Compiled by Dr.

Barkat Ali Khan & Iftekhar Ahmad

CHAPTER# 09: ALKALOIDAL DRUG ASSAY
Alkaloids
Alkaloids are basic organic compounds of plant origin, containing nitrogen in their structures.
Alkaloids are amines, so their names usually end in "ine" (e.g., caffeine, nicotine, morphine,
quinine).
Chemial !a"#$e o% alkaloids
1. Insoluble or slightly soluble in water
. !oluble in organic sol"ents which are immiscible with water
#. !alts of alkaloids are soluble in water
E&"$a"io! o% Alkaloids
$y the introduction of latest separation techniques and ad"ance information, the separation of
many alkaloids from the natural plant sources is carried out by the following steps
(i) !ample preparation
(ii) %&traction methods
(iii) 'urification
i' P$e(a$a"io! o% sam(le %o$ assa)
1. (he plant part (lea"es, fruit, bark etc) from which the alkaloids to be e&tracted is 1
st
of all
air)shade dried.
. (he dried part is then reduced in si*e by pul"eri*ation to the required finesse to facilitate
ma&imum effecti"e contact of the sol"ent with the alkaloid containing tissues and cells.
#. +are should be taken to a"oid the loss of water during the si*e reduction)pul"eri*ation.

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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
*ei+hi!+ %o$ assa)
(he accuracy should be within 1, mg for quantities equal to -g or o"er i.e . "ariation
is allowed.
It means that if you are taking -,, mg pul"eri*ed crude drug then the weighing limit
may be /0,1-1, mg
ii' E&"$a"io! me"hods:
%&traction is the process of isolating the acti"e ingredients from a crude drug.
(he alkaloid bearing drugs are e&tracted with one of the following methods
1. 2aceration.
. 'ercolation.
#. +ontinuous e&traction (!o&hlet e&traction).
/. !tas13tto 'rocess
-. 4ippenberger5s 'rocess.
(he sol"ent (2enstrum) used for e&traction may be6
7ater miscible such as methanol, ethanol, isopropyl alcohol etc.
7ater immiscible sol"ents such as n1he&ane, chloroform, diethyl ether, ben*ene etc.
Acidified distilled water.
,ae$a"io!
2aceration refers to soaking a substance so that it softens.
Accurately weighed powdered drug is dipped in a specified amount of sol"ent or mi&ture
of sol"ents.
It is then made alkaline with ammonia solution, mi&ed well and allowed to macerate for
/18 hours with occasional shaking.
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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
Afterwards allow the sol"ent to settle.
9ecant and press the liquid with the help of 1: layers of 2uslin cloth.
E-a(o$a"io!:
After maceration process, e"aporate the sol"ent in a ;otary e"aporator under "acuum.
(emperature of the sample containing flask is maintained at /,1-,<+ while that of condenser,
the temperature is kept at ,/<+. %"aporation is continued until one1third of the liquid is left
behind (e.g. if sol"ent before e"aporation is 0, ml, then e"aporation is continued until the
sol"ent is reduced to its 1)# i.e. #, ml.)
.i+: A /o""le sho0i!+ mae$a"io! ($oess
Pe$ola"io!
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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
'ercolation differs slightly from maceration. (he powdered drug is 1
st
dampened with the
menstrum, left for some time and then packed into a percolator.
'lace accurately weighed powdered drug is in a suitable container, saturate with suitable
sol"ent for -min
2ake it alkaline with Ammonia solution, mi& thoroughly and put in percolator
Allow the drug to macerate for 1,11 h
'ercolate it slowly until percolate is alkaloid free
Alkaloidal "es"
1To e!s#$e "ha" (e$ola"e has /eome %$ee o% alkaloids i'e' all o% "he alkaloidal o!"e!"s
ha-e /ee! e&"$a"ed2
(ake / ml percolate (of that coming out of the percolator, not that which has been
already percolated).
(hen e"aporate this percolate and dissol"e the residue in ,.-= >+l.
Add a drop of mercuric chloride and obser"e for turbidity.
If turbidity is not produced, percolate is free from alkaloid.
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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
?ig6 An e&ample of percolation, soluble compounds lea"es the powdered drug and @oins the
menstrum to form percolate. Insoluble compounds remain within the filter.
Co!"i!#o#s e&"$a"io! OR So&hle" E&"$a"io! P$oess
It is similar to percolation process with the modification that continuous e&traction is carried out
using !o&hlet e&tractor.
(he !o&hlet assembly is a continuous e&tractor which is generally suitable for the e&traction of
alkaloids from powdered plant materials with the help of organic sol"ents. In this case, the
powdered drug is usually moistened with dilute ammonia solution and then packed loosely in
the thimble of the !o&hlet apparatusA and the organic sol"ent gi"es a deep penetration of the
moist drug thus allowing the greatest possible e&traction of the alkaloids from the crude drug.
3nce, the e&traction is completed, the sol"ent is filtered and e"aporated in a ;otary %"aporator.
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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
?ig6 A !o&hlet e&tractor in"ented in 1B80 by ?ran* "on !o&hlet.
iii' P#$i%ia"io!
'urification of the alkaloid is done in two ways.
i. Addition of acid solution
ii. 'recipitation with precipitating agents.
i' Addi"io! o% Aid Sol#"io!
It is done in two steps6
In 1
st
step, dilute acid is added to the solution obtained after e"aporation. (hen add
suitable menstrum. In acidic en"ironment, menstrum will dissol"e impurities. >owe"er
if chloroform is used as a sol"ent (menstrum), then >+l should not be used for
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Compiled by Dr. Barkat Ali Khan & Iftekhar Ahmad
acidification because some of the alkaloids may form alkaloid hydrochlorides which
may also get dissol"e in the menstrum.
In the
nd
step, the solution is made alkaline by addition of =>
/
3>. (hen suitable
menstrum is added which dissol"e the alkaloids, lea"ing behind the aqueous layer.
ii' P$ei(i"a"io! 0i"h ($ei(i"a"i!+ a+e!"s
In this method, the alkaloids in the solution are precipitated by the addition of suitable
precipitating agents like tannic acid, mercuric acid etc. It is then filtered off and the precipitate
obtained is then decomposed to get free alkaloids.
%&ample6 +omple& formation by the addition of tannic acid which is then decomposed by
treating with Cead hydro&ide ('b(3>)

).
De"e$mi!a"io! o% alkaloids 1Ti"$a"io! me"hod2
%"aporate the Alkaloidal solution to dryness by steam
!often the residue with 1 ml neutral ether or alcohol
Add a "olume of standard acid and warm gently to make complete solution
Add the "olume of water to make - ml
(itrate the e&cess of acid by a standard alkali using methyl red as an indicator
If alkaloidal residue is to be weighed, dry it at 1,-
o
+ to a constant weight
If sol"ent is chloroform
;emo"e the traces with few ml neutral ether or alcohol followed by e"aporation
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