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Text book of
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·SIS

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by
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[Link] Sankar, [Link]., t_ M.B.A., Ph.D., FIC.

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Address for correspondence: •

Rx Publications •
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267 A/3, West Car Street, Tirunelveli - 627 006, India

Phone: 0462-2334856 e-mail: rxpublications@[Link]

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© All rights reserved

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No part of this book may be reproduced or transmitted in any form or by any means,
electronic or mechanical, including photocopying, recording or by any information •
storage and retrieval system, without written permission from the author or publisher. •

Edition : 1997, 1999, 2001, 2010

Price : Rs.200/-

Published by : Rx Publications, Tirunelveli, India

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For list of Book sellers, refer to Last Page

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 � • "'t BI ':;: order No. (O. 1. 2. 3, etc)· \
Colour corrected ,[Link]
9 = angle of deflection I diffraction
9lass for visible region.
�
� .

We can understand the working of gratlng with the following examp�.:., Polystyrene cells : are
.. .....
••
....•.
•
•
available for use wtth •cm , . Jcm....

,
• •
• •
. . •; "'-,;•
...·.'... ,·' I < aqueous solvents but
i ! Let d = & 0 = 6.89'. OQ=�
' ....
. 2000 cannot be used with
CC)��
:

'0=lk organJc solvents. For W ...

Cf)!·�
�

i.e. d = 5 x I o-4 1• 10• 'l)pcaofaanp-a-Ue

.-
v. e
region. thes cells must F'1 g
: dJ
'• 0 "' be made up of quartz since. glass. absorbs· W
-4
= 5 x 10 x sin 6.89> radiation.
i
m
..r{;- -
;

:· �
.
5 x 104 x 0.12
... .·
t .
= 600
cm. nm Detectors used in lN/Visible spectrophotometers can be called as
' I

. t.'
'.
.
.' m m .
photometric detectors. When a radiation is passed through a sample cell,,.
):'
.'• J-

Fot this 0, pifferei:it wavelength of light can be obtatned. depending part of it is being absorbed by the sample solution and the rest is being
[Link] order No. transmitted. This transmitted radiation falls on the detector and the intensity
'...., of absorbed radiation can be dete1·1nined or displayed. In these detecl0r.s.
'
Order No 1 2· 3 4 ••• the light energy is converted to electrical signal which can be read . or
..
recorded. The most commonly used detectors are
·).. nm obtained 600 600 600 600
1
=600nm
2
=300nm
3
=200nm
4
= 150nm •••
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1. Barrier Layer cell or Photo Voltaic cell
Thus a light radiation at any angle (0) or any order can be collected 2. Photo tubes or photo emissive ·cells and
and· used .m the instrument by either moving the grating and frxing the 3. Photo Multiplier tubes
slit or moving the slit and keeping the grating constant.
1. Barrier Layer cell or Photo Voltaic cell
C. Sampl,e cells These cells are the cheapest and are use d in inexpensive tnstruments
like filter type colortmeters. fluorimeters and nepheloturbldtmeters. -To�
Sample cells or cuvettes are used to hold a sample solution. Their
following Fig 1.11, shows the construction and working of the detectoft'.·
geometcy as well as material varies with the instrument and nature of
Tfie detector has a thin metallic layer coated with silver or gold and -ac�
sample handled. The material of sample cell should not absorb at the
as an electrode. It
wavelength being obseived. Cells are available which change with the c.1a,,
also has a metal base 1blo [Link] ol Sllft:r
following parameters.
plate which acts as
Sample [Link],ume Small· volume cells {0.5ml or less) and large volume another electrode.
cells (5-1Oml). These two layers are SeJawm
separated by a
- semiconductor layer
•
Shape of cell Cylindrical '1Jke test tube) or rectangular (Fig I. IO).
of selenium. Selenium
Path length - lcm (normally), upto 10cm (long pathlength) has extremely low
(internal distance) 1 mm or 2mm (short path length) cells are available electrical conductivity e
and hence the "- 1.11. l'lllllto .,.ltnlc e:?D

1-20 1-21
 J

. = • �.. e p ,. __
ua _.._
•
· •1· . --1
.u.i1 1a • ,.·•••
• material i 26. BLAME PHOTOMETRY
�, S: T o 8 n d Ole (p ut: •iitq ,
o w d er . tr an sfe r ta a . co n ic 8 1 fl�s k , a d d ind tq _ ato r Principle
Wei . a quantity o f p
as p er re q u ir em en ts an d ti tr a te a g ai n s t a ti tr an t.
and�ther , salu tlon s
., :Plnf) out the tttte value. Components of a Flame Photometer
Tth value x St. of tltrant x Eq. wt factor 1oo
u ri ty of gt v e n s u b s ta n c e = Wl of substance taken
x
Percentage · p Burner (with fuel and oxid ant)

e a,a ou at of drug pJesent In e ach tab le t

4. To ibid out tb Filter / Monochromator

Tbe: [Link] steps are followed: • Detector

W�t �ccur�iely 20 tablets. Readout Device •

Ftnd the average weight of one tablet

Schematic diagram of
(Total weight. of tablets divided by 20).
' Fihely powder all the 20 tablets. Flame Photometer •
I
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Weigh a quantlty of powder as specified in monograph. II Flame Spectrophotometer

f
Perform the titration and find out the titre value:. " Double beam Flame Spectrophotometer
f
The amount of drug in each tablet 'is calculated as foUows: Applications
. - Tit value x St. o f tltrant x Eq. wt factor x Av. wt of tablet
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.. . . · each [Link]-
Amount Of drug in .
wt. of tablet powder taken Qualitative analysis .

"• 5. To tlnd the % purity of tablets Quantitative analysis

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Methods: Direct Comparison Method
Follow the steps as given in Formula 4 (as above). Calibration Curve Method
. Standard Addition Method
( % purity of tablets = Tit value x st. of tltrant x Eq. wt. factor x Av. wt. of tablet x 100
Wt. of tablet powder taken x label claim in grams Internal Standard Method
•
Interferences and Methods to overcome

'. Preparation of Standard Stock solutions

26-·1
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S . 1> . i. .. . • ••• ,, • '.

O N
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�- ·A P P L I C A T
• • .
PHOTOGRAPH OF HPTLC The applications of HPT LC is very vast, since whatever be· the comp��nd.s

Computer controlled Densitometer/ Scanner -which can be analysed by TLC, be analysed using HPTLC technique. Moreov:er,
as the technique is much sensitive and small sample volume is -�ufficient for
detection and estimation, various applications are po ss ible in several fields.

The following are some of the categories of applications.

l
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i
. Pharmaceutical Applications:
es in fo x1n ul at io ns an d biological
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+ Quantitative analysis of drug substanc
l t . fluids
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+ Assay of active components and multicomponent analysis .
+ Content uniformity in dosage fonnulations
'·' .. . - .• . "" ..,,. ... . + Presence of impuritie s in drugs
-1 · . '

a n d fo rc ed d eg ra d a ti o n stu d ie s
+ Stability test in g
.'

. ., . . Samp�e applicator (Linomat 5 ) d H y d ro xy

B u ty la te
•
.' ,
B u ty la te d H y d ro xy A n is o le ,
+ - eg .
'

P re s er va ti v e s

To lu e n e , p a ra b en s in fo 11 n u la ti o n s
c ts : E x a m p le s a re p re s e n te d in
- p la n t e x tra
• •
-· .. ( + Phytoc o n s ti tu e n ts in
d U S H e rb a l P h a rm a c o p oeia ,
-. H e rb a l P h a rm a c o p o e ia an
- .
Indian in v a rio u s h e rb al
a tio n o f p h y to c o n stitu e n ts
where e s ti m ip er N ig ru m ,
S t. J o h n 's W o rt , P ip e ri n e in P
.. . ---
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p r o d u c ts a re m a d e . E g .
o, e tc .,
•

e e , N ic o ti n e in T o b a c c I.

.. t•
' •• .,., •
• Caffiene in Tea/Coff
,F'J',(Jj.... . .
� Adulteration of plant extracts
•

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>
'. .. . .. '* '
�-
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-�··•y: . n / fi n g e r p r in ti n g / s cr e en in g o f p lant
u th e n ti c a ti o
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+ Quality control / a
, a ll ic in ), v a le r ia n , a sh w a g a n d h a ,.
a rli c e x tr a c t (Alliin
extracts (eg) G
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';i : - "' •
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ginkgo, ginseng, etc.
Photographs : Courtesy: ANACHROM (CAMAG)
s : eg. E u g e n o l in C lo v e o il '
a ly s is o f v o la ti le o il
� Phytochemical an
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Clinical applications
to detect drugs and
f
ldn eti cs a n d m eta b olism
� U s e d in pharm aco .
g fo r d rug s o f ab u se�
metabolites, screenin
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29-8
29-7
 ,.,., .. . · ·- • .. . ' >il
The lower phase (solvent Y) of container 1. contains the pure component of "b".
it .. ,, i ',- ·'�·: 1 -J. ·,,' . ·Ji-....i �· JNTRO DUCTIO N . ·
,• •
. �·· ., 1,
' .., • .. ' i •
•o
;-·�it;:� .� •• ... . .# "
'·

Fig 1. Diagramatic representation of counter current extraction

Counter current extraction is a method of multiple liquid-liquid extraction
technique ,vhere separation of components having variable solubility in two equlllbratlon
1
immiscible liquid phases is achieved.
•
StepO

In a conventional liquid-liquid extraction, 2 components (eg. ''a" and "b") After

are distributed between 2 immiscible liquids, according to their partition co­ .(.
'-
.S? b
equilibration
I"
efficients. Still pure "a" and pure "b" are not present in these 2 liquids, even ,·

1 = (b+a)0
after reaching equilibrium. ;.
I
Before
In the counter current extraction, 2 immiscible solvents flow in an I•
I
b
equilibration

opposite direction, in multiple stages, equilibrium is established and after Step 1 l

several stages, pure "a" and "b" can be obtained. • r

I'
.wa .•·
�-- .
After
I equilibration
b2 ba
PRINCIPLE
,. �
b +a= (b+a)1
In counter current extraction, when 2 components "a" and "b", having •

varying affinity or partition co-efficient, is distributed between 2 immiscible Before

equilibration
ba
solvents (eg. X and Y) which are allowed to flow in opposite direction,
separation of pure "a" and "b" takes place, in multiple stages, as described Step 2

in the figure 1, in the next page. After

equilibration
b a2
In the first stage, when equilibrium is achieved, in container l, solvent X '
� b2 + 2ab + a 2 = (b+a)2
(lighter or upper phase) and solvent Y (heavier or lower phase) will have both
components "'a" and "b". Of course, based on their distribution coefficient, ••
Before
let us say, "a" is present more in X and "b'' is present more in Y. The upper b a2
equillbration

phase (solvent X) is transferred to next container 2, with similar composition Step ·n·
of solvents. Fresh solvent X is added to container 1.
After
equilibration
ba3
�ter achievement of equilibrium in container 2, now th e upper ph as e will
4
Pure ·b'--+-- b

conta1� less of "b", due to its low solubility in X

and more of "a". This upper
phase 1s then transferred to container 3 with The value of "n" depends upon various factors described later in this
similar composition of solvents.
Now, the upper layer of container 1 is then chapter. Also, the number of steps required to separate "a" and "b", depends
transferred to container 2 and fresh
solvent is added to container 1. The ab upon the difference in their distribution coefficient. When the difference
ove steps are repeated till the uppe r
layer contains pure "a" m· the "nth" container, where "n" is th between them is more, few steps are required. But when the difference in
e last container
31-2 31-3
 " ,, m o re st ep s ar e , -.
.
tween . a and "b " is less , then j • '

the distribution coefficient be

. ' Models are available·.. to contain about 20-25 tubes, which can be
required. ••• • connected in sequence and used for demo purpose (as given below).
. •• '4
.
. l

Instrumentation t
l
lt
,r

la bor atory sc ale is a C ra ig a p p ar a :' 1s

A simple type of apparatus on
· · iior separatin" g upper layer and transfer11ng
(Fig 2). This tube has provision
lvent on ly is pl aced. Fr es h so lv en t 1s ad de d to
. e heaVl·er so
f!·
to next tube, wher
tube 1.

Start of Cycle l
t
• On industrial scale, the design of extractors is different and depends
-/
upon no. of factors described below:

Factors affecting extraction

• Solvent selection
• Operating Conditions
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• Mode of Operation
•' • Extractor Type and
• Design Criteria
•
•
• Solvent selection, depends upon the type of solute mixtures. Other
factors affecting solvent selection are boiling point, density, interfacial tension,
Fig 2. Craig Apparatus viscosity, corrosiveness, flammability, toxicity, stability, compatibility with
product, availability and cost .

Applications

• Separation of components from synthetic mixtures .

• Separation of components from plant extracts .
• Purification of compounds (removal of impurities).
";

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