
icaCol
umn:

Thi
stechni
quer emovesinter
fer
ingcompoundsbypassingt
heextractt
hroughbasi
candaci
dic
col
umnandt henseparat
ingacti
veconst
it
uent
sspeci
all
ypest
ici
desonasili
cacol
umn.

Pr
epar
ati
onofSol
i
dAdsor
bent
s:

Thepr
epar
ati
onofsol
i
dadsor
bent
s–basi
cal
umi
na,
aci
dical
umi
naandsi
l
icagel
isasf
oll
ows.

i
) Basi
cAl
umi
na:

About100gmsofal umi
naisplacedinasi l
icadi
sh,heatedi
namuf f
lefur
nacef or4hrsat800oC,
cooledt
oabout200oCandt hentor oom temperatur
einadessicat
or.Water(4%,w/w)isadded
totheweighedpor
ti
oninastopperedflaskwhichisshakenwel
l
,sealedandthenstored.

i
i
) Aci
dicAl
umi
na:

Aporti
onoft healuminaiswashedwith1NHClbymaki ngslurr
yinabeaker.I
tisf
il
ter
edt hrough
asint
eredfunnelanddriedi
nasi l
icadishat150oCfor4hour sandcooledinadesi
ccator.Wat er
(4%,w/w)isaddedt oawei ghedport
ioninastopperedflask.I
tismixedthor
oughl
yandst ored.
Thesealuminapreparati
onsgetslowlydeacti
vatedonexposur etoai
randshouldbedi scarded
aft
ertwoweeks.

i
i
i) Si
l
icaGel
:

About100gmsofsi li
cagelisheatedi nasil
icadi
shinamuffl
efurnacefor2hoursat500C,
cooledandthenpl
acedinadesiccator
.Aporti
oniswei
ghedi
ntoastopper
edglasscontai
nerand
waterequiv
alentt
o3% oft hesil
icaisadded.Thesil
i
cageldeact
ivat
esmor erapi
dlythanthe
al
umi naandshoul
dprefer
ablybeprepareddai
l
y .
 Pr
ocedur
e:

Thecolumni spre-
washedwit
hacetonefol
lowedbyhexaneandal
l
owedtodry
.Theacid/base
aluminacolumnisprepar
edbyfi
rstaddi
ng2gms.ofacidical
umi
nathen1gm ofbasi
cal
umina
tothecolumn.Thecolumnist
appedtosett
le.

Thesili
cacolumni sfil
ledbyadding2. 5gmsofsi li
cagelpr eparedasstatedabov eandt appedto
sett
le.10mlofhexanei spassedt hroughthecolumn( t
owett hecolumn)andr unof finexcess
ti
llt
hehexanemeni scusi satlevelwiththecolumnmat er i
al.Theorganiclayer(obtai
nedaf t
er
extr
acti
onofmat r
icesf orpest
ici
desordr ugs)ist hentransferredontot hecolumn.Theact i
ve
consti
tuent
s( pest
ici
deordr ugs)ar ethenelutedfrom thecol umnbyhexaneordi ethy
letheror
suit
ableorganicsolvent.Theelut
ei scoll
ected.Iti
st henpassedt hr
ought hesil
icacolumn.The
el
uteiscoll
ectedanddr i
edoveranhydroussodium sulphate.Itisevapor
atedtodr yness.

3.
10.
2 Modi
fi
edmet
hodf
orCl
ean-
Upandsepar
ati
onusi
ngAl
umi
na/Si
l
icaGel
:

Pr
epar
ati
onofSol
i
dAdsor
bent
:

(
i) Alumi
na–About100gmsofalumi naisheatedi
nasi li
cadi shat500oCf or4hour sandthen
cooled.Toaweighedport
ioni
nast opperedglasscontainer,deionizedwaterequiv
alentto7%
(w/w)weightofal
umina.i
saddedandt hesampl ei
sagitatedtomi xthor
oughly.Thealuminais
keptinasealedcont
ainer
.Thi
sadsorbentisstableforonl yaboutaweekoncer e-exposedto
atmosphere.

(
ii
) Al
umi
na/Si
l
verNi
tr
ate:

Abat chofmat eri

alforaddingtothecolumnisprepar
edbydissolvi
ng0.75gm ofsi
l
vernit
ratein
0.75mlofwat erf
ollowedbyt headdit
ionof4mlofacetone.Tot hissol
uti
oninanunstoppered
conicalf
lask,10gmsofdr i
edaluminaisaddedandshakenthoroughl
y.Theacet
oneisal
lowedt o
evaporate.Thepr eparat
ionisstoredinthedarkuntilr
eadyforuse.Theadsorbentshouldbe
preparedfreshl
y.

Pr
ocedur
e:

Thechr omat ographiccol umni spluggedatt hebasewi thhexanewashedgl asswoolorcot t

on
wool.15mlofhexanei sadded.1gm ofal umi na/silv
ernitrateispouredandal l
owedt oset tl
e.
Then2gmsofal umi nai saddedandagai nallowedt osettle.Thisisthenchar gedwi thal it
tl
e
anhy dr
oussodi um sul phate.Hexanei srunof finexcessunt iltheli
quidleveli satt hetopoft he
column.Theconcent r
atedor ganiclayer( containingpesti
cidesordr ugsast heact iveconstit
uents)
i
saddedwi thri
nsingt ot het opoft hecol umn.30mlofhexaneordi et
hylet heroranysui tabl
e
organicsolvent(ast hecasemaybe)i spassedt hroughthecol umnandt heel uteiscollected.The
eluteisconcentratedf urt
hert o10ml .
Asi l
icacolumni st henpr eparedbyaddi ng2gmsofsi l
i
cat oapl uggedchr omat ographiccolumn
withal ayerofanhy droussodi um sulphat eatt hetop.Theconcent r
atedelut efrom thealumi na/
sil
vernitr
atecolumni saddedwi t
hrinsingt ot hesili
cagelcolumnandal l
owedt obeadsor bed.10
mlofhexaneordi ethyletheroranysui tableor ganicsolventorami xt
ureofsol ventsisaddedt o
thet opoft hecolumnandt heel ut
ei scol lected.Thisisdriedov eranhydr oussodi um sulphate
andev aporatedtodr ynessf orf ur
theranal ysis.

3.
10.
3 Cl
eanupbyaSi
mpl
eCol
umnChr
omat
ogr
aphi
cmet
hod:

Themet hodcanbepr ofi

tablyempl oy
edtopur if
yt hemateri
alafterextr
actionfr
om bi ol
ogical
material
sforpest i
cidesordr ugs.Theconcent r
atedextr
act( or
ganiclayer)iscleanedupby
passingthr
oughachr omat ographiccol
umn( dia.2.
5cm)containi
ngfrom topaluminalayerof5cm,
acti
vatedcharcoall
ay erof2.5cm (middlel
ayer)andaanhy droussodium sulphatel
ay erof2.
5cm
(bot
tom layer
)pr ev
iouslyarrangedaf t
erpluggingt hechr
omat ogr
aphiccolumnandwashi ngby
 hexaneordiet
hylet
heroranysuitabl
esolventormixt
ureofsol
vent
s(asthecasemaybe).The
el
utionismadewi thappropr
iat
eor gani
csol v
ent
.Theel ut
eisdriedov
eranhy dr
oussodi
um
sul
phateandevapor
atedt
odr y
ness.

3.
10.
4 Cl
eanupusi
ngSol
i
dPhaseExt
ract
ion(
SPE)Car
tri
dges:

Thereareavari
etyofcart
ridgesavai
lablev i
z.BondEl ut
ef r
om Analyt
icChem I nt
ernat
ional
.The
cart
ri
dgeisfi
l
ledwithachemi cal
l
ymodi fiedsil
icaadsorbentandtheappropriateoneisselect
ed
accordi
ngtot
henatureofthemateri
al(toxicant
)viz.aminopropy
lforor
gano-chlorocompounds.

Pr
ocedur
e:

Aselectivecartr
idgei stakenwit
hanadapt erfi
ttedont op.A10mlgl asssyri
ngeisfi
tt
edont othe
adaptor.5mlofmet hanolisputint
ot hesyr
inge.Itispassedthrought hetube.Thetubei sthen
washedwi t
h5mlofhexane.Thesy ri
ngeisdetached.Theconcent ratedorgani
clayercontai
ning
acti
veconst it
uenti saddedt ot het opofthet ubeandal l
owedt opass.Thecl eanextractis
coll
ected.Thet ubewi ththesyri
ngei swashedwi t
hthesamesol vent .Thi
sisaddedtot heclean
extr
actasabov e.Thecombi ned layerisdried overanhy dr
oussodi um sul
phate.Itist hen
evaporatedtodr yness.

3.
10.
5 Cl
eanupbyPr
epar
ati
veTLCMet
hod(
14)
:

Thisisapplicableift
hei denti
tyoft oxi
cantistobeknown.Ther eafter
,byscr eeningoftheTLC
plate devel
oped by appl yi
ng similarconditi
ons i
.e.developi
ng solv entsy stem,adsorbent
,
temperatureetc.Thespot scontai
ningt heact
ivecomponentsseparatedatdi f
ferentRfval
ueson
thedev el
opedpl at
esarescr appedof f
.Thescrappedmateri
alsatdifferentzonesar eel
utedwith
select
iveorganicsolv
ent.Thisiscollect
ed,dri
edoveranhydr
oussodi um sulphateandevaporated
todryness.

3.
10.
6 Cl
eanupbyHPLCMet
hod:

Int
hismet hodal so,theident
it
yoft oxicanti
stobeknownbynot i
ngret
enti
onti
meunderspeci
fi
c
chr
omat ographiccondi t
ionsviz.solventsystem,f
low r
ate,col
umnetc.AgainthesameHPLC
methodi sempl oy edandt heseparatedcomponentsatv ar
iousret
ent
ionti
mes( asest
abl
i
shed
ear
li
er)arecollectedforfurt
heranalysi
s.

Dif
ferentmethodsofext r
action/i sol
ati
onoft oxi
cantsindiver
semat r
icesandtheirst
ri
ppi
ng
methodshav ebeendescr ibedint hepreviousparagr
aphs.Themet hodsaret obeempl oy
ed
dependingont henatureoft oxi
cant,matri
cesandav ai
labi
l
ityofinf
rast
ruct
urefaci
li
ti
es.Int
he
for
thcomingchapter
st heanalysi
sofdiff
erentcl
assesoftoxi
cantswil
lbedescr
ibed.

Ref
erences:

1.Appl
i
cabl
eNot
eNos.
320&321onASE,
Diones,
USA.

2. BoughPJ,GasChr
omat
ogr
aphy–Apr
act
icalappr
oach,
p-174,
1st edi
ti
on,
Oxf
ordUni
ver
sit
y
Press,
NY,
1993.

*
3.Cur
ry,
Alan.
,Poi
sonDet
ect
ioni
nHumanOr
gan.3r
dEdn.Char
lesC.Thomas,
Spr
ingf
iel
d,19767.

4. Lundqui
st,
Frank.
,Met
hodsofFor
ensi
cSci
ence,
Vol
.I
-I
V,I
nter
ncl
encePubl
i
sher
s,N.Yor
k,1962.

5. Cl
arke,
E.G.
C.,
Isol
ati
onandI
dent
if
icat
ionofDr
ugs,
2ndEdn.ThePhar
maceut
icalPr
ess,
London,
1986.

6. Bamf
ord,
F.Poi
sons,
Thei
rIsol
ati
onandI
dent
if
icat
ions,
J/A/
,Chur
chi
l
lLt
d.,
London,
1940.
7. Stewart
,C.P.andSolman,
A.,
Toxi
col
ogy–Mechani
sm andAnal
yti
calMet
hods,
Vol
.2,
Academi
c
Press,N.York,1961.

8. St
ars,
J.S.
,Bul
l
.Acad.RoyMed.
,11,
304,
1851.

9. Ot
to,
J.,
Anal
.Chem.Phar
m.,
100,
39,
1856.

10.Jones,G.
R.andSi
nger,P.P.i
nAdv
ancesi
nAnal
yti
calToxi
col
ogy(
Bassal
t,R.
G.,ed.
),Vol
.2,Year
BookMedical
,Chi
cugo,1989.

11.Whelpt
on,R.i
nAnal
yti
calMet
hodsi
nHumanToaxi
col
ogy(
Cur
ry,A.
S.–ed)
,Macmi
l
lan,London,
1984.

12.Middl
edi
tch,B.
S.,Analy
ticalAr
ti
fact
s.GC.
,HPLC,TLC andPC,J.Chr
omat
ogr
.Li
br.
,Vol
.44,
El
sevi
er,
Amster
dam, 1989.

13.A.
O.A.
C.,
Off
ici
alandTent
ati
veMet
hods,
1980.

14.Ghosh,
S.andBagchi
,K.
N.,
Organi
candTaoxi
col
ogi
cal
Chemi
str
y,Ar
tPr
ess,
Cal
cut
ta,
Indi
a,1948.

15.Maehl
y,Andr
eas,
Chemi
cal
Cri
minal
i
sti
cs,
Spr
inger–Ver
lag,
Ber
li
n,1981.

16.St
ahl
,E.Thi
nLy
erChr
omat
ogr
aphy
,2ndEdn.Spr
inger–Ver
lag,
Ber
li
n,1969.

17.Gour,
T.H.(
Ed.
),Gui
det
oModer
nMet
hodsofI
nst
rument
alAnal
ysi
s,Wi
l
ey.I
nterSci
ence,
N.Yor
k,
1972.

18.Jenni
ngs,
Wal
ter
,Anal
yti
cal
GasChr
omat
ogr
aphy
,Academi
cPr
ess,
Inco.
,N.Yor
k,1987.

19.Ewi
ng,
Gal
enwood(
Ed)
,Anal
yti
cal
Inst
rument
ati
onHandbook,
Mar
cel
Dekker
,Inc.
,N.Yor
k,1990.

20.Set
hi,
P.D.
,Hi
ghPer
for
manceThi
nLay
erChr
omat
ogr
aphy:
CBSPubl
i
sher
sandDi
str
ibut
ors,
Indi
a,
1996.

21.Gaugl
it
z,Gunt
erandVO–Di
nn,Tuan(
Eds)
,HandbookofSpect
roscopy
,Wi
l
eyVCH,Ger
many
,
2003.
 SECTI
ON–4: GASEOUSANDVOLATI
LEPOI
SONS

4.
1 Ti
tl
e: Anal
ysi
sofgaseousandv
olat
il
epoi
sons.

2 Scope: Sy st
emati
canalysi
sofgaseousandvol
ati
l
epoisonsinvar
ious
t
ypeofformul
ati
ons,
biologi
cal
mat er
ial
sandnon-bi
ologi
cal
mat
ri
ces.

3 Pur
pose: Toi
dent
if
yandest
imat
egaseousandv
olat
il
epoi
sons.

4.
4 Responsi
bil
i
ties:
Gazet
tedof
fi
cer
sandassoci
atedsci
ent
if
icst
aff
.

4.
5 VOLATI
LEPOI
SONS:

Someoft
hev
olat
il
epoi
sonswi
tht
hei
rchar
act
eri
sti
csar
egi
venbel
ow:
-.

TABLE–4.
1

Name Sy nony m &Source Phy sical Proper ties Ot herChar act eristics
Acet
aldehy
de Acet ic aldehydeCol our l
ess, i nflammabl e Met abol ized t hrough
ethanal , Et
hyll
iqui d, B. P. 20. 2oC, oxi dat i
on t o aci d and
aldehy de. mi sci ble wi th wat er, ev ent uallyt o
Sour ce:By -
productinethanol andet her. car bondi oxide and
alcohol i
c wat er.Mor et oxi ct han
ferment ati
on. ethanol oracet icaci d.
Acet
one Cet ona, DimethylA cl ear, col ourless, Lar ge amount s ar e
Ket one, volat ile, i
nflammabl e excr eted unchanged,
2-Pr opanone. l
iqui d, B.P.56oc butsmal ldosesmaybe
Sour ce:A productof Mi sci ble wi th wat er, oxi dized t o car bon
dest ructi
ve ethanol ,chl or ofor m and di oxi de.Acet onei st he
distill
ati
onofwood,a ether . mai n met abol ite of
by-pr oduct i n t he i
sopr opyl alcohol ,
ferment ati
on of occur s nat ur ally i n
starch. blood and ur ine of
diabet i
cs, abused i n
gluesni ff i
ng.
Ani
l
ine Aminobenzene, Liqui d,col orless buton I ntoxi cat i
on occur s by
Pheny l
ami ne, keepi ng t urns t o br own i nhal ation,i ngest i
onor
due t o f ormat ion of cut aneous absor pt ion.
nitroso compound . Toxi c sy mpt oms
B.P. 1840C.Mi sci blewi th incl ude cy anosi s,
alcohol , benzene, met hemogl obi namea,
chlor oform and ot her v er tigo headache and
organi c solvent s. conf usion.
Insol ubl einwat er.For ms
saltswi thmi neral acids.
Benzene Pheny l
hy dri
de A cl ear, col ourless, Benzene i s absor bed
Source: In l i
ght oi
linf lammabl e li
qui d, from the gast ro-
fr
action obt ai
ned by char acteristi
c i
nt est inal t ract ,t hr o-
di
sti
llati
onofcoal t
ar. aromat ic smel l, B. P. - ugh ski n and l ungs.
80oC, pr acti
cal ly 50%ofi nhal edbenzene
i
mmi scible wi th wat er, maybe el imi nat ed v i
a
mi sci blewi t
hdehy drated t he lungs; smal l
alcohol , acet one, et her amount i s excr et ed
andgl acialacet icaci d. unchangedi nt heur ine
 mai nl
y as phenol ,
catecholand quinolin
theconjugat
edform.
Benzy
lal
cohol Pheny l
carbinol, Acol ourl
essli
qui
dwi tha Use:
Pheny l
met hanol fai
ntar omati
codour,B.P. Local anaesthet
ic
Source:As est erof - Disinf
ectant
.
cinnamic acid 203oC.Sol ubl
einwat er,
&benzoic aci d i n met hanolchl
orof
orm and
Balsam ofTol u and ether.
Peru.
Car
bon Carbon di sulphide, Acol ourlessl i
qui dwi tha Usedi nt hesy nthesi
sof
di
sul
phi
de Carbonicanhy dride faintaromat icodour ,B.P. diff
erent
Source:Coal tar. -203- 208oC sol ublei n dit
hiocar bamat es.
wat er, mi scible wi th
ethanol,chl orof orm and
ether.
Carbon Carboneum Aheav y,clear,col ourless Non- i
nflammabl e, I n
tet
rachl
ori
de tetr
achloratum l
iquidwi thchlor oforml i
ke contactwi t
haf l
amei t
medi ci
nale odour ,v ol
atil
e. Al most decomposesandgi v es
Source:Sy nthesisby i nsol uble in wat er,toxic productofacr id
chlori
nati
on of mi scibl
ewi thdehy drated odour , absor bed
hydrocarbonet c. alcohol,chl orof orm and t hrough ski n and by
ether. i
nhal ati
on. Ext racted
most lyf rom l ungs as
such,i nur ineasur ea.
Nephr otoxic.

Chl
oral
hydr
ate Aquachl oral, Col ourless or whi t
e Readi l
y absor bed by
Chl oradom, Chlo- cr ystals, i t l i
quef i
es or al admi nistration,
ralex, Dor mel , El ix- bet ween50oC andboi l
s r eadilymet abol i
sed by
Noct e Rec- tules(-t o at98oC,sol ubl ei net her,r eduction to
be checked) ,Mi cky , chlorof orm and par t
ially tri
chloroet hanol and
Finn, Knock- out sol ubl einwat er. fi
nallyt ot ri
chl oroacet ic
drops. Ethy lalcohol-sol ubil
ity? aci dbyst eps.
Sour ce: Synt hetic Used as hy pnot i
c as
well assedat ive.
Chloroform Chl oroformi um, A col ourl
ess v olat i
l
e Absor bed by or al
Anaest hesicum, l
iqui d B. P.-about61oC, admi nistration and
Chl oromor mum, Pr o insol ubl e in wat er,inhal ati
on.I tisexhal ed
nar cosi mi sciblewi thdehy dr ated unchanged bet ween
alcohol andet her . 20% t o 70%.Gener al
anaest hetic.
Cresols Synony m: An al mostcol ourlesst o Absor bed after
(Ami xtureofo-
,m Cresy li
c Aci d, pale br owni sh y ellow ingest ion and t hrough
-&p-cresols) Tricresol ,Pr opr i
etar y l
iqui d becomi ng dar ker ski n and mucous
Name-Ly sept ol. withageoronexposur e membr ane. I t i s
Sour ce: Occur s i n t o li
ght. Al most met abol i
sed by
coalt ar,inpi neand compl etely sol uble i n conj ugat i
on and
bleachedwoodt ar,in wat er, mi scibl e wi t
h oxi dation.
Lysol ( cresol and et hanol ,chl or oform and
soap solution et her.
cont aining 50% v / v
cresol ).
EthylAlcohol Ethanol , Methy l Transpar ent,col ourless Burnswi thbl uef lame.
Car binol, Spi ri
t of andv olatil
eliqui d,hav ing Usedassol v ent,
Wi ne. spirit
uousodourand conv ertedi ntoal dehy de
 Sour ce: As f r
ee burningtast
e. andaceti
cacid.
alcoholi nsomef r
uit Hygr
oscopic,boi
lsat Absol
utealcohol
-99.
95
j
uices, as est er in 78.
40C. %ofalcohol.
some eucal yptusoi l
,
produced by
ferment ati
on of
starch, mol asses,
grapeset c.
For
mal
dehy
de Formi c aldehyde, Acolourl
essgas,solubl
e Inf l
ammabl e,irritantt o
Met hylene oxi
de, i
n water,sli
ghtl
ysolubl
e mucous membr ane,
Oxamet hane i
nethanolandether. i
nhal ation thr ough
respi rator y t r
act may
cause br onchi t
is and
pneumoni a, rapi dly
met abol i
sedi nt hebody
tissue t of ormi c aci d
andmet hanol .
Hydrocyanicaci
d Hy dr ogen cyani de Smel lli
kebi tteral monds. Haemogl obi n is
prussi caci d Saltsar ehi ghlysol ubl ei n conv erted t o cy ano
Sour ce: wat er and al kaline i n haemogl obi n.
Nat ur al: Cher ry, natur e. Const riction oft hroat,
apr i
cot ,peachbear s, dizzi ness, verti
go.
bitter al
mond,
Cy anogenet i
c
glycosi des.
I
sopropyl al
cohol Dimet hylcarbinol , 2- A cl
ear , col our l
ess I nf l
ammabl e, r eadi ly
Propanol .Pr opr ietary v ol
at ilel iqui d B. P.-81 – absor bed by or al
names - Al cojel, 83oC, mi scible wi th admi nistrat ion, sl owl y
Av ant ineSt er ets. wat er, et hanol, met abol i
zed than
Sour ce: chlor oform andet her . et hanol , l
ar gely
Fer ment ation. conv erted t o acet one,
then t o acet ate and
for mat e.
Kerosene Coal oi l
,Var sol . Oil
y liquid of Inf l
ammabl e,
Sour ce: By char acter i
stic odour , consumpt ion or
fr
act ionat i
on of boi li
ngr ange: exposur e causes
pet roleum. 150- 300oC. i
r rit
at i
on t o ey es,
headache and bl ur r
ed
vision.
Methylalcohol Met hanol , Car binol, A col ourless l iquid,B. P. Ext remel y poi sonous
woodspi rit
. 64.7oC,mi xeswi thwat er &causesbl i
ndnessand
Sour ce: The l iquid and or gani c sol vents, ev en deat h.
fr
act ionpy rol i
gneous pecul iar odour and a Met abol i
sed by
acid i
n the bur ni ngt aste. oxi dation to
dest ruct ive for mal dehy de, f ormi c
distillation of wood aci d&f inal l
yf ormat e.
cont ai nsmet hanolas
a maj orconst ituent .
Can be sy nt hesi zed
commer ciall
y .
Naphthalene Mi ddl eoi lfract i
onof Col our l
ess t ranspar ent Bur ns wi th a smoky
coal tardi sti
llation. scales,M. P. -80oC,B. P.-f lame, used as
208oC, pract i
cally i
nsect icide and i nt he
i
nsol ubl einwat er,solubl e pr epar ation of dy es
i
n ethanol and such as i ndigo az o-
Chlor ofor m, highly dy es.
 solublei net her .
Ni
tr
obenzene Nit
robenzol, oil of A pal ey ell
ow oi l
yl i
qui d, Usedf orpr eparationof
Mirbane practical l
y i nsol ubl e i n ani li
ne.Poi sonous.
Source:produced by wat er,mi sci blewi thet her
ni
trati
onofbenzene. and met hanol , B. P.-
206oC, char acter i
st i
c
odourofbi tteral monds
(shoe- polish) .
Par
aldehy
de Paracetaldehy de Acl earcol our lessorpal e Used as hy pnot ic,
Source: yell
ow l i
qui d,B. P.123- r eadily absor bed on
Polymer i
zation of 126oC,sol ubl ei n wat er, admi nist ration,
acetaldehyde. miscibl e wi th et hanol , met abol ised to
chlorof orm andet her . accet aldehy de by
depol ymer isation&t hen
oxidizedt oacet icaci d
and f inal ly car bon
dioxide.
Phenol Carbolic acid,Fenol, Colour lessorf aint l
ypi nk, Phenoldenat ures and
Pheny lhydrate. deli
quescentcr y stals or pr ecipitat es cell
ular
Source: Li ght oi l crystalline mass,whi ch pr otein, whi ch may
fr
action of coalt ar becomespi nkonst or age, r apidly cause
dist
il
lati
on. B.P.181oC, partially poisoni ng. Rapidl y
solublei nwat er,sol uble absor bed t hrough G. I.
i
n et hanol , chl or oform t ractorpenet r
ateski n,
andet her. met abol ised t o pheny l
glucur onideandpheny l
sulphat e and smal l
amount s ar e oxi di
sed
to cat echol &qui nol
(conjugat edf orm) .
Tur
pent
ine OilofTur pentine,Oi
l Colour less oi l wi th Absor bedt hroughski n
ofPine peculiar odour , orbyi nhal at i
on.
Source: Pi
net ree. i
mmi sci blewi thwat erbut
miscibl e wi th al cohol ,
etherandchl orofor m.

4.
6 METHODOLOGY:

Thedisti
l
lat
ionmethodsf orisol
ati
onofv ol
ati
lepoisonhaveal
readybeendescribed(sect
ion-
3).
Thedisti
ll
atesintwof r
actions,aci
dst eam di
sti
ll
ateandalkal
inesteam dist
il
latearekeptto
under
takesy st
emat
icscreeningandotherdet
ail
edanalysi
sasmentionedher
eunder.

4.
6.1 Pr
eli
minar
yscr
eeni
ngofVol
ati
l
ePoi
sons:

Thescreeni
ngisdonei
nthr
eest
epsv
iz,
a) Smell
b) Col
ourtests
c) UV-spectra

Asmallport
ionofthedi
sti
l
latemayal
sobesubj ectedtoGC/ GCHeadspaceexami nat
ionandt he
ret
ent
ionti
meoft hepeakscanbecompar edwitht hest
andardref
erencesavail
ablei
nl i
ter
ature
andconfi
rmedbytheinj
ecti
onofacontr
ol.Forthispurpose,nor
malFlameIonizati
onDetectoris
usedandthecol
umnsusednor mal
l
yareCar bowaxorHellcomid.

λmax(
nm)v
aluesofsomecommonl
yknownv
olat
il
einwat
erar
egi
venbel
ow:
-
 TABLE–4.
2

Nameoft
hepoi
son λmax(
nm)

Anil
ine 280
Benzene 228,223, 238,242,248,254,266.
Camphor 282
Cresol 273
Kerosene 270( mayv arydependingonint
erbat
ch
vari
ati
ons)
Methy lsali
cyl
ate 238, 303
Napht halene 265,275, 283,206
Nitr
obenzene 274
P-Dichlorobenzene 270
Phenol 264, 271,279
Thymol 270

Col
ourt
est
sofsomecommonl
yknownv
olat
il
epoi
sonsar
egi
venbel
ow:
-

TABLE–4.
3

S.
No Reagent Obser
vat
ion Probabl
epoisons
1. Addit
ionofbr
omi
newat
er Decol
our
ati
on Anil
ine,
Phenol,Cr
esol

2. Addit
ionofMi l
l
on’
sReagent Red or Or ange r
ed Ani
l
ine,
Phenol
,Cr
esol
&heati
ng colour
ati
on
3. Addit
ionofAgNO3 Grey/Blackppt
. Cyanides,
Phosphor ous,
Phosphi ne.
4. Ani
li
ne + Al
cohol
i
c NaOH + Obnoxi
oussmel
l Chloroform, Chl oral
Heat hydrate, Carbon
tetr
achloride.
5. Pyr
idi
ne+Al
cohol
i
cNaOH + Redcol
our
ati
on Chloroform, Chl oral
Heat hydrate, Carbon
tetr
achloride.
6. Di
rectSchi
ff
’sReagent Rest
orat
ionofpi
nkcol
our Acetaldehy de,
Formal dehy de
7. Schif
f’
sReagentaddedaf
ter Rest
orat
ionofpi
nkcol
our Ethanol, Methanol,
Oxidat
ion Acetaldehy de,
Formal dehy de.
8. Dir
ect addit
ion of Pi
nk-
viol
etcol
our Formal dehy de.
Chromot ropicacid
9. Chromot ropic aci d added Pi
nk-
viol
etcol
our Methyl
alcohol and
aft
eroxi dati
onofsampl e For
maldehy de.
10. Chloroform +Al cohol
icNaOH Red col our
ati
on wi
th Pyr
idi
ne.
+Heat obnoxi
oussmel
l
11. Dir
ect addi ti
on of Deni ges Yel
lowppt. Acet
one.
Reagent+Heat
12. DenigesReagentaddedaf t
er Yel
l
owppt
. I
sopropyl al
cohol
,
oxidati
on Acetone.
13. Lead Acet ate + KOH soln.+ Bl
ackeni
ng Carbondi
sul
phi
de
Formal dehyde+Heat
14. PrussianBlueTest Bl
uepptorcol
our
ati
on Cy
ani
de.
4.
6.2 I
dent
if
icat
iont
est
sforv
olat
il
es:

Inbi
ologi
calmater
ial
s(v
iscera),
stomachcont
ent
s,ur
ine,
isol
ati
onbyaci
d-di
sti
l
lat
ionasdescr
ibed
earl
i
er.Thedi
sti
l
latei
ssubjectedtothef
oll
owi
ngtest
s:

4.
6.2.
1 Test
sforacet
one:

1. I
odof
orm Test
:

A. Wi t
hI 2+NaOH:
To1ml .oft hedisti
ll
ate,af ew dropsof10% NaOH i sadded.Thisisf ol
l
owedbydr opwise
addi
tiont i
llthesoluti
onbecomesbr own.Themi xtur
eiswar medonl ow f
lame.Af ew dropsof
NaOHsol uti
onar eaddedtochanget hecol
ourofthesolut
ionfr
om browntoyell
ow( addi
ti
onofa
few dropsofi odi
nesoluti
oni srequired,i
fonwar mingthesolut
ionbecomescol ourl
ess).I
tis
cool
ed.Thepr eci
pit
ateisobser vedundermicroscope.Cryst
alsarefor
medof tenonst andi
ng.
Characterist
ichexagonalcr ystal
sofi odof
orm areseen( al
sopositi
veinpresenceofet hanol
,
acet
aldehy de,iso-
propanol)
.

B. WithI
2+NH4OH:
Posi
ti
veonlyi
ncaseofacet onebyusingNH4OH,inplaceofNaOH,asabov
e,i
.ehexagonal
cr
yst
alsofi
odof
orm ar
elocat
edbymicroscopi
cal
exami
nation.

2. Legal
’
sTest:

To1ml .ofthedist
il
late,2dropsofsatur
atedsoluti
onofsodium ni
tr
oprusside(fr
eshlyprepar
ed)
and1dr opof10%sodi um hydr
oxideisadded.Ar edoryel
l
owishredcol ourisproduced.Af ew
dropsofglaci
alacet i
cacidi sthenaddedtoaci dif
ythesolut
ionwhichchangest hecolourto
carmineorpurpl
i
shred.Subsequent l
ythesolut
ionisheat
edwhichproducesv i
oletcolour
.

4.
6.2.
2 Testf
oracet
aldehy
de:

I
tcanbeisol
atedfr
om ti
ssueandotherbi
ologi
calmat
eri
albyaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
emay
besubj
ect
edtothefol
l
owingtest
s.

1. I
odof
orm Test
:

Wi
thI
2+NaOH–Posi
ti
ve(
Alsogi
venbyet
hanol
isopr
opanol
,acet
one)
.

2. Schi
ff
’sr
eagentTest
:

To1ml .ofthedisti
ll
ate,
2ml .ofSchiff
’sreagent(decolouri
sedbasicf
uchsine)isaddedandkept
for30mi nutes.A purplecolourisobt ai
ned( for
mal dehydealsogivespur pl
ecolour).Tothe
developedpur pl
ecolour0.5ml .ofconc.sulphur
icacidi sadded.Thepurpl
ecolourdisappear
s,i
f
acetaldehydeispresent.

3. To1ml.oft
hedisti
l
late,af
ewdropsoffr
eshl
ypr
eparedsodi
um nitr
opr
ussi
desoluti
onand
pi
per
idi
near
eadded.Theformat
ionofbl
uecol
ouri
ndi
cat
esthepresenceofacet
aldehy
de.

4.
6.2.
3Test
sforf
ormal
dehy
de:

For
mal
dehydeisisolat
edf
rom bi
ologi
calmat
eri
alsbyaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
ed
tot
hef
oll
owingtests.
 1. Schi
ff
’sr
eagentTest
:

To1ml .oft
hedist
il
lat
e,2ml.ofSchi
ff’
sreagenti
saddedandkeptasi
defor30mi nut
es.Purpl
e
colourisfor
med.Ifthecol
ourper
sist
sev enaft
eraddi
ti
onof0.
5ml .ofconc.sul
phuri
cacid,t
he
presenceoffor
maldehydei
sconfi
rmed.

2. Chr
omot
ropi
caci
dTest
:

To0.5ml.ofthedistil
l
ate,5ml.ofChr
omotr
opicaci
dsolut
ion(pr
eparedbydissolvi
ng0.5mg.of
sodi
um saltofChromot r
opicaci
din100ml.ofConc.sul
phuri
cacid)i
sadded.Thecont entsare
heat
edonahotwat erbathat60oCfor30minutesandthencooled.Vi
oletcolourisproduced
(λmaxofthecolouredcomplex=570mµ).

4.
6.2.
4 Testf
oret
hyl
alcohol
:

Ethy
lalcoholi
sisol
atedf
rom bi
ologi
calmat
eri
alsbyaci
ddi
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
o
thefol
l
owi ngt
ests.

1. I
odof
orm Test
:

Wi
thI
2+NaOH–Posi
ti
ve(
alsogi
venbyacet
oneacet
aldehy
deandi
so-
propanol
).

2. Sul
phomol
ybdi
cAci
d–(
1gm ofmol
ybdi
caci
din25ml
.ofConc.sul
phur
icaci
d):

To2ml .ofthedi stil

late,2ml.ofthehotr eagentisadded.Adeepblueri
ngappearsatonceatthe
j
unct i
onoft wol iquids.Onshaki ng,thewhol emi xt
urebecomesdeepbl ue.Itindicat
esthe
presenceofet hylalcohol( Thist
estisv erysensiti
veandisnegati
vewithacetone,acetal
dehy
de
anddi l
utesol utionofmet hylal
cohol
.St rongsolutionofmethylal
coholgi
vesonl yal i
ghtbl
ue
colouraftersev eralmi nutes)
.

3. Et
hyl
Benzoat
eTest
:

2dr opsofBenzoylChlori
deareaddedt o2ml .ofthedi
sti
ll
ate.Themixtureismadealkali
neby
adding10%sol ut
ionofsodium hydr
oxidedropbydr op.Onwar ming,i
rri
tat
ingsmellofBenzoyl
Chlori
dedisappearsandt hesweetfr
uityodourofethylbenzoateappears(methy
lalcoholal
so
gi
vest hi
stestbutitdoesnotgi
vetheiodof
ormt est
).

4.
6.2.
5 Test
sfori
sopr
opy
lal
cohol
:

Theaci
d-di
sti
l
lat
ionisusedt
oisol
atei
sopropylal
coholf
rom t
issueandot
herbi
ologi
calmat
eri
al.
Thedi
sti
ll
atei
ssubject
edtot
hefol
lowi
ngtests.

1. I
odof
orm Test
:

Withiodi
ne+NaOHSoln.
: Posi
ti
ve(
presenceofi
sopr
opy
lal
coholal
ongwi
thacet
aldehy
de,
acetoneandet
hanol
).

2. 2ml .ofdisti
ll
ateist akeni
neachoft wot esttubes.3dropsofKMnO4i nphosphor icaci d(by
dissol
v i
ng3gms.ofKMnO4and15ml .ofsy rupyphosphor i
cacidin85ml .ofwater)inoneoft he
testtubes.Thi sisal l
owedt ost andfor5mi nutes.Thecolour,ifanyleftafteroxidationi s
decolourisedwithapi nchofsodi um bisulphi t
e.1mlof10%NaOHand1ml .of5%f urf
ur alare
addedt obot hthet esttubes.Thecont entsofeachoft hetesttubesarefil
teredint
ot estt ubes
containi
ng2ml .ofconc.hy drochlori
caci d.Api nkr i
ngisformedatt hej uncti
on,(containing
oxidi
sedpr oductbyKMnO4)i ndicatesthepr esenceofi sopr
opanol.Apinkcolourintheot hertest
tubeindicatespresenceofacet one.
 4.
6.2.
6 Testf
ormet
hanol
:

I
sol
ati
on:
Byaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
othef
oll
owi
ngt
est
s.

1. Schi
ff
’sr
eagentTest
:

To4-5ml .oft hedi st

il
lat
e,0. 5ml .ofethylalcohol,2ml.of3%KMnO4sol uti
onand0. 2ml .Of
phosphori
caci dar eaddedandkeptasi def or10mi nut
es.The1ml .of10%oxal icacidisadded
foll
owedby1ml .ofconc.sulphuricacidandt hecont ent
sarecool edtoroom temper atur
e.5ml .
ofSchiff’
sr eagenti saddedt oi tandt hecol ouri sobserv
edaf ter30mi nutes.Purplecolour
appearsifmet hanolispresent( confi
rmst hepr esenceofmet hanolinpresenceofet hanol).The
blankrunisal somadesi debysi de.Thequant i
tationmayalsobemadebymat chingtheintensit
y
ofcolourdev elopedwi t
hdifferentknownst andar dsofmethanol.

2. Chr
omot
ropi
cAci
dTest
:

To0. 5ml
.ofthedisti
ll
ateinat estt ube,0.2ml .of5%pot assium permanganat esolut
ionisadded.
Af t
er5 minutes,saturat
ed sol ution ofsodi um bisulphit
ei s added dr op bydrop unti
lt he
permanganatecolourisdischar ged.Ifabr owncol ourst i
llper
sists,adropofphosphor icaci
di s
againaddedt oitadr opofsodi um bi sulphit
esol ut
ion.Tot hiscol our
lesssoluti
on,0.5ml .of
freshl
yprepar
edchr omot r
opi caci dsol ut
ion( preparedbydi ssolving5mg.ofsodi um sal
tof
chromotr
opicacidin10ml .ofconcent r
atedsul phuri
caci dandheat i
ngonawat erbathat60oC
for30minutesandcool i
ngt hereafter.Av i
oletcolouri
sobser ved.

4.
6.2.
7 Testf
orchl
orof
orm:

Chlorof
ormisisolat
edf
rom bi
ologi
calmat
eri
alsbyaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
o
thefoll
owi
ngtests:

1. Fuj
i
war
aTest
:

To1ml .ofthedi
sti
ll
ate,1ml .ofpyr
idi
neand2ml .of20%sodium hy
droxidesol
uti
onareadded
i
nat esttubeandthi
si sheatedonawat erbat
hfor1mi nut
e.Apinktor edcol
ourindi
catest
he
pr
esenceofchlor
ofor
m( chl
orofor
m andpoly
hal
ogenatedcompoundsrespondtothi
stest)
.

2. To1ml.oft
hedist
il
late,
1ml .ofalcohol
i
csolut
ionofpot
assi
um hy
droxi
de(10%)isadded,
fol
lowedby1dropofanili
ne.Themi xt
ureisheat
ed.Adisagr
eeabl
eodourofphenyli
socyani
de
evolves.

3. To1ml .oft
hedist
il
lat
e,1ml.ofNessl
er’
sreagenti
sadded.Nobr
ownpr
eci
pit
atei
sfor
med
(
dif
fer
ent
iat
ionfr
om chl
oralhydr
ate)
.

4. To1ml.ofthedist
il
late,1ml.ofst
rongl
yalkali
nesol
uti
onof-napht
holi
saddedandt
hen
heat
ed.Abl
uecolourt
urningtogr
eenandf i
nal
lybrownisobser
ved.

4.
6.2.
8 Testf
ort
urpent
ine:

Turpenti
neisisolat
edbyaciddisti
ll
ati
on.Thedist
il
lat
ei scol
lectedinani
cecooledrecei
verand
ext
ractedfourtimeswi t
h5ml .port
ionofdiet
hylether.Thi
sext racti
sdr
iedbypassi
ngthrough
anhydroussodium sul
phateandkeptreser
vedf
orthef ol
lowi
ngt ests.

1. 2ml.ofetherext
racti
sevapor
atedtodrynessonaspot
ti
ngt
il
e.1dr
opofconc.sul
phur
ic
aci
disadded.Adeepreddi
shbrowncolouri
sproduced.

2. 2ml
.ofet
herext
racti
sev
apor
atedt
odr
yness.Tot
hisr
esi
due,af
ew dr
opsofMar
qui
s
 r
eagenti
saddedandwar
med.Api
nk-
redcol
ouri
sobser
ved.

3. 2ml
.ofetherext
racti
sevapor
atedtodry
ness.Tothi
sresi
due,2ml.ofethanol
foll
owedby1
ml.of1%Vanil
li
ninconc.HClisadded.Themixt
ureisheat
ed.Greencol
ourati
onturni
ngtobl
ue
i
sobserved.

4.
6.2.
9 Testf
orpar
aldehy
de:

Paral
dehydei
si sol
atedf
rom bi
ologi
calmat
eri
alsbyaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
o
thefol
lowi
ngtests.

1. 0.
5ml .ofdist
il
lat
eisaddedto5ml .ofchi
l
led2%sodium bi
sulphit
esoluti
oninanice-
bath.
To1ml .ofthechil
ledmixt
ure,1dropoffr
eshl
yprepar
edp-hy
droxydiphenylsol
uti
onisadded(1
gm i
n25ml .ofhot2N.NaOHi saddedanddil
utedto100ml.
).Violetcol
ourati
on(λ-
maxat560
nm)isobserved.

4.
6.2.
10Testf
orcar
bondi
sul
phi
de:

Carbondisulphi
deisisol
atedbyacid-
dist
il
lati
on.Thedist
il
lati
onoccur
sslowly(duetopresenceof
car
bondi sulphi
de).I
tisther
efor
ebettertocoll
ect250ml .intwofr
acti
ons(lat
terpor
ti
oncont ai
ns
much higherpr oport
ion ofcarbon di
sulphi
de,ifpresent).The di
sti
ll
ateissubject
ed tot he
fol
l
owingt ests.

1. Leadacet
ateTest
:

2dropsofleadacet
atei
saddedtothesoluti
ontobetest
ed.Anexcessofpotassi
um hydroxi
de
sol
uti
onisaddedandboi
led.Abl
ackpr
ecipit
atei
sobtai
nedifcar
bondi
sul
phideispresent
.

2. Thi
ocy
anat
eTest
:

Toapor t
ionofthedist
il
lat
e,1ml .ofammonium hydroxi
deand1ml.ofethylalcoholareadded.I
t
i
st henboil
edfor5mi nutes,concentr
atedt
o1ml .andacidi
fi
edwithdil
utehy dr
ochlori
cacid.1
dropofferr
icchlor
idesoluti
onisadded.Thef ormationofredcol
ourindi
catest hepresenceof
carbondi
sulphi
de.

3. Cast
igl
i
oni
sTest
:

To1ml .ofdi
sti
ll
ate,1ml.ofal
coholi
csolut
ioni
sadded.Thef
ormat
ionofy
ell
ow pr
eci
pit
ate
i
ndi
cat
esthepresenceofcar
bondi
sulphi
de.

4.
6.2.
11Testf
orcar
bont
etr
achl
ori
de:

Carbont
etr
achl
ori
dei
sisol
atedbyaci
ddi
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
othef
oll
owi
ng
test
s:

1. Py
ridi
neTest
:

5ml .of30% NaOH and5ml .ofpur i

fi
edpy ri
dineareaddedt o10ml .ofdisti
ll
ate.I
tisgent
ly
heatedfor5to10mi nut
esonast eam bath.Api nkredcolourindi
cat
espresenceofcarbontet
ra
chlori
de(al
sogiv
enbychl or
oform,chlor
alhydrateandotherchlori
nat
edcompoundssuchasDDT,
tr
ichlor
oethanol
).I
fthedisti
ll
atei
sv erydi
lute,ext
ract
ionforcarbontet
rachlor
idemaybemade
withn-hept
aneandtestcarr
iedoutwiththeresidueofextr
act).

2. Pheny
lIsocy
ani
deTest
:

To10ml
.oft
hedi
sti
l
lat
e,1ml
.ofdi
sti
l
ledorpur
if
iedani
l
ineand2ml
.of20%sodi
um hy
drox
ide
 areadded.Themi xt
ureisheatedforafew minutes.Theevolut
ionoffoulsmel
lofphenyl
i
socy ani
deindi
cat
espresenceofcarbontet
rachlori
de(chl
oralhydrat
eandchlor
ofor
m al
so
respond).

4.
6.2.
12Test
sforchl
oral
hydr
ate:

Chl
oral
hydr
atei
sisol
atedbyaci
d-di
sti
l
lat
ion.Thedi
sti
l
lat
eissubj
ect
edt
othef
oll
owi
ngt
est
s.

1. Fuj
i
war
aTest
:

Asst
atedi
ncaseoft
estf
orchl
orof
orm.

2. Nessl
er’
sreagentTest
:

Afewdropsofthereagentar
eaddedtoafewdropsofthedi
sti
l
lat
e.Ay el
l
oworr
eddi
shbr
own
pr
eci
pit
atechangi
ngtogreyorbl
acki
sfor
med(negat
ivei
ncaseofchlor
ofor
m).

3. Resor
cinol
–Pot
assi
um br
omi
deTest
:

To2dr opsofreagent(
0.2gm ofresorci
noland1gm ofpot assium bromidein10mlofwat er)
,2
mlofconc.sulphuri
cacidisaddedcarefull
yf ol
lowedby2mlofdi st
il
late.Thi
sisthenheatedon
awat erbath.Changeofcolourisobser v
edasy ell
ow t
opi nkandf inall
yt oviol
etat900C.At
1000Cbluecolourappearswhichturnstoor ange-redbyaddi ng2mlofwat er.Onaddingstr
ong
KOH soluti
on,thecolouragainchangest ov i
oletandf inallytoar eddi
sht i
nt(notgiv
enby
chl
oroform).

4.
6.2.
13Test
sforbenzene:

Theaciddist
il
lat
ionisusedtoisol
atebenzenefr
om ti
ssueandotherbi
ologi
calmater
ial
s.The
disti
l
lat
eisext
ractedwit
hdiet
hylet
herandt heet
her
ealext
racti
sdriedoveranhy
droussodi
um
sulphat
e.

1. 2ml .ofnitr
atingmixt
ure(1ml .ofConc.nitr
icaci
d+1ml .ofconc.sulphur
icacid)i
scooledin
anicebat handthenaddedt or esi
dueobt ainedaft
erevaporati
onof2ml .ofetherealextr
actfr
om
thedi
stil
late.Thecontentsar
et r
ansferr
edt oahardglasstesttubeandt henheatedbykeepingit
i
nahotwat erbath.Afterthatt hecont entsarecool edanddi l
utedwi thwat er.Duetot he
for
mationofni tr
obenzene,bi
tteralmondodouri sobtained.

2. Thepr
ocedur
easaboveistofor
m nit
robenzenebynit
rat
ion.Thefol
lowi
ngtestmaybecar
ri
ed
outt
oconfi
rmthepr
esenceofni
tr
obenzenei.
e.thepr
esenceofbenzeneindi
sti
ll
ate.

The mi xtur
e obtai
ned afternit
rati
on i
sr educed on a boi
li
ng waterbath byusing t
in and
hydrochlori
caci
d.Af t
erthereacti
onisceased(usuall
yaft
er5mi nut
es),
1ml .ofanil
i
neand1ml .
ofalcoholicpot
assium hydroxi
desoluti
onareadded.Themi xt
ureisheated.Obnoxi
oussmellof
phenylisocyani
deindicat
esthepresenceofbenzene.

Gaschromat
ogr
aphy
:Ther
etent
ioni
ndexandr
etent
iont
imeofsomecommonv
olat
il
epoi
sons
ar
egivenbel
ow:

TABLE–4.
4

Compound Ret
enti
onIndex Retent
ionTi
me
i
nSystem 1 i
nSyst
em 2(inMinut
e)

Acet
aldehy
de 372 0.
70
 Benzene 660 14.
8
Carbont etr
achlor
ide 659 8.
6
Chloralhydrat
e 695 12.
5
Chloroform 605 6.
2
Ethanol 421 1.
9
I
sopr opylal
cohol 530 4.
0
Met hanol 491 0.
7
Paraldehyde 786 23.
2

Sy
stem –1:

Column–2.5%SE30on80–100meshChr omosorbG(aci
dwashedanddimet
hyldi
chl
orosi
l
ane
tr
eated)
,2m x4mm I
.D.gl
asscol
umn.Thesuppor
tshoul
dbeful
l
ydeact
ivat
ed.

Col
umnTemper
atur
e:100–300oC
Car
ri
erGas :Nit
rogenat45ml/mi
n.
Ref
erence :n-Al
kaneswit
hanev
ennumberofCar
bon
at
oms.

Sy
stem –2:

Col
umn–0.
3%Car
bowax20M on80–100meshCar
bopakC,
2m x2mm I
.D.gl
asscol
umn.

Col
umnTemperat
ure:
35Cper2minut
eandthenprogrammedat5
Cper
minut
eto175Candhol
dforatl
east8minutes.

GasCar
ri
er :Ni
tr
ogenat30ml
/mi
n.

4.
6.2.
14Test
sforHy
drocy
ani
caci
d:

Hydrocyani
cacid,maybedet ectedbyscreeni
ngtestswi
tht
estpaper
sbypl
aci
ngi
nai
rti
ght
condit
ionsi
nsi
dethejarcont
aini
ngbiol
ogi
calmater
ial
s.

(
1)Guaiacum –Coppersulphatetestpapert
urnsbl
ueafter30mi nut
es(t
estpaperi
spreparedby
di
ppingst r
ipsofNo.1f
il
terpaperfi
rsti
n0.1%aqueoussolut
ionofcoppersul
phat
eandt i
nctur
e
ofguaiacum (10%)
.

(
2)Picri
cacidtestpaperturnsfrom y
ell
owt otanorbr
ownwi t
hin5minut
es(
picr
icacidtestpaperi
s
preparedbydippi
ngstripsofNo.1fil
terpaperi
ntoasol
uti
oncont
aini
ng1gm ofpicr
icacidin10%
sodium car
bonatesolution)
.

Aft
erpreli
minaryscr
eeni
ng,hydrocy
anicaci
disisol
atedbyaciddi
sti
l
lati
onandt
hedisti
l
late
i
scoll
ectedinice-
coldcondi
tionin5ml .of1N sodium hydr
oxi
desolut
ion.Thedi
sti
l
lat
et hus
col
l
ect
edi ssubj
ectedtothef
ollowi
ngtests.

1. Pr
ussi
anbl
ueTest
:

To2ml .ofconcent r
atedalkal
inedi st
il
lat
e( concentr
atedbyheati
ngonwat erbath),af ewdrops
offreshlyprepared5% sol uti
onoff er
roussul phateand1dr opoff er
ri
cchl ori
desol uti
onare
added.Themi xtureisheat edjustt oboi l
i
ngf oll
owedbyt headdi t
ionofdi l
utesul phuri
cacid
dropwise to make t he sol
uti
on j ustacidicand war med.A Pr ussian blue colourf ormati
on
i
ndicatesthepr esenceofhy dr
ocy anicacid.Onkeepingt hecont
entov ernight,apr ecipi
tat
emay
beseen.

2. Ni
tr
opr
ussi
deTest(
Vor
tman’
s):
To5ml .oft heal kali
nedisti
ll
ate,af ew dr opsofaf reshlypreparedaqueouspot assium nitri
te
soluti
oni saddedf oll
owedbyt headdi tionoff ourdropsoff err
icchloridesoluti
on.Thendi lute
sulphuri
caci di
saddedwhi l
eshakingt i
l
lt hemi xtur
eassumesabr i
ghtyellowcolour.Themi xture
i
st henheat edt oboi li
ng.Subsequentlyiti scooledanddi lut
eammoni um hy droxi
desol uti
oni s
addedt opr eci
pitateoutexcessofi ronandt henf il
tered.Tot hecl earf i
lt
rat
e,af ew dropsof
dil
utesol uti
onofammoni um sul phi
dear eadded.A v i
oletcolouri sobt ai
nedwhi chchanges
graduall
yt obluegr eenandf i
nall
yt oy ell
ow (inpr esenceofv erysmal lamountofhy drocyanic
acid,abluishgreencol ourisobservedinst eadofv i
oletcolour).

3. Sul
phocy
anat
eTest
:

To2ml .ofconcent ratedal kal

i
nedi sti
ll
ate,1ml.ofy el
l
ow ammoni um sul
phidei saddedi na
porcelai
nbasinandev aporatedtodr y
nessonaboi li
ngwat erbath.Thentheresidueisdissolv
ed
i
n1ml .ofacidulatedwat er(containi
ng2dr opsofhydrochlori
caci d)
.Iti
swarmedandf il
ter
ed.To
theclearfi
lt
rate, afewdr opsofv erydil
utefer
ri
cchlori
desol uti
oni sadded.Abloodr edcolouris
obtai
nedi npr esenceofhy drocyanicacid.Onaddingaf ew dr opsofmer cur
icchl or
idesoluti
on,
theredcolourdi sappears.

4.
6.2.
15Testf
orPhenol
:

Phenolisisol
atedi
nbiol
ogi
calmat
eri
alsbyaci
ddisti
ll
ati
on(Pr
eferabl
ywit
h1:4sulphur
icaci
d).
Thedisti
ll
atei
scoll
ect
edin5ml.of1NNaOHsol ut
ionini
ce-
cooledcondi
ti
onandi
ssubject
edto
thefol
lowingt
ests.

1. Fer
ri
cchl
ori
deTest
:

Toabout2ml .ofdi
sti
ll
ate,
2dr opsofav erydi
lutesol
utionoff er
ri
cchlor
idesol
uti
onareadded.A
violetorbluishviol
etcol
ourati
onispr oduced.(cr
esol,otherphenolsandsali
cyl
i
cacidgivesthi
s
testwi ththeshadesofcol our–v i
olettobluishviol
et,beingdiff
erenti
neachcase).Anti
pyr
ine,
py r
ami donandsi mil
arnon-phenol
iccompoundsal sogi vethistest
.Incaseofphenol
,thecol
our
produceddi sappearsonaddingalcohol.

2. Li
ber
mann’
sTest
:

5ml .ofalkalinedi
sti
l
latei
sev aporat
edt odrynessoverasmal lfl
ameinapor celai
ndish.Itis
cooled.Api nchofsodium ni
tr
iteisadded.Thereaft
erconc.sulphur
icaci
disadded.Onmi xing
careful
ly
,blueorgreencolour
ationisobserved.Itt
urnstoredonaddingwat ert
oit,whichturns
greenifmadeal kal
i
nebysodium hy dr
oxideorammoni asolut
ion.(Cr
esolandotherphenolsalso
respondtothistest
).

3. War
e’sni
tr
it
e–ni
tr
ateTest
:

To5mloft heal kalinedi sti

llateinamor t
ar,10ml .ofconc.hy drochlori
caci dand0. 5gms.of
nit
rite-ni
tratemi xture( sodi um ni t
rit
e-1 part,sodium ni tr
ateorpot assium nitrat
e-1 partand
anhy droussodi um sul phat e2par t
s)areadded.Theni tismi xedt hor
oughlyt odi ssolv
et he
cont entsandt hereafterallowedt ostandfor2- 5minut es.
Int hepr esenceofcar bolicaci d,ari
chcr i
msonorbl oodr edcolourappear s.1ml .ofthisblood–
redorcr imsoncol ouredmi xtureisaddeddr opbydr opt o10%ammoni asol ut
ion.Adeepemer ald
greencol ouri spr oduced.
Alternati
v ely
, 2dr opsoff or mal i
nareaddedt o2ml .oft hebloodredmi xt
ureasabov e.Thecol our
changest opur ple.Thi smi xtur eisaddeddr opbydr opi nto10%ammoni asolution.Greenishblue
ordeepbl uecol ouri spr oduced.( Inpresenceofcr esols,nobloodr edcol ourisproduced.Onl y
brownorr eddishbr owncol ourappear s.Neitheraddi ti
onoff ormali
nnorpr esenceofammoni a
solutionchangest hecol ouri ncaseofcr esols).

4.
6.2.
16Test
sforCr
esol
:
 Cresolsareisolat
edbyaci
ddisti
l
lat
ion.Thedist
il
lat
eiscol
lect
edin5ml.of1N.sodi
um hy
droxi
de
solut
ioninice-col
dcondi
ti
on.Thedist
il
lat
ethuscoll
ectedi
ssubject
edt
ot hef
oll
owi
ngtest
s:

1. Fer
ri
cchl
ori
deTest
:

Asdescr
ibedi
nthet
estf
orphenol
.

2. Li
ber
mann’
sTest
:

Asdescr
ibedi
nthet
estf
orphenol

3. War
e’sni
tr
it
e–ni
tr
ateTest

Asdescribedinthedet
ectionofphenol
,br
ownorreddi
shbrowncol
ourappear
s(phenolgi
ves
bl
oodr edcolour
)whichdoesnotchangeonaddi
ngaf ew dr
opsoffor
mali
nor10% ammoni a
sol
uti
on(dist
inct
ionf
rom phenol
).

4.
6.2.
17 Test
sforAni
l
ine:

Asanili
neisbasic,i
tisisol
atedi
nbiol
ogicalmateri
alsbyalkal
inesteam dist
il
lat
ion.Thedi
st i
l
lat
e
i
scollectedi
nicecoldconditi
oni
n5ml .of1Nsodi um hydroxi
desoluti
on.Thealkali
nedist
il
latei
s
ext
ractedwiththree,10ml .port
ionsofdi et
hylether.Theet herisdriedbypassi ngthrough
anhydroussodi
um sulphateandi
susedforfoll
owingtests.

1. Di
chr
omat
eTest
:

Apor t
ionofetherext
ractisevaporatedtodr
ynessinapor
celai
nbasin.5dr
opsofconc.sul
phuri
c
aci
dand1dr opofpot assi
um dichromatesolut
ionar
eadded.Inafewmi nut
es,t
heedgeoft he
mixtur
ebeginst oshow abl uecolour,whi
chbecomesuniforml
yblueonaddingafew dropsof
water.

2. Pheny
lisocy
ani
deTest
:

Asst
atedi
nacaseofchl
orof
orm,
obnoxi
oussmel
lofpheny
lisocy
ani
deev
olv
es.

3. Hy
pochl
ori
teTest
:

Apor t
ionofetherextractisevaporatedtodryness.Theresi
dueist akenupinwat er
.Toitafew
dropsofsodium hypochlori
teorfreshlypr
eparedsoluti
onofbleachingpowderisadded.Apurpl
e
orviol
et-
bluecolourappearswhichchangest or eddi
shbrownordi r
tyred.Onaddingafewdrops
ofadiluteaqueoussol ut
ionofphenolcont ai
ningsomeammoni a,theviol
etordi
rtyredsol
uti
on
changestoblue( i
ndophenol)
.

4.
6.2.
18Testf
orNi
tr
obenzene:

Nit
robenzeneinbiol
ogicalmateri
alsisi
solat
edbyalkal
inedisti
l
lati
on.Thedisti
ll
ateiscol
l
ectedi
n
i
ce-col
dcondi t
ion.I
tisextr
actedfourtimesby10ml .port
ionsofdi et
hyletherandtheextr
acti
s
passedthroughanhydroussodium sul
phatewhi
chispreser
v edforthefol
lowingtest
s.

1. A portionofet herextractisevaporat
edt odr yness.Tot hedr
iedresidue,3ml .of
concentratedhydrochlor
icacidandmetall
icti
nar eadded.Thisi
swarmedonahotwat erbat
hfor
5mi nutesandf il
tered.Tothef i
lt
rat
e,2mlofchl orofor
m and2mlofal coholi
ccausti
cpotash
sol
utionar eadded.Themi xtur
eisheatedstrongly.Theevolut
ionofobnoxi
oussmellofphenyl
i
socy anateindi
catesthepresenceofnit
robenzene.
2. Apor ti
onofet herextractisevaporat
edtodryness.Tothedr i
edresi
dueinatesttube,5ml.of
ammoni um chlori
desol uti
onandzi ncdustareadded.Themi xtur
eisheatedonawat erbathfor5
minutes.Tothemi xture,1ml .ofammoni acalsi
l
vernit
ratesoluti
onisaddedandheatedonwat er
bathfor5mi nutes.Thef ormationofmi r
roronwall
soft estt
ubeorf ormati
onofblackpreci
pitate
i
ndicatesthepresenceofni trobenzene.

4.
6.2.
19Testf
orNapht
hal
ene:

Naphthal
eneinbiologi
calmat eri
alsisisol
atedbysteam di
sti
l
lat
ion.Thedi
sti
l
lat
eiscol
lect
edin
i
cecoldcondit
ion(Smellofnaphthalenemaybef el
t)
.Thedist
il
lat
eisextr
act
ed4timeswi
th10ml .
por
ti
onofet her.Thecombi nedet her
eallayer
saredr i
edbypassingthr
oughanhydr
oussodium
sul
phateandkeptreservedfortests.

1. Testwi
thpi
cri
cAci
d:

Aporti
onofetherext
racti
sevapor
atedtodry
ness.Tothedr
iedmass,
afewdropsofpicr
icacid
sol
uti
onisadded.Yell
owcryst
alsofnapht
halenepi
crat
earefor
med,whi
chshowcharacter
ist
ic
shapeundermicr
oscope.

2. UVSpect
rum:

A por
ti
on ofetherext
racti
s evapor
ated t
o dr
yness.The r
esi
due i
s di
ssol
ved i
n spect
ra
pur
eet
hanol
.Itshowsmaxi
masat266nm and288nm inUVSpectr
a.

4.
6.2.
20Testf
orKer
oseneoi
l
:

Ker
oseneoilinbi
ologi
calmateri
alsisi
solatedbyst eam di
stil
lati
on.Thedi st
il
lat
eiscollectedi
ni ce
col
dcondit
ionin20ml sat
uratedsolut
ionofsodi um chlor
ide.Thet oplayer(1cm.)oft hedist
ill
ate
i
stakenoutaf t
ersomet i
meandext ractedfourtimeswi th10ml .port
ionsofdi et
hy let
her.The
combinedether
ealext
ract
sar edriedbypassi ngthroughanhy droussodium sulphate,evaporated
atr
oom temperatur
eandsubjectedtot hefol
lowingtests.

1. UVSpect
ra:

Aporti
onofetherealext
racti
sevapor
ated.Tot
heresi
due,wat
erisadded.Onscanni
nginUV
Spect
rophot
ometer,maxi
masareobser
vedat223–228μm andalsoat366nm (
weakband)
.

4.
6.2.
21Test
sforNi
cot
ine:

Nicoti
ne(onlysteam v
olati
lealkaloi
d)isisolatedbyal kal
inedi
stil
l
at i
on.Thedi
sti
ll
ateiscol
lected
i
ni cecoldconditi
onin10mlof10%hy drochlor
icacid.Itismadeal kal
ineandextr
acted4t i
mes
with10ml .porti
onsofdiethylether.Theet herextractsarecombi nedanddri
edov eranhy
dr ous
sodium sul
phate.Theextr
actiskeptr eser
v edforthefoll
owingtests.

1. Roussi
n’
sTest
:

Totheetherealext
ract
,1ml.ofethersol
uti
onofiodi
neisadded.Abrownishredamorphous
pr
ecipi
tat
eisformedwhicht
urnst
ocr y
stal
l
inerubyr
edneedl
eshapecr
yst
alsaft
ersomet
ime.

2. Schi
ndel
mei
sterTest
:

Aporti
onofether
ealextr
actisevapor
atedt
odr yness.Tothe,1dr opof30% formal
dehyde
sol
uti
on(f
reefr
om f
ormicaci
d)andadropofconcentr
atedsul
phur
icacidar
eadded.Themixt
ure
becomesroser
ed.
 4.
6.2.
22Et
hyl
eneDi
bromi
de:

Sy
nony
ms:
1,2-
Dibr
omoet
hane;
Ethy
lenebr
omi
de,
EDB

Ethyl
enedi bromideisapest i
ci
deusedasf umigantforstor
ageoff oodgrains.Itisaheav yli
quid
havi
ngt heodourl i
kechlorof
orm,solubl
einabout250partsofwaterandmi scibleinalcohol,
ether
.
Ethyl
enedibromideispot ent
ial
lyacauseofav ari
etyofacutehealt
hef f
ectsi ncludi
ngdamaget o
l
iver
,stomachandadr enalcort
exalongwithsi
gnif
icantr
eproduct
ivesyst
em t oxicit
y,part
icul
arl
yin
test
es.

GasChr
omat
ogr
aphi
cmet
hod:

Column :Glasscol
umnpackedwi t
h10%SP-1000( 1.8mX4mm Glass,80-100mesh)
Detector :ECD Carr
iergas–CH4: Ar(5:
95),
F/R40ml /
mi n.
I
njectortemp:2000C I
nject
ionquanti
ty-5μl
Ovent emp. : 1150C
Detectortemp.3500C Li
mitofquanti
tati
on–2ppb(2)

4.
6.2.
23Pheny
l:(
Househol
ddi
sinf
ect
ant
)

Thesear ehomogenoussol uti

onsofcoaltaracidsorsimil
araci dsderi
vedf rom pet
roleum, wi
thor
wit
houthy dr
ocarbons,orotherphenol
iccompoundsi ncludingsubst i
tutedphenoliccompounds,
orami xtureoftheseandasui t
ableemulsi
fi
er.Iti
sblackincol ourandondi lut
ionwithwat ergi
ves
tr
anslucentof f-
whiteturbi
dit
y( 3).Phenylis used as disinfect
anti n houses,hospitals,and
l
aboratoriesforsani
tarypur
poses.Thetestsprescri
bedforphenol sandcr esol
sshouldbeappl ied.

4.
6.3 METHODSOFQUANTI
TATI
ONOFSOMEVOLATI
LESI
NBI
OLOGI
CALMATERI
ALS:

4.
6.3.
1Det
ermi
nat
ionofEt
hyl
Alcohol
inbl
oodandur
ine.

1. Modi
fi
edKozel
ka–Hi
neMet
hod:

Theapparatususedinthismet hodconsi stsoff ourhardgl asstubes(lengt

h20cm,di a
2.5cm)connect edwi theachot herbymeansofqui ckf i
tjoi
nt s.Inthef i
rsttube,5mlof2%
potassium di
chromat ei
nconcent ratedsul phuri
cacidi stakent hroughwhi chairisbubbledto
remov ethemoistureandotherorgani cvolatil
es.I
nthesecondt ube, 2ml.ofbloodsampl eor10
ml.ofur i
nesampl eistaken.Tot hissampl e,2ml .of10%sodi um tungstateand0.5ml .of2N
sulphuri
cacidsar eaddedt odepr otei
nizet heblood( deprot
einizati
onisnotr equiredf
orur i
ne
sampl e).

Inthethi
rdtube,5ml.ofsat uratedmer curicchlori
deand5ml .ofsaturatedsodium hy
dr oxi
de
solut
ionar
eaddedandmi xeduni forml
ywhenay ell
owpr eci
pit
ateofhy dratedmercur
icoxideis
obtai
ned(usedtotrapacetone,acet al
dehy deetc.)
.Thistubeisinter
posedbet weenthet ubeof
bloodorurinesamplesandt hef our
tht ube.Tot hefourt
ht ube,10ml .of0.1N potassi
um
dichr
omatesolut
ionand10ml .ofconcentratedsulphuri
cacidareadded.

Allt
hefourtubesarear r
angedi nsuchamannersot hatt
hef i
rsttuberemai nsoutofhotwat er
whil
etherestofthetubesaredippedi ntothehotwat erbat
h.Theai rissuckedwi ththehelpofan
aspi
rat
or(attherateof25ml ./mi n.)t hr
ought hetubecontai
ning2%di chr
omat e-sul
phuri
caci d
mixt
ure;then successi
velythrough t he tube ofblood sample,t he tube containi
ng alkal
i
ne
mercuri
cchlori
deandf inal
l
yt het ubecont ai
ning0.1N potassium dichromat e-
sulphur
icaci d
mixt
ure.Aft
eronehour ,
thedichromatesol utionist
akenoutoft hetubei.e.fourt
ht ubeandmade
 upt o100ml.wit
hdist
il
ledwat
er.Thi
ssol
uti
oni
sti
tr
atedi
odomet
ri
cal
l
ywi
th0.
1N sodi
um
thi
osul
phat
esol
uti
on.

Ablankexper
imenti
sal
soper
for
medsi
debysi
debyt
aki
ngal
lther
eagent
sexceptt
hebl
ood
sample

Cal
cul
ati
on:
- Consi
der
ingr
edoxr
eact
ion,

1.
0ml
.of0.
1K2Cr
2O7sol
uti
on=1.
15mg.ofet
hanol
.

Ther
efor
e,bl
oodal
cohol
%=1.
15x(
X–Y)x100mgper100ml
.ofbl
ood.
V
Where,

V= Vol
ume(ml
.
)ofbl
oodt
akenf
ort
heexper
iment
.

X= Vol
ume(
ml.
)of0.
1Nsodi
um t
hiosul
phat
erequi
redi
nbl
ankexper
iment
.

Y= Vol
ume(ml .
)of0.1Nsodium t
hiosul
phat
esol
uti
onr
equi
redi
n
t
heexper
imentat
ionwi
ththebloodsample.

2. Det
ermi
nat
ionofal
cohol
inbl
oodbyheadspacegaschr
omat
ogr
aphi
cmet
hod:

1. 0.5ml .ofbloodand0. 5ml.ofi

nternalst
andard(0.2%w/vsoluti
onofn-pr
opanolindi
sti
ll
ed
water)istakenintotheglassvi
alandplacedintotheturnt
abl
e,headspacesamplerther
mostatat
70oCf or10mi nutes.Theheadspacesampl eiswithdrawnandsubject
edtogaschromatogr
aphy.

2. Simil
arset
sofexper
imentaremadewithdiff
erentst
andar
dsofal
coholf
orcal
i
brat
ionpur
poseas
pert
hefol
lowi
ngchromatogr
aphi
ccondit
ions.

Chr
omat
ogr
aphi
ccondi
ti
ons

1. Column : 2m x1/ 8”O.D.St

ainl
essst
eel
col
umn. P/
W
3.
8%Hal
comidM-8onChromosor
b
W(mesh:
100-
120)
.

2. Det
ect
or :
F.I
.D.

3. Car
ri
edGas :
Nit
rogen(
32l
bs/sq.i
nch.
).

4. Temper
atur
e :I
nject
iontemperature :
100
C
Transf
ertemperature :125
C
Columntemperature :65C
F.
I.D.t
emperat
ure :150
C

5. Pr
ogr
amme :Constantther
most
ati
ngti
me:10minutes
I
njecti
ontime :0.
1minut
e
Pr
essurisat
ionti
me :0.1minut
e
Cycleti
me :4.5minutes

Quant
it
ati
on:

Donebypeakar
eamet
hodusi
ngst
andar
dsofet
hanol
upt
o400mg%t
oensur
eli
near
it
y.
3. Al
ter
nat
eheadspaceGLCmet
hodf
ordet
ermi
nat
ionofet
hanol
inbl
ood

A. Reagents: Cal
ibrati
onRef er
enceMat eri
als(Calibr
ator
s).
Ethanol:0.10,
0.20, 0.
40%w/ v(
1.00,2.
00,4.00g/L)aqueous
solut
ions.
MixedCal i
brat
or: Low( Acetone1.00g/ι ,
Et hanol2.
00g/ι ,
Isopropanol1.00g/ι ,
Met hanol-1.
00g/ι,
aqueoussol uti
ons).

MixedCali
brat
or:Hi
gh(Acetone1.
00g/ι
,Et
hanol
4.00g/ι
,
Isopr
opanol1.
00g/ι ,
Methanol1.00g/ι,
aqueoussolut
ions)
.

B. I
nter
nalSt
andar
dsolut
ion:
Acet
oni
tri
l
e:0.
15%,v/v(
1.5ml
./ι
aqueoussol
uti
on)
.

C. Sodi um Chl
ori
de.
Al
lther
eagent
sshouldbeofA.
R.Gr
ade.

Oper
ati
on:

Thel i
qui
dsampl e(20μlt o1ml .)isplacedi ntoaglasssept um vialtoget herwithsuf fi
cient
sodium chl or
idet oassuresaturati
onoft heliquid(
andaf t
erdilut
ioninfixedpr oporti
onwi t
ht he
i
nt ernalstandar dsolut
ionwhent hatproceduralmodifi
cationisused)Ref erencesampl esar e
treatedidenticall
yandal lseal
edv i
alsareinsertedi
ntothet hermostatedwat erbath.Aftera45
mi nuteequi l
i
br at
ionperiod,af i
xedquant i
tyoft heheadspacev apourofeachsept um v ialis
sequent i
all
ysampl edwi t
hagast ightsyri
ngeandi nj
ectedi nt
othepr esetgaschr omatogr aph.
Theanal ysispr oceedsf orafixedt i
mei nter
valandther esponseofdet ectorisrecordedasa
funct i
onoft i
me.

GasChr
omat
ogr
aphi
cCondi
ti
ons:

Col
umn :
Car
bowax1500(
0.4%)on60/80mesh.

Car
bopackC1.
8M x3.
2mm O.
D.(
6ft
.x1/
8in.
)st
ainl
essst
eel
col
umn.

Car
ri
erGas :
Hel
i
um :
Inl
etpr
essur
e :
55psi
g.

Fl
owr
ate :
45ml
./mi
n.

Temper
atur
e :
Col
umnOv
en :
80
C

I
nject
ionpor
t:175
C

Det
ect
or :
225
C

F.
I.
D. :
Hydr
ogen :
Inl
etpr
essur
e:20psi
g

Fl
owr
ate :
45ml
./mi
n.

Ai
r :
Inl
etpr
essur
e :
15psi
g.

Fl
owr
ate :
330ml
.
/mi
n.

Temper
atur
ePr
ogr
amme :
Isot
her
mal
.
 Char
tSpeed :
5mm.
/mi
n.

Pr
ocedur
e:

1. Awel lmixedali
quotoft hebloodspecimeni smi xedani denti
calvolumeofacet oni
tr
ile
i
nternalst
andardsoluti
onusingfullyquant
it
ati
ont echnique.Normall
ytherespecti
vevolumesar e
both500μl .Thesampl emeasur ementandinternalstandardsolut
ionmeasurementandmi xing
arecarri
edoutpr ef
erablywit
hanaut omati
cdiluteandt hesampleint
ernalstandardmixt
urei s
del
ivereddir
ectlyi
ntooneclear,drynumberedsept um vialcont
aini
ng1gm ofcr ystal
li
nesodium
chl
oride.

2. Ethanolormi
xedv
olat
il
esr
efer
encesol
uti
ons(
usual
l
ylowandhi
ghcal
i
brat
ors)ar
etr
eat
ed
i
dent
icall
y.

3. Aftert heminimum equi li

brati
onper i
od, afixedidenticalvolumeoft heheadspacev apouris
wit
hdrawnusi ngt hegas- ti
ghtsy r
ingeadjustedt ofixedv olume( usual
ly250μl )withthechaney
adapterorot herwise,t hesyri
ngehav i
ngbeenpr eheatedt o38oC int hei ncubatororsi mil
ar
devi
ce.Repeat edrapidpumpi ngoft hesyringeisempl oyedt oobtainafull
yrepr esentat
iveali
quot
ofthe headspace v apour,whi ch isthen immedi atel
yi nject
ed intothe gaschr omatograph
i
mmedi atelyfoll
owingt heheadspacesampl ewi thdrawal,eachsept um v i
ali sventedwi t
ha25
gaugeneedl etoreestabl i
shatmospher i
cpr essure.Thesy ringeandneedl earet horoughlypurged
byrapid,r epet
iti
veint akeandexpul sionofr oom airandt hesyringeisreturnedt otheincubator
unti
lit
smeetuse.

4. Theanal
yteisal
lowedt
opr
oceedf
orappr
oxi
mat
elyl
5mi
nut
es(
or3mi
nut
es)i
fet
hanoli
s
t
heonlycompound.

5. Thesamegast ightsy
ringewithvol
umecalibr
ati
onintacti
susedfortheentir
eunknownand
ref
erencesample.Eachspecimenisanalysedbythi
sprocedureinatl
eastindependentdupli
cat
e,
asineachcali
bratoraft
erall
owingeachseptum vi
altore-
equil
ibr
atef
oratleast20mi nutesaft
er
thepri
orheadspacesamplewi t
hdrawal.

6. Theanal
ysi
srecor
disappr
opr
iat
elyi
dent
if
iedf
orcomput
ati
onofr
esul
tasf
oll
ows.

A. Resul
tsComput
ati
onbyPeakHei
ghtMeasur
ement
.

R1
Bl
oodAl
cohol
concent
rat
ion(
BAC)=-
--
--
--XConc.Et
hanol
,Cal
i
brat
or
R2

Where,
PH= peakheightofthei
ndi
cateddetect
orr
esponser
ecor
ding(
e.g.PHi
.s.=Peakhei
ght
oft
heacet
oni
tr
il
eint
ernal
standar
dresponse)
.

PHEt
hanol = R1Forunknownsampl
e-i
nter
nal
standar
dmi
xtur
e
PHI
.S

PHEt
hanol = R2 Fort
heCal
i
brat
orsampl
e-i
nter
nal
standar
dmi
xtur
e
PHI
.S

B. Resul
tComput
ati
onbyPeakAr
eaMeasur
ement
.

R1
BACunknown=-
--
--
-XConc.Et
hanol
,Cal
i
brat
or. R2
 Where,PA=Peakareaoftheindi
cateddetect
orresponserecordi
ng
(
e.g.PAi.
s.–Peakareaoftheacetoni
tr
il
einter
nalstandar
dr esponse)
.

PAEthanol
R1= fortheunknown-sampl
eint
ernal
st
andar
dmi xtur
e.
PAi
.s.

PAEt
hanol
R2= P.
A.i
.s. f
ort
heCal
i
brat
or–i
nter
nal
standar
d
mi
xtur
e.

C.Resul
t,comput
ati
onmayal
sobemadef
orot
herv
olat
il
esaccor
dingl
y.

4.Met
hodForTheDet
ermi
nat
ionOfAl
cohol
InBl
oodByGC-
HS
Av ol
umeof0. 5mlofbl oodand0. 5mlofinternalstandard( 0.
2w/v
sol
uti
onofn-propanolindisti
l
ledwater)wast
akeni ntotheglassvialand
pl
acedintotheturntabl
eofheadspacesampler,thermostatedat70cf or
20 minut
es,vaporswer ei nj
ected i
ntothecolumnand anal ysed.The
condi
ti
onsforanalysisar
easbelow.
Gas–Chr
omat
ogr
aphi
cCondi
ti
on:

I
nst
rument
:-Per
kin-ElmerClari
usGasChromatographcoupled
with110HeadSpacesampl erandanal
y t
icaldat
a
Stati
on.Per
kin-
Elmer8410,
8600GCcoupl edwi t
h
HS-101isal
sousedf orthi
spurpose.

Col
umn:-1/8”
o.d.s.s.column2met er
,p/w3.8%halcomi
d
m- 18onchromosor
bw( hp)messrange100-
120
Det
ect
or:-Fl
amei onizat
iondetect
or.

Car
ri
erGas:
-Ni
tr
ogen30l
bf/
inch.

Temper
atur
e:-Inj
ecti
ont emp.=080c
Transfertemp.=087c
Columnt emp. =067c
FIDt emp. =150c
Sampl etemp. =065c

Programme: -Ther mostattime=20mi n.

I
njecti
ont i
me =0. 05mi n.
Pr
essurizati
ont ime=0. 1mi n.
Cycleti
me =4. 0mi n.
Calculat
ion:
-cubi c+or i
ginfittypemul til
evel usi
ngpeak
areaat5di fferentlevels.Thisensuresthe
l
ineari
tyfitcurv eto400mg%ofet hanol
checkst andar dar eusedt overi
fytheinit
ial
cali
brati
on

4.
6.3.
2 Gas Chr
omat
ogr
aphi
c det
ermi
nat
ion ofEt
hanoland ot
herVol
ati
l
es i
n Bl
ood/
Uri
ne/
viscer
al
di
sti
l
lat
e.

Isol
ati
onofvol
ati
lesinbl
ood:1ml.ofbl
oodisdil
utedwit
h4ml.ofwater.I
tisacidi
fi
edwi
thafew
dropsof5% tar
tari
cacidsolut
ionandthendisti
ll
ed.5mlofdisti
ll
ateiscollect
edini
cecold
condit
ionandanal i
quot(
10μl)ofitisinj
ect
edintothegaschromatographaspercondi
ti
ons
stat
edbelow.

I
solat
ionofv
olati
l
esinUri
ne:1ml.ofuri
neistakeni
ntoamicrocent
ri
fugeandcent
ri
fugedfor15
minut
es.5μlofsuper
nat
antli
qui
disinj
ectedi
ntothegaschr
omatogr
aphaspercondit
ionsstat
ed
bel
ow.

Col
umn–Por
apakθ– Pol
ymerbead,
80–100mesh,
5’X4mm.( i
d.)gl
asscol
umn.

Col
umnTemper
atur
e–160
C

Car
ri
erGas -Ni
tr
ogen

GasFl
ow -Ni
tr
ogen-50ml./mi
n.
Hydr
ogen-50ml.
/min.
Ai
r -300ml.
/mi
n.

Det
ect
or -F.
I.
D.

TABLE:
4.5:
Ret
ent
ionTi
meofDi
ff
erentVol
ati
l
eSubst
ancesRel
ati
vet
on-
Propanol
.

Sl
.No. Subst
ance Rel
ati
veRel
ati
ont
ime(
RRT)

1. Ethanol 2.30
2. Met hanol 2.80
3. Acet one 2.80
4. I
sopr opanol 2. 90
5. Ether 5.10
6. Chl or oform 6.00
7. Trichl oromethane 0.
44
8. Acet aldehyde 0.21
9. Par aldehy de 0.70
10. Benzene 0.76
11. Amy l Alcohol 0.98
12. Toluene 1.45

Thepr
esenceofav
olat
il
e/v
olat
il
esmaybemadef
rom t
heabov
est
udy
.

Quant
it
ati
on:

The quanti
tat
ive est
imat
ion ofv ol
ati
l
e substance i
s made bydeter
mining t
he area ofthe
cor
respondi
ngpeakandcal cul
ati
ngthequanti
tyofthesamplef
rom t
hefoll
owingrel
ati
onship.

Ca Cb
-
--
--=-
--
--
-
Aa Ab
Wher
e,
Ca=Concent
rat
ionofv
olat
il
esubst
ancei
nexhi
bit
.

Aa=Ar
eaofpeakofv
olat
il
esubst
ancepr
esenti
ntheexhi
bit
.
 Cb=Concent
rat
ionoft
hest
andar
d.

Ab=Peakar
eaoft
hest
andar
d.

Cb i
soncedet
ermi
nedbyi
nject
ing10μlofsuper
nat
antpr
epar
edbyst
andar
dbl
oodsampl
e
hav
ing150mg

Ab ofv
olat
il
esubst
anceper100ml
.ofbl
ood(
ther
ati
oremai
nsconst
ant
).

4.
6.3.
3 Det
ermi
nat
ionofcy
ani
de:

I
. ByAr
gent
omet
ri
cTi
tr
ati
on

Allsil
versaltsexceptt hesul phidear ereadil
ysolubl ei nexcessofasol uti
onofal kalicyanide.
Hencechl or
ide,bromideandi odidedonoti nter
fere.Theonl ydi ff
icult
yinobt ai
ningashar pend
pointl
iesi nthefactthatsilv
ercy anideisoft
enpr ecipit
at edincur dyforms, whichdonotr eadilyre
-di
ssolve,andmor eov ertheendpoi ntisnoteasyt odet ectwi thaccur acy.Therearetwomet hods
forovercomi ngthesedi sadvantages.Inthef i
rst
,thepr ecipi
tationofsi l
verargentocyanideatt he
endpoi ntcanbeav oidedbyt headdi ti
onofammoni asol ut
ioni nwhi chitisreadil
ysoluble.I fa
l
itt
lepot assi
um iodidesol ut
ionisaddedbef orethet i
trationiscommenced,t hesparingl
ysol uble
sil
veriodide,whichisinsolubleinammoni asolut
ion, willbepr ecipi
tatedattheendpoi nt.

Ag[
Ag(CN)2]+4NH3=2[Ag(
NH3)
2]++2CN
[
Ag(NH3)
2]++I =AgI+2NH3

Attheendpoi nt
,sil
veri odideinpreci
pit
ated.Viewingagainstablackbackgroundbestseest he
preci
pitat
ion.Inthesecondmet hoddiphenylcarbazi
dei semployedasanadsor pti
oni ndi
cat
or.
Theendpoi ntismar kedbyt hepinkcolourbecomi ngpal eviol
et(al
mostcol ourless)onthe
coll
oidalprecipi
tat
ei ndi lutesolut
ionbeforetheopal escenceisvisi
ble.Thecol ourchangeis
observedont heprecipit
atedpar t
icl
esofsilv
erargentocyanide.

Pr
epar
ati
onofsol
uti
ons:

0.
1NSilv
erNit
ratesol
uti
on: 8. 494gms.ofA.
R.gr
adesi
l
v erni
tr
ateisweighedaccur
ately
,
di
ssol
vedindisti
l
ledwaterandv
olumei
smadeupto500ml.vol
umetri
cfl
ask.Thi
sgives0.1N
si
l
verni
trat
esoluti
on.

Alt
ernati
vel
y,about8.
5gms.ofpuredrysil
verni
tr
ateisweighedoutaccur
atel
yanddissol
vedin
500ml .ofwaterinagraduat
edf
lask.Themolarconcent
rati
oniscal
culat
edfrom t
heweightof
si
lverni
trat
eemployed.

Di
pheny
lCar
bazi
deSol
uti
on:I
tispr
epar
edbydi
ssol
vi
ng0.
1gm oft
heDPC(
sol
i
d)i
n100ml
.of
et
hanol
.

Ont
heabov
ebasi
s,cy
ani
dei
sdet
ermi
nedasf
oll
owsi
nsal
tsaswel
lasi
nbi
ologi
cal
mat
eri
als.

1) Det
ermi
nat
ionofCy
ani
dei
nsuspect
edsal
t:

Pr
ocedur
e:

Thesampleisweighedaccur
atel
yanddissol
vedin250ml.ofwat
eri
nav ol
umetr
icfl
ask.I
tis
shakenwell
.Fr
om thest
ocksolut
iont
husprepar
edcyani
demaybedet
ermi
nedbyanyofthet
wo
methods.

Met
hodI
:
25ml .ofthestocksolutionistr
ansf er
redtoa250ml .conicalfl
askbymeansofabur ett
e(notby
apipetteduetotoxicit
yofcy ani
de).75ml .ofwater
,5-6ml .of6M ammoni asoluti
onand2ml .of
10% potassium i
odidesol ut
ionareadded.Thef l
askispl acedonsheetofbl ackpaperandt he
contentofthef l
askist i
trat
edagai nst0.1Nsil
vernit
ratesoluti
ons.Thesil
verni t
rat
esoluti
oni s
addeddr opwi setil
lsignofper sistenceofyell
ow colourofsi l
veriodi
deisobser ved.Whena
fur
therdropofsilverni
trateproducesaper manenttur
bidit
y ,
theendpointi
snot ed.

Met
hodI
I:

25ml .ofthestocksoluti
onofsampl eistransfer
redtoa250ml .conicalfl
askasabov e.75ml .of
water,5-
6ml .of6M ammoni asolut
ionand2ml .of10%potassium iodi
desol uti
onareadded.
Thecont entofthefl
askistit
ratedagainst0.1Nsi l
verni
tr
atesolut
ionusing2- 3dropsofdiphenyl
car
bazideasani ndi
cator
.Theendpoi ntisr ecor
dedwhenaper manentv i
oletcolouri
sproduced
bydropwi seadditi
onofsilv
ernitr
atesoluti
on.

Cal
cul
ati
ons:

Theamountofcy
ani
deorpot
assi
um cy
ani
demaybecal
cul
atedasf
oll
ows:

1ml
.of1M AgNO30.
05204gofCN
0.1304gofKCN.

2) Det
ermi
nat
ionofCy
ani
dei
nBi
ologi
cal
Mat
eri
als:

Pr
ocedur
e:

100gms.ofv iscer almat erialisplacedinadi stil

li
ngf l
askoft hest andar
df itdisti
l
lation
uni
tandaci difi
edwi tht artaricacidanddi sti
ll
ed.Thedi sti
ll
ateiscoll
ectedi nicecoldcondi ti
oni n
agr aduat edconi calflaskcont aining10ml .of6M ammoni asolution.100ml .ofdi sti
ll
atei s
col
lect ed.Whilecol l
ectingt hedisti
llateint hereceiv
er,tubeshoul dbedi ppedi nt
ot heammoni a
sol
ut ion.2ml .of10%pot assium iodi desoluti
oni sadded.I tisthenpl acedonasheetofbl ack
paperandt it
ratedagai nst0. 1M silv ernit
ratesoluti
onbydr opwiseaddi ti
onandshaki ngtil
lt he
persistenceofy el
low col ourofsi l
veri odideisobserv ed.Theaddi t
ionofdr opsofsi l
vernitrate
sol
ut ionisdonecar efully
.Whenadr opproducesaper manentt ur
bidity
,theendpoi ntisr eached
andnot ed.Ther el
ati
oncal culatestheamountofcy anideasf ol
lows.

1ml
.of1M AgNO30.
05204gofCN
0.1304gofKCN.

Theabov
eti
tr
ati
oncanal
sobecar
ri
edoutbyusi
ngdi
pheny
lcar
bazi
desol
uti
onasi
ndi
cat
or.

I
I. BySpect
rophot
omet
ri
cMet
hod:

Scoggi
n’
sMet
hod:

Cyanideinpost -
mor t
em mat er
ial
sisisol
atedbyaci ddistil
lati
onandhy dr
ocyani
caci dthus
l
iberatediscol
lectedeit
herin1% Na2CO3or0.2N NaOH ( r
ecov ery70%)or2x10–2M ni ckel
ammi ne( r
ecovery90%)directl
y.Pre-
washing ofthecollect
ed mat eri
albychlorofor
m gi v
es
minimalbackground.Bythi
smet hodcyani
deisreactedwit
hni ckelaminetoform ni
ckelcyani
de
compl exhavi
ngmaxat268. 8nm.Comput i
ngf rom Beer’
scur v
ef ortheknownamountof
cyanidesdoesthequanti
tat
ion.
 Pr
ocedur
e:

10gms.ofhomogeni zedv i
sceralmat terismi xedupwi th50ml .ofwat erandacidifi
edwi t
h1gm
oft ar
tari
cacid.I tisdi sti
l
ledov eral ow f l
ame.Thedi sti
ll
at e( 1st30ml .)iscoll
ectedatr oom
temper at
ureinami xtureof40ml .nickelami nesolution( prepar edbymi xi
ng20ml .of1M ni ckel
chlori
deand20ml .of30%ammoni asol uti
on)oral ternativ
el yin25ml .of1%sodi um carbonat e
soluti
on.Inei t
hercase,t hev olumei smadeupt o100ml .wi th15% ammoni asol uti
onor1%
sodium carbonatesol utionast hecasemaybe.Thedi sti
ll
atei swashedwi t
hchloroform.1ml .of
thedistil
l
ateismi xedupwi th1ml .of2x10–2M ni ckelami nesol uti
onandmadeupt o10ml .In
caseofdi sti
l
latecol l
ecteddirect l
yi nnickelami ne,furtherdi l
ution( 10ti
mes)wi t
hwat eri
smade.
Thebl ankinthecaseofabsor pt ioninsodi um car bonat esolutionhaspr acti
call
yzerobackgr ound
i
nt hespectralregioni nquesti
on.Wher easthebl ankint hecaseofabsor pti
onintonickelammi ne,
aslightbackgroundi sshownev enwhenCN–i sabsent .

Theabsor banceofnickelcyani
deismeasuredbyt aki
ngappropri
atevol
umeofstocksol
uti
onof
nickelcy
anide(preparedfrom thedi
sti
ll
ate)at268.8nm.From theBeer ’
scur
ve,t
husprepar
ed
theamountofcy anideinbiol
ogi
calmateri
al(unknownsample)iscomputed.

4.
7 GASEOUSPOI
SONS:

Var i
oustoxicgasesar
eknowni nforensictoxi
cology.Al
thought helisti
ncl
udesvar
iousty
pesof
toxicgasesofdiver
seori
ginorsources,casesinv
olvi
ngaf ewt oxicgasesar
eencounter
edinday
-
to-daywork.
Howev er,t
hetable–4.
6indicat
estheircharact
eri
sti
cs,or
igin,
pr oper
ti
esetc.

TABLE–4. 6:CHARACTERI STICOFGASEOUSPOI SONS:

NAME SOURCE PROPERTI ES OTHERCHARACTERI STI CS
Carbon Atmospher e Colourless, Sy mptoms ar e gi ddi ness,
Dioxide ( 0.
4%) ,l i
me ki l
n odourless, muscul ar weakness,
i
ndust rial soda sl i
ghtlyacidtaste,drowsi ness, st er
t oyous
wat er,dryice. heav iert han air.breathingcomaet c.
very sol uble in
water.
Carbon Decomposi tion Col ourless, Oxy gencar r
yingcapaci t
yof
Monoxi de of oxal ic and t ast
eless, blood i s lost due t o
for
mi c aci d by odourless gas,f ormat i
on of car boxy
sulphur i
c aci d,l
ighter t han air,haemogl obi
n. 240 t imes
i
ncompl ete i
nsolubl e i
n mor e affi
nity for
combust i
on i n water, combi nes haemogl obi
nt han t hatof
conf i
ned space,wi th chl ori
ne to oxy gen,sy mptomsi nclude
coalgas, form phosgene. sudden weakness,
automobi l
e dizziness,comaanddeat h.
exhaust s, Blood becomes deep
ref
iner i
eset c. cher r
y red, pink
discolourati
onofski n.
Hydrogen By Colourlesshaving Conv erts met haemogl obin
sulphide decomposi ti
on odour of r ot
ten to sul phamet hemogl obin.
ofor ganic eggs. Sy mptoms include
 substances conj
uncti
vi
tis, headache,
containi
ng cough,anemi a,off
ensi
ve
sulphur,by
- smelli
sfeltonopeni
ngthe
productinsome body.
sulphurbased
i
ndust ri
es&in
sewers.

Ammoni a In industries Colourless, Sympt omsi ncl

udechoki ng,
(ferti
l
izer s, i ce pungent smel l
,coughi ng, pulmonar y
cream, solubleinwat er. odema f
oll
owed by
refri
ger ation). bronchopneumoni a.
Phosphi
n Phosphi desused Col ourless, Highlyt oxic,af fects CNS
e as r odent i
cides,f l
ammabl e wi th and reacts with
phosphor ous- odour ofhaemogl obin.
basedi ndust r
ies.decay i
ngf i
sh.
Phosgene Indust r
ies Colourless gas,Suf focat ion, coughing,
(manuf acture ofsuf focating. edemaofl ungs.
i
nsect icide, war
gaseset c.).
Sul
phur Bur ning ofHeav y, Ir
rit
abl etot heai rpassage,
di
oxide sulphur ,sul phide colourl
ess, suffocat i
ng, sympt oms
oresofzi nc,iron pungentsmel l
ing i
ncludecoughi ng, sneezing,
and l
ead,gas sol uble i n bronchopneumoni a
volcanoes. wat er
. oedema.
Chl
orine Bl eachi ng Greenish y ell
ow I r
rit
at es t he mucous
powder i
n gas, unpl easantmembr ane &respiratory
disinfect i
ng odour,sol ublei nt r
act .Causescy anosisand
formul ation. wat er
. suffocat i
on.

4.
7.1 Ident
if
icat
ionofsometoxi
cgasesbyConwayMi
croDi
ff
usi
ont
echni
que(
blood,
st
omachwashorur i
ne)
:

1. Conwaymi
crodi
ff
usi
onassembl
y:

I
tisofabr i
nktype,polypr
opy
lenecellswit
hcl earpoly
styrenecov ershav
ingan
outer
mostannul
arseal i
ngwell
,anintermedi
ateannularwel lforthesampleand
theli
ber
ati
ngagentandacenterwellforthereagent(detecti
onr eagent
)whichi
s
usedtotr
apthediff
usinggasorvapour.
 Gener
alpr
ocedur
e:

1. Inthecent
erwell(
sampl
ewell
)1ml .ofsampl
e(uri
neorblood)i
sint
roduced
i
ntotheonehal fand1ml.ofl
iberat
ingagentint heotherhalf(
sampleand
l
i
berati
ngagentshoul
dnotbemi
xedatt hismoment)
.

2. 2ml.ofseal
ingagent(sampl
easthel
i
ber
ati
ngagentasst
atedabov
e)i
s
addedintotheouter
mostseali
ngwel
l
.

3. 2ml.oftr
appingagent(
absor
bsv apourorgasl
iberat
edbydi ff
usionand
i
ndicatesthe pr
esence ofvol
ati
l
e componentorgaspr esentin sample by
specif
iccol
ourchangeorfor
mati
onofcolouredpr
eci
pit
ate)istakeni
nthecenter
well
.

4. Thecoveri
snowpl
acedandt
heassembl
yisr
otat
edt
oef
fectanai
rti
ght
l
i
qui
dseal
.

5. ThesealedorclosedConwayisti
lt
edbackandf
ort
htomi
xthesampl
eand
l
i
ber
ati
ngagentuni
formlyt
hroughoutt
hesamplewel
lar
ea.

6. Theassembl
yist
henpl
acedonat
abl
efort
her
eact
ion.

7. AnotherConwayassemblyist
akentocar
ryoutt
heabovest
epf
ort
hecont
rol
i
.e.bytaking1ml.ofwat
erinst
eadofsamplef
orcompari
son.

8. Thesampleandthecont
rolcont
aini
ngassembl
i
esmaybepl
acedonwat
er
bat
htofaci
l
itat
ereact
ion.

9. Attheendofthereacti
on,theConwayassemblies(sampleaswel
las
cont
rol
)areremoved wit
h greatcare so as t
o prev
entanyseali
ng agent
spl
ashi
ngordi
ppi
ngfr
om theedgeofthecoverint
othecenterwel
l.

10. Thecont
entsofthecenterwellcontai
ningthereact
ionpr
oduct
sfor
medmay
nowberemovedforcol
ori
met r
icorotherappropri
atemeasur
ement.

Using t
he fol
l
owi
ng tabl
eindicat
ing seali
ng and tr
appi
ng agent
s and
detect
ionthereofmayconv
enient
lycarr
youtt hedetect
ionoftoxi
cgasesor
vapours

Tabl
e–4.
7:MI
CRO-
DIFFUSI
ON-ANALYTICALCONDI
TIONSFORTOXI
C
GASES/VOLATI
LES.

Poi
son Seal
ing/
Liber
ati
ng Absor bent/ Detect
ion
Agent reactant
Car
bon 10%Sul phuri
cacid Palladium Pall
adi
um Chlori
de sol
uti
on
Monoxi
d chlori
desol ut
ion tur
nsgr eyt
obl
ack.
 e (0.5%)in2NHCl -
actsasdet ect
ion
reagent.
Et
hanol Sat
urat
ed sodium Pot assi
um orangetogr een.
car
bonatesol
uti
on dichromate
solution i
n
sulphuricaci
d.
Cy
ani
de 10%Sulphur
icaci
d 10% NaOH Al kali
neext racti ssubj ected
solution (
absorbt o Prussian blue t estusi ng
HCNgas) . FeSO4and HCl– Pr ussian
blueppt.orcol ouration.
Sulphur 10%H2SO4 10%NaOH Formati
onofbl ackpptwi th
Dioxi
de leadacetate..
Phenol 10%H2SO4 10%NaOH Byaddi ng Fehl ing’
sr eagent
to alkal
ine ext ract – bl ue
colourat
ion.
Par
aldeh 10%H2SO4 10%NaHSO3 Byaddi ngp- hydroxydiphenyl
yde inH2SO4. -bluecol our

4.
7.2 I
dent
if
icat
ionofsomeGaseousPoi
sons:

1. Phosphi
nei
nBi
ologi
cal
mat
eri
als:

25gms.ofbi ologicalmat eri

alist akeninaconi calflaskfil
ledwi t
haguar d
tubecont aini
ngl eadacet atesoakedcot ton.A f ew ml .ofcadmi um sul phate
solutionisadded.I ti sacidif
iedwi thdi l
utesulphuricacid.Themi xtureisheat ed
gentlyonwat erbat hat40- 60 C.Thegasev olvedi sall
owedt ocomei ncontact
withAgNO3paper .I tturnsgr eyory ell
owishbr ownorbl ack.( Duet or eacti
onof
phosphi newi thsi l
verni tr
atesol ution).Thepr esenceofphosphi dei sindi
cated.
Thepaperi sdriedandcuti ntopi ecesanddi ssol v
edi ndilnit
ricacid.Theext ract
i
sev aporatedt odr y nessf or2- 3times.Ther esidueist akeni naf ew dropsof
concent ratedni t
ri
caci d.1ml .ofammoni um mol ybdatesol uti
oni saddedand
war med.Thef ormat ionofcanar yy el
low precipi
tateconf i
rmst hepr esenceof
phosphi de.

A) I tmay be est imated by trapping the gas i

n bromine waterorsodi um
hydrochl ori
tesoluti
on,andaf tereliminati
onoft hebr omine,determinati
oni s
donef orthepresenceofphosphat e.Itmaybedet er
minedingasesbyuseoft he
reaction,
PH3+3HgCl 2P(HgCl )
3+3HCl ,thequanti
tyofacidbeingpr oport
ionalto
thev olumeofphosphi nepresent.
2. Phosgene(COCl 2)i
nBiol
ogicalMater
ial
s:(4)
10gms.ofv iscer
aistakeninaconicalf
laskfi
tt
edwi t
haguardtubewhosebul
bisfil
ledwi
thlead
acetatesoakedcotton.Theotherendoft het ubei sputint
oar ecei
vingfl
askcontai
ninga
soluti
onasabsorbentfortheevol
vedgas.20-25ml .of1(
N)sul
phuri
cacidisadded.Thef
laskis
 nowheatedsl
owl
yandt
heev
olv
edgasi
scol
l
ect
edi
nanyoft
hef
oll
owi
ngsol
uti
onf
orcar
ryi
ng
outt
est
s.

1. Theliberat
edgasist r
appedi n5ml.of5%si l
vernit
rat
esoluti
on.Thesoluti
onturnsblackor
yellowish-brownorbrowni ndicatingpr
esenceofar si
neorphosphineorstil
bine(Theblackening
ofleadacet atecott
oninguar dt ubeshowsi nter
ferenceduetosulphi
de).Tothesolut
ionisadded
af ewdr opsofconcentratednitricaci
dfol
lowedby0. 5mlof5%ammoni um molybdat
esolutionin
nitri
caci d(Reagent:Appendix393Vogel ’s).Canar yyel
low col
ourat
ionisobservedwhicht urns
blueonaddi nganalkal
inesolutionofbenzidine.

Sensi
ti
vi
ty:
0.1mgphosphor
ous
2. Thegasev ol
vedistr
appedinasoluti
onconsi
sti
ngofamixtur
eofp-ni
trobenzy
lpy
ridi
neand
n-
benzy
lani
l
ine.Theformati
onofredcolouri
ndi
cat
esthepr
esenceofphosgene.

4.
7.3 Det
ect
ionoft
oxi
cgasesi
nai
r:

Thedet
ect
ionoftoxi
cgasesinai
risanarearel
atedt
oai
rpol
l
uti
on.Howev
er,
for
thet
oxi
col
ogical
purposes,
mult
igasdet
ector
smaybeused.

4.
7.4 Det
ect
ionofCar
bonmonoxi
dei
nbl
ood:

Carbonmonoxi dehasmor eaffi

nityforbloodtoformt oxi
ccarboxy
-haemoglobi n.Carbon monoxi de has an af fi
nit
yf orcombi ni
ng with
hemogl obi
n mor ethan 240 times greatert han oxygen.This causes
sinist
ertoxicit
ybyprev enti
ngnormaltr
anspor tat
ionandsuppl yofoxygen
tocel l
s.Further,apersonsurvivi
ngsev eralhoursafteranexposurehas
alreadyeli
mi natedamaj orper
centageofcar bonmonoxidefrom hisbody.

I
dent
if
icat
ion:

1. Taket wosmallporcelai
n-evapor
atingdi
shes.Pl
ace1mlof
nor
malbloodi ntoonedishand1mlofsuspect edbloodinanot
herdish.
Heatbotht hedishesgentl
y.Thenor malbloodwil
lchangetoabr own
bl
ackwher easthebloodhavingcar bonmonoxidewil
lbecomebrickred.
Thi
stestissensit
ivet
o40%car bonmonoxi de.

2. Takeadropofbl
oodanddi
lutewith10-15mlofwat er
.Compare
wi
thblooddil
utedi
nthesamemanner.Bloodcontaini
ngcar
bonmonoxide
i
spink.Thi
stesti
ssensi
ti
vet
o50%car bonmonoxi de.

3. Mi
crodi
ff
usi
ont
echni
que(
Fel
dSt
ein)
:

Outercompar
tment: 1 mlon one si
de pl
us 1 mlof10%
sul
phuric
aci
donothersi
de.

I
nnercompar
tment: 2mlofpal
l
adi
um chl
ori
de(
11mg
pal
l
adium
 chl
ori
dein25mlof0.01Nhy dr
ochlor
ic
aci
d;gentl
yheatto di
ssol
ve)prepare
fr
eshonneed.Sealwit
h10% sulphur
ic
aci
d.

Atr oom t
emper
ature( 25-
300C)dif
fusi
on ti
me istwo hour
s.
Reducti
onofpal
ladi
um chlori
detogreyorbl acki
sposi
ti
vefor
car
bonmonoxi
de.Putr
efacti
onofbl
oodint
erf
eres

Thi
stesti
ssensi
ti
vebet
ween5and10%sat
urat
ion.

4.
7.4.
1 Quant
it
ati
veest
imat
ionofCar
bonMonoxi
dei
nBl
ood:
-

Met
hod-1

The met hod depends on t he f actt hatnormalbl ood cont ains

several f orms of hemogl obin, e. g.,t he r educed f orm, t he
oxygenat edf orm,andasmal lamountofmet hohaemogl obin.Ifa
reducingagentsuchassodi um di thioniteisaddedt ot heblood,
both t he oxy genated f or
m and t he met hohaemogl obin ar e
quantitati
velyconv ertedtot her educedf orm,whi chhasav i
sible
spectrum.Car bon monoxi de has a much gr eateraf fi
nityf or
hemogl obint hanoxy genandcar boxy haemogl obi ni snotr educed
by sodi um di thionite.Thus,ev en when t reated wi t
h sodi um
dit
hionite,car boxy haemogl obi nr etains i t
s nor malt win-peaked
spectrum.Thewav elengthofmaxi mum absor bancedi f f
erencef or
spectraAandBi sat540nm,whi lstat579nm,t hespect r
ahav e
thesameabsor bance( i
sobest i
cpoi nt).Theper centagecar bon
monoxi de-saturationofabl oodsampl ecanbecal culatedf rom
measur ement soft heabsor banceatt hesewav el
engthsoft he
carbonmonoxi de-saturatedsampl e( A) ,thecar bonmonoxi de-free
sampl e( B),andt heunt reatedsampl e(C),aft
err educti
onofeach
withsodi um dithionit
e. (
5)

Pr
ocedur
e:

Dil
ute0. 2mlofthebloodsampl e(mi xedwell)with25mlofa0. 1%
sol
utionofammoni aanddi vi
det her esul
ti
ngsol utionint
ot hree
approximatel
yequalpar t
slabeledA,BandC.Sat uratesol
utionA
wit
hcar bonmonoxidebybubbl i
ngt hegast hroughi tinawi de
containerandatar ate,whichmi nimizesfrothi
ng.Af ew minutes
bubbli
ngshouldsuff
ice.SaturatesolutionBwi t
hpur eoxygenfor10
minutest odi
spl
aceallthestrongl
yboundcar bonmonoxi de.

Addasmal
lamountofsodi
um di
thi
oni
tet
oeachofthesol
uti
onsA,
BandCandalsot
o10mlof0. 1%ammoniasol
uti
on,andmixwell
.
Placemat ched1cm cel lscontaini
ngt heammoni asoluti
oni ntothe
sampl eandr eferencebeamsofaspect rophotometerwhi chhas
beensett or ecordbetweent hewav elengths500and650nm, inthe
superimposed,r epeat-scanmodeonar angeof0t o2absor bance
units.Recor dthebasel ineforthedet erminati
on.Thenr ecor dthe
absor bancespect raforsolut
ionsA,BandC.Washoutt hesampl e
cellthoroughlybet weent her ecordi
ngsandwasht hecel lwi t
ha
l
ittl
eoft hesolutionwhoseabsor banceisabouttober ecorded.

Ift
hebl
oodsampl
ewasfrom apersonwhodiedfr
om i
nhal
ati
onof
car
bonmonoxi
de,
asetofspect
rawill
beobt
ained.

Measuretheabsorbanceofeachsol
uti
onat540nm and579nm
andcalcul
atet
heratioofabsor
banceat540t
othatat579nm f
or
eachofthesol
uti
onsA, BandC.

The per
cent
age car
boxy
-hemogl
obi
n-sat
urat
ion i
s cal
cul
ated as
fol
l
ows:

rat
ioforC–rati
oforB
%sat
urat
ion= _ _
_ _
___
___
_____
___X100
rat
ioforA–rati
oforB

Approximat
e normalv al
ues f
orthe r
ati
os ofabsorbance are:
sat
uratedcarbox
y-hemogl
obi
n1.5,
andreducedhemogl
obin1.1.

Not
ethatthehemoglobi
ncontentofbloodcanv ar
yandt her
efor
e,
t
hevolumeofdil
uentmayalsoneedtobev ar
ied.Adil
uti
ongivi
nga
maxi
mum absorbanceofabout1i
sideal.

Carbon monoxi de in blood may also be quant

if
ied using an
automat edvisi
blespectrophot
ometer(IL282Co-oximeter
),which
uti
l
izesf ourwav el
engthstomeasurehemogl obi
n,oxyhemoglobin,
carboxy-hemoglobinandmet haemoglobi
ninsmallbloodsamples.
Referencestandardsarecommer ci
all
yavail
abl
e.

Met
hod–2

Pr
inci
ple:

Car
bonmonoxi deisl i
beratedf rom carboxy-
hemogl obinby10%
sul
phur i
cacid.Thegasi str appedbypal l
adium chloridesoluti
on
and reduces yell
ow tan pal ladi
um chlori
de sol
ution t o metall
ic
pal
ladium,whi ch fl
oats as a bl ack si
l
verf i
lm on t he sol
ution.
Car
bonmonoxi deisoxidizedt ocarbondioxidei
nthispr ocess.The
unr
eact edpal
ladium chl
oridei smeasured.
 Mater
ial
s:1)30%COHbbl oodstandardb)UnknownSeal
ingagent
–2ml .of10%sul phuri
cacidsample.Li
berat
ingagent–1ml .of
10%sulphur
icaci
d( v/
v).

Trappi
ngagent:2ml.ofpall
adi
um chl
ori
desoluti
on(0.
22gmsof
pall
adi
um chl
ori
dei
n250ml .of0.
01Nhydrochl
ori
caci
d).

Pr
ocedur
e:

1. Thereagentsaremi xedformicro-
dif
fusionasstat
ed
earl
i
eri nt he generalprocedur
ef ormi cro-di
ff
usi
on and
al
lowedtost andforonehour.

2. Theextentofreact i
oni snotedat10,20,30and60
minutesint
ervalbytakingt hecontentsoft
hecenterwellby
adropper.I
tisfil
ter
ed.To0. 1ml.ofthefi
lt
rat
e,1ml.of15%
potassi
um iodide,8ml .ofwat erand1ml .of1% starch
sol
utionareadded.Itismi xedthoroughl
yandabsorbanceis
measuredat500nm.

3. Abl
ankr
uni
smadef
oral
ltheobser
vat
ions.

4. Theper
cent
ageofcar
bonmonoxi
dei
scal
cul
atedas
fol
l
ows.

1.
0ml
.ofPdCl2sol
ution0.
528mgofpal
l
adi
um
1mgofpal
ladium 0.
21mgofcar
bonmonoxi
de

Assumingat
otalhaemogl
obi
nconcent
rat
ionof15g/100
ml
.ofbl
ood,

O.
D.ofreagentbl
ank–O.D.ofsampl
e
%COhaemogl
obi
n= X9
O.D.ofr
eagentbl
ank

Ref
erences:
1. J.For
ensi
cSci
ence6:
401,
1961.

2. AOACof
fi
cial
met
hodsofanal
ysi
s986.
20chapt
er10,
p-47-
48,
(1995)
,

3. Bur
eauofI
ndi
anSt
andar
dsSpeci
fi
cat
ion,
IS1061:
1997

4. A SCur ry
,PoisonDetect
ioni
nHumanOr
gans,Char
lesC.
,Thomas,
Spr
ingf
iel
d.,
England,
1963.
4. E.
J.v
anKampenandH. Kl
ocwen,Ned.Fi
dschr
.Geneeskd,
195498,
161164,etalRee,
Trav
.Chi
m.desPaysBas,1954,
23,119
128.

5. Lundqui
st,Fr
ank,Met
hodsofForensi
cSci
ence,Vol
.It
oIV,
Int
er
Sci
encePubl
isher
s,N.Yor
k,19962(
Ref.No.
2inSect
ion3).

Ot
herr
efer
ences:

1. Cl
arke,E.
G.C.,I
sol
ati
onandi dent
if
icat
ionofdr
ugs,2ndEdn.
,
ThePharmaceut
icalPr
ess,London,1986.(Ref
.No.
15insection1
and2)

2. Fei
gl,F.
,SpotTesti
nOr
gani
cAnal
ysi
s,7t
hEdn.El
sev
ier
,
Amster
dam,1966.

3. Sunshi
ne,I
.(ed)
,Met
hodol
ogyf
orAnal
yti
calToxi
col
ogy
,CRC
Pr
ess,
Clevel
and,
1975.

4. Braun,
R.D.
,I
ntr
oduct
iont
oChemi
calAnal
ysi
s,McGr
awHi
l
l,N.
Yor
k,1982.

5. Safer
stei
n,R.(ed)
,Cr
imi
nal
ist
ics:AnInt
roduct
iont
oFor
ensi
c
Sci
ence,
6thEdn.
,Prenti
ceHal
l
,NewJersey,
1998.

6. Siegel
,J.A.,Saukko,P.
J.andKnuffer
,G.C.
,Encycl
opedi
aof
For
ensi
cScience,Vol.
1,AcademicPress,
N.York,2000.

7. Maehly
,Andr
eas,ChemicalCri
minal
i
sti
csSpi
nger–Ver
lag,
Ber
li
n,1981(Ref.
No.
13insect
ion3.)

8. Ghosh,S.andBagchi,K.
N.,Organi
candToxicol
ogi
cal
Chemi
str
y,Ar
tPress,
Indi
a,1948.(
Ref
.No.12insect
ion3)
.

9. Jungr
eis,
Erv
im.
,SpotTestAnal
ysi
s,JohnWi
l
eyandSons,
N.
Yor
k,1984.

10. Cur
ry,A.S.
,AdvancesinFor
ensi
candCli
nicalToxi
col
ogy
,CRC
Pr
ess,Cl
eveland,1972(Ref
.No.
4atPage1)

11. Lundqui
st,Frank.
,MethodsofForensi
cSci
ence,Vol
.I-
IV,I
nter
sci
encePubli
shers,N.Yor
k,1962(Ref
.No.2atPage2).

12. AOAC,
Off
ici
alandTent
ati
veMet
hos,
1980(
Ref
.No.
11atPage
2)
.
13. Fei
gl,
Fri
tz.
,SpotTesti
nOr
gani
cAnal
ysi
s,El
sev
ier
,Amst
erdam,
1975.
 SECTI
ON–5:ANALYSI
SOFI
NORGANI
CPOI
SONS
(
CATIONSANDANI
ONS)

5.
1 Ti
tl
e: Anal
ysi
sofi
nor
gani
cpoi
sons(
cat
ionsandani
ons)
5.
2 Scope:Anal
ysisofi norgani
cpoi
sonsincri
meexhibi
tsvi
z.hai
r,nai
l
,skin,bone,biol
ogical
materi
als,foodandfoodproduct
s,meat
,mil
kandmilkproducts,dr
inks,cer
eals,
gr
ains,wat eret
c.

5.
3 Pur
pose:Todet
ectanddeter
minei
nor
gani
cpoi
sons(
met
alcat
ionsanddi
ff
erentani
onsor
r
adi
cals)i
nexhi
bit
s.

5.
4 Responsi
bil
i
ties:
Gazet
tedOf
fi
cer
sandot
herassoci
atedsci
ent
if
icst
aff
.

5.
5 DESCRI
PTI
ONOFMETALLI
CANDNONMETALLI
CPOI
SONS

Thei mpor tantpoi sonsi nt hegr oupofmet alsincl udear senic,ant imony ,
mer cur y,l
ead, thal
lium, zinc, manganese, bari
um andal umi nium.Thesal t
s
ofthesemet alsar et oxic.Non- met alsorspeci ficallytoxi cani onsi ncl ude
borat e,br omi de,chl orate,cy anide,f luori
de,hy pochl or it
e,i odide,ni trate,
nit
rite,oxal ate,br omat e,i odat e,sul phide,thiocyanat eet c.Gener all
y ,the
anionsi nsal tsar er esponsi blef ort het oxicact ion.Thus,t heirdet ect i
on
giv
esacl earidear egardingt hesour ceofpoi son.Somet i
mes, toxicani ons
aredi rectlyusedasact i
v especi esv iz.cyanide,oxal ate,bor ateet c.These
cationsandani onsar et obei solat edespeci allyi ncaseofbi ologi cal
mat er i
alsf oronwar ddet ectionandest imation.Ther ear esomecl assi cal
met hodsv i
z.Rei nscht est ,Gut zei ttest,Mar sht estt hatar est i
llfollowed
fort hedet ect ionofcat ions.Thepr esentdayanal ysisi ncludesmi cro
met hodsandi nstrument alt echni ques.Bef oreelabor ation,thedescr ipt i
on
ofcat ionsandani onsar egi v enbel ow.
5.
5.1Char
act
eri
sti
csofsomet
oxi
cCat
ions:

NAME SOURCE SI
GN&SYMPTOMS/TOXI
CITY
CHARACTERI STI CS
Ar
seni
c Differentcompounds Al lar seni calsi nhibit Ar seni ci sagr eysubst ance,
of ar seni c: Ar seni c sul phy dryl enzy me sy stem whi ch i s sai dt o be non-
tri
oxi de (As2O3) necessar y for cel l
ular poi sonous.Asi tisi nsol uble
(Whi te ar seni c), met abol i
sm, sy mptoms of i n wat er and t her efore
chlor i
de, sul phi de poi soni ng i nclude f aint ness, incapabl e of absor ption
(Orpiment ), red depr essi on, nausea, sev ere f or m t heal iment ar ycanal .
sulphi de (As2S3) bur ning pai n,const riction of Howev er ,itiscont inuousl y
(Real gar )
, copper t hroat ,increasedsal ivationand changi ng i nt o ar senious
acet oar senite ( Par i
s st omat i
ti
s.Sev er et hi rst and oxi de whi ch i st ast eless
Green) , copper pr oject ilev omi t
ing.Vomi tmay and most poi sonous.
arseni te (Schl ee’s cont ain st reaks of bl ood Ar seni ccausest oxi cityby
Green) ,arsenat es. (distinct i
onf rom chol er a),ur ine combi ning wi th sul phydr yl
i
s suppr essed,ski n becomes enzy mes,t hus i nt erfering
Organi c compounds col dandcl ammy . wi th cel l met abol i
sm.
ofar senic:Cacody li
c Inchr onicpoi soni ngr ed Poi soni ng i s most ly done
acid, sodi um pi gment ation ( rain dr op t ype) by ar seni ous oxi de.Such
pent apheny lar sonat e onski noccur s. type of poi soni ng
(Atoxy l
), dioxy- account edf or90%ofcases
diami no ar seno- i
nt he past .Thi si s not
benzene common now a day s.
dihydr ochl ori
de Howev er ,animalpoi soni ng
(Salvar san) Silver by arseni c occur s
arphenami ne ( Silver frequent ly.
Salvar san)et c. pent aval entar senici st obe
Propr ietory ar ticles reduced t o t he t r i
valent
cont ainingar seni c. stat ef ori tsdet ection.
Rough on r ats, f l
y Theor gani car seni cal smay
paper ,weedki ller,f l
y cont ainar senici nt r i
valent
wat er, fl
ypowder . and pent avalentst ate.As
anant i
dot eBALi sused.
Ant
imony Ant i
mony occur si n Sy mpt omsar esi mi l
art o Ant imonyasamet ali snot
thef or m ofanoxi de ar seni cpoi soni ngbutappear s consi der ed as poi son but
and sul phi de and i s somet i
me l at er than ar seni c. wheni nhal edi nt hef orm of
present as an Thesy mpt omsi ncludeast r
ong v apouri ti s sai dt o hav e
i
mpur ity i n many met all
ict astef oll
owed by a pr oduced danger ous
mi ner al ores. bur ningsensat i
oni nt hemout h sy mpt oms. Poi soni ng by
Ant i
monyi s used i n and Oesophagus,Const riction t artar emet ic due t o
all
oy s, f oil, t y pe oft hroatf ollowed bynausea, ov erdosemayoccur .
met al , pl ating incessantv omi t
ingwi thpai ni n Acci dent al poi soni ng by
bat t
er ies, Cer ami cs st omachandabdomen.Vomi t ant imony t r i
chlor ide i s
andpi gment s,saf ety may cont ain bl ood i n l atter known ( used i n ar tsasa
mat ches and ant st age.Respi r ati
oni sl abour ed. br onzi ng l i
qui d). As an
past e. Deat hoccur sbycar diacf ailure ant idot eBALi sused.The
mechani sm ofpoi soni ngi s
Inorgani c simi larwi tht hatofar seni c
compounds: poi son bycombi ning wi th
Ant i
mony pot assi um sul phy dr y l enzy mes and
 tart
arat e (tartar t
hus i
nter
fer
ing wi
th cel
l
emet ic), ant i
mony met
abol
ism.
tri
oxide, ant i
mony
tri
chloride ( but
terof
anti
mony )
, ant i
mony
tri
sulphide (
black
anti
mony )
, ant i
mony
hydride( sti
bine).
Organi cCompounds:
Prepar ati
ons:
Sti
beny l
, St i
bami ne,
Urea sti
bami ne,
Hey denet c.

Mer
cur
y Met allic Mer cury:I taf f ects cel lularmet abol ism Const ri
ction of t hroat is
Brightsi lvery,heav y and f unct ion.The sy mpt oms mor e mar ked.I r
ri
tation of
l
iqui d used in are due t o cor rosi v e subl ime. ki dneyi spr onounced.
ther momet ers, Sympt omsst ar twi t hinhal fhour Met alli
cmer cur ycanhar dl
y
bar omet ers,mer cur y of i nt ake. The sy mpt oms be consi der ed t o be a
vapourl amps. i
ncl udeacr id,met alli
ct ast e,a poi son.
Inor gani c feeling of const ri
ction or I ti s notabsor bed when
Compounds: choki ng. The mout h,t ongue t akenbymout h.
Mer cur ic chl or i
de andf acesar ecor r oded, swol len Mer curyi sr eadilyabsor bed
(cor rosi vesubl imat e), andcoat edwi thagr eyishwhi t
e t hrough ski n when r ubbed
cyani de (as coat ing. Hot bur ning pai n ofal lcompounds.Mer curi
c
fungi ci de) , ni trate, mout h ext endi ng t o st omach chl oride and ni t
rate ar e
sulphi de ( Cinnabar , &abdomenf ollowedbynausea, r esponsi blef ormostoft he
Hingul , Si ndoor )
,r etchi ng and v omi ting.Vomi t casesofpoi soning.
sulphat e. may be accompani ed by Ant i
dote: BAL.
Organi c Compounds: mucousandbl ood.Thi smaybe
Dimet hy l mer cur y
,f ollowed by di ar rhoea wi th
mer cur ochr ome, bloodst ain.Ur inei ssuppr essed
organi cmer cur ial. and scant y. Pul se becomes
Prepar ation: Nept al
, quick, smal l and i rregul ar.
thiomer in sodi um, Spasms, conv ulsi ons may
mer cur ophy ll
ine precedet hedeat h.
Mer cur ousmer cur yi s
nott oxi c.
Copper Inor gani c Salts: Afterswal lowi nganast ri
ngent Ski nmaybey ell
ow duet o
Copper sul phat e, tast ei sf elt.The sy mpt oms j aundi ce Greenish- bl
ue
carbonat e, sub- incl ude met all
ict ast ei nt he f orthmaybecomi ngoutof
acet at e. mout h,sal i
vat i
on,bur ningpai n mout handnost r
il
.
i
n st omach,nausea,r epeat ed Thecont ent sofst omachi s
vomi ti
ng. Vomi ted mat t
er i s gr eenorbl uei ncol our.
blueorgr eent urnst odeepbl ue Ant i
dote:Cal cium EDTA or
on addi ng ammoni a sol ut ion BAL.
(distinct i
onf rom bi le).Ol igur ia,
haemat uria, al -
bumi nur ia,
uremi amayoccur .Jaundi cei s
common i n sev ere cases.
Cramps and conv ulsion al so
occur .
Lead Inor gani cSal ts:Lead Sensat ion ofbur ni ng,dr y ness Chr onic poi soning ar e
acet at e,sub- acet ate, of t hr oat , sal ivat ion, i nt ense mar ked byCol i
c,lead l i
ne
nitrat e, sul phat e, thirst af ter swal lowing t he ongum, anemi aet c.
chr omat e, chl oride, poison. Vomi t may cont ain Ant i
dote: Cal ci
um EDTA.
 monoxi de.The f ind bl ood.Col ickypai n.Theot her
use i n oi lpai nt ing, sympt oms i ncl ude nausea,
calico pr i
nti
ng, dy e headache, v er ti
go, cr amps,
andpi gmentet c. conv ul
sion,numbnessf ol l
owed
byhemol ysisandol i
gur i
a.
Thal
l
ium Assal t: Joints pai ni nl eg and f eet , Poisoni ngi srare.
Thal lium acet ate, stomat i
tis,drowsi ness,dr yness The f al l
ing ofhai ri st he
sul phat e ofmout h,v omi ti
ng,di arrhoea, t ypicalsy mpt oms.Fusi form
pain, pul monar y oedema, swel li
ngoft her ootofhai r
cyanosisandr espi r
at or
yf ailure. occur s.
Antidot e: Pr ussi an bl ue,
potassi um i
odi de or
chlor i
de.
Zi
nc AsSal ts: Met all
i
c t aste, sal ivation, Poi soni ngi srare.
Zinc chlor i
de v omi ti
ng, abdomi nal pai n, Wi t
hzi ncchloridecor r
osi ve
(sol der ing fluid), purging conv ulsion, col lapse sy mpt omsoccur .
sul phat e (
wood anddeat h. Phosphi ne i s t he act ive
preser vat i
ve), oxi de const ituent, whi ch i s
(pigment ),phosphi de l
iber atedbyact i
onofaci d
(rodent icide)etc. i
n body sy stem when
phosphi deisconsumed.
Bi
smut
h Assal ts: Met all
i
ct aste, salivat i
on, paini n Poi soni ngi srare.
Car bonat e( used f or throatandabdomen,v omi t
ing, Bi smut h subni trate( used
treat ment of purging,scant yur ine col lapse f orX- rayexami nat ion)
diar rhoea) , sub- anddeat h. In bismut h poisoni ng face
nitrat e ( cosmet ic). i
sbl ackordar kbr own i n
Or gani c sal ts of colour .
bismut h. Bi smut h Antidot e: BAL.
salicy lat e( dermat ol )
,
orphenami ne
sul phonat e
(bismar sen).
Manganese Assal ts: Bur ni
ngpai ni nt hemout hand Poi soni ngi srare.
Pot assi um throat,abdomen,di ff
icultyi n Tongue and phar ynx ar e
per manganat e ( as swal l
owing, cont i
nuous st ained ( black or dar k
bleachi ng agent i n vomi ti
ng,di f
ficultyi nbr eathi ng br own)
text i
le, abor ti
fi
ci ent, deat hoccur sduet oci rculat or y Antidot e: Calcium
oxi dant ) ,dioxide ( in fail
ure. gluconat eoredenat e.
drycel l).

Chr
omi
um Assal t
s: Bitt
er met all
ict ast
e,i ntense In chroni
c poi soni
ng
Potassium chromat e pain i n stomach, gi ddiness, ulcerat
ed sor
es and
(dye) dichr
omat e vomi t
ing, diarrhoea, v omi t
ed eczemat ous der
mati
ti
s
(dye,furni
turest
ainer mat t
er isy el
low,st ools are ( chromehol e)occur.
etc.)
. yell
ow somet i
mes t inged wi t
h
bil
e or bl ood.Respi rat
ion is
slow and gaspi ng.These ar e
foll
owed bymuscul arcr amps,
ment alconfusi
on,cy anosisand
coll
apse.
Al
umi
nium Sal ts: Bur ning pai n i n t he mout h, Phosphi ne i s t he act ive
Alum – doubl esal t s throatand st omach.Vomi ti s const i
tuent, whi ch i s
of al umi nium and mi xed wi th bl ood,dy spnoea, pr oduced i n t he body
pot assi um or r apid pulse, l oss of system when phosphi de
ammoni um ( mor dant , coor dination, conv ulsion and consumed.
pur ifi
cat ionofwat er ) deat h.
Phosphi de ( uses as
rodent icide) .
Bar
ium Assal ts: Sev ereabdomi nalpai n,nausea Sol uble sal ts ar e hi ghly
Bar ium chloride met alli
c t ast e, sal i
vation, poisonous. Among t he
nitrate(py rotechni c), v omi ting,intenset hi r
st ,
pur ging, solublesal t
s,chl oridesar e
car bonat e(rodent i dilati
on ofpupi ls,di mness of most poi sonous. Bar ium
cide)sul phat e( forX- v ision,cr amps,t remor sanur i
a, i
ons ar e al so hi ghly
ray conv ulsion,col l
apseanddeat h poisonous. Poi soning
exami nai tion of GI duet or espirat oryf ailure.Sal ts casesar eacci dental.
tract )
, sulphat e actascar diacpoi sonandt hey
(depi l
at or y). alsopar aly seCNS.
Cadmi
um Asmet als:Sof t,light I ncreased sal i
vat i
on, nausea, Poi soningmayoccurf rom
whi te used i
n sev erev omi ting,cr ampsi nt he t hei nhalati
onofCadmi um
wel ding, abdomen,di arrohea,col lapse dustorf ume.
elect r
opl at ing butr arelydeat h. Antidote: BAL.
manuf act ure of
elect r
odes, cont rol
rodsi nat omi cpiles.
Sal t: Cadmi um
sul phide ( colour ed
glass, pai nt s,plast i
c) .
Ni
ckel Ni ckel t etracarbony l Pul monar yoedema. Poisoningi srare.
(mobi l
el iqui d).

5.
5.1.
1 Det
ect
ionanddet
ermi
nat
ionoft
oxi
ccat
ions:

Thetoxiccati
onsaretobedetectedinbiol
ogi
calmateri
alsi
ncaseoffatal
poi
soning.Thisisdonebydi gesti
on(dryorwet).Theext r
actobt
ained
aft
erdigest
ionisusedforchemicalt
estandquant
it
ation.

5.
5.1.
2ARSENI
C:
A.Chemi
cal
Testf
orAr
seni
c:

1. Rei
nsch’
sTest
:

About20ml .ofconc.HCl( pur

ef ortoxicologi
calwork)and100ml .of
wateraret
akeni naporcel
ainbasi
ni nwhi chabrightcopperfoi
l(about3
i
nchesby1/ 4inch)ispl
acedwithoneofi tsendsbeingf i
xedontheedge
ofthebasi
nint heform al
oop.Iti
sboiledf orabouthalfanhourtoseeif
thecopper
,basinandtheacidarefreefr
om t hemetaltobetested(herei
t
i
sar senic).Ifast ainoncopperf oilappear s,theblankexper imenti stobe
carried outagai n wi thf resh mat eri
als.I ft he bl ank i s negat i
ve,t he
suspect edmat eri
al(bi ol
ogi calornon- bi ological
)isaddedandboi l
edf or
aboutanhourormor ewi thoccasi onaladdi ti
onofwat erandaci dt omake
upf ort hel ossduet oev apor at i
on.Ashi ningst eelgr ainst ainappear sina
few mi nuteswhi chbecomest hickgr adual ly.Thest ainedcopperst rip
obtai nedbyRei nscht esti swashedcaut i
ouslywi thwat erf oll
owedby
alcoholandf inall
ywi thet hert or emov et headher ingf at,ifthemat ri
ces
arebi ol
ogi calmat erials.Thest r
ipi sdr iedbykeepi ngi tbet weenf i
lter
papersheet s,cutinsmal lpiecesof0. 2mm x0. 2mm si zeandt akeni nt o
Reinscht ube.Thet ubei sheat edsl owlyont heflameofspi ri
tlamp.The
blackdeposi tont hecopperst ri
pv ol
atili
zesandget sdeposi tedont he
cool erpar toft hetube.Thet ubei scool edandv iewedundermi cr oscope.
Char acteristicoctahedr alcr yst al
sofar seni ousoxi dear eseen( sensi ti
vity
10g) .Ther ear ecertainl i
mi tat i
onsoft het estviz.negat iveresultmaybe
obtai nedi foxi di
zingagenti spr esentorAsi sin+5st ate( iti st obe
reducedt o+3st ate)byt reat mentwi thsodi um sul phiteorpot assium
i
odi deorst annouschl ori
deorf erroussul phat easr educi ngagent .Or gani c
arseni cals do notr espond i for ganic mat teris notdest r
oy ed.Some
organi csul phurcompoundspr oducesbl ackst ainsofcoppersul phide,
whichmayber emov edbyoxi dati
on.Theconcent rationofHClshoul dnot
bet ool owort oohigh.Thi st estisgener all
yusedf orr apidscr eeni ngofAs,
Sb, Hg.
2. Gut
zei
tTest
:

Thesol uti
onobt ainedfrom theWetDi gest
ionpr ocessist estedbyt hi
s
method.1ml .ofthesoluti
onistakeni nt
oaGut zeitapparat
us, 2pel l
etsof
purezincmet alareputintoit
.5ml .ofdil
.sulphuricacidispour edov erthe
contents.Theev olvedgasispur i
fiedbypassi ngov erl
eadacet at
epaper
(t
oabsor bH2Sgas)andi sreactedf i
nall
ywi t
hmer curi
cchl oridetest
paper.Ay el
lowst ainonthepaperindicatesthepr esenceofar senic.

3. Mar
sh’
sTest
:(
Quant
it
ati
ve)

Electrol
yti
cMar shBerzeli
ust estisdoneov erconv ent
ionalzinc-sul
phuri
c
acidmet hodf ortheevol
utionofnascenthy drogenast hesereagentsare
oftencont aminatedwithar senicetc.Scantymat eri
alslikebur ntbones,
hairandnai lpeeli
ngscontainingmi nutet
racesofar senicandf ortesti
ng
thef eebl
et r
acesofarseni
cpr esentasanaturalconsti
tuentintissues,t
he
Mar sh’stestappearst
obet heonl yreli
abl
etechniqueav ai
labl
e.

Thet estisperf
ormedi nt hesolut
ionobtai
nedf r
om t hewetdigesti
on
process.The solut
ion contai
ning ar
seni
cint he pentaval
entstateis
reduced tot r
ival
ent stat
e by boi l
i
ng with pyrogal
lolsolut
ion and
sulphurat
edwater.Onemloft hetestsol
uti
oni stakenintoaporcelai
n
basin,mixedwit
h2t o3dr opsof0.5% py
rogal
lolsolut
ionand1.0mlof
sat
uratedsulphur
atedwat er(wat
ersat
urat
edwithSO2)andboil
edf
or30
minutes.Thepent
av al
entarseni
cisr
educedtot
hetri
val
entst
ate.

A blankcont rolt estbyt akingdi l

utesul phuricacidi nt hecat hodeand
anode chamber s oft he Mar sh appar at
us by passi ng a cur rentof
approx.4.5Aat6v olt
sf or30mi nut es.Thereshoul dbenost ainont he
Mar sht ubeatt hisst age.Thet estsol uti
oncont ainingar senici nt he
tri
valentst agei st hen pour ed intot heappar at
usand t hepr ocessi s
repeatedf or30mi nutes.Ami r
rorofbl ackmet al
li
cl ustrei sobser vedat
thecool ersideoft heMar sht ube,ifar seni
ci spresent .Forquant i
tat
ive
esti
mat ion,thecont rolmi rr
or sofar senicint heMar sht ubear epr epared
for0. 001,0. 002,0. 006 and 0. 008 mg ofar seni
ct r
ioxide.Themi r
ror
obtained wi t
ht het estsol uti
on iscompar ed witht hesecont rolsand
quant i
tyofar senici scalculated.

Toconf ir
mt hatt hemirr
ordepositedint heMar sht ubeisonlyduet o
arsenic,
boththeendsoft hetubearesealedaft
erreplacinghydr
ogenwith
air
.Thet ubeisthenheatedslowlyonthef lameofspi ri
tlampbykeepi
ng
thenar r
owerpor t
ionoft hetubecooler.Themi rrordisappear
sandthe
octahedralcr
y st
alofar seni
oust r
ioxi
deget sdeposi ted on t
hecooler
narrowerendoft hetubewhichareidenti
fiedunderthehi ghpoweroft
he
microscope.

4. Gr
oupanal
ysi
s

Theext r
act
spreparedf
rom t
hedigest
edmater
ial
s(dr
yorwetdi
gestionof
biol
ogicalmat
eri
als)ar
esubject
edtogroupanal
ysi
stodet
ectarsenicby
separati
on.

1. Test
sforAr
seni
c(V)
:

Thefoll
owingt est
smaybeper f
ormedwi
tht
heextract
sprepar
ed
fr
om thedi gested mat
eri
als(
dryorwetdi
gest
ionofbiologi
cal
mater
ial
s).

A. Testwi
thSi
l
verNi
tr
ateSol
uti
on:

To2ml .oftheextr
act,
silv
ernit
ratesoluti
oni sadded.Browni
sh-red
preci
pit
ateisf or
med ( di
sti
ncti
on from ar seni
te and phosphate
whichyiel
dy el
l
ow precipi
tat
e).Thepr eci
pitateissolublei
naci ds
andammoni asolut
ionbutinsol
ubleinaceticacid.

Alt
ernat
ivel
y,pl
aci
ngaf ewdr opsoft
estsoluti
oninami crocruci
ble
maycar r
youtt hetest.Itiswarmedafteraddingaf ew dropsof
concent
ratedammoni asoluti
onand10%(v/v)ofhydr
ogenper oxi
de.
Iti
sacidifi
edwithaceticacid.Onaddi
ng2dr opsofsi l
vernitr
ate
 sol
ution,t
heformati
onofabrowni
shr
edpr
eci
pit
ateorcol
our
ati
on
i
ndicatesthepr
esenceofAs(
V).

B. Ammoni
um Mol
ybdat
etest
:

To2ml .ofext ract,ammoni um molybdateandni t

ri
cacidar eadded
i
n consi der
abl e excess. A y el
low cr ystall
ine precipit
ate of
ammoni um ar senomol ybdateisobt ai
ned on boi l
ing (
disti
ncti
on
fr
om ar seni
tes,whi chgi venopr ecipit
ate,andf r
om phosphat es,
whichy i
eldapr ecipitat
eint hecoldorupongent l
ewar ming).The
preci
pit
ateisi nsolubl ein nit
ri
caci d butdi ssolvesin ammoni a
solut
ionandi nsol utionsofcausti
cal kal
is.

2 Test
sforAs(
II
I)
:

Thetest
swit
htheextract
smayalsobeperf
ormedasinthecaseof
As(V)asst at
ed hereunder
.Itmaybement ioned t
hatAs( I
II
)
compoundar
ewi del
yusedforpoi
soni
ng.

A.Testwi
thSi
l
verNi
tr
ateSol
uti
on:

Yel
low preci
pit
ateofsil
verar
seni
tei
sfor
medi
nneut
ralsol
uti
on
(di
sti
nct
ionfrom ar
senat
e).

B.Testwit
hStannousChl
ori
deandConcent
rat
edHy
drochl
ori
cAci
d
(Bet
tendor
f’
sTest)
:

Adr opoft estsoluti

oni smixedupwi t
h1-2dr opsofconcent rat
ed
ammoni asoluti
oni nami crocrucibl
e.Tot hi
s,2dr opsof10%( v/v)
hydrogenper oxi
deand2dr opsofmagnesi um sulphatesol
ut i
onare
added.I tis ev aporat
ed slowlyand f i
nallyheated unti
lf uming
ceases.Ther esiduei streat
edwi t
h1- 2dr opsofasol uti
onof
stannouschl ori
dei nconcentratedhy dr
ochlori
caci dandwar med
sli
ghtly.Abrownorbl ackprecipi
tateorcolourat
ionisobtained.By
thi
st est,
arseniccanbedet ectedinpresenceofant i
mony.

5.Testf
orOr
gani
cAr
seni
cal
s:

To2ml .oft
heextracti
nat esttube,3ml .ofr
eagent(
stannous
chl
ori
de+concent
ratedhy
drochl
oricaci
d)isaddedandwarmed.A
l
emony el
l
owpreci
pit
ateorcol
ourisobserv
ed.

B.Quant
it
ati
on:

1. Est
imat
ionofAr
seni
cinbl
oodbyUVSpect
roscopy
:
Mat
eri
als:

a.St
andar
dAr
seni
cSol
uti
on:

2.4196gms.ofar senictri
chl
ori
deisdissol
vedinsuffi
cient1N
hydrochlori
caci
dt oproduce1000ml.Thissol
uti
oncont
ains1mg.
ofar senicperml.Thi sisdil
utedseri
all
ywithwatertopr oduce
soluti
onscontai
ning0.5,2.
0,5.
0and10.0gofarseni
cin1ml .

b.Si
l
verDi
ethy
ldi
thi
ocar
bamat
eSol
uti
on:

A 0.
5% solut
ion ofsilv
erdiet
hyl
dit
hio car
bamat
ei n pyr
idi
ne i
s
pr
epared.Thi
sreagentshoul
dbestor
edinamberbott
les.

c.St
annousChl
ori
deReagent(
40%)
:

40gms.ofstannouschlor
idedi
hydr
atei
sdi
ssol
vedi
nsuf
fi
cient
hy
drochl
ori
caci
dtoproduce100ml.

d.Di
gest
ionMi
xtur
e:

Amixt
urecontai
ning3v
olumesofnit
ri
cacid,1v
olumeofsul
phur
ic
aci
dand1volumeofperchl
ori
caci
disprepar
ed.

Met
hod:

1. Sampl
ePr
epar
ati
on:

5ml .ofbl oodsampl ei splacedi na125ml .fl

ask.5ml .ofthe
digesti
onmi xtureisadded.I tisheatedgent lyatfirst,t
henatabout
150oC.Whent hesolutionboi l
sandbegi nst ochar ,2ml .ofnitric
acidisadded.1ml .ofnitri
caci dandaf ewdr opsofper chlori
cacid
areadded cont i
nuouslyunt ilacl earst raw coloured solutioni s
obtained.The t emper atur
ei s mai ntained untilwhi t
ef umes of
sulphurtri
oxideareev olvedandt hesol uti
onisf r
eef rom nitr
icacid.
Itiscooled.Thecont entsofthef l
askist ransferr
edquant it
at i
vel
yto
a10ml .vol
umet ri
cflaskanddi lut
edt ov olumewi thwat er
.

2. 3ml .ofdi gest

edsampl eist r
ansferredtothear senic-
gener
atingvessel.Suffi
cientwat eri
saddedt opr oduce35ml .5ml .
ofhydrochl
ori
caci d,2ml .ofa15% sol ut
ionofpot assium i odi
de
and0.5ml .ofst annouschl or
idereagent( 40%)ar eadded.The
sol
uti
oni sswirledandal lowedt ostandfor15mi nutes.Apadof
gl
asswoolmoi stenedwithl eadacetatesol
utionisinsertedintot he
l
owert ubeoft hegener atingv essel
.3ml .ofsi l
verdiethyldithio
 carbamat esoluti
oni sintr
oducedi ntotheabsor bertube.3g.of
granul
ated zinc is added tot he f l
ask.The t wo parts oft
he
apparatusar eassembl ed.Theev oluti
onofar senicfor1houris
al
lowed. Si lver diet
hy l di
ethyl
dithi
o car bamate sol ut
ion i
s
tr
ansferred fr
om t he absorbed tube t o a 1 cm cel land t
he
absorbancei smeasur edat540nm usi nginther ef
erencecel
la
blankpreparedbyt r
eati
ng5ml .ofwat erinthesamemanner .

3. Theprocedureasaboveisrepeat
edusing5ml.eachof
di
lut
edst
andardsolut
ions.Theabsor
banceofeachoft
hesolut
ions
i
smeasured.

4. Theconcent
rati
onofarseni
cint hesampleofbl oodis
deter
minedfrom t
hecur v
eobtai
nedbypl ott
ingtheabsorbanceof
eachofstandar
dsolut
ionsagai
nsttheconcentr
ati
onofarsenic.
2. Det
ermi
nat
ionofAr
seni
cinur
inebyat
omi
cabsor
pti
onspect
rophot
omet
ry:

I
nt he met
hod the arseni
c gener
ator(
as st
ated i
n 5.
9.1)i
s
connect
edt oatomi
cabsorpt
ionspect
rophot
ometer.
Materi
als:

1. St
andar
dAr
seni
cSol
uti
on:

Asdescr
ibedear
li
eri
n5.
9.1.

2. St
annousChl
ori
deReagent(
20%)
:

20gms.ofstannouschlor
idedi
hydr
atei
sdi
ssol
vedi
nsuf
fi
cient
hy
drochl
ori
caci
dtoproduce100ml.

Met
hod1:

To25ml .ofur i
nesampl e1ml .ofa20% sol uti
onofpot assium
i
odideand0. 5ml .of20% st annouschl ori
dereagentar eadded,
mixedandal l
owedt ost
andf or20mi nutes.Thear senicgener ator
i
sisolatedf rom t hespectrophot omet erand100ml .oft hesampl e
sol
ut i
onisi ntroducedintot hev essel
.0. 5gm ofgranulatedzi ncand
1ml .ofhy drochloricaci
dar eadded.Thet wopartsoft hegener ator
are assembl ed immediat ely and st i
rred for2 mi nut es witha
magnet i
cst ir
rer.Thear senicgener atorisnow connect edt ot he
spectrophot omet erandtheabsor bancei srecor
dedat193. 7nm.

Theaboveprocedurei
srepeatedusi
ng25ml .ofeachoft
hedi
l
uted
st
andardsol
utionsand25ml .ofwat
er(
blank).
 Theabsor banceofeachofst
andardsol
uti
onofarseni
cispl
ott
ed
againsttheconcentr
ati
onofarseni
candtheconcentr
ati
oni
nthe
unknownsampl eisknownthereaf
terf
rom t
hecalibr
ati
oncur
ve
(non-l
inear
).

5.
5.1.
3ALUMI
NIUM
A. Chemi
cal
Test
sforAl
umi
nium:

1. Testwit
hAl uminonReagent(
aSat
urat
edsol
uti
onofAmmoni
um
sal
tofAur
inet
ri
carboxyl
icacid)
:

To1ml .ofextract,ammoniasolutioni sadded.Whi tegelati

nous
preci
pitateappear s.Thepreci
pit
ateist akenupi nmi cr
ot esttube.
2M hy drochlor
icaci disaddedt o di
ssolv ethis.1ml .of10M
ammoni um acetateand2ml .of0.1per centaqueoussol uti
onof
thereagentar eadded.I tisshakenandal l
owedt ost andf or5
minutes.Ammoni calammoni um acet ate soluti
on is added i n
excesst odecolorizetheexcessdy estuffandpr eventi
nterference
ofchr omi um,sili
ca etc.A bri
ghtr ed pr eci
pit
ateorcol ouration
persi
stingi nal
kali
nesoluti
onisobtained.
2. Testwit
hAl
i
zar
inS(
0.1% aqueoussol
uti
onofsodi
um al
i
zar
insul
phonat
e)
r
eagent
:

1dr opoft heextract(whi

chhasbeent reatedwi t
hj ustsuffici
ent
1M sodi um hy dr
oxidesoluti
ont ogivethet et
rahydroxoaluminate
i
on)i spl acedonaspotpl ate.1dr opofr eagenti sadded.Then
aceticacidisaddeddr op-
wiseuntilt
hevioletcolourjustdi
sappear s.
1 drop ofaci dis added in excess when a r ed precipit
ate or
colourat
ioni sobserved.

B. Quant
it
ati
on

1. Det
ermi
nat
ionofal
umi
nium i
nvi
scer
awi
th8-
hydr
oxy
qui
nol
i
ne:

Pr
ocedur
e:

25ml .oftheext r
act( obtai
nedf r
om aci ddi gesti
on)ist akeni na
500ml .beaker.1ml .ofconcent ratedhy drochl ori
caci di sadded
andst ir
red.Itisdil
utedt o200ml .5-6ml .ofoxi mer eagent( a10
percentsolutionin20per centacet i
caci d)and5gm.ofi sadded.
Thebeakeri scov eredwi t
hacl ockgl assandheat edonanel ect
ri
c
hotplateat95oCf or2.5hour s.Thepr ecipitat i
oniscompl etewhen
thesuper natantli
quid,originall
ygr eenishy ell
ow,acqui resapal e
orange-yell
ow colour.The pr ecipitatei s compactand can be
fi
lt
eredeasi ly.I
tisal l
owedt ocoolandt hepr ecipi
tatei sf i
l
ter
ed
throughasi nteredglass-f
ilt
eringcr ucible( por osit
yNo.3orNo. 4).
 Thepreci
pit
ateiswashedwithal
itt
lehotwat
erandfi
nal
l
ywi
thcol
d
water
.Iti
sdriedat130oC.Thepr
ecipi
tat
eiswei
ghedas
Al(
C9H6ON) 3.

2. Det
ermi
nat
ionofAl
umi
nium i
nvi
scer
abyspect
rophot
omet
ry:

Met
hod:

a. Sol
ochr
omeCy
ani
neRSol
uti
on:

0.
1gm.oft hesol
idreagenti
sdissol
vedinwateranddi
lutedto100
ml.andfi
lt
eredt
hroughawhatmanNo. 541fi
l
terpaper
,ifnecessar
y.
Thissol
uti
onshouldbeprepar
eddail
y.
b. St
andar
dAl
umi
nium Sol
uti
on:

1.
3192gms.ofA.Ral
umini
um pot
assi
um sul
phateisdi
ssol
vedi
n
wateranddi
l
utedto1000ml.1ml .oft
hissoluti
on75g.of
al
umini
um.
c. Buf
ferSol
uti
on(
Concent
rat
ed)
:

27.5gms.ofammoni um acet
ateand11.
0gms.ofhy
dratedsodi
um
acetatearedi
ssol
vedin100ml .ofwater
.1.
0ml.ofglaci
alacet
ic
aci
di saddedandmixed.
d. Buf
ferSol
uti
on(
Dil
ute)
:

Toonev ol
umeofconcent
ratedbuf
fersol
uti
on,5v
olumeofwater
i
saddedandt hePH isadjust
edto6.1byaddingacet
icaci
dor
sodi
um hydr
oxi
desol
uti
on.

Pr
ocedur
e:

1. 20ml .ofali
quoti
st r
ansferredtoa250ml .beaker.5ml .of
5v olumehy drogenperoxi
dei saddedandmi xedwel l
.ThePH
oft hesol ut
ionisadjustedt o6( usi ngeit
her0. 2M sodium
hydroxideor0. 2M hydrochloricacid).5ml .ofSol ochrome
Cyani neR.soluti
onisaddedandmi xed.50ml .ofthedi l
ute
buffersol ut
ion i
sintroduced and di lut
ed t
o 100 ml .i na
volumet r
icflask.Theopt i
caldensi tyi smeasured after30
minut es with a spectr
ophot ometer at535 magai nsta
reagentblankina5mm.cel l
.

2. Thepr
ocedur
easabov
eisf
oll
owedt
omeasur
eopt
ical
 densi
tyofstandardsol
uti
onofal
umini
um f
orconst
ructi
onof
cal
ibr
ationcurveusing0,1,2,3,4and5ml .ofst andar
d
al
uminium sol
uti
on.

3. Theamountofalumini
um intheunknownsampl
eis
measur
edusi
ngthecal
i
brati
oncur
ve.

5.
5.1.
4 ANTI
MONY

A.Chemi
cal
Test
sforAnt
imony :
1. Rei
nschTest
:

Thetestisperformedasstatedincaseofar senic.Abl ui
shblackdepositonthe
copperst r
ipi ndi
cates t
he presence ofant imony .The st ai
ned stri
p aft
er
necessar
ypr ocessi
ngasst at
edincaseofar senici sheatedintheReinschtube
andthesublimateproducedinobservedundermi croscope.Charact
eri
sti
cneedle
shapedcrystal
sofSb2O3ar eobser
ved.

2. Mi
croTestwi
thSt
ainedCopperSt
ri
pinRei
nschTest
:

Apor t
ionofthestai
nedcopperstr
ipobt ai
nedfrom t
heReinschTesti
stakenina
porcelai
ntil
e.Theblackstai
nisdissolv
edbyt heactionofafewdropsofdilute
hydrochl
ori
cacid.Thesoluti
ont
husobt ainedi
sspottedonapieceoffi
lt
erpaper.
Thepaperi sdri
edandexposedt ohy drogensulphi
de.Anorangespoti
ndicates
thepresenceofanti
mony.

3. Mar
shTest
:

Theprocedur
easst at
edi ncaseofarseni
cisfoll
owed.Themir
r orfor
medon
subl
i
mat i
ondoesnotgivestheoct
ahedr
alcr
yst
al(di
sti
nct
ionf
rom arseni
c).

4. Gr
oupAnal
ysi
s:

Groupanal
ysi
sisdonewiththeext
ractspr
eparedf
rom t
hedi gest
edmat
eri
al(dr
y
orwetdigest
ionofbiol
ogicalmat
erial
sorblood)todetectanti
monybygroup
separ
ati
on.

5. Rhodami
neBReagent
:

1ml .oftheextr
actist
akenandmadest r
ongl
yacidicwithhydrochl
ori
cacid.A
l
it
tl
e ofsol i
d sodi
um nit
ri
teis added.1 ml.of0. 01% aqueoussol ut
ion of
RhodamineBi st
henadded.Thebri
ghtredcol
ourofthereagentchangestoblue.

6. Phosphomol
ybdi
cReagent
:

1mlof t he extr
act i
st aken i
n a semi-mi
cro test t
ube.0.5-1 ml.of
phosphomol
ybdicaci
dreagenti
saddedandheat
edforashortti
me.Thereagent
i
sreducedtoabluecompound,whi
chcanbeextr
act
edwi t
hamy lal
cohol
.

5.
5.1.
5BARI
UM
 A.Chemi
cal
Test
s:
Resi
dueobtainedbydr
ydi
gest
ioni
sdi
ssol
vedi
n2mlofconc.hy
drochl
ori
caci
d,boi
l
ed
andfi
lt
ered.

(
1)Fl
ameTest
:Aper
sist
enceappl
egr
eenf
lamei
sobser
ved.

(2)Aportionoftheacidif
iedsolut
ioni
sboiledwit
haf ewdropsofconc.nitr
icaci
d,made
alkal
i
ne wi th ammoni um chlori
de and ammoni um hydroxi
de solut
ions,fil
ter
ed,if
necessaryandt othecl
earsoluti
onisaddedanexcessofammoni um carbonat
esoluti
on.
-Awhi t
epr eci
pit
atewhichdissolv
einaceti
cacidi
ndicat
esthepresenceofbari
um.

(3)Thepreci
pit
atedi
ssol
vedi
nacet
icaci
dasobt
ainedi
ntest(
2)i
sdi
vi
dedi
ntot
hree
port
ions.

Theoneportionisaddeddi
lutesulphur
icaci
d,whichgivesawhit
epr
eci
pit
ate
ofbar
ium sul
phat
einsol
ubleincon.ni
tri
candhydrochl
ori
caci
ds.

Toanotherport
ioni saddedaf ewdropsofpot
assium chr
omatesoluti
on,which
producesay el
low preci
pit
ateofbari
um chromate.Itisi
nsolubl
ei ndi
lute
aceti
caci d(disti
nct
ionfrom str
onti
um andcalci
um)butr eadil
ysolubl
ei n
mineralaci
ds.

Thet hi
rdpor ti
oni sev aporatedtodr yness.Ther esiduedissolv
edinwat er.A
dropisspot tedonapi eceoff il
terpaper ,dr
iedandt oitispouredadr opof
thesoluti
onofsodi um rhodizonate.Abr ownorr eddishbrowncolouredspot
i
ndicatest hepr esenceofbar i
um whi chi si nsolubleindilut
ehy dr
ochlori
c
acid(dist
inctionfrom stronti
um) .Theabov etestmayal sobeperfor
medon
drop-
reactionpaper .

B.Quant
it
ati
on :
1.Det
ermi
nat
ionofBar
ium i
nvi
scer
a:

Met
hod:

100ml .oftheextract(prepar edfrom aciddi gestedmat eri

alofv i
scer almat terofknown
weightwi t
hami xtureofconcent ratedni t
ri
caci dandconcent r
atedsul phur i
cacidas
describedi nsection3oft hismonogr aph)i stakeni nabeaker .Af teraddi ngasl i
ght
excessofhot1Nsul phuricaci ditisheatedt oboi l
ingslowl
yandwi thconst antst i
rr
ing.It
i
st hendi gestedont hest eam bat huntilthepr ecipit
atehasset t
led.Iti sfil
teredthrough
No.4v it
reosolfil
ter
ingcruci bleandwashedwi thal it
tl
ewat eruntiltheaci di sremov ed.
(8or10washi ngsareusual lynecessary).Thecr ucibl
eisdriedandheat edinanai rov er
at100-110oCandt henignitedi namuf flefurnacef or15mi nutesuntil constantweighti s
obtained.Theper centageofbar i
um iscalculatedi ntermsofBaSO4.

5.
5.1.
6. BI
SMUTH

Chemi
cal
Test
sforBi
smut
h:

1. Rei
nschTest
:

Agr
aydeposi
tonthecopperst
ri
p,whi
chdoesnotsubl
i
meonheat
ingi
naRei
nscht
ube,
i
ndi
cat
esthepr
esenceofbi
smuth.
 2. Mi
croTest
:

Aportionofthestai
nedcopperstr
ipfrom theRei
nscht estistakeninaspottedt
il
e.The
depositi