32a1eafb-eb3f-4b69-9899-1605e833d486.pptx

| Pharmaceutical Analysis

Fourth Edition

by
Dr.S.Ravi Sankar, MPharm. M.B.A., Ph.D. FIC.

Address for correspondence:

Rx Publications
267 A/3, West Car Street, Tirunelveli - 627 006, India
Phone: 0462-2334856 e-mail: [email protected]

© Al rights reserved

Na part of this book may be repreduced or transmitted In any form or ty any moans,
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storage and retrieval system, without written permission from Ihe author or publisher,

Edition = 1097, 1999, 2001, 2010
Price : Ra200%
Pubished ty: Rx Publications, Tiruneivall, India

For list of Book sellers, refer to Last Page

Dedicated
to
Mike, aba
Guus and Almighty

Preface to the Fourth Edition

Dear Readers, lt han been a decade Jong since | have revised the ard
edition. In spite of thin, you have been à source of constant support and
encouragement by patrosising this book, which has helped you in your career
‘at various stages. I used to get feedback that this book Is useful during
the course al the study, for competitive examinations like GATE/CIPAT, job
interviews and pre-Ph.D examinations.

Keeping in view the requirements of various universities and feedback, I
have added 7 chapters, vis., HPTLC, Gel Filtration, Counter Current extraction,
‘Ultencentrifigation, Radio immunoassay, X-ray diffraction and ICH. | will add
¡gore chapters in future editions, keeping in line with the changes In the
sylabus of various unlventies in India, technologies! improvements in the
field of pharmaceutical analysis and based en your feedback,

Presse bear with me, for an increase in the cost of the book, which la
very meagre compared to the inflation over last 10 years,

My beartful acknowledgements to my well wiäher and thanks to
Mr.G,Rajasekar, M.Sc, M.Phil, Tirunelveli, who has been supporting me in
distributing this book throughout India without any scarcity, Thanks 10 my
younger son Master Rakahith, who was keen to include his name In this
book, which indirectly presturined me in bringing out this 4th edition,

Wish you all the best pot only in examinations, but also in ether
competitive cxama, pre-Ph.D exams and job interviews, towards a best
career,

Dr. 8. Rast Sankar

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INTRODUCTION TO
PHARMACEUTICAL ANALYTICAL TECHNIQUES

Pharmaceutical Analysis plays a major role today. and it can be
considered as an lnterdisciplinary subject. Pharmaceutical Analysts derives
hs principles frum various branches of Science Mec Chemistry. Phynica,
Microbiology, Nuclear Science, Electronics, ete. Pharmaceutical Analytical
techniques are applied mainly In two areas, viz Quantitative Analysis and
‘Qualitative Analysis, although there are several olher applications.

Drugs and Pharmaceuticals are Chemicals or lke substances, which
are of organs, inorganic or other origin, Whatever may be the origin, we
use some property of the medicinal agent to measure therm quantitatively
‘or qualiairvely, Pharmaceutical Analytical techniques, which are being used

A Spectral method where we use light absorption or emission
characteristics of deugs (ea) UV spectroscopy, IR apectroscepy, NMR
spectroscopy, Plouritmetsy, ete.

B. Chromatographie methods: where we use allinity or partition

A nous Ld a, DIE Pap

3 Lquié (HPLC), Paper
Chromatography, etc.

© Hiectre analytical ;-based on the electrochemical
of drugs, eg , Conduetometry, 7
electrophoresis, etc,

ang. Fem

D. Biological & Microbiological methods: where we use either artinalt
oF microorganisms for analysis. (eg) Biological assay of some Vitamin,
microbiological assay of antibiotics and vitamins,

E. Radioactive methods: like Radio Immuno assay and related
techaiques,

where E = Energy of radiation
= Planks constant (6424 x 10% Jseq
v= Frequency of radiation

cof light im 210 pl
Frequency = É or ASPE naci „ 3: bae

Hence Bete Kon

where Y = wavenumber

‘Therefore the energy of a radiation depends upon frequency and
wavelength of the radiation,

in unit time.
‘passing through a given point in
Prequency la measured in Ha (Hertz) or cps (cycles per

second)
‘The higher unite used are 1 Kücherte or 16M = 10ŸHx:
1 Megaherts or MHs= Of 1 Frensel = 1017 He
WAVELENGTH = Wovelongih is the distance between two successive
maxima (or) minima or distance between two successive
E troughs or peaks.
‘can be measured in metres, centimetres
cm ar 107m), millimetres (mm or 10m}. mierometres

la or 10m), nancmetres (am or 10%] or LAngstroen)
CA oF 107m,
Were Number » I is the number of waves per cm.

LE

vie

Wave nuenber ls expressed in cuy! or Mayser.

‘Wave number ts espectally used in IR spectroscopy where small
wavelength measurements are made, to diferentiale frequency of vibrations
in meules.

Typen of Spectroscopy

Spectroscopy can be conveniently divided into following types based
on

1. Whether the study ds made at atomic or molecular level.

a. Atomic Spectroscopy - where the changes in energy take place
at atomic level.
Eg Atomic absorption spectroscopy, Flame plotometry + where
cither atomic absorption or atomic emission of radiation ts being
studied.

b, Molecular Spectroscopy - where the changes in energy take
place at molecular level

14

spectroscopy. Colortmetry, Infra Red Spectmscopy.
act where he morales absorption, asco ar ME
1s being studied,
2. Whether the study in based upon absorption or emission of EMR,
a Absorption Spectroscopy - where absorption of radiation ts being
studied,

Rg, UV apectroseapy, Colorimelsy. Indra Red Spectroxepy, NMR
Spectroscopy. ‘Spectroscopy,
b. Emission Spectroscopy - where emission of radiation is being
studied,
Eg, Flame photometry, Fluorimetry,
3. Whether the study la at electronie oc magnetic levels,

a Electronic Spectroscopy
Eg, UV spectroscopy, Colorimeuy, Fluorkmetiy » where the study
in done using electromagnetic radiation only (without the
Influence of magnetic field)

b. Magnetic Spectroscopy

The energy of a molecule can be due to electronic, vibrational or
rotational energy, They are in the following ratio:

Rotational energy : Vibrational energy : Electronic energy = 1:100:10,000
Wien any electromagnetic radiation la passed on to a tolecule, the

following energy changes take place in a molecule/atom which can be
measured,

|

D Serien Wt = Intensity of incident light (lol
‘Therefore na absorption Le le = lo No change in energy takes
place and hence no Information about the molecule can be

dertved,
2. Reflection, Refraction or Scattering, (scattering of light by particles)
where some studies like Nephlometry or Tusbidimetry are being made.

3 Ketel trio gr iste un Ses Oe
ls absorption of energy. Here some information can be derived.

Absorplion of energy or light followed by transition from ground state
to excited state ls called as exeitatlon process.

Ground state is nothing but the siate of a molecule or an atom
which ts most stable and has least energy.

Excited state ls u state which ls least stable but contains more
energy. The life time of excited state la

Relazation Process

As the excited state ls not the stable form, an atom or molecule
Iooten energy and relurma lo ground tate, This process is called as
relaxation process.

‘The absorbed energy can be lost by any one or more of the following
way:

1. Production of heat (colistonal denchmation)
2. Decomposition into a new species (Photochemical reaction)
3. Emission ol radiation of
a. Spectllc wavelength, characteristic of excited species (as in Flame
photometry).

b. Longer wavelength immediately las in Fluorescence).
€, Longer wavelength ater a short time bag (as in Phosphorescenee),

as Relaxation
Me — Mo 1. Het
Bess |! A 2 Emission of specific
= E
A rc a
re à Photochemical reaction

1, VISIBLE SPECTROSCOPY (COLORIMETRY)

‘= Prtneiple in Calorimetry

® Laws governing ubsorplion of Radiation
[Beer-Lamberts Law}

7 instrumentation

# Source of light - Tungsten Lamp, Carbon arc
370 450 450 400 550 500 650 780nm

]1=Te[+To1r]

Mill indigo: Blue Green Yellow Orange

4 Files + Absorption filter
= Interference fer

Monochromator - Prism - Refractive (iltsperaive)
+ ReBeetive [Litiraw type)
- Grating = Transmission grating

- Diffraction grating
% Sample Celle - Cylindrical, quadrangular
» lam path length
à Detectors + Photo Voltate cell or Barrier Layer cell
- Photo tubes

= Photo Multiplier Tubes [FMT
Read out Device (Digital Display, Recorder, Plotters)
= Single beam & Double beam instruments

=” Deviations from Beer's Law

se Applications in Fharmacy

18

PRINCIPLE BY COLORIMETAY -

lorimeuy ls concerned ‚ein te ae et e
rate me hose wavelength ranges from 400nm800nm. Any coloured
Subslance will absorb radiation in this wavelength regon. Coloured

ean
A Unique Pallem far every coltured lee lo Ula ADEME ve,
the wavelengih at which mmdmum absorption of radiation takes place te
called as Jeux, This deman ls characteristic or unique for every coloured
substance and this is a qualitative aspect, useful in Adentifjing the

— =
of same subetancs
| 1240
i
ea =
Weredength (rend Wercheng (nan

ya

5 lois mm
¿Concentration (yt/l)

19

The

changes,
7g 1. ut de concern (Y) Abrrbancs We feta Cabra
Curve or Standard Curve (Fig.1.3), This Calbraiion Curve ls useful in
Gelee the concentration or amount of à drug substance in the given
sample solution or a formulation. by extrapolation or inirapolatien ang
calculation.

LAWS GOVERKINO ANSORPTION OF RADIATION
‘The two laws related to the absorption of radiation are:
LL Boer law (relnted 10 Concentration of absorbing species)
2 Lambert law (related to thickness/path length of absorbing species)
These two laws are applicable under the following condition:
ml + ke
= Intensity of incident tight
= Intensity of absorbe tight

Intensity of transmitted ight and

1
la
hk
No /ecatieriey, of light takes place
‘The

e
!

law states (hat ef a beam of monochromatic
with increase in the concentration of

arithmetically".

i:
i
|

Accordingly, «= J = 1 ie decrease in the intensity of

incident light {1} with concentration (e)
ls proportional to intensity of incident
Aight 0

Pee (rearranging termal

Anl = ke + b Equation |
(On integration, b la constant of integration)
When concentration = 0, there ts mo absorbance. Hence | = lo
+ Substituting in Equation 1,

chlo xO ed

Aloe b
Substituting the valve of b, in equation 1,

tel m ke Ink,

Ike + bel ke

{uince-HogA tog B= log Sy

Bae (removing natural bogarithen)

ET TS mc)

Equations 2 and 3 can be combined to get
Tabet

. (converting natural logarithen
a to base 10 & K = k x 0.4343)

toe (rearranging terms}

+ 1
we can be learnt that Transatance (7) = q and Absorbance (Al = 1

Mence À og fe
ne À Equation 5

Using Equation 4 & 5, Since A = dog Y and tog À = Ket we can

Inder that
A= Ket Instead of K, we can use €)
Amt
(Mathematical Equation for Beer-Lamberts Law)
where

= Absorbance or optical density or extinction co-efficient
= Molecular extinction cocfficient

= concentration of drug (mmol/l)

= path length (normally 10mm or lem)

-n

© can also be expressed as follows:

cout, Molecular weit
Les 10
where Bin, means the absorbance of 1% wi solution, using a
path length ef tem.

Elo, al a wavelength la a constant value for each drug and can be
seen in Pharmacopocins and standard hooks on the subject, This value la
uselul in determining the concentration of drugs in sample formulations
Or in solutions. Emar ls the value at das
INSTRUMENTATION

Before describing different parts of instruments, we should know the
different invtrumenta used. They are:

Colarimeters - which are usually Inexpensive and lesa accurate. They
ensure either Absorbance or Transenittanee or both and have filters for
use with different coloured solutions, The range of wavelerath used is
usually small eg, 400-700em,

npectrophotemeters - which are little more expensive than colortmetern.
They can be used for a wide wavelength region eg. 360nm-200nm or

radiation over the entire wavelength can be used The
requirements of a source of light for colorimeter are

LIL should provide continuous radiation from 400nm = 00m
ik Mt should provide adequate intensity
Ak It should be stable and free from Buctuallons

‘The following are the sources of light used esemmendy

E ‘As it satisfies the above criteria, this lamp finds

Vcr in won edt and peeps. The imp
consists of a tungsten filament in à vacuum) bulb nu
‘ones used damestically. But it offers aulficent intensity.

Carbon ; Por a source of very high Intensity, carbon are
* a us be un I ao prods an erie range able spectrum

B. FILTERS AND MONGCHROMATORS: The source of light gives

Filters are of two kinds. They are:

1. Absorption filters and
2. Imterference filters.

Monochrematora are of two kinds,

1. Prism type (Dispersive type 4 Littrow typed
2 Grating type (Dillraction grating & Transmission

pang
.
Im Anti en
These fiers are made up of glass, coated with pigments or they
are made up of dyed gelatin. They absorb the unwanted radiation and
tranamit the rest of the radiation which is required for colortmetry.
‘These filters can be selected according to the procedure given below:

1. Draw a filter wheel (eircle with 6 parts). (Fig LA)

2 Wnie the colours (VIBGYOR) in clockwise or anticlockwise manner,
‘omitting Indigo.

3 1 the colour ef the solution ls Red, we have io use Green Alter
and if the colour ef the solution la Green, we have to use Red
Mier: (The colour of the fier ts opposite to the colour of the solution
Le, complimentary in nature),

4, Stelarty. we can select the required Aller in a colorimeler, based
upon the colour of the solution

Fig 1.4 Ascanio pares

3, Selection of Aller la easy,

Demerits

À: Less accurate since band pass is more (280nen) (Le. M we have lo
Measure at 50Onm, radiation ranging from 470m to Sam falls
am the sample). (Band pass ls the difference in wavelength between
Se Pants where the Fansites ome af Ue maxim)

2 intensity of radlation becomes less due ta absorption by tes.
(M) Interference Filters

‘This filter la otherwise known as Fabry-Perot Mier (Fig 1.6). The
features are

1. It has diclectric spacer Mm made up of CaPz, MgF2 or SiO, between
two parallel reflecting silver files,

2 The thickness of dielectric spacer Alm can be 1/24 fist order,
2 1/2 (nd onder}, 3 4/2 (3rd order), ete.

3. The mechanism is that, the radiation refleted by. the dad fim und

the incoming radiation undergoes to ge . Monochromators
a monochrome radiation, which in governed by the following Manochreenalora are better and more efficient than filters in corwerting
equation, a pehychranatie lyst or heterochromatic light into monachromale Mgt. À
monochromator has the following unite:
A= Anb/m
where) = wavelength: of ght obtained
m = dielecinie constant of layer material
b = layer thickness
mo» order mo, (0,123, ete)
INgLAINTERFERENCE MUA
on
mn
="
Er
Mi
so

4 Band pass is 10-18 nm. be. if we select Blönm, the obtained
radiation ranges from 4ó0nm to 51001)

5. Maximum transmission is 40%.

Merits

1, Inexpensive.

2. Lower band pass when compared to absorption filters and hence
more accurate,

3, Use of additional filter cuts off undesired wavelengtis.

1. Peak transmission is low, and becomes so when additional filters
are used to cut off undesired wavelength.
2 The band pass is only 10-16nm and hence higher resolution obtained

FRA Tara a AN IRS CNRS ee. ÉORENT

A). Reflective type (Littrow type mounting)
‘The principle of working ls similar =
to the refractive type except that, a
reflective surface is present on ene side na
of the prism, Hence the dispersed e
radiation gets reflected and can be h
collected on the same side wa the source 8
of light (Fig.1.7). ; M

2, Grathogs

Gratings are the most efficient
ones in converting a to monochromatic light in the real

sense, As a resolution of + Olnm could be achieved by using gratings,
they are comnonly used in spectrophotometers.

ratings are of two types: (.Difiraction grating (ul Transmission grating

(D. Diffraction grating
En u

‘ratings are
nothing but rulings
made on some material

{}
De
A

These gratings are replica made from muster grating, by coating the
original master grating with epoxy resin and are removed after setting. To
make the surface reflective, a deposit of aluminium is made on the surface.
‘The diagram of diffraction grating is shown in Fig 1.8.

‘The mechanism is that diffraction produces reinforcement. The mays

FE eee ue en Se a a ie ie oe,

and hence the resulting radlalien has wavelengih which la governed Sy
the equation:

ma = b (sin À 2 on

|

m onder No, 10, 1, 2, 3, ete)

4 = angle of deflection / deffraciion &
Wo mat mn cp a nl
fl

1
Let d= a & On GBP fi

Le de Sx 10%

4
4
El

1. E 104 x 6,89
ET

510% « 012 Be]
wem m a

For this. diferent wavelength of Might can be obtained, depending
upon order Na.

Onder No
om obtained

and used in the Instrument

C. Sample cells

wavelength being observed. Cells are available which change with
following, parameters.

Sample volume = Small volume cells (0.5001 or less) and lange volume
cells (5-1.

Shape of cell Oylindrical (ike test tube] or rectangular (Fig 1.101.

Fath length - Tem (nornaally). upto 10cen flog pathlengih)

(internal distance) Im or Ann (short path length) cells are avallabie

1. Barter Layer cell or Photo Voltale cell

These cells are the cheapest and are used Ir Inexpenatve instruments,
Whe Alter type colorimeter. Gortmelers and. 1

if
i
i
i
3
|

can trons,
falls on it. Thin flow of electrons towards anode
to the intensity of light radiation, Composite

fi
i
y

E
Ln
ih
fie
|
i LA
ul

AR
3
pH
Hit
ga
3

el
i
:f

is maintained ‘at 76-100 higher than the preceding one. AL each singe,
the electron emission la mullplied by a factor of 4 or 6 due to secondary
mission of electrons and hence an overall factor ef 10 ts achieved

PMT can detect very weak even 200 times weaker than that
could be done using Photovoliale cell. Hence it ls useful in Muorescence
measurements, PMT should be shielded from stray light in order to have
accurate results,

Different types of instruments

‘The variations in the source. fliers or moneehroenators, detectors,
read aut / recording devices and design lead to various types of instruments
like colorimeters, spectrocolerimetess und apectrophotometes. Although they

are used for similar purposes, Ihe cost, sophistimation, options, automation
ete, cilfer significantly,

Colorimeters contain tungsten Insp, absorption filters and photovoltaic
cell They are designed to read either % transmittance or absorbance,
normally single beam instrumento and are of non-recoeding type. Wavelength
accuracy are normally £30nm,

Spectrocolorimeters contain tungsten lamp. prisms as monochromators
and either photo vollaie cell er photo tube as detector, They are also single
beam, nor-recording type and are designed ta measure % transmittanee
er Absorbance. Wavelengih accuracy la Sam,

Spectrophotometers are expensive and more sophisticated and are
designed 15 rend % Tranamitiance or Absorbance. record the absorption
spectrum using a plotter or recorder, ure of double bear type where we
can use sample and reference sebutión at a time. Storage of spectrum,
comparison of spectra, quantitative techniques, rapid warelengity scanning,
data manipulation, derivative spectral mode, etc can also be present as
options in more automated Instruments. They can be ether microprocessor
based of software driven using computer, Wavelengih accuracy of much
instruments {s #D.Inm. These instruments are hence more accurate and
reliable than other types.

‘The following pages describe different types of instruments

H

BU veus colectas avs trough sample ween hl and fl one den
This constata of a tungsten amp ax source of Mi Tio ght ration ' rem out la in Absorbance or Tranamitiance, obtained

la focused on ta à allt by using a concave mirror, This light passes rough after electronic manspulation of the above 2 detectors (Pig 1.18)

a simple absorption Mller where only the required wavelength of light passes

through it and falls on the sample cell where the schitlon to be analysed

is present. The sample or standard solution absorbs à part of Ihe radiation

and the rest is tanseitied. The intensity of the transmitted or the

unabsorbed radiation is determined using a photo voltaic cell using a

digital display or an analogue display (Galvanometer type). The diagram of

be nd pes: FS

À

The method of construction and principle of operation la similar
doable beam colorimeter except that grating menechremator tad
photommultipler tube wre used in the place of Mlter/prises monochromator

1, The readings are affected by Guctuations in the intensity of source.

2 Rapid scanning to get a spectrum In not possible, wince OMT and
1OOWT has to be adjusted at every wavelength.

3, Recorder cannot be used with single beam type.
Double beam colorimeter

Double beam colorimeter is similar Lo that of single beam instrument,
Here the light beam after passing through or monochromater ts

DEVIATIONS FROM BEER'S LAW
A system Is said to obey Beer's low, when |
a plot of Concentration Va Absorbance gives a
straight line, The straight Une la obtained by
tuning line of best Mt or meted of bemat squares À | Lumany
=

using a colorimeter/spectrophotameter. When a
straight line ts not obtained, Le, a nonlinear curve is obtained in a plot
al Concentration Vs Absorbance. the system is said to undergo deviation
from Beer's Law, Such deviation can be positive deviation or negative
deviation. Positive deviation results when a small change in concentration
produces a greater change in absorbance. Negative devintion results when
a large change tn Concentration produces smaller change in Absorbance.

It is normally seen (at several system obey Beer's Law only in a
concentration range, above which may show deviation (eg 10-SOpg/ml, It
may obey. but may exhibit deviation above Bing

Several reasons for the observed deviations from Beer's law are as
follows:

1. Instrumental deviations

Factors Ike stray radiation, improper slit widih, fluctuations in single
bear and when monochromatic light ts not used can influence the deviation,

2. Physicochemical changes In solution

Factors like Association. Dissociation, tonisation (Change in pH), sanity
development of colour (incompletion al reaction) refractive index at high
concentrations, can Influence such deviations.

il

blue at concentrations of 10M exists as monomer and
ef 660mm. But Methylene blue at concentration above 10%
dimer or trimer, bu has Aa 6 BOOM, Hence when Une shit
la not observed for absorbance measurements, devialions occur,

Rag
it

Cr? + 1190 + 2H? + 20904"
(orange) peon
Pags + 480) east + 4100)

Pulasaiur dichromate in high concentration exists as orange solution
eax ~ 450mm, ut on dien, dichromate fons are dissociated into
chromate lona which is yellow coloured Pasas - 4100m). Hence when 4500m
ls used for absorbance inensuremenis, deviation from Beers law is seen.

Incomplete reaction

When suffictent time ls not allowed for making absorbance measurements
‚or when Ue readings are made when the colour has faded any due to
instability of colour, deviations can cecur (eg) Determination of (roe using
Ahioglyeslic acid before completion of reaction.

Some of the important terms used la colorimetry
Chromephorr
Chromophore or chremopheric group Is a group or part of a molecule.

Auzochrome
Auxochromes are / unsaturated. They do

net have any characteristic ubsorption on their own, but can modify the

‘absorption of chromophore. like OH, “Na, halogens ete

Aukochrommes
have non bonding valence electrons which do mot absorb above 200nm,

a substance present along with impurities,
‘When tsobestic point is used, relevant 45m
absorption due to impurities Es eliminated,

li can be determined by recording the
curves of same substance al different pH on the same Rape
paper and noting Le potnt of intersection. (eg) the following figure the
isosbentie point of the gen substance is at SO and 710mm.

CLS
‘Extinction or Absorbance of 1% w/w solution of substance using m
Jom path begin cell

called as red alhifl. It cun occur due to inerease In conga
al double or triple bends). addition of alkyl substituents in Use molecule,
ete.

‘Aypsochromle shift: SU in Amar towards shorter wavelength. Also
called as blue ah It can occur due to removal of double or triple bonds
by saturation, Geadkylation, ele.

Hyperhromie effect: Increase in the intensity of absorption
Hypochromie effect: Decrease in the intensity of absorption

Chromogenis agent: Chromogen or Chromogenic agents are those
‘whieh ts capable of forming a chromophore or colour by complexation,
chemical reaction, lonisation, ete. (et) Ferric chloride when added to ralieylic
acid produces a viet colour with Jamas at S80nm. So Ferrte chloride
(reagent) ds called as chromogen.

APPLICATIONS IN PHARMACY
1. Quality Control af purkty

Colonel can be used te detect the impurities, because of their
additional peaks or more absorption at particular wavelength.
led Gyanocobalamin (Vi Ba) absorbs at 27800, Mini and 580m, The
ratio of absorbance at 2780m / 36inm = 0.57 and the ratio of

absorbance at SSinm/IGlnmed.3. If lmpuniies are present, the ratio of
the absorbances change and can be known,

2. Quantitative analysis

This aims to determine the concentrallón and amount of drug in
sample schition and thus the percentage purity can be determined.

For the quantitative analysis ef drugs by calorimetry, the following,
characteristics must be kn. rn

1. Light absorption characteristics = Le. teas to be known. This wavelength
has to be used because of high sensitivity. specificity & accuracy.

2. Validity of Beer's law - Le. Concentration range in which the system
obeys linearity (e.g) 10-SOpg/ml, 2-10ug/el, ete.

3. Solvent, reagent, other conditions, Elliy - if available free literature
ete.

‘The several quantitative methods are:

a Ging Elfi: values

This method can be used for estimations from formulations. ar raw
materíal, when Reference standard is not available.

eg Elo for Paracetamol at 257m ts 715, (obtained from Pharmaeoporta
or Journals or text books)

To find the % purity of paracetamol tablets, the following formula
can be used, *

ss mew „ Obaerved Absorbance 10)

7 hn this example, the absorbance of 0.00092% wi solution at 2571
vas 0068.

. 25,0.
Hence % Purty « DE poa Dam ww

bd. Elli, la not wvallable, but Reference standard available

In this case Eth, value can be determined by observing the absorbance
at different standard solutions and the average E value is taken. Aller
calculating the E value, the method as In (a) can be followed.

Aternatively we can use any of the following methods:

(1) Single standard or Direct comparison method

ln this method, the absorbance of a standard solution of ‘known
concentration and a sample solution is measured, The concentration of
unknown can be calculated using the formals

Ay = tet

Aa = eet
where Aj, Aa - Absorbance of standard and sample
el, ea - Concentration of standard and sample
E - Mol. ext. coellictent

£ - palhlengtb (len)
On dividing we get

ay eet
À eet
1,4
A a

. a

Aaa

Since Ci, As and Ay arg known, Ca can be calculated,
concentration of the sample. the amount as weil as % purity of

toy plotting curves using different ratios and observing the linearity of the
curve.
4. Structure elucidation of organic compounds

“The absorption spectrum of an unknown compound can be compared
with known compounds, 50 that the meat probably structure may be
obtained. Since compounds of similar structure have analogous spectra.

nes,

leg) Two structures 1 and H can be proposed for Tyrosine

Mt was observed that absorption spectra of cempourd II was similar
lo Phenol [CAHSOH], Hence structure If can be proposed.

8. Determination ef pKs value of indicators
Determination of dissociation of an acid-base indicator (eg) Methyl
orange Methyl red. ete.
Donised]_
pla + pit - tonic

The value of fog ¡Lech can be determined sptetrophotometriealy,

Le. Concentration (Va) Absorbance al different pH and from the equation,
ka can be eniculated.

8. Determination of Molecular weight of amines

A known weight of amines Is taken and converted to amine plerate
and the absorbance of the solution is found. By using the equation, the
molecular weight of the amine can be calculated.

We know already,

A
Asad and
eax Molwt, Mon À

For most amine drugs, € of amine picrates at 380mm ts 13,400.

where A = Absorbance of a solution of concentration °c’ using
pathlength Y,

7, Determination of elements, lona or functional groups

Several elements, fons or finctional groups can be determined
quantitalively even at low concentrations, by using specific or non-specific

8. Determination of organic substances and pharmaceuticals

Only few drugs are coloured and meat of the drugs are while or
colourless In nature. Hence we add reagent or chromogenic agents Le such
drugs and form a colour, whose intensity is proportional ta the concentration
of drug

The selection of chromogen plays a major role in colorimetry, whieh
depends upon the functional groups present in the molecule.

‘The following table shows some of the functional groupe and the
reagents used.

dye & ración of complex tn orgue obren
Basic dye 6 extraction of complex in organic sobra |

Conditions to be observed:
& Elther (he substance, utrant or product should absorb,
Beer's law must be obeyed under experimental conditions.

© Titrent must be stronger, as the error due to volume change must
be minimised,

Absorbance is monitored at Amex Of Ulsani. reactant or product.
Examples of titration curves

CHCOOM
Water
Wer
“Tartarte act
Foc acid 10 NaNOg + NINED 550 nm
(Hprtrochioeothianide 10 [NaNO3 + NI-NED 518 nm
nocarbosande Ho molybdate 420 om
|Onyteurncyriine 140 + Het [Ferric chloride 400 ru
¡Rea yO + CHCOON = Ht nm
[Allampsein Acai pi 74 475 nm

Tram only abat Proton amet
= =
‘A dame me Carr

CT
nt ib ed

‘Sutetance rent only abat Codere ra ah Cured tra

ce
N ¡3
== pr

a aed
Ph ida Te

2. ULTRAVIOLET SPECTROSCOPY
© Introduction
#7 Principle in UV spectrophotometer
sr Electronic transition and excitation process
er Types of electronic transitions
© Instrumentation

Source of Mit = Ma dixcharge lamp:

= Deuterium lamp
+ Xenon are
= Mercury vapour
* Moncehromator - Grating - Transmission grating
> Diffraedon grating
% Sample Cells - Cylindrical, quadrangular
= lem path tenga
Detectors - Photo tubes

= Photo Muluptier Tubes (PMT)
® Read out Device (Digital display, Recorder, Plotters)
À Single beam & Double beam instruments

© Applications in Pharmacy

INTRODUCTION

Ultraviolel spectroscopy ts concerned with the study of absorption of
UV radiation which ranges from 200rm te 400nm. Compounds whieh are
colbured, absor' radiation from 400ne-800nm, But compounds which are
colourless absorb radiation In the UV region. In both UV as well aa visible
3 only the valence electrons absorbs the energy. thereby the

méleeule undergocs transition from Ground Male 19 excited state. This
absorption la characteristic and depends on the nature of electrons present.
‘The intensity of absorption dependa on the concentration and paihlength
as given by Beer-Lambert's law.

‘The types of electrons present in ary molecule may be conveniently
dasaibed as

I. ‘a electrons: These are the ones present in saturated compounds,
‘Such electrons do net absorb near UV. but abso vacuum UV
‘radiation (<200nwm).

2, ‘x electsona; These electrons are present In unsaluraled compounds
(eq) double or triple bonds leg) >CeC<, -CaC>

3, ‘a electroms: These are non Bonded electrons which are nat
involved in any bonding leg) lone pair of electrons lke in 5. O, N
& Halogen 00.

FRINCIPLE,

Any molecule has either n, x or a or a combination of these electrons.
‘These bonding lo & x) and non-bonding (n) electrons absorb the characteristic
radiation and undergoes transition from ground state to exclted state
By the characteristic absorption peaks, the nature of the electrons present
and hence the molecular structure can be elucidated,

ELECTRONIC TRANSITIONS AND EXCITATION PROCESS

Ik was stated earlier that o. m and a electrons are present in a
molecule and can be excited from the ground stale by the absorption of
UV radiation. The various transillons are nor, mex, mot and 0-00,
‘The different energy states associated with such iransuons can be given
by the dingram (Fig 2.1),

1

Docta
ig 21. Energy levels of electronic framailioos
The energy required for excitation for different transitions are: nest
< sant < niet < 0-0, OF these transitions pex" required lowest energy
And ge requires the highest energy for excitation in the UY region.

=

fed 2020 + > — 0 + oc — ot

Polar solvents shift n-*
pe and nto to shorter wavelengths and <-+*

TYPES OF ELECTRONIC TRANSITIONS
À, Bova: Of all the types of transition, n-e* transition requires the

lowest energy (longer wavelength), The peaks dı transition,
also called as Rbands, This pap »

i
i
i
323
a
-
+
|

&

ath |
|

nu à nbs

|

the band disappears if mex" has been
present.

The presence of other hetero atoms can be identified by comparison
‘ith a similar compound without hetero atom, These technique can be
used tn structure: elucidation.

2 myn: This type of transition gives rise to B, E & K bands,

Type I Bea te
bands (benzenoid bands) [aromatic & hetero aremailc aysteens

E-barsde (Evtrytenic bands) tens
Rebate (e+e! Conjugated mysterma

‘The energy requirement of thls transition la between n-+0* and n-vn°,
But extended conjugation (aidition of more double/triple bands) and ally!
substituents shifts the Meee towards longer à MBathochromic shift} Also
trans isomer of olefin absorbs at longer À with more intensity than Cis
isomer, (Bathochromic shift and hyperchromic effect), Extended conjugation
land alky! substitution] sbifis dom 19 such an extent that Ue Dax falle
in the colorimetric region (eg) Plant pigments luke P-carotene, lycophene.
ic. The Imax of some chramopliores and other systems are given below,

“The presence of n-ve* transition can
be identified easily by comparing the UV e
spectrum of the substance with the | =
spectrum recorded in the acid solution \'
of the same substance. In an acid solution,
Pa present ‘Peak dinappeaes

Chromephore nn
>CaCe V7änen
fethylerue)
<=c- 178m

dor

‘This transition occurs in saturaled compounds, with hetero atomis)
ike 5, O. N or Halogens. It requires lesser energy when compared lo 0-30"
transition. Normally the peaks due to this transition occurs from 280mm

24
——_—

4. 0-+e% Of all the electranie transitions, this type of transition
requires the highest energy. This ls observed with saturated compounds
(especially hydrocarbons), The praks do not appear ln UV region, but occur
in vacuum UV or for UV region. Le. 125-13%am. Some of the compounds
with such transition are Methane (122nm). Ethane (135em), Propane (1 380m]
and Cyclopropane (190nm). Since UV spectrophotometers are operated above
20am, saturated like eyetohexane (1O50m] can be used as
neapelar solvente, as It does not give solvent peal,

Beer-Lambert's Law
Beer-Lamberts law is applicable to UV radiation also, The laws and

for the same has been already destved and shown in Chapter 1.
SY Ught:

‘The best source of light ts the one which is more stable, more intense
and which gives range of spectrum from 180-360nm fupio 400nm), The
different. sources avaliable are: -

Hydrogen discharge lamp: It is more stable, robust and widely
used. li gives radiation from 120-3500m, The lamp consists of hydrogen
under high pressure. >

25

UV spectroscopy since glass cells will uv radiation. The pathlength
of the ces are 10mm ar tem,
4. Solvents
‘Solvent plays an important role in uv spectra, since compound peak
could be obscured by solvent peak. Hence the solvent for a sample la
selected in such a way that the solvent neither la the region of
affects the

Stagle team and double beam UV spectrophotometer

The design, construction and working of single and double beam uv
ters are similar to that of colorimeters (Chapter 1) except

apectrophotomel

few changes, as mentioned below:

Source of light - Hydrogen discharge or deutertum lamp in the piace
of tungsten lamp

Monechromators - Grating moncebramator made up of quartz in the

place of filters and priam monochromators,

Sample cell Quartz sample cell in the place of glass or
polystyrene cell

Detector + Photormultiplier tubes instead of photovoltate cell
oF photo tubes.

Deviations from BeerLambert's law

Al of the reasons mentioned in colorimetry (Chapter 1) are applicable,
but only the wavelength region ls from 200 to 400nm.

1. Qualitative analysis

a. Detection of impurities: To limit Ihe presence of impurities, we
can use uv messurements. Additional peaks can be due
Ms ee sete a Gel be cone ah Ut a sec
raw material. absorbance measurements ak specific wavelengths,
the impurities can be detected.

leg) The presence of phenones (310nm) in Adrenaline (280nm) can be
determined. The extinetion of a 1-em layer of 0.2% ww solution tn
GOIN HCI at Sión ds not more than 0.2. Mil Is mare, then
phenones as impurities are present
». Structure elucidation of organic spectroscopy
helps In structure eluetdation of organte molecules, The or absence
of unsaturation, the presence of hetero toms bike 5, O, N or halogens
can be determined. The following figure will show how ihe presence of
peaks correspond to the unsafuration/saturation and presence/absence of
hetero atoms,

2 UV

nen
nel Ge n= hag

be obtained. À
the spectral studies help tn structure elucidation, whether the compound
/

la saturated / unsaturated, hetero atom is present/absent, ete.

e. Structural analysis of orgunie compounds: Sorme of the aspects
of structural analysts are already discussed under electronic transition. Wee
dentifiation of n-ss* transition, efect of eanjugation, ete. Other phenomena
dee effect of alkyl substitution, effeet of cross conjugation. effect of geometric
isomerism will be described briefly.

saturation of double bonds leads to the oppasiie elfeet Le. hypsocliromie
shift (blue obit > De abifted to aborter A

Au) Ritect of Geometric isomerism
Of the Cis and tans isomers, trans isomer absorbs at longer
wanelength than Cis Isomer.
eg Calciferal (Cia isomer) mx + 265
som (all trans) demas + 2870m
Hence cormersicn from Cis to trans tsomer resulta in hathochremie
shift and hyperchromic effect. Conversion from trans to Cis isomer results
in hypscebromic shift and hypochromie effect.

(UN Eéeet of cross conjugation

Cress conjugation has ne

fect 00 due which can be

shown by the follomng

OS
m m
Conjugation Cross Conjugation

as Prednisone nnd Predutsolone Hi a
hve ture (D, bul ta Joe natal
is al 26Inm and remains unaflected. Hence cross conjugation has no

‘effect on imax,

2. Quantitative analysis
© using Eta vadues

HL gia not available, but raw malertal la available
fi, Single standard oF direct comparison method

tv, Calihation curve method or multiple standard method

‘Apart from these methods briefed in Chapter 1, the following methods
can be used for quantitative analysts, especially for the determination of
mixture of medicinal substances.
0) Simultaneous multicomponent method

If a mixture of two components a and b are present in x w/w and
yA w/v respectively, by measuring the absorbance of mixture at two
Valens Ay and a, the concentra or amount of components à and

BE

b can be estimated. ay and az and bi and da are El values determined
at Ay and da far component a and b respectively.

(li) Derivative spectrophotometrie method

tn this method, spectral isolation ls achieved rather than
chromatographie Isolation. In a derivative spectrum the change in absorbance
with respect to wavelength (vs) wavelength is recorded. Ist or 2nd derivative
spectrum is recorded and the characteristie peak for the individual component
can be identified and quantified. using a calibration curve of pure substance.

UM} Difference spectraphotometric method
‘This method ts especially useful to quantify a substance when interfering,

achieved by using pH manipulation using a pair of bullers, This method
is used te quantity drugs in biological fulda,
3. Determination of molecular weight (as in Chapter 1)

4. Determination of dissociation constant of acids and bases
(pHa determination as In Chapter 1)

6, Keto-esol tautomerism
Keto and enol forms of a substance have different uv absarplion

pattern, Using this principle, the percentage of keto and enel form in a
„mixture, the presence of enol in ether or alcohols can be calculated.

7. Quantitative analyals of pharmaceutical substances
substances

Drug im
nana NET = |]
[Auspurisat tableta Er) 250 ES
[Biacody! tablets cnc 264 108
[Cartuemasepine tablets [Alcohol ES 0
(CRoramphenical capsules [Water ‚ara 3
[Chiorphineesenine Maleate [0.58 HaSOu 202 147
tablets

se jez = er
|Cypéohéptadine tablets [Alcohol 206, 35
Diloxarsde Furoate [Alcoba 258 2e
Diazepası tablets [0.5% WSO tn MeOH) 254 40

212

dl

= Factors influencing Nourescence intensity
"© Effect of concentration on Flourescence intensity
Mr Quenching of Buorescence - types
© Instrumentation

% Source

de Filters and Monochromalera

& Sample cells

à Detectors

& Singe beam and double bear instruments
sr Advantages and limitations of flourimetry
‘= Applications

INTRODUCTION

Absorption isíble radiation causes transition of electrons from,
A exit sale As this state 1 not stable, de
the energy in the form of av viable radiation and returns Lo singe
Pound state. This study or measurement of (his emitted radiation fen
(ransition from singlet excited state to singlet ground
y Phosphorescence ls also à related
phencatenon. which is the study of emitted radiation when electrons
undergo transition {rem triplet state to singlet ground state,

i
|

Before understanding the principle, I la important to understand some
dectronic states,
1, Singlet state: a state in which all the electrons In a molecule
ponga

2 Doublet state: a stale in which an unpaired electron is present
"tag fred radical Y or 4

[unpaired and same spin).

4, Singlet exelted state: y state in which eleetrons are unpaired but
of opposite spin like 1” (unpaired and opposite spin).

Absorption of uv/visible radiation causes transition from singlet ground
state to singlet excited sthte As tus excited state ts not stable, it emits
the excess energy and returns back to ground stale, To achieve this
transition here are 3 possíbililes:

desctivatica: in which the entire energy ls lost due to
Collisional deactivation and no radiation is emitted.

„2/frurseee: À pat energy 1 ot due o rational canins
and the remaining energy Is emitted as uv/visible radiation of longer
wavelength than the incident light. This ts because the energy of

emitted radiation is lesser than that of incident or absorbed
radiation, because a part of energy ts lost due to vibrational

3. Teiplet state: a mate in which unpaired electrons of same spin
present. 1

32

"The different elecironie transitions. energy level, states and time delay
are mentioned in the following diagram Fig 3.1.

Fig Floumscenee à Phasphomacence

A]
10%. 101% soc

‘The following table gives a summäfy'ef transitions and the resul
tle produced. ty: the anne: ee D
‘vibratios

TIFES OF FLOURESCENCE dlectromagnetic radiation und partly by thermal energy.
Luminescence is the phenomenon of emission ef ght radiation by racrol RS DOLUENCICO TLOURESCENCE INTENSITY.
substances, when excitation occurs in any form. E
Some of the factors which influence / affect the Oourescence intensity
of compounds arc:
A Conjugatica
A molecule must have (x electrona, 1e. conjugation) so
‘that uv/vis radiation can be abs ‘there is no absorption of radiation,
Flourescence and phosphorescence are examples of photo luminescence. eevee E
Flourescence can be classified into following types: à el ment gueno
1. Based upon the wavelength of emitted radiation when bd
= compared Liane cer cree Ha}, drang (OH), groups
2 Stoke's Mouresceace: The wavelength of emitted radiation is Electron withdrawing groups Mike Mtro group
longer than the absorbed radiation (eg) as in ñ
mg a) conventional reduce Saurescence Inlenaliy. a u.
Groups like SOSH or NiLa* have no effect on flourescence intensity.
b. Anthstoke’s flourescence: The wavelengthi of emitted radtation ‘The eect of several substituents on the emisstve wavelength and Hourescence
is shorter than (he absorbed radiation. This type ls uncommon intensity is shown in the following Table.
and seen in Thermally assisted fourescence. a
© Resousace flourescence: When the wavelength of the emitted : ee ==
ciao Le equal o the absorbed radiation fe) Mercury vapour pe] No fes ‘Stight increase lo À
4
d BEER ‘CHO, COOR. Y (nereaseh Idrereasel
‘OMe, OB ? finereasel Y linerense)
= Seasitised ourescence: When elements like Thallium, Zine,
Cadmtum or an alkali metal are added Lo mercury vapour, these > “= —

elements are sensitized and Uuus gives flourescerice.
t. tes 34 35
AS

DICO
—

haar et mrctures
* (regain + More flourescence intensity

Flexible structures -+ Less flourescence tntenstly

- O
Sa GR

Increase in temperature leads to increase in collisions of malecules,
therefore deviation, which results in decrease in Plourescence intensity.
On the other hand, decrease in temperature leads to decrease in collisions
of mobecules, which results in increased flourescence intensity.

CS
= in wiscosity lends to decreased collisions of molecules, which

Oxygen decreases the flourescence intensity In two ways:
5 36

D) Ik axidises flourescent substance to non-flourescent substance.

MM) IL quenches (decreases) Nourescence, because of the paramagnetic
properties of molecular energy, as it has triplet ground state,

7, Effect of pit

+ The effect of pit depends on the cbernical structure of the molecule.
+

Detain to ment or allas mats ves
In ac condivona gy Taurencence In uy

_ Pesa in mei; condition are undisocaied and do net ge
but in alkaline condition,” they are disscciated tonic)

Mi oo
jotochemical decomposition

Vin abserption sometimes leads to phatochemical reaction. In such
cases. flourescence chnmot be seen Hence a wavelength with is noi
strongly absorbed should be chosen to avoid auch a reaction. Olherwise

errors upto 20% is possible.
EFFECT OF CONCENTRATION ON FLOURESCENCE INTENSITY
It is quite obvious that less number of molecules absorb lesser radiation
radiation.

flourescence but

by
law has to be known,
Fiourescence intensity = Q x ly
where Q = Flourescence efficiency

‘loureseence quanta emitted
EMR quanta absortiod

la = Intensity of absorbed light

Flourescence efficiency =

Since emission la proportional to absorption, la has to be known
leek
where la = intensity of incident light and kk = int. of transmitted light
Nt Bown that, t= leet [by Beer-Lamberts law)

la = le Wett (subatitultng for li in above equation) +

la = dh (eto

la = tfifiaetl
fbecause e's I-act, using Taylors serien)

la = lo lite
te = lex act
+ Flourescence tntenstty = Q x I, = Okact
Le. F = Oläct
In this equation,
9 = constant for a particular substance/aystern
le = constant for an instrument
A= Molecular extinetion coefficient, whieh is constant for a substance
t = pathlengun la constant for a sample cell}
€ = concentration of substance

5 P = RC, where represents the combination of all
constants Q. lo a and t

ar Flourescenice intensity « Concentration
Le. Plourescent intensity is directly proportional to concentration of

the substance. But this is true in low concentrations (ug or rut (Fig 3.21.
But in high concentration (mg/m) 1 doen not obey Hnearity (Fg 3.3),

determined experimentally, For any experiment, only the linearity range
should be used.

QUENCHING OF FLOURESCENCE AND TYPES

‘Quenching is the decrease in intensity due to specille
effects of constituents of the solution Hsell. These effects may be due to
various factors lke concentration, pH, presence of speciBe chemical
substances, temperature, viscosity, ete. Various types of quenching are

1, Self quesching or concentration quenching

AL low concentrations leg or ng/ml, Linearity ls observed, At high
concentrations (mg/mil of the same substance. proportionate increase in
Mourescence intensity does not occur. This phenomenon la called as self
Quenching. or concentration quenching.

39

2. Chemical quenching
In this type, quenching is due to various factors like change tn pH,
presence of oxygen, halides or heavy
a pH: Aniline at pH 5 to 13 gives blue flourescence when excited ap
200nm. But al pH <S5 (exists as calor) and pH > 13 (exists ay
anionl, it does net exhibit Mourescente.

b. Oxygen: Presence of oxygen leads to quenching because of uy
paramagnetic property (triplet ground state)

© Halldes and electron withdrawing groups: Halides like chloride,
bromide, lodide and electron withdrawing groups like Nitro and
carboxylic group leads to quenching (Le. 4 flourescence intensity)

de Heavy metals: Presence of heavy metals also leads to quenching
because of collisions and triplet ground state.

3. Static queaching

‘This occura because of complex formation, (eq) cafflene reduces the
Aourescent intensity of ribolayine, by complex formation.
4. Colllsional quenching <

Ms the result of several factors like presence of lalides, heavy metals,
tnereased temperature and decrease in viscosity, where number of collisions
place.

1. Source of light:

‘The source of light stiould emit a radiation over a contínucus region
and be of adequate intensity and stability.

= Mercury ‘vapour lamp: Mercury vapour at high pressure (8

[pres Intense lines on a continuous background above

360nen. Lines are seen at 305, 398, 438, 846, 579, 690 and T34nm.

Low presaure mercury vapour gives an additional line at 25dnm. tt
la used as source In Alter type of flourtmeters,

ae 210

. are lamp: It gives a more Intense radiation when e
By men kop CS uel ss Oo De Al
meters,

€ Tungsten lamp: if excitation has to be done in visible region, this
can be used. It does not offer uv radiation, more over the intensity
of this lamp la 100 low.

2. Filters and monochromatora

In flourtmetry two things are important, Le. excitation wavelength and
emission wavelength, As these two wavelengths are different in most cases,
a Miter cr monochromaler ls used for the purpose. In an Inexpensive
instrument like filter flourimeter, peemary filter and secondary filter are

present.
Primary Miter -+ absorba visible radiation and transmits uv radiation
Secondary Mer -+ absorbs uv radiation and transmits visible radiation

In spectrofiourtmeters, excitation moncchromators and emission
monochromator are present which have gratings. In spectroflourimeters.

Exetation monochromator + provides m suitable radiation far excitation of
molecule (radiation which is absorbed by
molecule}

Emission monochromator -+ isolates only the radiation emitted by the
Bourescent molecule

(For description regarding Gites and monochromators - refer Chapter 1),
3, Sample cells

The sample cells are cylindrical or polyhedral (quadrangular) like those
used in colorimetry. The cells are made up of colour corrected fused glass
and pathlength ds normally 10mm or lem. NL need mot be made up of
quarts, since we are measuring only the emitted radiation and not the
absorbed radiation, Hence even tf there ls absorption of radiation by glass.
M will not affect the results,

ln colorimetzy, in quadrangular cells, two polished surlaces (Uwough
which light ts Iransmilted) and two ground surfaces (for handling the cells)

en

7
|

are present, ln contras, al the surfaces are polahed in fourietry, because
emission measurements are made at 90° ange,

4. Detectors
emitted radiation is mostly visible radiation and sometimes uv
rata. To measure the Intern of such radiation, Photomalate ee,
Photo tubes or Photo multiplier tubes can be used. But since we use low
concentration of substances and the intensity of emilicd radiation ts weak,
only Photomultiplier tubes are the best and accurate, In incxpeniaive
Mafuments Me Pheishayimeera, Polo tubes can be used Desriplion
el all Photometric detectors are grven In Chapter 1),

5, Instruments

The most common types are

a Single bears (Alter) flourimeter

b. Double beam lier) Gourtmeter

© Spectrofiourimeter (double beam)
2. Single beam Alter Dourkmeter

‘This ls an inexpensive instrument, contains tungsten lamp as source
‘of light and has an optical system composed of primary filter. The primary
Jitter absorbs visible radiation and transmits us radiation which excites
‘the molecules present in sample cell. The emitted radiation (Nourescert

radiation) is measured al 90°, by
using a secondary ler and a detectar, 7M 94, gle beam (iter Houriemeter)
absorba

ens seis by ths copa

1. A ie not possible to use sample and reference solution at a me,
3 Rapid seanning to get excilaion or emission spectrum of the compound
tn not possible. The diagram of single beam Gourtmeter is given In Fig 3.4,
».

Double beam (fiter) Mourimeter

AL ds similar to singe beam, except that the two incident beams from
a single light source pass through primary flters separately and fall on
esther sample ar reference solution. The emitted radiation from sample or
reference pass separately through secondary Alter and produces response

solution can be analysed skmultancosisly, Dut the demerit la that rapid
seanniié ls pot possible due to the use of filters. The diagram ls given
tn Fig 28

NEAR Done e mar matter

The primary filter in deuble beam ler) Mlourtmeter la replaced by
excitation Imonociromalór and the secondary (er ls replaced by emission
wanschremater. The incident beam is split into sample and reference beam
by using bean splitter. Normally the detector is photo mulupler tube. The
construction and working of spectrodourimerer ts gven in Fig 26, The
advantages are: 1), rapid scanning to get excitation and emissice spectrum,
Al. more sensitivity and accuracy when compared to Miler type instruments.

ADVANTAGES AND LIMITATIONS OF FLOURIMETRY
Advantages
1. Sensitivity: When compared to absorption techniques, flourimetric
technique ls more sensitive since concentrations as low as g/ml
or even ng/ml can be determined.
2 Precision; Upio 1% can be achieved easily in fourimetry.

3. Bpeclflcity: As both excitation and emission are
ancre, 8 mare ec Gan abro metas whee
absorption maxima may be same for two compounds.

4, Range of Applications: Even non flourescent compounds
be convert to Dourescent compounds by chemical rcllen

3-14

‘Limitations
1. Change in pH allects flourescent intensity
2 Dissolved oxygen may affect Mourescent intenany
3 Traces of halides, heavy metals, ete can affect flourescent intensity.
4. UV absorption somejimes causes chemical changes.
5. Al elements and compounds may not be Mourescent, inspite of
ebemical

reaction,
APPLICATIONS
1. Determination of Inergunic substances
[ ton ‘Reagent tr os 8 7
| | excitation | mourescence | Mal
A [Aliana garnet 470. 500 0007
Zn [Benet = Green 100
St [Flavonot 400 470 o1
lsinydroxy quinoline | 370 500 oa

ot
= Irapbtholc acid 300 460 2

‘2, Determination af organte substances

Aromalle polycyclic hydrocarbons, indoles, naphthels, proteins, plant
pigments, steroids, ele, ean be determined at low concentrations by

flourtmetey.

3. Pharmaceutical applications
a. Compounds which are flourescent

‘The examples are described briefly:

Diphenyfiydantoin on oxidation with EMOS In alkane medium Lo
benzoghenone, which la Gourescent.

xy a Ann
cé or y”
spent sony
12 pe hy elation a ia sac. iene een
Oro
y
A,

(ye
Pr
M Thiamine or Aneurine = cxidation with Potassium ferrocyanide in
alkaline medium and extraction in isobutane

was.

e. Mixture of medicinal substances
Various options available are:
4 A wavelength at which end ont drug de exited la chosen,
A Using separation methods and analysing the compounds,

le. Preparation of dertrations
feg) Hoxin with Atropine forms a choreform soluble complex which

#
4, Miscellaneous applications

‘Plourescent indicators: The intensity and colour of many flourescent
substances depends on pH range and hence are used in acid-base titrations,
Seene examples of such flourescent indicators are

4. NEPHLOMETRY AND TURBIDIMETRY ©”

Introduction
= Principle
© Choice of Method
sr Factors responsible for producing uniform turbidity
sr Factors responsible for intensity of Scattered radiation
#7 Effect of Concentration and Wavelength on scattering
‘© instrumentation
% Source af light
# Piliers and Monochromators
à Sample cella
% Detectors
de Neplilo-Turbidimeter
se Comparison of Colocimetry with Turbidisetry
#7 Comparison of Flourtmetry with Nephlometry
+ Applications

Hight by particles sumpended in a liquid, The
refractive index different from that of the medium. This le lo
Tyndal effect, where a light cone is seen due to reflection or scattering of
Light when viewed at right angles to the incident beam passed on a
suspenalon,

Nephlometry ls the measurement of scattered light as a funcion
el concentration of suspended particles (leas than, approximately 100mg/ litre},

Turbidimetry ts the measurement of transmitted light as à function
of concentration ef suspended parues (more than 100mg/lItre. high
eancenurations).

Nephlometry: At low concentrations of a suspension, there ls uniform
scattering. Hence thr intensity of scuttered light is proportional to
the concentration. This is the principle En Nephiometry. The intensity of
scattered light is normally meanured at 90° ike in | lt can
also be measured at any convenient angle like 45°, 60% 135%, ete.

Turbidimetey: At high concentrations of a suspension, scattering
ls nat uniform and light in scattered In all directions, Hence it becomes
difficult to measure the intensity of scattered radiation at all angles. Hence

less and when concentration is less, I ls more, This ls the principle
in tusbidimetry,

‘The cholce of method, vie. Neptilometry or Turbidimetry depends on
the concentration of suspension. When the concentration of suspension
ts less, seattertng of ght is less and hence can be accurately measured
at 90° or any convenient ange. This makes Nephlometry ns the choice
ta low concentrated suspensions.

— ir

FACTORS RESPONSIBLE FOR PRODUCING UNIFORM TURIIDITT

For Nephlometric or turbidimetic measurements, it la Important to

have uniform turbidity of suspension, otherwise accuracy may not be
obtained, Hence to produce untlarm turbidity, the following factors must

be considered.
1. The manner. order and rate of mixing of substances
2. Agitation of suspension.

3. Temperature: As temperature affects solubiity of substance and
viscosity of medium.

4, Presence or absence of inert electrolytes, protective colletds toe
gelatin, acacia, dextrin ete which affects flocculation and deflocculation.

5. Concentrations of solutions mixed, to get a suspension.
FACTORS RESPONSIBLE FOR INTENSITY OF SCATTERED RADIATION

In nephiometry and turbidimetry, even after producing uniform turbsdity,
the following factors are to be considered which affects the intenalty of
seatiered radiation, appearing al any angle.

1. Number of suspended particles (concentration).

2. Sie and shape of particles: The size should be equal to or greater
{han the wavelength of the incident light.

3. Wavelength of radiation used: It should be selected In such a way
there ts no absorption, but only scattering

4. Dillerence tn the refractive index of particles and the medium,

EFFECT OF CONCENTRATION AND WAVELENGTH ON SCATTERING
Concentration

The intensity of transmitied light is expressed using an
similar to that of Beer-Lambert's law. Le.

peer
where P = Power of transmitled beam
Pa = Power of incident beam
T = Turbidity or Turbidity co-elBcient
b= Path length

Pa
¿dd

T was found to be proportonal to the concentration (e) al suspended

Hence, as T = ke, keb = log Le

Werelength
Eis expressed by the following equation.

1.8

Y

where T
a for a given system
à = Wavelength
U = depends on site of particles and ls '4 when
particle size 1 smaller than wavelength.
INSTRUMENTATION
Specifk instruments are available for use as eu

Alternatively, modification. of colorimeter
nib an «lids emule eet ee

In a colorimeter, when a blue Alter ‚or 30cm 18 used iM timcemes à
‘Turbldimeter, When a visible Alter is used as secondary Miter, Ihe Flourimeler
becomes a Nephlometer, Whatever be the design of instruments, the following,

lamp ly used when a polychromatie ight ls used.
arc lamp is used when a monochromatic light la required. This le
any light absorption, since we require only scattering of light

il

When m white ft or polychromatie light Is used. Miles and
monochremators are not required. But when manochromatie ght Is required,
a Gker or monochromator Is used.

response. Special cells (Fig 4.1) arc also used to measure scattered light
‘at different angles Uke 45°, 20°, 135° and 180°, The cells are made up
of glass.

yla Aretamgudar genial ore

4, Detectar -
Photometric detector lhe Photovoltaic cello, Phototubes or

source of Ii and the sample Tener
cell Mat bottom) ls placed on
top of source Light passing
through ter falls on suspended
particles. These particles scatter Sample
the light. The ight scattered by
the particles ure collected by
curved mirror and reflected to sé
the Photoveltalc cell, kept at
the bottom of the instrument. ei
This model ie nimple,
inexpensive and easy to operate.
It has reasonable precision and bad
accuracy. GA Mephdemeter
Mg 4.3, Merlo. A flourimeter can also
be converted to m

Nephlometer by using a
4 A visible filter as secondary

fer as given in the
wma: diagram [ig 43.

‘neta o

he io i te

(UY Nephloturbldimeter

Ma suspension has to be analysed. It is important to know whether
At can be analysed by nephometry and turbédimetry, But in practice. It
ds not essential to know this, because recent instruments are à combination
of the above two Le. Nephlo-turbidimeter. These Nephlo-turbidimeters have
Abe design, as shown in the Fig 4.6. They have two detectors. one for
measuring Use scattered light at 90° and the other at 180? for
the transmitted light The ratio of the response of the two detectors la
displayed as Nephlo-Turbidimetric units (NTU), which ls proportional to the

[Analyte to be estimated

[Phosphorous —

Has [ie decrease in intensity of incident|The decrease in intensity of
ugha ts due Lo abserption of incident ight is due
(scattering el radiation
COMPARISON OF FLOURIMETKY WITH NEPHLOMETRY
Flourimetsy Nephlometzy |
Similares fus of emergent radation Anlintenmity of emerges radiate)
at In mensisred at 66°
Diflerenen |, Entensity ef emitted mática to [1. intensity of séattered
measured in measund
Bmitied radiation is of langer (2 Seattered git bas sume
twovelength than tnesdent Ma as that of reagent lo produce turbiduy and NTU
inciden ght. (NephloTwstidemetse Unita) ls measured mu
using Nephloturbidimeter, The sample
APPLICATIONS ‘ solution ls treated tn the same way as Ee
|. standard and from the ealibration curve,
1. Analysis of water the concentration of the ton in the cms

Per determination of darity and for determining the concentration of
various ens by adding selective precipétanta, nephloturbtdimetry Is applied,

2. Determination of carbondloxide

‘The sample of yas i passed through bartum salt. preciphated an
barium carbonate and ls determined by Nephlo-turbidimeisy.
3. Determination of Inorganic substances

Inorganie elements / ions Uke phosphorus, ammonia, suite.
Chloride, Carbonate, flouride, Cyanide, Calcium, Zinc, ete can he estimated,
by precipitating them using peecipitanta. The apaleecence / turbidity ean
be measured by using Nephlo-Turbidimeter.

DES

utikpewn solution can be determined.
5. Miscellaneous

Nephloturbidimeuy is applied in water
power and steam generating plants, breweries, botlling industry, petroleum

products, clarity of citrun juices. ete.
6. Turbidimetzie titrations

It is similar lo spectrophotemetssc
ratlos. In tls, the NTU (Nephio-
‘Turtadimetric Unit) is monitored against
the volume of tétrant added, The Utrant
and titrate gives a product which ls turbid.
The end point of the Urban can be
known from the point af inflection In the
‘graph (Fig 4.7).

5. INFRARED SPECTROSCOPY (IR)

7 Introduction
@ Principle
er Types of vibrations
% Streiching - Symmetrical & Asymmetrical

di Bending - In plane bending - Selssortng & Rocking
+ Out of plane bending - Waging & Twisting

Mr Instrumentatson

# Source, Sample handling, Monschroenater, Detectar,
Recorder / Plotter, Types of Instruments

sr Absorptions of commen functional groups
= Applications
Tig 5.0. infrared Spectra of Ibuprofen (Kr dise)

a 1600 Ma ID NN Ak

‘waren dc

INTRODUCTION

—Anfrared spectroseopy or vibrational spectroscopy ls concerned with
the study of absorption of infrared radiation, which resulis in vibrational
transitions. IR spectra is mainly used inf structure clucidadlon to determine
the funcional groups, I is already known that,

(Energy of m molecule = Electronic energy + Vibrational energy + Rotatinal energy

In this chapter, the study is focussed towards the changes in the
vibration of molecule or absorption of energy dur lo vibrations.

PRINCIFLE

ln ary molecule. 1 la kam (hal Alam or groupe olaaa ae
ialogous to springs (Fig 5.1)

enge in rte, Elmo of Uhe cowinnoas imei of the
they malntain some vibrations with seme frequency, characterialic to every
portion of the melecule (This bs called the Marural

When energy in the form of infrared Se een ots ud deu

spablied infrared frequency = Natural frequency of vibration,

absorption of FR radiation takes place and a peak is observed.
Fig 5.1. Infrared vibrations of Ethane!

Every bond or portion of a molecule or functional group requires
different. frequency for absorption. Hence characteristic peak ts observed
for every functional group or part of the molecule. In other words, IR
spectra la nothing but a finger print of a molecule. Eg. IR spect of
Touprofen ts given in Fig 50.

In Pharmaceutical analysis, we use tnfrared radiation. (mid-IR) 2
wavelength 25y to 25p or waveriwsters from 400em' to 4000cm'*. There WW) Selssoring: in which bond angle decreases

are other regons Uke pear-iR (0.84 to 254 and far-IR (254 to 1000y) ll) Rocking: in which bond angle is maintained, but both bends
which are not used tn Pharmacy. ‘moves ‘within the plane
in IR spectra, we use wave numbers instead of wavelengths for b. Out-of-plane bending: (outside the plane of molecule}

mentioning Uke characteristic peak, because wave numbers are larger values

and easy to handle than wavelengths which will show only small differences 0 Wagging: in which both atoms move to one side of plane.

‘between functional groups. (ii) Twisting: in which one atom is above Ue plane and the other
is below the plane.
Wave number ls nothing but the number of waves present per cm,
‘whlch can be calculated from the wavelength. PAG Different types of vibrations tn u mclecale
1 x . 4 Mrechlag
F5 10% = wavenumber per em or cm
Criteria for a compound to absorb IR radiation
1. Change in dipole moment, „ > DA
2. Applied IR frequency should be equal to the natural frequency Xx
of radiation. ¿
Otherwise compounds de not give IR peaks.
heating
TIFES OF VIBRATIONS ‘tmplane bending (Ot at plane Deming

‘There are different types of vibrations (Fig.5.2) st

su pue ru
e A À À À £ ! x * =
1, Stretening vibrations
‘These ure vibrations in which the bond length is altered Le. increased
ar decreased, There are 2 sub-types:
stretching: ln which two bonds Increase or decrease | \ | % Ny

a
in length, syminetrically, + an
b. cal stretchlag: in which vien one bond length increases, ee
“he other one decreases. if a molecule contains “e” atoms, the total number of fundamental
vibrations can be expressed as
2, Bending vibrations Pal
(Gn-6] in a Non-linear molecule

is
‘angle Bending of bonds takes place within the same plane En] in a Linear molecule

1, Source: A source of IR melation like Nernst glower, sabar
or Nieluoene wire la used.

2 Sample handling: A
M Solid: By pressed pellet technique in which solid samples are
nie with Potassium bromide and compressed into a thin transparent
pellet using a Toydrmulke press and la used for anahrik- Allernativehy,

beg Liquid: Samples can be held using a liquid saniple cell made
ides.Aqueous solvents cannot be used as they will
ane D lb ere ns ae

3. Monochromator: Filters made (ap of Lim fiouride or made
dass test Canoe Sea li: aon
ide are es

Diffraction gratings made up of alkali halides
Are also Used,

eee ne mm cy dinars if futon,
etectors like Thermocoupe, Golay cel. eg
electric detectoew are used.
5. Recerder/Plotter: They are used to record the IR spectrum, on
white paper or transparen| sheets,
& pes af Instruments: Singe bem IR spectrophotameter and Doub
are valable Also FT-IR (Fourier

Fr

matching of spectra. ca uen Rroup/cempound

Weg ge eta Dan ep
PIAR over dispersive instrument ls that I In rapid, more serie,
accurate and has more computational capabilities,

1, Identification of functional group and Structure elucidation:
The entire IR region ls divided Into

Group frequency region + 4000em À to 150010"!
Finger print region - 1500cm 1 to 400em'*
In the group frequency region, the peaks corresponding to different

groups can be observed. (cg) Amino group, alcoholic group
ele. Table 3.1 lists some of the common groups and their absorption

y

Standard can be compared to identify a substance. If the spectra
are same, then the identity of the sample can be confirmed. This
technique is called ns spectral matching Eg The IR spectra of
sample Ibuprofen is compared with the standard as in Fig 5.0.

3, Hentifying the Impurities in a drug sample: Impurities have
‘different chemical nature when compared to the pure drug, Hence
these impurfiies give rise to additicnal peaks than that of the pure
‘Grog, By comparing these, we can identify the presence of impurity,

4. Study of hydrogen bonding - whether it ts of intermelecular or
intramolecular type, 4

8, Study of polymers,
6, Rabo of Cu-trans tomers in a misture ef compatnds,

6. NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY
(NMR)

© Introduction
© Principle

© Relaxation Process

& RP transmitter
& RP Receiver / detector
% Sweep generator
% Recorder
& Sample Cell
SF Salvent Requirements
se Shielding and Desbielding
© Chemical Shift
# Reference Standard

© Applications

ee

«Any proton or nicleus with

8
3
F
t
E
2
El
2
SE
il
E
E
Fs
E
H

When energy in the form af Radiofrequency ls applied and when
Applied frequency = Precesslonal frequency,
absorption of energy occurs and a NMR signal ls recorded. (Fig 6.1).

Pg A aad

absorption of energy, the mueleus moves from ground state
to excited state, which results in Spin reversal or antiparallel orientation
- in which the magnetic field caused ty the spin of nucleus opposes
{aligned against) the externally applied magnetic field, When Use application
dm ny sony ppal, the nus ree to ound fake
PA E

it should be noted that increasing the strength of magnetic field does
not cause (risisition from ground state to exctted state, but Ik merely :
increases the precessional frequency. application of
es is RS Gee en cee eg et, oe
radio frequency absorbed. Therefore,
magnetic field and radio frequency la necessary to cause a NMR spectra.
(rig 623,

RELAXATION PROCESS
process of transition from excel state to ground stat,
cher de RS ner rdreueney enr ca be lol y ho war.

1. Radiation ersisslon - with emisalon of radiofrequency radiation ttself,

2 fndiationiess transition (without radiation) - By two ways
Spin-Latiice or Longtudinäl relaxation process » where the energy
"Pont by means of translational / vibrational / rotational energy.
b. Spin-spin or Transverse relaxntion process - where the energy la
Jost to the neighbouring nuclei.
[INSTRUMENTATION
in any NMR instrument (Py,65}, the main components are:

1. tramas - which is used 10 apply a radieaueney ration

ka J09MB:. 220MHs, SO, AQUMH= depending

an pa ro

4 2. RF receiver/datecter - to imeasure the intensity of unabsorbed radio

| frequency energy.

4 3. Sweep Generator - To vary the strength of the applied magnetic
field, Le, to sweep magnetic Meld, A Beld strength of 14,092 gauss,

21,140 gauss or 23,490 gauss etc is used, depending en the Radio
frequency region employed,
4, Recorder - to record the NMR signal obtained from detector.
5. Sample cell - sample test tube which is about 25em long and Simin
sul ir ep naa sl cory sl gs Sr

(revolutions per second) so as Lo provide urafarım
the sample solution.

A combination of GOMER radio frequency and a magnetic field
strength of 14,082 gauss ls used. For high resolution instruments, other

fa we are naling Gr compe fc the rare, type, mr

and eretroninent of Protons (Hydrogen), the solvent used in the NMR
spectroscopy should not contain Hydrogen atoms. Hence we use solvents
like carbon tetrachloride (CCl), Deuterated Chloroform (COCK, Deuternted
water (020), Deuterated Methane! (CD30D), Deutermted dimethyl sulphoxide
(CDAJSO, Deuterated acetic acid [CD9COOD), Deuterated trilouro acetic acid
(crscoopl.ete..

Also the solvents should have the following properties:
1. Chemical inertness“

2 Magnetic isotropy (magnetically neutral) #*

3. Volatility (to fncilitate sample recovery} =~

1) ceeibinations are used.
7 4. Absence of Hydrogen atoms

{ In peactice, radiofrequency is kept constant and the strength of
magueio eld le vored stv vice ver la dificult In aclare. 5. Eaally available and Inexpenstvo =
64 1 65

are required for excitation. Therefore we get different peaks or signals for
each kind of proton,

Chemical shift is the difference between the absorption position
of a sample proton and the absorption position of the reference compound.

Chemical shift la measured in à values, The value ranges from 0 to
108 for most compounds. (Fig 6.5)

Pig 68. Sate of muaa

2. Magnetic tsotropy [magnetically neutral]

3. Give a single sharp peak

4. Easily recognisable peak

5, Miscible with a wide range of solvents

6. Volatility - to facilitate recovery from valuable samples.

tandard, Tetra Methyl Silane (TMS) at 0.5% concentrat
TA
sample itselí, It la called as reference

o

<5,

TMS has 12 protons which are unjformly shielded because of the
highty electropositive nature of silicon al the centre, Hence these 12 protons
(pve a single sharp peak at 06, which require maximum magnetic fleld
dan 1 of most of Ihe orgarde compounds.

1, Structure elucidation of organic compounds

Organic compounds Invariábly have Hydrogen atoms in their structure
and the environment of each proton is not same. Hence from NMR spectra,
following can be known about the structure.

a Types of protans: Fram the no.of peaks recorded, no. of types of
Protons can be known. (eq) Two peaks means two types of protons
and 3 peaks means 3 types of protons, ele.

b. Environment of protons: Whetlser the proton is shielded ar deshieldes,
from the position af the peaks. Shiekied protons require high
magnetic fled and destuelded protons require low magnetic Dei.
(eg) Aliphatic proton - 0:25 [High magicld),

Aromatic proton - 6-95 flow tag fleld),

Absorption position of same protons '

Alphatic/alicyclic - 0-28 Aromatie & heteroaromatic - 6-98
ue Aldehydic - 9 - 10 8
Olelinle - 45 - 7.55 A

2 bevestigation st drummic properties of the molecules. ike

rh mando. eb aries conte tags booing.

3. Determination of optical purity. .

4. Study of molecular interactions like micelie ia ig

cl of Gupdtepur imine on

5. Quantitative analysts

a Assay of components: Single component or multicompanent
without separation of companents, ean be quanltalively eatnated.
Specific peak for cach component ls idenußed and the peak
aea/height ratio ¡ven ty imegral value Is Found ving aan
and sample and the quanuty can be estimated,

b. Surfactant chain length determination: This can be determined
from the proportion of Hydrogen atom in the poly-oayethylene

© Hydrogen analysis: Percentage of Hydrogen in the compound
can be determined.

4. Tofine value: which is a measure of double/tiple bond can be
known from the proportion of olefinic protons.

© Moisture analysis; Since water (H30), can ge characteristic

peak, the % can be known from peak ratio of water peak and
component peak.

ae

7. ELECTRON SPIN. RESONANCE (ESR)

© introduction
© Prneigle

$ Source
dr Microwave bridge
de Magnete Medd
de Sample cavity
# Detector
# Amplifier / Recorder
#7 Reference standard (internal reference)
© Derivative curve
se Determination of’ values
sr Applications

+1

INTRODUCTION
Electron Spin Resonance (ESR) In also called as Electron Parumagnetic
Resonance (EPRI,
ESR spectroscopy Is the study of spin changes at the electron level
when a microwave frequency is absorbed in the presence of a magnetic

PRINCIPLE

Unpaised
earth ions. odd molecules and triplet electronic states.

Analogous to NMR. when no magnetic Meld ts applied, there ia only
one spin state of electron. But when magnetic Beld is applied, there are
two states viz, Ground stale (mes) and Excited state (mess li, The
energy required to undergo this transition from Ground state to Excited
state is absorbed in the form of Microwape radiation {Fig 7.1).

Fig 7.1. Excitation process in SR.

Mite ts 5. Detector; Usually m silicon crystal detector Is used.
6. Amplifier & Recorder: To record the derivalive spectra, tt ts used.

REFERENCE STANDARD (INTERNAL REFERENCE)
ESR spectrophotometer consists of the following:

Any one of the following ts used as Reference standard in ESR.
1. Source: A source of microwave radiation with constant frequency 1, DPPH 1.1-Di Phenyl2-Peryl Mydrasyl free radical which has
and variable amplitude is used. It contains value of 2.0036, lá

A Kiystros - which offers microwave of 9500 MHz. (If 35,000 MHz |
resolution) |

da used lt gives 20 times more À (O) NO,
b. Inolator - to isolate a narrow range of microwave. DPPH ‘6-10,
© Wevemeter - to measure the radiation. ©) ÑO,
d. Attenuator - similar to m Alter in a spretrophotometer, 2 Ce tn a ny éhip of ruby crystal with ag value of 1.4 can also

used.
2. Microwave bridge (Circular-T of MagloT] (Fig 7.2), bid
FG? 2chamate ag of Mises tigo DERIVATIVE CURVE
panda ha Derivative curve 1s used instead of an absorption curve, to get more
information. À typical absorption curve (Fig,7.2) and a derivative curve (Fig
74) bs gen.
Fig 7.2. Absorption curve NE TA. Destrativo curve
= o
{0 D |
et
e Mae Bed sweep from o 500 gauss or 0 Me ge
DETERMINATION OF ‘g VALUES.
4. Sample cavity: Samples in the form of gus, 1 sola
used, Solvents of low dit constan! Are pla, AR : '£ value lo called as Spectroscopie spliting factor’ or Landes spliting
Concentration of 10 M to 19% la used in sample cells, winch are factor, Determination: of this '£ value ts important as this gives an idea
Dal and can bold 0.08m to Om. about the electronte environment.

int) :
where Hi Resonance frequency ised
AH = field separation between standard and sample
qua =f value of Internal standard
Some common € values
Unbound electron - 2002310
DPPH, - 20006
oF la
Organic tree radicals - 2.0023 (nppraxtmately) |

instead of if values, Muperfine splitting constants On gauss) are |
also used for comparison. |

e HË-H HOM OCH
3 15 137
APPLICATIONS.
1, Study of free radicals, including reaction velocay and mechanism, 1°
2 Structure elucidation of organic and inorganie molecules.
3. Study of biological systems, using spin labelling techniques.
4. Quantitative analysis: it bs done ln instrument hing dual sample

cavity. Reference standards like DPPH or Peroxylamine disulphonate
Is used [Ig of DPPH contains 1.53 x 10% unpaired electrons),

2. Separation of the positively charged fragments formed, based on their
masses, by using electrical or magnetic fleld or both,

‘The sample Is bombarded with high energy electron beam (7DeV),
‘where an electron ts knocked off from every molecule. Hence the molecule

{accelerating potential)
somone Sr nr m 2
travel with great speed, in a straight pall =
= Kinetic energy of molecule Px À wince H & V are maintained constant
= 1/2 mt 2 Mass se (radius of fof path)?, since © = 1 (unit positive Charge)
where e = charge of lon
INSTRUMENTATION
Vo acceleration voltage
‘The diagrarts of a single focusing, Electron impact ‘Spectrometer
mo mans la shown in Fig Bil. wee
y= velocity after acceleration le ni
magnetie field or an electric Mel la applied, the positive
ged fragments wich were toweling in straight pa, no travels in
m curved path. When they travel in a curved path under dhe infuses of
eld. the’ fragments are separated into different masses because
radius of curvature dependa upon heir respective: masses.
Under magnetic field. ‘vacuum de
my
wu fon
Sample ja? a To amplifier
we tell a 6
ee {sm home its dpe sn dad te ine. ben
Inlet, electron bombarding source and areelerating plates on one
al ba present. AL the curvature of the

|
:
E
E
i
5
i
a
i

i
|

with electron beam at ZOeV. This knocks off one
electron from every molecule: As‘ these molecules become positive charged,

collected using a cailecting all. The different fragments fall on
and m mass spectrum la recorded (Fig 8.2), à
‘Fig 4.2. Masa epectral pattern.

e temeraaty,
%

A. Single focussing type - Where only magnetic field Is used

# ts of different masses, These have resolut
which means that It is possible to recotd mass of

components/fragmenta with a difference of 0.0003 (or 1/3000),

masses with a difference of even 0.000025 (Resclution = 40,

2. Cheaulesl loalsation Mass Spectrometer (CIMS}: in which n

das ls used to obtain positive charged ons from neutral

leg) Methane, Ammonia or tsobutane ls used as reactant gas.

m

3, Field Tonlsation Mass Spectramieter
4. Field desorption technique
5. Plasma desorption technique

6. Laser Mass Spectrometer (Laser induced ionisation or
desorption ionisation),

PES OF Frans
1. Parent peak, Mass peak or molecular lea peal: (often called Mr
os FE The peak normal with highest m/e value Dems) pa

=

"
A

2 Huse peak: The most intense

hers Peak (tallest ent y-axis) is called

3, Mel pen Ik cecure due to the presence of YC, Ah, Uy, 335
4. M#2 peak: It occurs due to the presence of Mo, Mg, Sc, lp,

4 Drug metabolism studies: By recording the mass spectrum of -
> Anetabolite and that of pure drug, the metabolism of the drug can
be know.

_5 Cilnleal, and Foremale appllestions: Very minute
~—‘fquantities of the drug can be used to find out mass and henee the
substance can be identiled from the mass spectral pattern.

ELECTROCHEMICAL METEODS OF ANALYSIS

INTRODUCTION

of chemicals or drugs can be done by using their

Some of these techniques are Potentiometry, Conductometry,
Polarography and Amperometry.

Potentiometry consists of measuring the potential of a solution in
mV. using a set of indicator and reference electrode. The Potential of a
solution depends on the nature, concentration and temperature of the ane,
‘of the drug substance. As potential ts related to pH. N can be measured
tuning a pH meter, Recent pH meters are designed to measure both pH
as well as potential of a solution.

Potentiometric titration is the one in which the potential or pH of
a solution is montlored during a titration, During any titration. as
concentration of fons changes, potential and pH of the solution also changes.
When potential or pH changes are monitored, the end point af such Utration
can be found out, The end point is the point at which the rile of change
of potential is the maximum, which ean be found out graphically, by using
ration curves,

Conductometry is the technique of measuring the conductivity of a
solution fin mba} using a conductivity meter, The conduetivtty of a solution
dependa on the sist of Jans, charge of lons and no, of fons (Le, concentration)
and temperature, The lona (eatlons and anions) move towards respective
electrodos and hence a solution with ions has conducting nature,

During a Utration, as the number of conducting tons (concentration)
changes, conductivity also changes. Wien conductivity changes are monitored
in a titration, there is sharp change in conductivity at the end point. Such
ration where conductivity ls monitored, to detect end point la called as
Conductometric titrations.

Polarography ls the technique in which a varying negative potential
ls applied between a polarisable clecirode (dropping mercury electrode) and
a reference electrode and the resulting cument ls recorded, The obtained
current-voltage curve 1s called us Polarogramn, from which Half wave potential
(2/2 and Diffusion current (id) are determined. Half wave potential is à
qualitative aspect which helps ta Identify the substance. The diffusion

33

current ls proportional la the concentration: of the substance
py various methods, quantity of a substance can be nat anes

ln Amperometric tltration, the voltage aj
and reference electrode la kept constant and the changes In the Aha
current during the Utration Is monttwred. The difhision current ch
red on die ion af fone Gat to ai of Uren. 44

iy the u on. & sharp change in dior current. cura,
following table summarises all the techniques desertbed,

‘Variable controlled
(kept constant)
Currents

Currents

Mame of the
method

[PoAeeaiocnetry

==

‘Parameters measured

Hydrogen electrode
Antimany-Antimony oxide electrode
Glass electrode

"© Membrane / ion selective electrodes.

© Measurement of emf & pH

Simple potentiometer
Commercial potentiometer (pH Meter)

indicator electrode responds to changes in emf or pH of a solution,
tris used to indicate the emf or potential or pH of a solution, (eg) Antimony

‘The potential (E) of a metal eitetrode at 25°C tmmersed into a solution
‘of tts own fons ls given by

BE. 1

loge

where E°- Standard potential of the metal
n= Valeney of tons
€ - Concentration of ions

1. Factors like purtty of hydrogen and pressure of hydrogen affect the
potential of this electrode,

Ly Hit fected by the presence of xs or ducing substance
3. Potential in affected due to potsoning of platinum surface.

2, Saturated calomel electrode

It consists of an inner jacket and outer
sleeve (Fig 9.2). The Inner tube (jacket) has
wire contact wilh mereury and plugged with
a mixture of calomel (Hg2Cla) and KCL This
Is surrounded by an outer sleeve and Ihe
Up ls fled with crystals of KCI and porous
Plug of wabestos. The space between te
inner jacket and outer sleeve ls lcd with
either saturated KCI or IN KCE or LAN
KCL on which the potential of the electrode
depends upon, The potential of this half
cell depends upon i) Concentration of

3, Sllwer-Sitver ebloride electrode

le ts simply a silver wire coated electrolytically with aûver chloride
and dipped into potasslur chloride. The potential ofthis half cell also depends
upon lemperalure as well as concentration of potassium chloride used.

tt has the advantage that Ht ls easy to use, but the demerit is that
tt ia difficult to prepare.

INDICATOR ELECTRODES
Indicator electrode indicates the potential or pH of a solution in

1. Hydrogen electrode: Hydrogen clectrode has been described in detail
under reference electrode. I Is used as reference electrode when
dipped into a standard acid solution. Hydrogen electrode ls used as
indicator electrode when dipped into a unknown solution whose
Potential or pH has to be determined.

2. Antimony-Antimany oxide electrode: It consists of a Antimony’ rod
dipped into a sctution, whose potential or pH has to be determined,
Antimony oxide (50203) is formed on exposure to air.

Reaction:
So + HO + SbO* + 2H? + de

Potential:
Eu e+ SE y ison (HI
+ EB = 0288 - 0.0882 pH (since E* for $b = 0.255 &
simplifying other terma)
Advantages

1, it can be used from pH 3 to pli & IL can be used even upto pH
12, if saturated solution of oxide Is used,

2 lt is not cantly poisoned or damaged.
3. It can be used even with viscous fluids,

Disadvantages

‘This clectrode cannot be used In the presence of dissolved oxygen,
oxidising agents, highly acidic or alkaline conditions or complexing agents.
97

pity = pH of setutlen inside the ‘bulb
pla = pH of test selutlon (outside)
SE = ke + 0.0592 pHy - 0.0802 pity
E = K- 0,0502 pia
since pHi = constant, ik + 0.0502 pil ls also another constant,
‘Advantages
1, dt gres a rapid response.

2. His chemically resistant to casdising and reducing agenta, dissolved
(gases, collolda and salts, ete.

3, When Hiia-ailien glasses are used, it can be used over the entire
DH range,

‘Disadvantages
1, HE in extremely frage, since contains a very thin bulb,

2. Minute abrasions on the surface of the Up, damages the electrode,
SR cannot be used with simple potentiometers, because of high
resistance.

MEMBRANE / 10N SELECTIVE ELECTRODES

The glass electrode can be modified suitably to a selective ton
membrane electrode. The composition of the membrane Is changed
adding specific fons or synthetic substances or pelymera thereby
electrode can be used for measuring cations or anions. For example,

1. Cation selective glass electrode is used in the determination of H*,
Ut, Na, Ag for) K°

2 Divalent cation activity electrode can be used to measure the
total hardness of water (response due to calcium / magnesium ce
other divalent ions) and for complexometrie titrations using EDTA.

3. Heterogenous membrane or solid state electrode can be used la
measure concentrations of CT, Br, L CN, 84, ete

MEASUREMENT OF emf AND pH

‘The measurement of potential or emf of a solution in m¥ is made
using a simple of electronic (commercial) potentiometers. Now-a-days, the
instruments are designed to measure the emf as well as pit of a solution
simultaneously. Hence they can be called as potentiometer or pli meter,

Simple Potentiometer (ull polnt)

The principle in simple 1,0
potentiometer Is that the potential of an
unknown solution is matched with a
nown emf (standard cell Fig 9.4 gives a
the eireult diagram of a polentiometer
il point), 1 contains a power supply
unit (C) Usrough which current Y avs.
Wire AB is of uniform dimensions and
ls of known resistance. When a solution

ME

fo und. Promi be ue al el lo town an i
determined.
CENT
where B= emf of unknown sctution
ty = length (Ab!)
1 current
Story, Ea = thy
where Ea emf of standard cell
te = engl (AD)
r = resistance/unét length

2-4

(emf of unknown solution) = E « i

‘Merits
1, Simple,
2, Ease of construction.
3, Cheap.
Demerits
1. Calibration to be done every time with cel of known cen

2 Temperature correction, slope correction, ete are not avadable.
sia

ren

These
commercial potentioneters or ahy

indicator electrode. In recent A
instruments, these two electrodes are also combined to given an appearance
of a singe electrode,

The diagram of a commercial potentiometer la shown in Fig 95, The

‘Greuit diagram ts similar to simple potentiometer, and constata of a Kheostat
foe temperature compensation for adjusting to the room temperature.
‘Standardisation and calibration ts done using bullers of pH 7 and pli 4
er pH 9.2, A slope correction kneb ls also present. The potential in Y or
mV and pH can be read directly by digital display because: of automatic
balancing to set null deflection of galvancmeter,

Advantages/Features of commercial potentiometer (pl meter)
ra of camer pointes es don pomme
of the folowing features:

1. Temperature (compensation) control knob.

2 Calibration kneb.

3. Slope correction knot,

4. Temperature display.

5. internal calibration. without external calibration using bulle.

1, Coloured solutions, cute solutions or turbid suspensions can be

Strate.

2, Actual potential of reference electrode need not be known since I
la maintained as constant throughout the titration

3, Mira can be automated (as in au er)

‘tration ce redox titratson, ete,
In most ttrations, saturated
calomel electrode la used an

reference electrode, Only the indicator electrode, varies with respeet Lo the
type of titrant (Fig DE.
Method of detecting end point

When indicator method ls not sullable, we use potentiometric method
ol determining end paint. I} ls done graphically by’ using

1. A normal titration curve Le, a plot of emf (va) vol. of titrant or pH

fen} vol. of tirant
2. First dertwalive curve Le a plot of AB/Av or plan (val vol. ef
Un.
3. Second dertrative curve Le m plot of APEJa or ARpH/An ten]
volume of titrant,

In a potentiometric titration, at che end point, dx rate of change

ef potential is maximum. The method of detecting the end point 19,

mal atin curve Pt dertratne cure Bern dertrative uve
si Sen]
Ar wi
y e E E
cl]
q = wy
Yok of rant tof trent sai ait
ig 8.7, Methods of determining end point

APPLICATIONS OF POTENTIOMETRIC TITRATIONS
“The following types of trations can be done by Potentiometris method.

A Acid-base titrations (aqueous and nor-aqueotal
B Redox Utrations

€. Diazotisation trations

D. Precipitation titrations

E Comglexomeute Ulrations
F. Dend stop end point technique
Each application ia discussed in deiaf as follows:
A Acid-base titrations
Acid-base titrations can be done in aqueoua as well as by non
guess: medium,
Indicator electrode : Glass electrode
Reference electrode : Saturated calomel electrode
‘The potential (EJ of auch system ts gen ty the flowing, equation.
E = k + 00592 pH

ll, Mixture of acids (val bases (ej) ICHSCOOH + HCI vx) NaOH
ar bones fw) nei (9) NOR + DIO Im MN

A. Poly basic acids (vs) bases (eg) Citrte acid fra) NaOH
fei) Tartaste acid (al HAC

> Nomagueous medium
‘The same electrode system used for titration jn aqueous medium ta

‘Titrations

L Weak acids (va) Potassium (KOMe) or lithium methode (LiOMej
(eg Bartiturio acid (va) LOMe

ii. Weak bases (va) Perchloric aci
(eg) Epbedrine Hydrochloride, Quinine sulphate, Metronidazale or
Chloroquine Phosphate, (vs) Q.IN HEI,
B. Redox Titrations
‘The general equation of a redox titration can de given by
Reductant + ne =» cxidant
fea) Cot + pet + co + Fe

ce ts an agent which cxidises Fe?* (ferrous) to Fe™ (ferric
Jon), ln this process Ácerric) gets reduced to Ce (eerreus) ions,

‘The potential (E) of redox titration can be represented by
0.052
BR ag

where E°- standard potential
A = no, of electrons involved in the reaction
fag cone. of oxidant

Red] - conc, of reductant

Reference electrode : Saturated calomel electrode or
‘Silver-aidver chloride electrode

Indicator electrode : Platinum wire or fall
For end point detection, mV scale is used and not pH.

Indicator method is by potentiometric method of determining end point.
Reference electrode : Saturnted calomel electrode
Indicator electrode : Glass deetrode

Examples

Alealolds, Amines, Sulpha drugs and cther drugs which contain
aromatie primary amine group can be titrated against 0.1N Sodium Nitite
in hydrochloric acid,

D. Prectpttation titrations

For the quantitative determination of several tons or elements,
precipitating agents are used as titrate and the end pont ls determined
by petentiometric method, The potential of such system is given ty

pure 2 tg pe

where (MI- refers to the concentration of tana.
m» electronic: state
Relertace electrode : Saturated calomel electrode
Hydrogen electrode
Silver «silver chloride electrode
Indicator electrode : Silver wire electrode

Examples: Determination of Mercury, Silver, Lead. Copper and several
other lona using precipitants to form insoluble salta,

E. Complezometrlo titrations
Several metallic tons (divalent, ete) can be Utrated against disodium
edetale solutions by Potentioinetric method. Measurements are made kn mi
scale, The Reference electrode used la Saturated calamel electrode or any
reference electrode. The Indicator electrode used ls Silver electrode or
Mercury electrode, Eg. Divalent lona. trivalent tons, CN” ete (val EDTA

This can be explained by using the following circuit diagram. Classical
¡cumple ls the determination of water [Moisture conten!) by Karl Pischer

reagent (Pig 9.8). N contains toe platinum

depolarisation of ne md

‘The reactions involved in the titration of water using Karl Fischer
reagent la given below:
oo]

PE
UN A 7

10. CONDUCTOMETRY

® Introduction
"© Measurement of Conductivity
7 Determination of Cell constant
© Applications of Conductivity measurements
© Sally of sparingly soluble salts
fone product of water
Banicity of organic acids

Purity of water
Quantitative analyse

Conductometiy ls the mensurement of conductivity of à solution due
to the mobiity of cations and anions towards respective electrodes,
Conductivity (CI Is inversely proportional to resistance (RI of a solution
€» fy The unit of conductviy la mios, Conductivity of a solution depends
upon no. of ans (concentration), charge of ions, size of fons and temperature,
‘All the laws which are applicable to solid conductors substances alse ca
be applied to the solutions containing fons. Accordingly, the resistance (Rj
ñas b pan ty
net
where E = potential difference and
1 = een which fous through

‘The unit of Resistance (R) is ches, Potential difference (E) ts vata
ani that of Current (I) in amperes,

‘The resistance (R) of a solution depends upon the length 1) and cross
section (a) of the conductor through which conductivity takes place. Therefore,

af

where p = specific resistance
ve p= tt

‘Specific resistance (pl is the resistance offered by a substance of
em length and 1 1q.c surface arca. Unit of measurement ls ohm cm.

Specific contuatiaty (ua la the condustiviy or ty n subelance

oon go sr Ono ert mh

1
ken

Equivolent conductivity (xj In the conductivity of à sebutien
equidad We of che seit Dee crade, Ea Spur and 1 ag
surface area. Unit of measurement Ir WdE em

nen emductiy Specie conducey la) x volume fl sen
containing 1 gram equivalent weight of electrolyte

Molar conductivity Im] la the conductivity of a sebutlon containing
molecular weight of the soluto between electrodes 1 em apart and 1 sq.em
surface area.

Molar conductivity = o A Ei
‘ecotaining one moleculas weight of Ihe eleetrotyte,

‘uniform coating. The different electrodes used depends upon the

Of the solution, Le. whether the conduciviy of ie SE
achution la bag ar low. The commonly used platinum
electrode la shown in Figure.

‘The whentstone bridge circuit consists of a standard
resistance in one of lts arms and the other arm contains =

of the tsnknown solution with that of standard resistance.

de

Wien mul deflection la obtained, the flowing equation hold pod,
of BC WE
pto E
where, Ra= Resistance of unknown solution
Ri = Standard resistance

fa Be eR

BA
Hence, Conductiity of unknown solution = Ge * Rt

‘Tina the conductivity of a solution can be determined,

observed comfuetiity In not always the Specific conductivity,
een roles ofthe platinum electrode of various nänuleturen
are not same Le Use distance between the electrodes (1 em) and the
surface area of the électrode (1 seem}. Hence the value cell constant (x)
la calculated.

cat constant (x) = À

where, 1 = distance between the clectrodes (em) and
a = area of the electrode (quem),

‘The rebaticaship between specific conductivity and observed conductivity
can be derived as follows:

Accra 1 equation for Resistance, R « À

Specific conductivity 5
Gbrervedcenductity ~ *

Specific conductivity = x x observed conductivity
Le. Bpeclle conductivity = Cell comstant x observed conductivity
(DETERMINATION OF CELL CONSTANT

Cell constant of a conductivity cell is determined by measuring the
conductivity of a known strengih of Potassium chlarde at a speetiie
temperature,

Dy determining the conductivity of 0.02 KCI at 28°C, Cell constant
can be calculated.

a ——
se observed conduetrrtty of 0,02 KEI at 28°C in jambas

Alternatively, the conductivity of 0,01 KCI at 18% can also be
determined. Then the Cell constant la given by the following equalton:

1220

a observed conductivity of 0.01 KO at 16°C in paros:
(Sp. conductivity of O.02KC1 at 250-2765 A that cl O.0IKCI at 18°C=1221)
Conductivity water: Conductivity water ts water purtled by a technique
10 that water is free from conducting ions (both cations and andonsh
APPLICATIONS OF CONDUCTIVITY MEASUREMENTS
1, Solubllity of aparingiy soluble salts

Mary salts like, Silver chloride, Barium sulphate, Lead sulphate are
sparingly soluble and their solubilities can be determined by making

conductivity measurements. (Solubility 1s the amount of salute present in
1000 cc of a saturated solution).

A saturnted solution of the salt in conductivity water ls made and
thespecificconductivity at 28°C la determined by usthg conductivity meter,

Since sparingly soluble salt fully dissociates into lona, de » de

here Am = equivalent conductivity at infinite dilution
dy = equivalent conductivity

de = = he x Y there Vovolume containing, Igrumenmt of the soie)
teeta the
de hag ther
de = 6192 + 7035
de = 138.27 mhos at 25°C

de
AS

As de = de and dor are known, Y ean be calculated,
Y ec of solution contains Igram cquet of He salute,
x 1000ce ol solution écetains ent x 1000/V
à Saab» SEE 1000

„rn 1000 x le dy,
Soluble EASA los vn)

1435 » 1000 «
sony o EI (eat of ARCA in 1435

In the above equation, as all the values except le (Specific conductivity)
are known, solubility of silver chloride can be
“The specific conductivity of the salt can be calculated as follows:

iy © cell constant x observed conductivty of the salt solution
nina altro table o
Cell constant * Sytrredcanductivityol KCI at the mensuredtemperature

2, Tonle product of water (hte)
It in the product of lonie concentrations of H* and OH expressed In
h gram moles per litre at constant temperature.

140 = HT + ON

"hers
[MO] ls constant as HzO ls slightly lonieed
Specific conductivity of pure water at 25% ts 554 x 10% mhos em?

RICHT = ky

A = Auen + Destin

de = 10853408

dy = 548.3 at 25°C
kv st
weit VaRAE

oof gr eg of ae» a BEES IO 101» 107

mo, of gram eq. of OM = 1.01 x 107
ke = BE] LOT
ky = 1.01 x 107 x 1.01 x 1071.02 x 10% »
ke 1 10 at 250
3, Bamelty of organte acids
The basicity (8) of organic aciós ts nothing but the no. af carboxylic

groups present in the molecule. eg. the basicity tartare
Site en dat of ou acl te 2 “au aan

neutralise

with water, ‘This solution 1a N/S2 solution, This solution la further «ute
to 1/04. 8/128, N/25G, N/812 and to N/1024. By mensuring the conduit
of N/1024 and N/32, the basicity ls calculated

4. Purity of water

pure water has a specific conductivity of 5 x 10% ohm’! emt Y the
determined value la more or lesa, the quality of Ue water ean be tnferred,
‘Thus the qualty of distilled water and deionised water can be known,

5. Quantitative analysis

8, Sallnity of sen water:
‘This could be known by conductivity measurements.
7. Equilihriam la tonic reactions

Since conductance of sotution changes during such reactions, the
progress of tonic reactions can be determined.

point of the titration, there ls a sharp
solution shown by the interscetien of the lines i the graph of conductivity
Vs volume of titrant added.

Advantages af conductometste titrations
¿Ae Determination of specie conductivity la nat required.
+2. Mis not necessary Lo use conductivity water.

, 2 No indicator ts necessary,
à 108

4 Timiions can be done wilh colored or ute solutions or turted
suspensions,

5. Incompletion at the end point dors not affect thie resulta ns few
meaaurements before and aller the endpoint are sufficient.

6, The principle that conductivity dependa upon the na. and mobility
of the tons in used and curves are mostly straight Unes. Hence few
measurements are mullet,

Te ha the end point ts determined graphically, errors are minimised
and accurate end palm can be

8. The cell constant peed not be determined provided the same electrode
lo used throughout the experiment.

4. Temperature need mot be known provided I la maintained constant
droughout the titration,

‘Apparatus required à Procedure

A titration vessel la beaker}, a stirrer for mixing, automate oF manual
buretle to deliver the titrant are sufficient. A conductivity meter with m
conductivily cell (Platinum electrode) la used to make conductivity
measurements. The conductivity ls measured An miflimhes (10 enbos or
micrombhos (10% mbos), The Utraril la added in small increments Wee OS
ar Ami, Ube seluticn 1s mixed properly and the conductivity readings are
taken. Several readings are taken, few before and few alter the approximate
end point (stoichiometric). A graph of conductivity Ve the volume of Url
la drown and the polnt of intersection of lines is found eut, This corresponda
La the end point or the volume of titrant required io neutralise the renctanta
or sample present in the ttrations vessel.

Precautions to be observed

1, The initial volume of the titenting substance and the Anal volume
after Utration are not same. Hence the conductivity measurements
made during the titration are subject to error. Hence à eaerection
factor ts included to know the actual conductwity rather than the
observed conductivity.

‘aca concu + Observe codi | SH a re ne

108

>

ACID-MASE TITRATIONS
a Strong acid Ve Strong base
eq. Hydrochloric acid Vs Sodium Iydrosiée
ac + Mato > Meter” + #0

A ci ken fo be ra
the initial conductivity la high, oe
strong. acid completely dissociates into
fons and the dono corductnty of H° ts 250. um dor,

hb. Strong seid Va Weak base
eg. Hydrochloric acid Ve Ammonium hydroxide
HCH" + NH, "OH -+ NH *CI" + HO
—
‘Strong Acid Va Weak base.

cenufuetivity gradually un,

Vel. of NOM

into OFF and Merce: te fy remains constant, A plot of conductivity
vs volume of NHAOH added la shown in Dgure. The Brat part of the curve
shows sleep fall in conductivity because of decrease in H* Donde conductivity
- 350) and second part of the curve shows a plateau,

CHICOO H + Na*OH + CHsCOO™ Na’ + HzO

When CH3COOM Is taken in beaker as
rate, the tnitial conductivity ts low, because
weak acid does not disscciale into H* tons.

‘Walk Acid Ve Strong base

NaOH causes increase Ih the no, of OH-ÿ | formation

and bence the conductivity atarta to increase ig
steeply llonie conducuwty of OH ts 185). >
A plot of conductivity Vs volume of NaOH
added is show in figure, The first part of
the curve shows a gradual increase and second part of the curve shows
steep increase because of increase in OH” onic conductivity - 199.

10-11

Vol of NaOH >

CHyCOOH + NHSOR => CHyCOONIY* + 130
CMICOOH ls taken in beaker an
rm tna cadet la low, beste y PESA Wk De
‘weak acid does not dissociate into H* tons,
When NIOH Is added as Utrunt. Armenordum Pi
reiste salt is formed which has better iat
than fer of the substances and
hence atl the comdsctivty increases
after every addition. Alter the end point, when [en
all the CHSCOOH has reacted, the addition seen
‘of NAOH causes no increase in the ao +

Utralions are pol as sccurnie as acid base titrations
Because of slow precipitation and adsorption of schutes on to precipitates,
An this titration, any one of the product of the titration can be a peecipttate
and the other soluble ar both products can be in the form of precipitate,

1. Only one product la a precipitate

Example: Potassium chloride Ve Silver nitrate
KG) + ANO] + Agel + KMD,

‘RCI ls taken in a beaker and Saver nitrate Is the titrant, When silver
nitrate ls added, the first part of the curve (given below) shows no increase
in conductivity as there ls only replacement of chloride ions with nitrate
Jor. (lone conductivity: Chloride - 76, Nitrate - 71), As the silver chloride
is peecipitaicd, it does not contribute to conductivity. The second part of
the curve, Le. afler end point, conductivity increases because of increase

An the concentration of Silver as well as nitrate fons,
aay

wire L 1
= fac. oh ao
akong em point
ua vane, =
Preetptlathen Utrethone

When magnesium sulphate Is the trate and bartum hydroxide ta the
titrant, Dou the preduels, Le. ‚magneasum hydroxide and bartum sulphaie
are sparingly soluble and are preciptinted. Mence these da net contribute
to eonduetisty and the mt part of the curve (as shown in previous page)
shows decrease in ccexhuctivity because of decrease in the concentration
of condweting lores (MgSO), The secund part of the curve shows a increase
in conducivay due to the addition of conducting sons (Ba{OHa).

©, DISPLACEMENT TITRATIONS
‘These are titrations in which there is displacement of one tan by the
other, The fellowing are examples of such titrations
m. Salt of wrong acid und weak base Va Strong base
KIEL + NaOH -+ NILLOH + Nach

ln this titration, Ammonium chloride ts used as titrate and Sodium
Ihydrexide as lteunt, The Arst part of the Utration ls a plateau because
there la caly displacement of Ammonium and chloride tons with sodium
and chloride lona UN the end point. Aller the end point. the addition af
sodium hydroxide comes u steep increase in the conductivity,

10-14

lt of strong Dane and weak hd Vo Strong, aci
‘cricoome + HI + COOH + HACI

la ths ttraica, Sodium acetate la used as trate and Hyrdrochlaie
cid is used an titrant, The Art part of the ftralon is gradual increase

condo because Chere la diplacement of
Ms ul ine end point. Ale the end point, the addon of Hrdrochlrie

‘Vol of HEL +

detect the small change in conductivity due
lo change in hydrogen fon concentration,
redox titrations eun be done by making

eg Titration of ferrous fons with dichromate fons.
GF + Orr? + 14H + GFe™ + 2Cr* © 710

Ferrous fons trated with dichromate ions shows the above graph of
conbuctivity wa Vol. of dichromate.

E. COMPLEXOMETRIC TITRATIONS

Like redox Utrauens, the conductivity
«change (hat could be observed or measured
near the end point as small, The following

‘The tuo inflections indicate the formation end ond
of MEU? and the completion of the paint,
reaction respective. Vol of Helly

F. NONAQUEOUS TITRATIONS

Like in aqueous medium, non-aqueous titrations ean be carried out
by making conductivity measurements, Titrations of weak acids or weak
bases can be done.

eg 1. Titration of weak organi acids in Methanol, Pyridine or Dimeikyl
formamide Vs Tetramethy! ammonium tyttrexide in methanol-bensene or
pyidine bensene.

2, Titration of weak bases Va perehlorte acid in diaan-formie acid.

Comparison with potentiomatrie titrations

Poteatiometsle titration | Condueiometzie titration
[Comductriy in mba

11. POLAROGRAPEY

#7 introduction
me Principle
sr Advantages of Polarography
© propping Mercury Electrode (DME)
Advantages & Disadvantages
sr Folarographie Maxima
2 Method of Analysts
mr Different types of current in Polarography
© Factors affecting diffusion current
"© Half wave potential
‘ Methods of Quantitative analysis
À Direct comparison method
À Calíbration curve method
3% Internal Standard or Pilot fon method
ds Standard addition method
‘© Applications
À Inonpanie and Organic
Qualitative and Quantitative

Polarograplie technique ts applied for the qualllalive or quantitative
ddisabie

particular compound Quantitative ‘Thus polarography can be used
for both qualitative or quantitatrve analysis of compounds.
ADVANTAGES OF POLAROGRAPIIY

¿LAmanic and inorganic samples can be analysed.

A o al

AL Rapidity of the technique Dean time is sulflctent,

3 Qualtatie and quantitative analysts can be performed easly

+ Mbaure of compounds or elements could be analysed without
separation.

119

¡Puementes: Rapid qualltaine and. quantitative ayes of mécture |

of organic. and inorganic samples without separation even at tow
concentrations (10% M to 10% .

"The size of the droplet depends upon the bore sise of the capillary,
Mama its are made aide the tubing where the mercury fows through.

Advantages of DME
Surface arca ls reproduetble
2 Constant renewal of electrode surface eliminates potsoning effects
3. Mercury forms amalgams with most metal tons and alkai metal ions
7 whieh are reducible
4. ln ds useful over the range of +04 to -1.8V
v
Disadvantages of DME
‘The electrode cannot be used above +0.4V (Va SCE). because He
Y Tecorded Lemer

dissolves and anodic wave ts than -1.8V, hydrogen
la liberated, =

2. The capillary ls difficult to maintain since dust or other particulate
matter ean block the eapilary.

bees tad
POLAROGRAPIIC MAXIMA
‘When a current voltage - curve ls À
recorded, a hump ts normally ween tn the

cecura la nöl known. The presence of this

polaregraphle iman leads to error tn Getertiining half ware

and difusion current. Herve maxis suppressors which increases

‘the viscoslty Îlke gelatin (0.000% Lo 0.014), dyes (methy! red) or surfactant.

(Triton) ets can be added, Although the mechanism of polarographic maxima
la ot clear, ft da not seen An very dilute solutions,

METHOD OF ANALYSIS

HABEN

HF a

LH TH =

In que i
i

Migration casrent (lal: It} due to migralson of cations from the
ue sagan e tardo cao du odie force, esc
‘This depends on the proportion of the

migration current, large proportion of supporting electrolyte (RCH
ran 100 tones te amount of lectroredueble ion ln added so
Bit the entire current recorded is only die current.

piston current (la: Diffusion current la dive Lo We actual cifusien
. aro the bulk of the sangle o the murice

of the mercury droplet Curent ed
by such loma under such condition ls called as diffusion curren

| Limiting gursent (hi: Beyond a certain potential. Ihe current renches
! ec 8 the hing coment, AL tin pa. We ae
Cf diffusion of lens is equal to the rate of réduction and the state

| Of electrode la said to be concentration polarised (surface saturation,

‘The diffusion current at Hs limiting value using DME is given by
kouie equation as follows.
iy = 607 n CD mi 1e

‚rent. due ta electroreducible lona
cea eae ET
mobecule
. cone Sn mme
= lors eovefficient of one
. mercury wing, Uco caplry Img/sec
= drop lime in seconds (2 to 7 seconds)

where id =

concentration of the electroreducibie tons. This forma the baala
quantitative analysis. Le If concentration ls

current ln less. If concentration la more, then dillumien Curr da
also mere,

2. Temperature: Diffusion of tons la being affected by temperature
Mence diffusion current also warten with respect to lemperuture
(ireethy proportional),

3, Viecoalty of the medium: Duffuston co-e(Meient depends on he
viscosity of the medium. Hence diffusion current also warte. ef.
Gelatin at very low concentration is used an maxkmam suppressor.
UE very high concentran of gain la added, then I cun allect the
Alfualon co-efficient and difusion current

4, Caplilary characteristics: The bore size of the capillary. drop time
An seconds and the pressure af mercury will all allect the diffusion
current as they alter the flow characterises of mercury droplet.

5. Presence of maximum suppressors: Maximum tie
gelatin, dye stalls and surfactants wil affect the difusion current,

‚Rölatlonshlp between applied potential, diffulon current and
half ware potential

00802 (un
Egg + STE dog

were Esp = applied potential

E® Half Wave potential

m = no, of electrons involved
M = diffusion current

U current al applied potential

HALF WAVE POTENTIAL te")
‘The Half wave potential ls the potential at the point of inflection

in the currentoottage curve E is characteriatle or specille lor
electroreducible on or functional

3

ou, At_this potential 50% of the

a of the oxidised for are ‘The chained E
lua Ria pond le the ‘values from the text
or reference and the substance can be identified. Thus Half wave patenta

ls the qualitative aspect which serves lo identify a substance.

METHODS OF QUANTITATIVE ANALYSIS

Quanutauve analysis is performed te find out the amount
ane aan ofan cio reduce an or funcional group. The parameter
la Le. diffusion current ts proportional to the concentration of the
analyte, There are several methods af quantitative analysts, They are

1, Direct ésmpartsoe method

2 Calibration curve method (Multiple standard method)
3, internal standard or Pilot lon method

4, Method of standard addition

1, Direet comparison method

In this method, the diffusion current of a standard solution of known
concentration and that of the sample is compared under identieal conditions
of temperature, concentration of supporting electrolyte, capillary cimentar,

‘The equation for the diffusion current of standard and sample ane:
given by

har © 607 n Cy DY mn de
La = 607 n Ca DY mn 14
where las & lez are défusien currents of sample and standard
Cı & C are concentrations of sample and standard

mn. D, m and t are kept as constant for both sample and
standard and hence can be Ignored. d
Hence oc dividing, one equation by the eter, the equation reduces la
in „Sı
la a 3
Since lar and laa are found experimentally and C2 la known. Ch
feencentration of unknown sample solution) can be determined.

9. Callbration curve method (Multiple standard method]

ln direct comparison method, the resulta are subject to error. To
mintmise the error, several standard solutions are prepared. The diffusen

3. Intersal standard or Pilot tea method

‘This method la used to determine the concentration of a lan, whose
mapdard solution carnet be prepared or not available or may be expensive
lo prepare etc. A pilot len ls Used to seasure Use concentration of the
analyte by indirect technique. For example, the concentration of guid or
platinum or an expensive fon in selution can be determined without having
OF preparing the standard acluticns of the analyte.

The diffusion current constant [0 ts independent of capillary
chameteristics provided temperature and esocentratice, ef supporting
electralpte are constante.

ho TC te
where, = 607 nD
For the test ton, day = ly Cy mt de
Por the pot Son Ka ta Ca ami 1%
Dividing ome equation by the other, we get
da Wer
tn ay
since the same capillary characteristics are used for übe test and pilol ice

‘Toe value [to called as the pilot lon mio and om be ebtanes

from standard text books or erature, Since the 141 and laz are determined

experimentally and Ca is known, the value of Ci, Le concententica of the
‘test hon la eadeulated,

4, Standard addition method

ln Uds method, the polarogmm of the teat solution bs recorded ang
the diffusion current ts determined, To the same sample solution, à
mani gumriy of ander mts la mi al oe ad Sara
la determined by recording the pelaregram once again. By wring the
following equation, the concentration of the tons in the given sample can
be determined,

— ann _

Corot 0e AA ET mn

where € = concentration
= diffusion current and
¥ = volume of test solution & standard added respectively

Maximum precision ls obtained when ty = 211

arrears

ager tape al rm) Ad
samples can be done even in low concentrations without separation. They
are given in detail as follow,

1. Inorganie applications (Qualitative und Quantitative)

2 Composition of alloys
b. Purity of elements

. y

mp Là
hii element ts

Pree la of mr

tr

(Mena seem [Ma were pecas |
ay ano cum |
3. Organic applications
Kisstrorseuchhle ar groupe can be determined by
polarographic technique” The functional
"quantity of the substance can be deteriáned (rom
diffusion current measurement, Mostly it is the electroreducthle functhonal
groups which are determined by plarographic technique by
using BME. cg, fictional groups lke Niro & Niiroso groups. nzo À diana
compounds, aldehydes, ketoces, organ peranides, lactones, organic halogen
compounds, disulphides, actvued C=C, some acid and organo metalic
compounds.
Multi stage reduction of groups Mike Nitro Lo Nurose in Hydraxylasiie
to Amino group can alsa be achieved,
RIO + ONO e FONHOH 9 Roy
(ed 4d td

‘The following ts a schematic representation of reduction reaction
H+ nt + ne + RH,
eh COCHO + Zr 920 -+ CoHsCHOM
The following table gives cumples of 24 of some compounds

[compound / Fasctional group eM

evaaldetyde “Lav
AS Ar

so peep pent hetone ET =
[tcburyt ping Arter AN

lcartory! group |BEITERTTT TH,
JAldehydes & Ketones 130 1e LOW

aro comprando ETE |

11-10

Aldehydes und ketones
‘Varin

p-Benoquinone

Vitamin K and its derivatives
‘steroids:

Aso and lazo compounds

‘Ketorolac Tromethamine

AE

12, AMPEROMETRIC TITRATIONS

a Introduction
© Principle
#7 Conditions for performing amperometric Utralion.
7 Apparatus for amperometric titration
© Rotating Platinum Micro Electrode
ax Types of amperometric titrations
% Trate la electroreducible
de Titramt ts electroreducible
de Both Utrate and titrant are electroreducible
& ecke titrations
% Dead stop end point technique
mr Advantages. of ampercmetste trations
#7 Applications of amperometric titrations

wi

ln the following curve, lead tons (titrate ts electroredurisie 14 Utraied
Va sulphate tons (Utrant is non-redueibie). Diffusion current la cbaerved
due to lead only. The first part of the curve Lan Wu
Abows a decrease in current. due to decrense na

|
i
4
E
$

ad
does not cause any change ln callos ne

4. Bither the utrate or titrant ef both should be electroredueible
2 The potential applied should correspond to the Mmiting current.

APPARATUS FOR AMPEROMETRIC TITRATION
‘The instrumental requirements for carryirg eut ameperonmetric Utratson

or RPME (Rotating platinum micro electrode), —
‘The construction and working of dropping
mercury electrode has been discussed in CES

previous chapter. Hence the construction and

working of rotating platinum electrode wil be
dincummed here.

ROTATING PLATINUM MICRO
ELECTRODE (RPME}

Rotating plate electrode consists of a
glass rod with a bent platiowm wire at its
Up and rotates at about 6Otkpen, Wire contacta

123

> .
3. Titration of electro reducible jon Vs electro reducible Ion
lead (Pb) Vs Dichromate ions (Cr207 2)

4. Redox titrations where oxidant and reduelant give diffusion current
og Ferric tons (Fe) Vs Titanoub lons FT

5. Dead stop end point technique: Determination of water using Kart
Fischer reagent

in solution. This is due to precipitation as
124

not contribute lo diffusion current. After

a
the end point, the addition of allver tons ee

Vat ag

by the difference in the slope of the diffusion eo-eificiemt of ferric and
Ulanous ions.
6. Dead stop end polnt technique (Mamperoenetry)
‘The method is applicable when u Pond stop end point technique
redox system ls present before and after
he end point. An example ts Une titration
of water using Kast Fischer reagent. A
small polential Is applied between two
similar platinum electrodes. i §
when water is present both electrodes
are depolarised, The addition of Karl
Fischer A & B (Solution of lodine and
sulphur dioxide in pyridine and
methanol) is continued ull the end poet, i
‘where the diffusion current decreases. EA

AL the end point, only one electrode ts
depolarised and the diffusion current is ‘Vol. of Karl Fischer reagent

almost zero or nil.

ADVANTAGES OF AMPEROMETRIC TITRATIONS

L/Both dlectroreducible as weil as non-reducible lons or funcional
groups can be determined,

¡2 le solutions can be analysed.
SETHE resctona camted out can be reverie or treverible.
A/T apparatus ln imple and a polaropraph Is sufctent,

5. Electrode characteristics need not be known provided they are
constant throughout the Utration.

6, The range and the sensitivity of the technique are higher than that
ef potentiometric or conductometric technique. By using amperometric
detector tn HPLC (High performance liquid technique,
hat or ng/ml of the substances could be analysed.

7, incompletion of reaction at the end peint does not afleet the results
since few measurements before and after the end point are sullcient.

8 Temperature need pot be known provided it te Kept constant.

3, presen of puren or ober ect des ot let the esla

lin fact they act as supporting electrobytes

10, Non reducíble tons which cannot be determined by polarography can
be determined easily by choosing m titrant which is reducible. An
example 15 shown by the estimation of chloride Jana (non -reducsble)
using silver lors as tirant (eectroreductble).

APPLICATIONS OF AMFEROMETRIC TITRATIONS

1. Amperometrle titrations: They are quantitative in nature They are
used to determine the end peint of such trations. All the Eupen
ef titrations discussed cartier in this chapter can be done using
amperometric tration,

&-Betermination of water by using Karl Fishoer reagent,

3, Amperometria detector: In HPLC, amperometric detectors can detect
very low concentrations of eectroreducible tons, ng/ml or pg/ml
quantities can be easily determined.

fp Suamuscation of tone or mixture ol fons

127

CHROMATOGRAPHY

INTRODUCTION ,
Chromatography la the separation of a mixture into india |
components using a stationary phase and a mobile phase.

‘TPES OF CHROMATOGRAPHT
L Based upon the mature of stationary und mobile phase

There are different types of chromatography based on the type ep
stationary and mobile phase used. They are

1, Gas + Solid Chromatography

2, Gas = Liquid Chromatography

3. Solid - Liquid Chreenatography

IColuma chromatography, Thin layer chromatography,
HPLC (High performance liquid chromatography}

4. Liquid = Liquid Chromatography:
Et peor penny i
IL Based on the principle of separation
The principle of separation can be either adsorption or partition, Hence
hey can be called as adsorption chromatography or partition chromatography,

1. Adsorption chromatography |
When a mixture of compounds (adsorbate) dissolved in the mobile
‚phase (eluent) moves through a column of stationary phase (adsorbent),
they travel according to the relative affinities towards stationary phate.
The compound which has more affinity towards stationary phase travels

Examples where sdserption la the principle of separation
Gas-Solid chromalograpkıy, Thin layer chromatography, Column
cbromatogagty and HOLE OU proue igual cnica:

2, Partition chromatography

‘When two lamiscible liquids are present, a mixture of solutes will
be distributed according to their partition co-efficients, When a mixture of
compounds are dissolved in the mobile phase and passed through a column
el liquid stationary phase. the component which la more soluble in the
stationary phase travels slower. The component which 1a more setuble In

|
E
i
E
?
i
i
i

la effective and thus solute mixture is separated into individual components,

Examples where partition la the principle of separation
Parution Chromatography, ete,
IL. Based en the modes of chromatography
There are two types, They are based upon the polarity ef the stationary
phase and mobile phase used. (Piense also refer page 18-3 and 18-4)
1. Normal Phase chromatography: ln this the stationary phase ts Polar
and mobile phase Is Mon-poler, This ts not widely used in Pharmacy.

2 Reverse phase chromatography: In this, stationary phase ts
Mompolar and mobile phase is Polar. This la most widely used in
Pharmaceutical analysis

—
Polar ‚Non-polar

== = =

(Compound eluted first and retained tess) Noo-polar Polar

‘Chiral chromatography: ln this (ype of chromatography, optical tsomers
Devo and dexiro form) can be separated by using chiral stationary phases.

‘The following chapters wall deal with different types of chromatography
in detail,

133

13. COLUMN CHROMATOGRAPHY

© Principle

% Stationary Phase

% Mobile Phase

% Cobumn charactertsties
‘Preparation of Column
À introduction of sample

# Development technique
# Detection of components

À Recovery of components
© Factors affecting column efficiency
© Applications
© Advantages of Column Chromatography
"7 Disadvantages of Column Chromatography
© Partition Coluttt Chromatography

INTRODUCTION

‘ectumn of stationary ts used, the technique ls calleg
nn en bie nature of talonary phase, Le,
Shahar tls solid or quid, It 1 called an column aidsorpticn elromalopraphy
or chine y Most of the discussions in this chapter

will be devoted to column adsorption , since column partition
Chromatography t+ nat being used widely.

A solid stationary phase and a liquid moblle phase is used and the
‘adsorption. When a mixture of components

components
The compound wrth lesser affinity towards the stationary phase (adsorbent) |

moves faster and hence it Is eluted out of the column first, The one with

renter Mit towards the stalonmry phase [adearbent) moves slower down
the column and bence it ls cluted tater. Thus the compounds are separated,

| The type of interaction between the stationary phase (adsorbent) and the
solute is reversible in nature. The re of movement of a component [RY

de ven as follows:
= jacana ld pe

This equation can be simplifed ax follows:
wi mowed by the solute

Distance
Dintance moved by ihe solvent
‘When a liquid mobile phase ts used, the equation ls written as

Ru —
An + SA

gone. in sutlonary phase
where a is the partition co-elficient = ne. mobile

Am ls the average cross section of mobile phase
Aa is the average cross section of stationary phase
|

pee 1

1. STATIONARY PHASE (ADSORMENTS)

‚An adsorbent used in column
aaa chromatography should meet the following

u Particle alse and geometry: The pares should have uniform atze
distribution and have spherical shape. Particle size: 60-200

b, Should have high mechanical stability.

€ Should be tert amd should met react with the solute or otter

4, Insoluble in the solvents or mobile phases used.
e. It should be colourless to facilite abserwation

À of zones and recovery
L lt should allow free Gow of mobile phase.
EM should be useful for separating for wide variety of compounds.
he Above all tt should be freely available, inexpensive. etc.

‘The mest commonly used adsorbent is Silica gel of 80-100 mesh or
100-200 mesh size which has a partido aise of 00-200.

Selection of stationary phase
‘The success of chromatography depends upon the proper selection af
stationary phase, The selection of stationary phase in column chromatography
depends cn the followeng:
L Removal of kmpusities: When a small quantity of impurity in present
and there is difference in any when compared to the major
component, a weak adsorbent Is mificient.

ik. No. of companents to be separated: When few components are to
be separated, weak adsorbent is used. When more components. are
to be separated, a strong adsorbent la selected.

úl AlMalty differences between components: When componente have
simitar affinities, a strong adsorbent will be effective. When there de
more difference in afficitlcs, a weak adsorbent ls selected.

iv. Length ol the columa used: Wien a shorier column is used, strong
adsorbent has lo be used, When a longer column ts used, a weak
adsarbent can be used.

v. Quantity of adsorbent used: 20 or 30 Uses Ihe weight of the
adsorbent ls used for effective separation.
‚Adscrbate : Adsorberst ratio = 1:20 or 1:30

13-7

2. MOBILE PHASE

Mobile phase ts very important. ad
set u sobe, dle an bn cana Te cea ice ay
ae Sanctions of u cota plana
To introduce the misture into the colore - As solvent
To develop the roves tee separation - An dí

‘Te remove pure component out of the column + As eluent

, 3 Alcobala
(Methanol, Ethanol. ee]. Water, Pyridine, Organic acids (Ncctc
Mixture of acids or Bases with ethanol or pyridine etc. hs

‘These solvente can be used in ciber pure fer or as mixture of
solvents of varying. compositions,

3. COLUMN CHARACTERISTICS

‘The material of (be column is mostly
good quality neutral glass since it should not
be allected by solvents, acids or alkalies. An
eedinary bureile can also be used as column
fer separation. The column dimensions are
Important for effective column separations, The
length : diameter ratio ranges from 10:1 to
20:1. For more elfietency, the berg: diameter
ratio can be 100:1, The length of the column
depends. upon ie

+ Allinity of compounds towards the
adsorbent used,

+ Number of compounds to be separated.
+ Type of adsorbent used,
+ Quantity of the sample.

the solvent and the stationasy phase and the column may not be
undformiy packed. Cracks appear in the adsorbent present in the

low characteristics and clear band of
not be obtained.

component may

technique: This is the ideal technique. The required

quantity of the adsorbent ls mixed with the mobile phase solvent

in a beaker and poured into the column. The stationary phase setles

‘uniformly in the column and there ls no entrapment of air bubbles.
de

6. DEVELOPMENT TECHNIQUE (ELUTION)

Aller the introduction of the sample, by elution techniques, the
individual components are separated out from the column, The two techniques
ae

130

en techaugen zone lo means same or sa) In thie
PE la used throughout the proc eee coe of se
54, Chrom any, Petether: Bensene = 1.1 any, ee,
11 Oradient elution technique: (Gradient
technique, solve + gradually) ln this elution
aa of ‘gradually increasing polarity cr increasing
en re Wed during the proces of separation Inávally

v. Evaporation of the solvent and weighing the residue
ML By monitoring the fractions by thin layer chromatography
‘Any one of the above techniques can be used for detection of compounds

#9 that It can be used Sor qualitative analysis and for Isolation of compounds.
$. RECOVERY OF COMPONENTS

Earlier, recovery of the components were done by cutting the column

Into several distinct zones. Later, extrusion of the column into sones were

13-10

by à process the
solvent called as eluent and the process of removing the componenyy
trem the column 1 called as elution. The different elulon techniques gg
isceratie elution technique

detect
earlier, Similar fractions are mixed 50
each type is obtained in a pure form, If
components, It can be resolved by using another column.

ASFECTING COLUMN EFFICIENCY

For any separation, efficiency of the column ls important. Unless the
factors alfecting the column efficiency are known, efficiency cannot be
Improved. They are:

ae can be reduced and hence the adsorbent activity increases.

Bl, Mature of solvent: The flow rate of solvent ls affected by its viscostty, |

‘The flow rate ls inversely to viscosity, Hence less viscous
solvents are better efficient than more viscous solvents,

lv, Temperature of the colma: Speed of elution is increased at higher
temperature. But adsorbent power ts decreased at higher temperatures.
Hence à compromise is made between apeed of elution and adsorbent
power.Normally room temperature ls used for all samples. Difficult
samples are separated at higher temperatures.

v, Pressure; High pressure above the column and low pressure below |

the column increases the efficiency of separation. High pressure
above the column is achieved by maintaining a column of liquid on
the top of the column (reservoir) or by using pressure devices

4. Molation of metabolites from Mological Guide: eg. 17-ketostervide
trom urine, corto, other drugs te ren Biologen] Tide Ike blood
plasma or serum, ele.

5. Estimation of drugs in formulations or crode extracts

4. Determination of % w/w of stychrane in syrup of ferrous
with quinine and strychnine. 7 =

IL Determination of primary and secondary glycoside in digitalis leaf
li, Determination of phytomenadicne in injection and tablets,

lv. Determination of Flucinelene actonide or Detamethasone 17-
valerate in formulated produets.

v. Separation of geometrical lnomers: Cis and trans forms of ban
and crocetin dimethyl ether using, aluména.

M. Separation of diastereomers,
vil, Separation of tneeganic tons lke copper, cobalt . Nickel. etc.
vin, Separation of tautomers and racemates,

19-19

ADVANTAGES OF COLUMN CHROMATOGRAPHY 4
1. Any type of mixture can be separated by column chromatograpty,
2. pny quanl of he eure can be separate (ng 1 tof uba
3. Wider choice of mobile phase,

4 In preparative type, the sample can be separated and reused.
8. Automation la posable,

DISADVANTAGES OF COLUMN CHROMATOGRAPHY
1. Time consuming method,

2. More amount of solvents are required which nre expensive, 1
3. Automation makes the technique more complicated and expensive, |
PARTITION COLUMN CHROMATOGRAPHY

‘The technique is similar to column adsorption chromategraphy except À
that, the staonary phase ls liquid. A solid support lhe ailica gel or

used, since the

14. THIN LAYER CHROMATOGRAPHY (TLC)
#7 Introduction
er principle
© Advantages of TLC
er Practical requirements
Stationary Phases
Ginss plates
Preparation asd activation ef TLC plates
Application of sample
Development tack |
Mobile phase
Development technique - One dimensional development
- Two dimensional development
> Hortrontal development
= Multiple development
Detecting or visualising agents

ADVANTAGES OF TLC
1. Simple method and cost of the equipment is low,

2. Rapid technique and not time consuming like column chromatograpty.
3. Separation of ag of the substances can be achieved,

fl
|

of the parce sls since Ik ls not coved cola in panar
(ype having thin layer of

6. Detection la easy and not tedious,

7. Capacity of he thin layer can be altered, Hence analytical and
preparative separations can be made.

142

8, Corrosive spray reagents can be used without damaging the plates.

9. Needs leas solvent, ataienary phase and time for every separation
when compared lo column chrematepepty: dE

PRACTICAL REQUIREMENTS

1. Statlonary phases
2 Glass plates

3. Preparation and activation of TLC plates
4. Application of sample

5, Development tank

6. Mobile Phase

7. Development technique

& Detecting or visunlising agente

1. STATIONARY PHASES

“There are several adsorbenta which can be used as stationary phases.
Some of the stulionary phases, their composition and the ratio in which

hey have to be mixed with water or other solvents to form a slurry for
preparing, thin layer chromatographic plates are given in the followirig table:

Microscopie slides can also be used for some applications luke monitoring
the progresa of a chemical reaction. The development ine ls much shorter
like 5 minutes.

Glass plates of diferent dimensions can also be used when the TLC
plates are prepared without the use of TLC spreader. In general, the gis
plates should be of ood quality and should withstand temperatures used

pouring, dipping, spraying and spreading,

In pouring technique. the slurry ls prepared and poured on to a
ass plate which is maintained on a levelled surface. The sluny ls spread
‘unfeemly on the surface of the glass plate. After selling, Use plates are
dried in an oven. The disadvantage is that uniformity in thickness eannot

Wig 14.1. TLC Sprenter

Activation of TLC plates ls nothing but
sd de dote es bee Ghia ER de
heating at high temperature so Uhal adsorbent activity in retained. The
setivated plates can be stored in thermostatically controlled oven oF In

desiecator and ean be used whenever required.

4. APPLICATION OF SAMPLE

Usually to get good spots, the concentration of the sample or standard
solution has to be minimum. 2-51 of a 1% solution of either standard
or test sample ls spotted using a capillary tube or micropipette. The spots
can be placed at randem or equédistant from each other by using a
template, with markings. The apots should be kept atleast 2em above the
base of the plate and the spotting area should ot be immersed in the
mobile phase tn the development tank. Auleast 4 spots can be spotled
conveniently on = quarter plate (Gem x fem).

5, DEVELOPMENT TANK

For the purpose of development, a developing tank (Fig 14.2) or
lumber of different sites to hold TLC plates of standard dimensions are
used, These require more solvents for developing. the chromatogram. When
a new method in developed, it ls better to develop tn ¡dass beakers,

specimen Jars, ete, 10 avoid more wastage of
ete e ander etd aed ele e
New of development tanks (Fig 14.3) have

Meet ees solvent: The development chamber or tank should be
inside: with filer paper molstened with the moble phase so an to satu
the atmosphere. If this kind of saturation of the atmesphere la rot
"edge effect” cecurs where the solvent front in the middle of TLC
moves faster than that of the edge. Therefore the spots are distorted
not regular (Fig 14,4),

BE

aid pe ew type

Fig 14.2. Developing tank

f{ 6 Matte Phase

1 ‘The solvent or the mobile phase:
used depends upon various factors
as mentioned din column
chromatography. Some of the factors
a

L Nature of the substances to
be separated

lik Mode of chromatography (Normal phase or reverse phase)
lv. Separation to be achieved - Analytical or preparative

Pure solvents or mixture of solvents are used, The following gives a

list of solvents (of increasing polarity)

, Petroleum ether, Carbon tetrachloride, Cyelohexane, Carbondisulfide.
Elber, Acetone, Benzene, Toluene, Ethyl acetate, Chloroform, Alcohols

i
Hf

141

Multiple development
One dimensional la this the
Pllc ars spa vesical ond ie ban ve appt grat, hoc

& collar aan. Mon separation done pra ie of ti pe
only,

Two dimenslonal technique: Although one dimensional technique
la sulficient for most samples, for complex mixtures two dimensional
technique is used. First, the plates are developed in one axis and
the plates afler drying are developed In the other axis. When large
umber of compounds cannot be separated by using one dimensional
Technique, this technique ts followed. Fig 14.5. explains the two
dimensional development of separation of mixture of several amino

i |

8. DETECTING OR VISUALISING AGENTS wie

Añer the development of TLC plates, tie spols should be visuadiseg |

‘Detecting coloured spots can be donc visually, But for detecting colo
spots, afıy one of the following techniques can be used.

a. Mon specific methods: Where the number of spots can be detecteg
bui not the eut nature or type of compound,

Faamples

À lodine chamber method: where brown or amber spots are
observed when the TLC plates are kept tn m tani with few aim M
coystals at the bottom.

à Sulphate weld spray reagent: 70 = 80% v/v al sulphurie ac
SA few mE el ether potassium dichromate or potasa 1
permanganate or few ml of nitric acid as axidising agent la used, M
‘This reagent after spraying on TLC plates is heated in an oven,
Black spots are seen due lo charring of compounds. 1

il. UV chamber for flourescent compounds: When compounds are à
viewed under UV chamber, at 284m [short A) oF at 365em (long M
A, Bourescent compounds can be detected. Bright spots are seen |
under a dark background. 8

lv. Using Mourescent stationnry phase: When the compounds are
not flourescent, a Bourescent stationary phase is used, When the
plates are viewed under UV chamber, dark spota are seen on a
Aourescent background. Example of such stationary phase ts
Silica gel GF

Specific methods: Specific spray reagents or datecting agents or
visualising agents are used to find out the nature of compounds
er far identification purposes, Examples are

Ferric chloride - for Phenotle compounds and tannins
Ninhydrin in acetone = for amino acids
Dragendrolls reagent - for alkaloids

35 - Dinltro bensote acid - for cardinc glycosides

. 24 + Dinttrophenyl hydrazine + for aldeliydes and ketones

ELLE

‘The detecting techniques can ks he ger an

L Dewtructive technique: eq, Speci
Spray reagent, ele where the uamples are ea See 964

QUALITATIVE ANALYSIS
‘The Ry value (Retantation factor) ls calculated for identifying the spots

Le. in Qualitative analysis, Ay value is the ratio of distance travelled!
by the solute to the distance travelled by the solvent front.

Re Distance travelled by solute _
Distance traveled by seven rem

‘The Ry value ranges from O to 1, Bust ideal values are from 03 to
04. Re value ts specific and constant for every compound in à particular
combination of stationary and mobile phase. Wien the Rr value ef a sample
and reference compound la same, the compornd 1 identified by its standard
When the Rf value differs, the compound may be diferent from Ws reference
standard

Ry value

Re value ls nothing, but the rato of distance travelied by the sample
and Ihe distance travelled by the standard Re value is always closer to 1.
Ra values

Rim value ls used in qualitative analysis to Und out whether the
compounds belong to a hamelogous series. they belong, to a homologous

of

series, the ARa values are constant, The Afm values for a pair of adjacens
series Is determined by 7

member of a homologous series I] CARTES

Ez
Es
3
E

—
Direct method: (Using densitometer) The quantity of the india
spots can be determined by using densitametrie method. Denstlormetrie

technique is called a Int method und is described éarller tn detecting

APPLICATIONS OF TLC

The applications are wider and there ls no limitation to Use compounds
that can be analysed by TLC, Anyhow different types of applications are
sted below,

1. Separation of mixtures of drugs of chemical or biological origin, plant
extracts, eie.

2. Separation of carbohydrates, vitamins, antibiotics, proteins, alkaloids,
Bycontides, ete,

seid : Butyl alcohol [march indie reagent

14-10

1411

— ue

IGM PERFORMANCE THIN LATER CHROMATOGRAPHY (HPTLC)

HPTLC ls m sophisticated and automated form of TLC. The following
are features of HPTLC:
1. The use of precoated plates with stationary phase particle aire of
less than 104 in dlameter,

2. Wide cholee of stationary phases Uke Silica gel, for Normal phase
and C18, C8, etc. for Reverse phase mode.

3, Auto sampler instead of manual spotting and streaicing for preparative
purposes. 4

4. New type of development chambers which requires lesa amount of
solvents for

5. Mere efficiency because smaller and uniform sire of adsorbents.

& The use of UV/Vis/Muocescence Scanner which scans the entire
chromatogram qualitatively and quantitatively. The scanner ls an
advanced type af densitometer,

7. Improved Data processing capabilities by the use of computers,

Preparative TLC

‘The apparatus, principle, procedure and other requirements are similar
lo that of analytical TLC, The thickness of adsorbent layer used is 2mm
and a non-destructive detecting technique lke UV or lodine chamber method
la used. The spots are cluted with solvent after scrapping the distinct
spots. The solvent ls evaporated leaving behind pure component.

14-12

INTRODUCTION ‘ 002%, ash - 0.01 - 0.07

Nos. No, Nod, No, Pv an er augen of dillerent grade bee

Na ete
chromatography ts defined as Use technique it which the anally Aer la sizes, shapes, Perenlien und wre used. These papers
ef unkown tanos I cared cut many ty the flow f seni cn = Mi Parcs.
specially designed filter paper. There are two {ype Paper chromatography, Choice of (ler paper
Dom purty, cc er PONS upon tea, Dow rue
paper adsorption chromatography: in which paper Impregnated with «7 mous
sien où ina acta ns adsorbent (stationary phase) and solver as tab te a PAPE Aci or base washed Mer paper. ase
phase. type paper,
Paper partition chromategrapby: in which motsture/water pres ‘© Hydrophilic papers - Papers
in the pores of cellulose Mibrea present In fer paper acts as stationary De = O

phase and another mobile phase Is used as solvent,
1 sr Hydrophoble papers - Areiylatien of OM groups leads to

in general, Paper chromatography refers lo paper partition h
i tame asa semis oe based: on partion Upa rer mature’ bees can be uned for reese phase
only. lography. Silicone pretreatment and organic non-polar
(polymers can also can be impregnated to give reverse phase
PRINCIPLE OF SEPARATION ‘chromatographic mode.
‘The principle of separation ts mainly partition rather than adsorpuice, sr impregnation of
Cellulose layers in filter paper contains malsture which acts as stationary re tate aa ll mm red
phase, Organic solvents or buffers are used as mobile phases. Instend of
water 3 née solvents
se mir ps, eter rw can be used by suitable, ws af Le popa nd ae fay ae cn e u
Paper should be kept in a chamber of suitable sine.
PRACTICAL REQUIREMENTS.
2. APPLICATION OF SAMPLE
1. Stationary phase and Papers used
une The sample to be applied ts dissolved in the mobile phase and applied
2 Application of sample 3 using capillary tube or using micropipette. Very low concentration la used
o voi larger zone.
3, Mobile Phase
3, MOBILE PHASE
$ Pene ee Pure solvents, buffer solutions, er mixture of solenta are used. Some
GB Detecting or visunlising agents al the examples of Mydrephille mobile phases:
1. STATIONARY PHASE AND PAPERS USED Isopropanal : Ammonia : Water - 212
n-Butanol: glacial acetic at: water - 4:1:5
Paper of chromatographic grade consists of a-ceifulose - 98-20%. Methanal : water + SL or del
Preellulose 03-19, pentosans - 0.4-0.8%, ether soluble matter - 0.015 - t-Butanel ; water : Formic acid - 40205

ET 153

Examples of Hydropbobie mobile phases
Kermsene : 70% lsopropanol

ether: cyclohexane
aries phase or tires phase sent systems are aso used

|
|
|

w

HH
I HH!
ot

i
seis
Í

5
i
i

il
y
U
x |
Hi
il

I
!

ih
i
N

but not the exact nature ar type of compound.

Examples

L lodine chamber method: where brown or amber spots are
‘observed when the TLC plates are kept in a tank with few loctine
erystals at the bottom.

li UV chamber for flourescent compounds: When compounds are
viewed under UV chamber, at 254nm (short A) or al 268nm Hang
N. fourescent compounds can be detected. Bright spots are seen
against a dark background.

165

a. Bünde in acetone = for amino acids
Drager agent fr alkalis
Ww. 35 - Dintiro benzole acid - for cardiac glycosides
¥ 24 - Dinätrophenyl hydrarine - for aldehydes and ketones
‘The detecting techniques can also be categorised as

L Destructive technique: of, Specific spray reagents, etc where the
samples are destroyed before detection. eg. Ninhydrin reagent,

IL Mon-Destructivo technique: Like UV chamber method, lodine chamber

For radicactive materials, detection is by using autoradiography or
Geiger muller counter.

For antbiatics, the chromatogram ts layed on nutrient agar inoculated
with appropriate strain and the zone ef inhibition ts compared.

QUANTITATIVE ANALYSIS: (Direct and Indirect techaiques)

‚et technique: Denzel en. firmen: shih meses
the density of the spots, When the optical density of the
spots forthe standard and test solution are determined, Une quantity of
the substance can be calculated. The papers are neither destroyed nor
tuted with solvents to get the compounds, This method is also called as
Inaitu method.

Indirect technique: In this technique, the spots are cut into portions
and eluted with solvents This solution can be analysed by any conventional
techniques of analysts like spectrophotometry, electrochemical methods, etc.

Re value ts nothing but the ratio of distance travelled by the sample
an the distance trae by mandat Re malos la always dover 1 1

Fm values

Ras value 15 used tn qualitative analysis to And out whether the
compounds belong Lo a homelogous series. if they belong to a homologous
series, the ARm values are constant The AR values for a. a, pat facet
member of a homologous series la determined by using the formula:

sed
APPLICATIONS
linia yo cee ee ee
that can be analysed by paper chromatography.

Paper chromatography la
tore Use for Che matron‘ por cas Ike ie as sugars,
natural products, etc, The diferent types of applications are listed below.

1. Separation of mitetuires of drags of chemical or biological organ. plat
extracts, ete

N

Column material und dimensions
Type of lon exchange resin and Physical chameteriaties

| Mobile phase Detecting agent ] a
M acetato; ete derriere, 2
| a a a ee Deon! a be demsgun ent etn
cule
[Esgotanine tection \Cnlaruforı ; methanol {dace elt Oy + ‘Regeneration of the be exchange resin
(Doman ; meihanol : water UV 366 © Factors affecting
2 BHA. TOL Soe eae
‘Nature and Properties of ton exchange resina
6. Identification of foreign substances in drugs a Pe;
7. Wentifieation of decomposition products Valeney of teas
& Analysts of metabolites of drugs in blood, urine ete, rien)

Concentration of setution
Concentration and charge of tons

"© Applications.

cs. 161

stenlar dons present in cation exchange resin, a Solid matrix. The exchange
can be represented by the following equation:

M + Sol Mt + HY
(Solution)

The cations retained by the solid matrix of fon exchange resin can
be cluted by using buffers of different strength and hence separation of
cations can be effected.

Aalon exchange

‘Similarly, separation of anions using anion exchange resin can be

sch

The exchange can be represented by the following equation:

Sold - OH + A7 + Solid - A + OH

(Solution)

|. CLASSIFICATION OF Resins

According Lo the source they can be clasificó ns
Natural: Cation - Zeolytes, clay, ete
Anion = Dolomite
Syathetle: Inorganic and Organe resina

Of the above types, organic resins are the most widely
hence they will be discussed in detail Eee

Organic lan exchange resins are polymeric resin matrix containing
exchange sites, The resin Is composed of Polystyrene and Divinyl benzene.
Polystyrene contains sites for exchangeable functional groups. Divinyl
benzene cin an cross linking agent and offers adequate strength Le,
mechanical stability.

‘Structure of Styrene and Diviayl benzeno

66

runeticaal groupe present In different Jon exchange resins
Strong Catlon exchange resin + SOA
‘weak entlon exchange resin + COOH, OH, SH, POata
Strong anion exchange resin - N'Rs No
Weak anion exchange resin = NHR, NHa

‘The following tabular column ges list of the fon exchange resina
the pH range to be used, nature and applications of the resins In durent

‘separations.

(Clas of resim
[Catton - Strong

(cation + Weal

Structural types of lon exchange resins
a Pellicular type with fon exchange fm: The particles have a se
of 30 - 40, with 1 = 2p film thickness, These have very low exchange
capacity lo separate the tans. Their lon exchange eflictency le 0.01

- 0.1 meq/g of ton exchange resin.

b. Porous resta eoated with exchanger beads: The size ranges from
5 + 104 They are totally porous and highly efficient. Their exchange
capacities are from 0.5 - 2 meq/g of ton exchange resin.

separation of ions of different stzes is difficult-as Ubey Cannot pau
through the pores present.

linking agent ls present, Uhey are less rigid,

Wino fos cree 4 ell rit De
as exchange af functional groups does not take place due to wie
pore. Hence an optimum quantity of cross linking agent should be
added to the polymeric ton exchange resin for the separation ta je

att at ay
a
$] a; 4

I= Oy = CC

nak sade
wir a

|, Column material and dimensions: Columns used in the laboratories
are made up of glass. But those used in Industries are made up
of either high quality stainless steel or polymers which are resistant
to strong acids and alkalis. The columa dimensions are also important

and a length: diameter ratio af 20:3 to 100: 1 for higher efficiency
can be used.

166

|
i
I
3
i
i
-
}

lesa useful and only acids, alkalis and buffers of different pH are
used. There are two elution techniques. They are isocratic elution
and gradient elution technique. In tsoeratic clution

sxchanigenble
functional groups are ost. But due to the cost of the lor exchange
resins, they cannot be disposed off, Herice like
regeneration of the resin is most important. Regeneration makes the
used don exchange resin to be as efficient ns a virgin resta.

Regeneration relers to the replacement of the exchangeable catians

strong acid like hydrochloric acid. Regeneration of anion exchange
resto ts done by using streng alkall like sodium hydroxide or
potassium hydraside.

The factors alfecting lon exchange separations are
A Nature and propertles of lon exchange resins
B Nature of exchanging tons

A. Nature of lon exchange resin: Crosalinking and mvelling is important
factor which depends on the proportion of cross linking agent (divinyl
bensene) and When more cross linking agent is present,
they are more right. but swells less. When swelling ls less, separation
of loma of different ares da dificult as they cannot pass through
the pores present and it becomes selective to tans of different seres.

dl. Size of lows: For alar changed
decrease in the ate of Ita ion” di

Ut < HY < Nat < NH € nt € Rh < Cut

tv. Concentration of setutlon: In dilute solution, polyvalent anions
are generally ndsorbed preferentially,

y. Concentration and charge of loma: If resin has higher eve
change and solution has lower eve charge. exchange ls faveured
at higher concentration. Wf the reain has lower sve charge and
solution has high +ve charge. then exchange ts favoured at low
concentration.

APPLICATIONS
1. Softening of water: Removal of monovalent and divalent sons ike
sodium, potassium, calcium, magnesium, ete.
2. Demineralisation ar delonisatica of water: Removal of different
fons to get demineralised water,

3. Purtfeation of some solutions to be free from tere impurities.
4. Separation of inorganic loss Cations and anions.

8, Ion exchange column in HPLC: For separation of compounds of
mixed nature lke acidic and basic substances. on exchange column
ls used tn HPLC [High performance Liquid chromatography),

© Dertratssation of sample - Precolumn & Post column
#7 Pretreatment of solid support
@ Parameters used In OC

% Retention lime, Retention volume, Separation factor
Resolution, Theoretical plate, Efficiency,

Asymmetry factor
só Applications of Gas Chromatography

a substance distributed between two tmmniscible Isquids at a constant
temperature)

CRITERIA FOR COMPOUNDS TO BE ANALYSED BY
GAS CHROMATOGRAPHY

‘Two Important crtterta are

1. ‘Volatility: Unless a compound ls volatile, It cannot be mixed with
mobile phase. Hence volatility is important.

2. Thermostablilty: All the compounds will mot be in the form of
vapour, There will be solid as well as liquid samples, Hence to
convert them to a vapour form, they have to be heated to a higher
temperature. AL that temperature. the compounds have to be
thermostable. If they are not thermostable, the compounds cannot
be analjed by Gas chromatography, since they will be decomposed.

The chales of carrier gas determines the efficiency of chromatographic
separation, Most widely used camer gases are Hydrogen, Hettum, Nitrogen
and Argon.

Hydrogen: IL has better thermal conduetrty, low density, Ii is useful
and flame fontsation detector, The

Hellum: lt also has extellent thermal conductivity. but it la expenabve,
lia a good carrier gas when used wich thermal conductivity detector.

Nitrogen: It is inexpensive but has reduced sensitivity:
173

As carrier gus ts compressible, gases are stored under high
in oylinders and used when required. er

2. FLOW REGULATORS AND FLOW METERS

As carrier gases are stored under high pressure, flow regulators
used to deliver the gas with uniform pressure or flow rate. m

H ía placed conveniently before the column infet. It has
an ‘ass tube (like burette) with a Moat held on to a spring. The
level of the Dont in determined by the flow rate of carrier gas and is

3. BUECTION DEVICES

‘Samples for Introducing into ‘
er gas. quid OF wald A nature” UB CA be OÙ any type Le,

ases can be Introduce into the column ty waive

A Depending om Its use

L Analytical column: Analytical

metres and an outer diameter of 3-6 mm, They are packed columns

and are made up of glass or stainless steel. Only small quantity of
samples can be loaded on to the column.

ll Preparative column: Preparative columns are larger when compared

to analytical columns since large amount of sample has to be loaded.

‘They have a length of 3-6 metres and outside diameter of 6 + Bam.

174

uses some property by which tt can detect the difference between m pure
carrier gas and à eluted component.

‘The requirements of an Ideal detector are:

L Applicability to wide range el samples,

il. High Sensiuvity to even small concentrations,

i, Rapidity of response.

lr. Linearity: Le. less response to low concentration and proportional
response to high concentration,

y, Response should be umnaflected by temperature, flow rate ce
charnctertstics of carrier gases.

vi Non destructive to the sample in case of preparative work.
‘vik Simple and easy lo maintain
‘ili, Inexpensive,
The different detectors used commonly are
) a Katharometer or Thermal Conductivity Detector (TCD)
b. Flame lonisation Detector (FID)

© Aygen lonisation Detector (ID)
d. Electron Capture Detector (ECD) =

CLIP

ome, 17 os

ASSESS DA

el the column passes. The two platinum wires are heated electrically and
pence assume equilibrium conditions of temperature and electrical resistance.
‘When pure carrier gas passes through both of them, there ts mo difference
in temperalure or reststance and hence baseline ls recorded, When a
component emerges from the column, It alters the thermal conductivity
and resistance of the wire. Hence this produces a difference in resistance
and so conductivity between two wires, which is amplified and recorded
as à signal.

‘The thermal conductivities of some carrier gases are ¡ven ns follows:

rise to negative peaks Because of lower thermal conductivity,
‘Advantages of Katharometer

1. Applicable to most compounds,

WL Linearity la good,

A ‘The sample la not destroyed & hence used In preparative scale,

iv. Simple, easy to maintain and inexpensive.
‘Disadvantages of Katharometer

1. Low sensitivity.

il. Affected by Muctuations in temperature and Bow rate.

ML The response ls only relative and not absolute.

te. Biological samples cannot be analysed.

1, This detector Is extremely sensitive and background noise is low,
Mence mg quantiles of the solute can be detected.

2. Stable and Insenaitive to posall changes tn the flow rate of carrier
gas and water vapour,

3 Responds to most of the organie eemnpounds.

4, Linearny is excellent.

€. Argon lonisation detector (AID)

‘Tuts type of detectas depends on the exitation of atoms 10 à
inestable site by using radoat energy. Ths a che by trading
the carrier gas with tither à particles or fi particles. a particles can be
‘sbiained form radium-D, f particles can be obtained fron % Sr of tritium,

Arratia a
ES

A
fe «GA
Advantages
1. Responds to moat of the organic compounds.
2. Sensitivity is very high.
Disadvantages
1, Response ls mot absolute and it is relative,
2. Lincarity la poor.

3, Sensitivity 1a affected by water and ts much reduced for halogenated
compounds.

4. The response varies with the temperature of the detector and for
high temperatures Like 240°C, voltages of 1000V or less are usually
necessary,

4, Electron Capture Detector (ECD)

The electron capture detector (Fig 17.6) has
two electrodos, with the column effluent passing
between them. One of the electrode la treated =
with a radionctive Isotope which emits electrons
As it decays. These emitted electrons,
secondary electrons which are collected by
anode, when a potential of 20V ls applied:
them. When carrler gas alone flows
all Use secondary electrons are col

Gite

he ponitively polarised electrode. Hence a steady baseline Is recorded.
Efftuent molecules which have afMalty for electrons, capture these electrons
hen they pass through the electrodes. Hence the amount of steady state

‘ference ts ampliled and recorded as output

‘This
The carrier gas used in this type of detectar depends upon the electron
aflaity of the compounds analysed. For compounds with high electron
alfinity, argon is used as carrier gas. For compounds of low electron
a Se RS Cm a
ss.

‘The advantage of this type of detector ls that it ls highly sensitive,

DERIVATISATION OF SAMPLE
Dertvatisation is a technique of treatment of the sample to improve
by

4. To improve separation factor.

Example: Carboxylic acids, sugars, Phenols, alcohols, etc can be
converted to fess polar compounds by using reagents Mike BSA reagent
(Bis trtincthyl SUyl Acetamide reagent.

la increased.
Bowrate is netiher stopped nor altered.

PRETREATMENT OF SOLID SUPPORT

‘Solid support is used to hold the stationary phase liquid as a thin
flm. But sometimes due to some defects, un¥ormity and stabil

flim of liquid stationary phase may not exist. In auch eases, taling of
peaks and low separation elllelency can be observed. Therelore to overcome
Such demerits, His best to do pretreatment of the stationary phase.

Sey ele en aonb es y
the following, techniques:
1. By using, more polar quid stationary phase.
2. Increasing the amount of liquid phase on the support.
3. By selecting a less active support.
4. Pretreatment of the solid support to remove active sites,
Example: By using hexamethy! disilazone or dimeday! allyl dichloride,

‘This converts the hydremy groups (polar) to dimethy! group (non-
polar) and hence the active adsorpuon sites are deactivated,

xx

Retention time (Ry
Retention time ls the difference in time

Bh Kg roped fo BO ou component Ll
to be cuted from a column. Retention
teamed la mimos ur wem: Bas
time la also proportional to the distance
moved on a chart paper, which can be
measured in em of men.

aie

Reteatlon volume (Vi)
Retention volume is the volume of carrier gas required to elute 50%
oe compe fem he conn A de peda ne
rate,

[Retention volume = Retention time = flow rate

Separation factor (5)

Separation factor is the ratio of partition co-efficient of the two
components to be separated. It can be expressed and determined by using
the following equation:

gee.
KOR ey
where to Retention time of untetained substance
Ky, Ka = Partition coefficients of b and a

thy te = Retention time of substance b and a
8 = depende on liquid phase A column temperature

{f there in more difference in partition coefficient between two compounds,
the peaks are far apart and the separation factor la more, Wf the partition
ceefBctents of two compounds are similar, then the peaks are closer and the
rn

un e
ans separation cto Mere separation Fear
‘Resolution

Resolution is a measure of the extent of separation of two components
and Use baseline separation achieved. It can be determined by using the
‘following formula:

„Rt - Rt)

pen

‘Theoretical Plate (Plate theory)

‘A theoretical plate is an imaginary or hypathetical unit of a colima
where distribution of solute between stationary phase and mobile phase has
attained equihrium. A theoretical plate can also be called as a functional
‘unit of the coltama,

ETP - Height Equivalent to a Theoretical Plate

‘A theoretical plate can be ofany height, which decides the efficiency of

separation, If HETP la less, the column la more efficient. If METP is more,

the column in lesa efficient, HETP can be calculated by using the following
Jeng of te colores

o lesa platen

‘HET i gen bythe Vas Deseret

meras Ze

‘where A = Eddy diffusion term or multiple path diffusion
‘which arisen due 10 packing of the column. This
ia unaffected by carrier gas velocity or flow rate.
This can be minimised by uniformity in packing.

B = Longitudinal diffusion term or molecular diffusion
which depends on flow rate,

e = Effect of mass tranafer which depends on flow rate

u = Flow rate or velocity of the mobile phase,

|

ißeleney (No, of theoretical plates) ==
Efficiency of a column la expressed by
the number of theoretical plates, It can be
determined by using the formula: je
[1
neu
Der

an Wöscelen triangle, But In practice. due to some factors, the
Mol symmetrical and shows tailing or fronting ns shown in the following
Sparen.

Fronting ls due to saturation of stationary phase and cun be avolded
by using leas quantity of sample. ‘Falling is due to mare active adsorption

sites and can be etiminaled by support pretreatment, more polar mobile
phased increasing, the amount of quid phase.

Asyometry factor (0.95 to 1.05) can be calculated by using the
formula: AF = b/a fo and a calculated at 5% or 10% of the peak height)

APPLICATIONS OF GAS CHROMATOGRAPHY

1. Qualitative analysis: It ts nothing but Mentilleation of a compound,
This ts done by comparing the retention time of the sample as
well as the standard. Under identical conditions, the retention time
of the standard and the sample are same. If there ls a déviation
then they are not the same compound.

2. Checking the purity of a compound: By comparing the chromatogram
of the standard and that of the sample, the purity of the compound
can be reported, 1 additional peaks are obtained, impurities are

present and hence the compound la not pure. From the percentage
area of the peaks obtained. the percentage purity can also be
reported.

4. Quantitative analysis: The quantity of a component can be determined
by several methods ke

peak areas, the quantity of the sample can be determined,
‚Area of the peak = penk height x width of peak at the half height

TAR

Mor

fo 8
where A1 and As are peak area of sample and standard

Wy and Wa are weight or concentration of sample
and standard

a ls the response factor

hb, Callbration curve method

In calibration curve method, standards of varying concentrations are
used to determine their peak areas. A graph of peak aren Ve concentration
of the drug Is platted. From the peak area of the unknown sample, by
intrapolation. the concentration of the sample can be determined. This
method has the advantage that errors, if any are minimised.

e. Internal standard method

In this method, a compound with similar retention characteristics is
used. A known concentration of the Internal standard in added separately
lo the standard solution and sample solution whose concentration Is mat
known. The chromatogram is recorded and the peak area ratio of standard
and internal standard ts determined. By using the peak arca ratio of
sample and internal standard, the concentration of the unknown solution
la determined. This method ls useful when more extraction Mepn are
involved in sample preparation and the sample matrix ts complex.

5. Multscormponent analysis or Determination of mixture of draga:

Similar to the quantification of a single drug, multicomponent analysis
‘can also be done easily, The quantity of each component ts determined
‘by using any one of the above methods. Marketed formulations are
avallable which contain several drugs and each component can be
determined quantitatively.

6, Isolation and identification of drugs or metabolites in urine, plasma,
serum etc can be camied out.

7. lsolation and identification of mixture ef componente lke amino
acids, plant extracts, volatile als, etc.

1. Purtty of compounds
{Dre Column | Temperature | Internal Standard
|Atropane a ‘Homatre pene
pu |trydirotsreemice
Fenfluramene 20 | 200"
[tablero Lu
|Prenaytenetty! mr ‘sulphate
indice

Drug Purpose. Column |Temp|toternal Standard
littmlorstzenet |17-ethytestran-17p-ct henytmettt| 200" alcobal
tablets Inshente
(Cunelidine went [Porapak @ | 128° [etui alcohol

220° |4brome aniline HCI

100* [Propanol

18. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)

‘© Introduction
‘© Comparison of classical column chromatography with HPLC
‘Types of HPLC techniques
4 A. Based on modes of chromatography
Normal Phase and Reverse Phase mode

% B, Based on Principle of Separation
Adsarption, lon exchange. lan pairing, Sire exclusion
or Gel permeation, Afinity and Chiral Phase
# ©. Based on Elution tochnique
1. Isocratic separation
2. Gradient separation
# D. Based om the sale of operation
Analytical and Preparative HPLC
% E, Based on the type of analysis
Qunktattve and Quanttative analysis
Mr Instrumental requirements
de Pumps - Solvent delivery system
Mixing unit, gradient controller & solvent degassing
Injector + Manual or auto injectors (Rheodyne injector)
Guard column
Analytical columns
Detectors
UV, Refractive index, Flourimetric, Cooductivty,
Amperometric and PDA detector
de Recordera & Integrators:
se Parameters used in HPLC
se Applications of HPLC

‘The development of HPLC from classical column chromatography ean |
be ntirbuted to the development of smaller particle sizes, Smaller particle
nize is important since they offer more surface area aver the conventional
langer particle sise

1960% - 40 to 60,
19708 = 10 to 204
10608 - 510 10%
1990% 1 to u

A porous particle of Sy offers a murface nrca of 100-600 9q.metres/gram
with an average of 400m*/g, These offer very high plate counts upto
1,00,000/metre.

COMPARISON OF CLASSICAL COLUMN CHROMATOGRAPHY WITH HPLC

Classical Colusa MPL |
Chromatography
‘Stationary Phase > Large ‘Small ’
Particle mise On 3-20
Column sise Lange ‘Saal
length x totdiameter| Oe x Ob tom id | 550m x 1-10mm 14
Column material Glass, | Mostiy metal
Comm packing | ‘Slumy packed at Low | Slurry packed at Mi |
Pre pressure - Often gravity ne
Operating pressure | aw (6 20 pad High (500-3000 pas)
Plow rales | Low lo very tow Medium - High,
fee > Sok min)
182

HPLC TECHNIQUES
on Modes of Chromatography

There are two modes vte - Normal phase mode and Reverse phase
mode. These modes are based on the polarty of stationary and mobile
phase. Before explaining the modes, 1 la Important to know the interactions
‘which occur between solute, stationary and mobile phase,

Polar-Polar - interacuon or affinity is more
Nonpotar-Nonpolar - interaction or affinity is more
Polar-Nonpolar - interaction or affinity in leas

These hold good, whether the interaction is between solute and
stallonary phase or solute and mobile phase,

1. Normal phase mode: In normal phase mode, the stationary phase
leg. Silica gel) 1s polar ln nature and the mobile phase is nonpolar. in
ls technique, non-polar travel faster and are eluted first. This
M because of less affinity between solute and stationary phase, Polar
‘compounds are retained for longer time in the column because of more
afliity towards stationary phase and takes more time to be eluted from
the column, This im not advantageous In pharmaceutical applications since
most of the drug molecules are polar En nature and takes longer time to
be eluted and detected. Hence this technique ts not widely used tn pharmacy.

as a

The principle of separation ls ton exchange, which is reversible
exchange of functional groups. In ion exchange chromatography, an kon
exchange rosin ts used to separate a mixture of similar charged ons. Foe
catigos, m ention exchange resin is used, For anions, an anion exchange
resin is used. The principle of ion exchange separation, techniques and
factors affecting ion exchange separations es discussed tn the chapter of
exchange chromatography is applicable 10 ton exchange chromatography

‘Tetramethy! or Tetrnethy! ammonium hydroxide, ete.

4. Size exclusion or gel permeation chromatography

In this type of chromatography, a mixture of components with diferent
melecular sixes are separated by using gels. The gel used acts as molecular

rigid gels like polystyrene. alley!
used. The mechanism of separation is by sere and diffusion effects,

5. AMinity chromatography

Affinity chromatography uses the affinity
stationary phases. This technique is mostly used
Microbiology, Biochemistry, ete.

6. Chiral phase chromatography

‘Separation of optical tsomers can be done by using chiral stationary
phases. Different principles operate for different types of stationary phases
and for different samples, The malonmy phases used for tts
chromutography are mostly chemically bonded silien gel.

©. Based on elution technique

Isocratie separation: In this technique, the same mobile phase
combination la used Uiroughout the process of separation. The same pobartiy
OF elution strength ts maintained throughout the process.

Gradient separation: in this technique, a mobile phase combination
of lower polarity or ehition strength la used followed by gradually increasing
the polarity or elution strength.

se

es a

D, Based on the scale ef operation
Analgtical HPLC: where only analyse of the samples are done,
Recovery of the samples for reusing la normally not done, since (he sample
used ts very Sow, eg. jé quantities.
HPLC: where the individual fractions of pure compounds
can be cellected using fraction collector. The collected samples are reused,
eg. Separation of few grams of mixtures by HPLC.

E. Based on the type of analysis

(Qualitative mnalyale: which Is used to identify the compound, detect
the presence of Impurities. to And out the number al components, ete.
‘This la done by using retention time values.

(Quantitative omalyals: which ls done to determine the quantiry of
the ncividual ar several component in a mixture. This ln done by comparing
the peak area of the standard and sample.

Principle of separation in HPLC

‘The principle of separation in normal phase mode and reverse phase
mode is adsorption, When a mixture of components are introduced tn lo
a HPLC column, they travel according to their relative affinities towards
the stationary phase. The component which has more affinity towards the
Adserbent, travels. slower. The component which has less affinity towards
the viationury phase travels faster, Since no two components have the
same affinity towards the atlonary phase, te components are separated.

INSTRUMENTAL KEQUIREMENTS

1. Pumps - Solvent delivery system
2 Mixing unl gradient controller and solvent degassing
& Injector - Manual or auto injectors

4, Guard cola

5. Analytical columns

6. Detectors

7. Recorders abd integrators

‘The schematic diagram of HPLC ls given tn the following fgure:

1. Pump - Solvent delivery system

The salvents or mobile phases used must be passed through the
column al high pressure at about 1000 to 3000psi, This ts because as
the particle size of stationary phase in few p 15-10), the resistance to the
Now of solvent is high. Hence such high pressure is recommended. There
are different types of pumps available. They are mechanical pumps and
poeuimatic pumps. Mechanical pumps operate with constant Dow rate and
uses a sapphire piston, This type of pump ls used in analytical scale.
Prcumatle pumps operate with constant pressure and use highly compressed
gas. The solvents used must be of high purity, preferably HPLC grade and
Altered through 0.48. filter.

Check valves: These are present to control the flow rate of solvent
and back pressure.

Pulse dasmpoces: These are used to dampen the pulses observed (rom
the wary baseline caused by the pumps,

2. Mixing unit, gradient controller and solvent degassing

Mixing unit ds used to mix solvents tn different proportions and pass
through the column, There are twa types of mixing unils. They are low
pressure mixing chamber which uses helum for degassang solvents. High
pressure mixing chamber does not require helium for degassing salvents.
Mixing of solvents 14 done either with a static mixer which ts packed with
beada or a dynamic mixer which uses magnetic stirrer and operates under
‘igh pressure,

used for such separations,

Solvent degassing

Several gases are soluble in organic solvents. When solvents are
pumped under high pressure, gas bubbles are formed which will interfere
‘wih the separation process, steady baseline and the shape of the peak,
‘Hence degassing of solvent is important. This can be done by using any
one af the following technique,

a Vacuum Mtration - which can remove the alr bubbles, But it ia
‘tot always reliable and complete.

b. Helen purging - Le. by passing helium Uhrough the solvent. This
is very effective but Helium is expensive,

2. Septuma Injectors - for injecting the
‘This In mot commen, since the San ee tee es

Column material: The columns are made up of either stables steel,
ass, polyethylene and PEEX (Poly ether ether ketone). Most widely used
are stainless steel which can withstand high pressure. Latest ones ore
PEEK columns,

Columa length: Varies from Sem to 30cm

Column diameter: Ranges from Zmm to SO
Particle sise: From In to 206

Particle nature: Spherical, uniform sited, porous materials are used

Surface area: 1 gram of satioansy phase provides surface area ranging
from 100 » 860 sq.m with an average of 4008q.m.

Functional ; The functional group present in stationary phase
pends en the pe dl camion aparatos In normal phase mde
it contains the allanol groups (rydraxy group), In reverse phase mode it
contains the following, groups:

Cis - Octa Decgl Silane (ODSI column
Ca + Getyl column
Ca - Butyl column
CN + Kite column
ily - Arno column

For other modes of chromategraphy, lon exchange columna, gel columns,
chiral columns, affinity chromatographic columns, ete are available. A model
of column la given below,

E
6. Detectors

Detectors used dependa upon the property of the compounds to be
separated. Different detectors available are

A UVedetecter: ‘This detector in based upon the light absorption

warelength detector which can be opened fem Len to BOOM.

b. Retractive index detector: This ls a non specific or universal
detector, This ln not much used for analytical applications because
of low sensitivity and specificity,

© Wiourimetrie detector: This detector Is based en the flourescent
radiation emitted by some class of compounds, The exiation wavelength
‘and emission wavelength can be selected for each compound. This
detector has more specifiy and sensitivity, The disadvantage ls

(Geel come commends ara mes Oc

d Conductivity detector: Based upon electrical conductivity, the
response ls recorded. Thin detector is used when the sample has
conducting lons like anions and cations,

e. Amperometrie detector: This detector ls based on the reduction or

eluted. This is applicable when compounds have functional groups
which can be either cxidised or reduced, This is a highly senaltive
detector,

7, Recorders and integrators

Integrators provide on
days computers and primers are used for recording and processing the
Gbtalned data and for controlling several operations.

8. Parameters used In HPLC
Refer ali the parameters as given in Chapter on Gas liquid
hromalagraphy.

3, Presence of impurities: This can be seen by the presence of
additional peaks when compared with m Reference standard or
reference material, The percentage of imgurities may also be calculated
from peak areas.

4. Quantitative analyala: The quantity of n component can be determined
by several methods lke

By injecting a sample and standard separniely and comparing thelr
peak areas, the quantity of the sample can be determined.

Area of the peak = peak height x width of peak at the half height

Am
where At and Ag are peak area of sample and standard
Wi and Wa are weight or concentration ef sample
and standard

a is the response factor

tner

rag la plotie, Prom the peak area al the unknown sample, by nirapelaion,
the concentration of the sample can be determined, This method has the
advantage that errors, If any. are minimised.

e. Internal standard method

5. Multicomponent analyals or Determination of mixture of drugs:
Similar to the quantification of a single drug, multicomponent analysis
can also be done easily, The quantity of each component ts determined
hy using any one of the above methods, Marketed formulations

6. Isolation and Identification of drugs or metabolites In urine, plasma.
serum ete can be carried out,

7. Ielation and identification of mixture of components of natural
er synthetic origin.

& Biopharmaceuikcal and Pharmacokinetic studies.

Stability studies.

10, Purification of some compounds of natural or synthetic origin on
preparative scale.

‘Although an exhaustive Bst with hundreds or thousands of compounds
‘which are analysed by HPLC can be prepared, only few of the pharmacesticas
applications are deserved below:

RRP-18 - ODS ar Octn Decy! Silane er Cin colima (5-10p)

RES - Octyl or Ca column (5-104)
Normal Phase - Porous síica colina 05-104)

19. NON-AQUEOUS TITRATIONS INTRODUCTION... “04

#7 Introduction Non-nqueous Ulrations are those in which the ttration of weally
acidic or basic substances are carried out using non-aqueous solvents so
as to get sharp end potnt. Such Utrations can also be used for the titration

‘© ‘Types of Solvents
of substances mot soluble In water,

* Saly The role of solvents in making a substance strongly/weakly acidic or
apace strongly/weakdy basic can be well understood from the following example:
‘Amphipcotic Solvents HG tn water is strongly acidic, where as HCI tn acetic neid is weakly

neidic. Acetic acid in water is weakly acidic whereas acetic acid in ammonia

2 Interference due to water in Non-nqueous titrations ls stronly acidic. Thus the ncidic/basic character can be altered by the
use of appropriate solverst,

= la el air ani Before understanding the significance of the use of non-aqueous

aqueous titration polenta, some Important terms and concepts have to be understood. The

a ol wat bases with erctlodo. acid reactions which occur in these nan-aqueous solvents can be explained by

% Principle using Lowry + Bronsted 3 an Acid is a proton donor
Preparation of 0.1N HCIO4 di
‘Standardisatin of 0.1N HCIOS Examples are
Applications of Non-aqueous titrations to weak bases hen

Direct titration of 1°, 2° & 3° amines atea
Titration of halogen cid salts bases DS
a te
= Non-aqueous titration of weak acids with alkoxides 4 HNO => H° + NOS
‘TYPES OF SOLVENTS

% Preparation and Standardisation of 0.1N NaOMe
Preparation and Standantisation of 0,18 KOMe Solvents are classified according to their properties as follows:
Preparation and Standardisation of 0.1N LIOMe k te sob
Preparation and Standardisation of 0.1N t-butyt N huge ces
mens bydrande 3. Protophilie salvents

4. Amphiprotic solvents

1. Aprotic solvents:

These are solvents which are chemically inert and are nat involved
in any chemical reaction, They have low dielectric constant and do not
favour jonisation. eg, Chlorofarm and bensene.

y 191 192 =

% Applications of Non-aqueous titrations to weak acids

These sclvents are of two types. They are strongly basic or weakly
basic tn nature.

A strongly baste dent na. stronger tendency to accept proton. A
‘weakly basic solvent has weaker tendency to accept proton.

A atrongly baste solvent ls called as m levelling solvent (for weak
and strong acids), because it can abstract proton from any acid, whether
it ts strong acid or weak acid, Thin effect is called levelling effect. because
It does not dilferentinte between strong or weak acids.

On the other hand, a weakly base solvent has weak tendency to
accept proton. Hence this solvent ls called ns differentiating solvent,
because it can abstract proton from strongly acidic substances and not

from weak acids. This effect la called na differentiating effect. Differentiating
solvents are not useful in nonaqueous titrations,

Sienilasty, strongly acidle solvents are levelling sciventa for weak bases
and wrong, bases. Le, Strong acidic solvents can donate proton to strong

bases as well as weak bases equally weil. Hence strongly acidic sobvent
Mad DEEE aid los anita ald I weed e sb Thron. of wei

4. Amphiprotic solvents:

These have
e ag Wer, nee

Since water has both
wicks BO A pe

INTERFERENCE DUE TO WATER IN\ NON-AQUEOUS TITRATIONS

When a weakly basic drug ts present, water (OH) acts as stronger
base and preferentially accepts proton from an acid. Thus there ts interference
tm the reaction of weak base with an acid.

Similarly when a weakly acidic drug ía present, water (M') behaves
ie m strong acid and preferentially donates proton to the base. Thus
there in Interference in the reaction of weak acid with a base,

Hence in the presence of water, Utration of either weakly aekdie
substances with stronger base or weakly basic substances with stronger
acid ds not possible,

NON-AQUEOUS TITRATION OF WEAK BASES WITH PERCHLORIC ACID
Principle

Weak bases are dissolved tn acetic acid and are Utrated with acelous
perchloric acid, The various reactions which occur are given ns follows:

“Acetic acid alone behaves as a weak acid, because of poor dissociation
into H*

CHCOON === CHyCOO + H*

But when a strong acid (Perchloric acti) ls added to acetic acid, there
is formation of Onium fons, which has mare tendency to denate protons,

HOIO, => H° + Cou
CH,COOH + Ht ==> CH;COOH, *
{Ontum jan)
When weak bases lice pyridine are disscived in acetic actd,

amount of acetate ions are produced which have more tendency to
accept pretons.

Cli + CHyCOOH === OsJlgNH" + CHaCOO!
Ulumately, the titration of weakly baste drug acid

in acetic
Aceloua perchlorie acid yields necurate end poll The series of reactions
are given as follows:

|

i

HCIO4 + CHSCOOH 5 CH3COOH2* + ClO4”
(Ondum tons)

CHHsN + CHSCOOH + CsHsNH * + CHICOO"
(acetate ton)

CHyC0OH* + CHsCOO" -+ ZCHSCOOH

(Burette) (Cortical Flank)

‘The net reaction la given as
HCIO4 + CyHgN + CoHiNH* + CIO4

‘Tus we have seen that, on one hand the tendency of acid to
donate proton ls increased and on the other hand, the tendency of
base to accept proton is increased. These lead to the sharp
in on-aqueots tration

/ Preparation of 0.1N perchloric acid

Mic Anl of perchloric acid (72%) with SOO!
and imi of acetic anhydride, cool and add glacial acetic
1000. Alternatively, mix lll of perchloric acid (60%) wit

i
1

i
i
E

5)
E
E
T
!
i
tt
|

in nce acid}, Each ml of 0.1N perchloric acid = 0.02042g af potassium
Hydrogen
WI. of Potussoum Iydregen phthalate taken
Strengih of O.1N perehlorie acid = "Vor perchloric acid » 0.02042
204.24 of CaHs04K = IN 1000m MCIOu

0.02042g of CatiO4K = Iml O.IN HCIOS

OS

METHODS OF DETERMINING END POINT IN
NON-AQUEOUS TITRATIONS
1. Potentiometric method: The end paint is determined by using
indicator electrode (glass electrode) and reference electrode
calomel electrode). A graph of dE/dV is plotted and the end point
ls determined. The peak maximum corresponds to the end point.
secondary derivative curve can also be drawn lo detect the
and

a>

point.

2. Indicator method: Several indicators are available and are used

detect the end point. Most of them are prepared in acetke acid and
sometimes in dins or methanol
r | Indicator |
basic neutral actdle
valet (0.5 percent in| violet ‘lue-green | yellowinh- greets
acetic ach
Naphthalbensesn (0.2 percent] blue or orange dark green
[in glacial ncetic acid) biumgreen
[ae Boe 5 106 pere ‘ue purple pi
Bars} acetic acid) —
(Ouinaldine Red (0,1 percent in) magenta - almost

¡Can

Applications of mon-queous titrations to weak bases
1, Direet titration of primary, secondary and tertiary amines
2 Turatson of halogen acid salta of weak bases

1. Direct titration of primary, secondary and tertiary amines

For raw materials, weighed quantity of powder ts dissolved in gincial
ncetic acid and acetic anlıydrkde. Few drops of indientor solution is added
and the contents of the flask are tried against standardised ON

ln this method, weighed amount of the sample ls dässehved ln warm
daca acetie acid, mi of Sw/v mercure acetate in glacial acetic acd
la added followed by indicator The contents are titrated against ON
pereblerie acid.

Teethoride, lithium methoxide, tetrabutyl ammonium hydraude, etc tn
sente like toluene-methanol The principle ls similar to the tration of
wenk bases against perchiate acid.

Preparation of 0.1N Sodium methoxido

‘Add 2,54 of freshly cut sodium into 150ml of methyl alcohol previously
coed in Jos water, When sodium has been dissolved completely. add
sufficient toluene previously dried over sodium wire to make upto 1000ml.
Protect the container from molsture and carbon-dlecde,

‘Standardisation of 0.1N Sodium methoxide

0Ag of benzale acid PS. is dissolved In 80m! of dimethy! formamide.
add 3 drops of thymalpthalein and Utrate with ‘sodium methoxide solution
lo a blue end point. Perform a blank utration without bensale acid and
subtract the volume of sodium methoxide consumed by Semi of dimethyl
formamide Ench mi of 0.012218 of benanic acid = Iml of O.1N Sodium
methoxide.

Preparation of O.1N Potassium methoxide
AMG Ag of fresbly eut potassium, de at a time to a mixture of 40m
of methanol and 80ml of dry toluene. When potassium has been dissolved.
Add sufficlent methane! to produce m clear solution. Add toluene with
becomes

Standardisation of 0:1N Potassium methoxide
formamide and 3 drops of thymol

y lia mea iad E

at wath 0.1 ha
se faa an O dd at once 0.004, of bensole acid ang
am

Pipette out 1Oenl of dimethyl formamide and 3 drops of thymot blue
in a conical Mask and utrate with 0.18 Lithium methode until the acidic
impurities are neutralised. Add at once 0.06, of benzoic acid and tirate
‘with Lithium methoxide. Take care to see that motsture and aimospherie
carbon-di-axide ls not absorbed,

Preparation of 0.1N tetrabutyl ammonium bydroxide
40g of tetrabutyl ammonium icdide ts dissolved

by using nitrogen. Each ‚mine 0.0: tetrabuty! ammonium
ydraxide = 0.012215 of benzeke neid.

20. REDOK TITRATIONS

sr Principle
sr Cerrie ammonium sulphate (CAS)

# Preparation of 0.1N CAS
‘Standardisation of 0.1N CAS

sr indicators used in redox titrations (Redax indicators)

de Self indicators
External indicators
Internal indicators
instrumental technique

‘© Applications of redex titrations using CAS
= Applications of redex Utrations using CAS
= Titanous chloride
% Preparation & Standardisation of tilanous chloride

% Types of titrations
Direct & Back ttrations

Oxidised form + ne + Reduced form

‘The redox potential of such redox system can be measured by using
redox solution as one half cell and reference electrode as the other
‘Wien normal hydrogen electrode la used as reference electrode,

CelSOuh, 2INHalaS04. 2430, (63.268 In 100ml]. Weigh
66g of Cerric ammontum sulphate and dissolve in a mixture of 30m) of
sulphurte acid and SO0in! of water. Cool and (ter the solution, I necessary
and make upto 1000m! with water.

Standardisation of 0.1N cerrie ammonium sulphate
Method 1 (using Arsenle trioxide)

Weigh accurately about 0.2g of Amenic oxide previously dried at
105°C for ane hour, Transfer to 500ml conical Hank. Add 25mi of BW w/¥

y, External indicators
instead of adding Indicator to the titration solution Nach, few drops
of the solution la removed pertodially, placed on a tie and mixed with

‘ih potassium ferrkyanide. At the end paint. only ferric fons are present
Sei Hence it does nat give pruasian blue colour with ferteyanide solution.
This method is not used because external monitoring 19 mot accurate,

TY» N sree — Menta
= = CE
AS €
CE ie

‘An example of such indicator ls orthophenanthraline ferrous complex
or 1,10-Phenanthroline or ferroin solution. Thus form is bright red tn colour.
This complex ls cxidised wt the end point by Utrant to
ferric complex, whieh is pale blue In colour,

Redax potential of Fett / Fe - 0,77V

Redex potential of Celt / Ce + LASV

Redox potential of Fe? indicator / Fe™ indicator - 1.14¥
Ferrol indleatoe ls suitable in this titratlon because ite redox potential

LAW la ln between tat of tirant (1.45V) and Utrate (0.7V1-

|
|

By using potentiometer or conductometer, the end point can be

In conductometer, a plalinum electrode ts used. But the
tion of end point ls dificult as sharp results are not obtained.
‚use, the change in conductivity at the end point la leas because
lo be maintained in redox titrations, In

nil
E

12 Tocopheryl ncetate
TITANOUS CHLORIDE

‘TManous chloride ls a strong reducing agent. The principle of redox
trations apply to the titrations involving Atanous
indicator ta done ustng E? values (11 / T1 0.064). Tuanous chloride

used in the estimation of derric salta, azodyes, nitro and mitroso compounds
and quinones,
Preparation of 0.1N titanous chloride

‘Add 109m of titanous chloride solution to 100ml of hydrochloric acid,
dute to 1000ml with recently bolled and cocled water, and mix. Standardise,
immediately before use.

Standardisation of 0.1N titanous chloride

Place an occurntely measured volume of about $0cni of standardised
OAN ferrte ammonium sulphate in à flask and pass tn a rapid stream of
carbon-di-oxide until all the air has been removed, Add the Utanous chloride
solution from a buretie and in an atmosphere of carbon-dl-axide until near
the calculated end point, then add Sel of ammonium thiocyanate solution
and continue the titration until the solution ls colourless, Each ml of 0.1N
fente ammonium sulphate ls equivalent to 0.01543g of TICla.

‘Types of titrations

1, Direct titration: Ferric salta, azodyes and quinones can be estimated
dy this method. When coloured substances are titrated, sometimes Indiontorn
fire not necessary. (eg) In the titration of Methylene blue with 0.18 titanous

indicator Is not used. But the disappearance of blue colour ls
taken as end point.

Other substances estimated by direct titration are Indigscarmine and
Nitrandie weht. ” “

2. Back titration: A know excess volume of 0.1 ttanous chloride
in added and the excess ls back tried with ON ferric ammonium
sulphate using ammonium thiocyanate as indicator, This kind of back
tration ts done with compounds which are not easily reduced.
fe) Drugs with Nitro groupe and Nitroso compounds
‘Drugs like Chlorasmphenical,
E ph, Metronidale, Dyes like brillant

_ ro

|
\

21. DIAZOTISATION »TITRATIONG

Principle
# Preparation and Standardisation of 0.1M NaNO2 solution
=F Procedure for Diazotisation titrations
‘© Types of Diazotisation titrations
fr Direct titrations
fr Reverse method
# Special method
‘© Applications of Diazotisation titrations
% Direct titrations
% Conversion to amino group by chemical reactions

By reduction
By Hydrolysis

(point technique of determining end point can also Le carried out,

‘The principle can be explained with the help of titration of Benzocaine
An local anaestheti) by this technique.

The colour change of Indicator paper in because of the reaction:

M + HC) + KO + HI
HI + ZHNO + 12 + 2NO + BHO

PREPARATION AND STANDARDISATION GR: P.1M SODIUM NITRITE
SOLUTION

(6.9 g In 1000m) 7.5 y of sodium nitrite is dissolved tn suflicient
water to produce 10000) and Is standardised. About 0.54 of Sulphanilamide
pS, previously dried at 105°C for three hours ts transferred to a suitable
beaker, 50ml of water and 20ml of hydrochloric acid is added, stirred untl
il dissolves and cooled to 15%, The contents of the benker are tiimied

it 0.1M Sodium nilrite solution, Each ml of O.1M sodium nitrite

olution = 0.01722 € of Sulphandamsde.

PROCEDURE FOR DIAZOTISATION TITRATION

Auhough procedure for individual drugs are different, a general
procedure la presented in brief.

Speeified amount of the drug ls dissolved in about S0mi of water
and 20m! of hydrochloric acid. The solution ls stirred and cooled to about
15°C. The mixture ls titrated against O.1M Sodium nitrite solution. The
end point la determined by using any one of the techniques discussed
earlier, le, external indicator method using starch todide paper or by
‘ising electrometric technique by using platinum electrodes.

TYPES OF DIAZOTISATION TITRATIONS

a Direet titrations: Direct titrations are carried out by treating | mole
of the drug with 3 moles of acid solution. Ice can be used to lower
the temperature to about O - 8°C. 0.1M Sodium nitrite solution bs
added in small amounts and the titration Is curried out. The end
point bs determined ty any one of the techniques mentioned eurtier.

b, Reverse method: In this method, a solution of amine and sodium
nitrite are run imo a solution of acid. This method Is used when
the diaronfum salts are insoluble. eg. Naphthylamine sulphone acids
form insoluble diazontum salts due to the formation of zwitter tons,
Hence reverse method is used in such cases.

© Special method: Aminophenals are readily exdcised by nitrous acid
16 quinones. For such substances, the titration ts carried out in the

presence of copper sulphate which forms dinzo-oxide, These
diazp-enddes are more stable and undergo coupling reaction.

5 Procaine HCL

6. Sodiuin amino salicylate, tablets, granules

7. Suramin

8 All sulpha drugs containing free aromatic amino group le
Sodtum, » Sulphadaxine,

‘These drugs contain aromatse Nitro group, which can be reduced by
using reducing agent to get arcmatic amino group. This primary aromatic
fuming group can be diazotised by using nitrite solution.

bo Uydrotyais

1. Paracetamol (Acetyl dertvative)

2. Pithalyl sulphathiazcte (Fahaly dertvative)

Y Succinyl aulphathiaole (Suceinyl dertvative)

‘These drugs ace dervatives of amino groups lke acetyl or phihalyl
destvative. After

eee a neta, cba Lo free amino group, these drugs

Inocartozazid - Aci solution of ernten. benzyihydrasine

‘an be died to Die bento e

gs

i

22. COMPLEXOMETRIC TITRATIONS

«e Introduction
ar Werner's Co-ordination number
ee Complexing agents
‘© Structure of complexing agents
* Structure of complexes formed by EDTA with Polyvalent tons
#7 Stability of complex
© Need for maintenance of pH
sr Types of determining end point in Complexometric titrations
% Potentiometric method
Conduetometrie method
Amperometric method
Spectrophatometrie method
Using pM indicator
‘© pM indicators used in Complexametric titrations
= How does an indicator In Complexometric titration work?
© Types of Complexeretric Utrations
# Direct Titration
Back Titration
Replacement of one complex by another
Alkcalimetsie titration of metals.
Masking and Demasking agents

% Addition of preeipitants
Addition of complexing agents
pH Control

"© Preparation and Standardisation of 0.05M disodium edetate
© Applications of Complexometric Utrations

INTRODUCTION

Complexometsic titrations are those in which a complexing agent iy
used to estimate polyvalent tons (divalent, trivalent, ete).

A complexing agent is an electron donating ion ce molecule (gang)
capable of forming one or more covalent or co-ordinate bonds (dative bee)
‘woth metal tons. The complex thus formed has properties which are different
from that of the metal fon. I a single bond ls formed between a complexing
agent and metal, then it is called as a ligand. Such complexing agent ts
unidentete in nature and called an coordination compound.

IF a complexing agent can form more than one bond with polyvalent
fon, then it is considered as polydentate and called ax chelating agent.
1 die complex formed is soluble In water, then it 15 called an sequestering
agent.

WERNER'S CO-ORDINATION NUMBER (WEN)

Le ge ed 5 De
ha ‘This ds no way related to the valency of the
too, but related to the sterte factor or the space available for attachment.
Elements of the And period can aceeenmadate 4 groups, Jed period - 6
groups and Ach Period - 8 groups. A knowledge of WEN ls required to

now the maximum number of groups wh
A central don. can be accommodated around

COMPLEXING AGENTS

inne oe are af different type which depends upon the

3. Sequentering
Nena he poupe Ike COOH, SOSH, Mita & OH.

Dimethylglyoxtme Jaidoxime are examples for chelating agents,
er Bye Diamine Tet dese eld 1 an example (or

sequestering agent,
STRUCTURE OF COMPLEXING AGENTS

MOOO-CH za
N-CHy-CH-N,
1o0c-cH, Y Netty-cooit
Ethylenedtamine tetraacetic acid
N-OH
CHig-CNOH
pe as
Dimnethiylgiyoxime ‘Salleytaldaxime

EDTA la the most commonly used complexing agent or sequestering
UNS Le en cola
form complexes, Irrespective of the valency of the Jon, it forms 1:1 com;
wich fons, The number of rings formed depends upon the valency of the
‘an,

MÍ" [divalent jon} + [HO -+ 2H? + IMAI? (3 rings
M (trivalent fon) + [SXF + 2H" + IMJ (4 ringal
MP" (tetravalent lon) + IHaXI? + 2H? + [MO (5 rings)

STRUCTURE OF COMPLEXES FORMED BY EDTA WITH POLYVALENT 10NS

ae E

mone CH,
Cats beta

a

STAMILITY OF COMPLEX

stability constant el a metal lon complex la given by K for the
san M 4 X DO, where M isthe meal fon and X I the chelating
fon. The square brackels represent the activities of the tans.

ma
Stability constant 1K) = ¡ppp

he set of the complex decreased by acidic pH, increase in
temperature and due to the presence of efectrlyte having no lon In common
‘with the complex The stability of the complex lo increased by cian
due to the suppression of fonisation,

EDTA (Edetic acid) tonises tn four stages due to the presence of
COOH groups. Le, pKix20, pKa=2.67, pKonë,16 and pK4=10.25.
delle acid (EDTA) ts less soluble in water, all pharmacopoeias specify
use of disodium edetate which is mare soluble in water, in the
EDTA. in disodium edesale, two COOH groups are present. Since pl
10.26, complexes formed from metal lon and discdium edetate are
cely in alkaline solutions. The moat commonly used buffer is Ammonia -
‘Amenonium chicride solution which has a pH of 10.5 At this pH. the
complexes formed are more stable and hence sharp end point can be
obtained, (The greater the siabllity of the complex. larger is the change tn
er een, Rie ll pole ae Sane AEP

end pata

NEED FOR MAINTENANCE OF pit
pH maintenance is required for two reasons.

Fae

E

1. pH allects the stabeity as well as the formation of the complex with
‘metal ton.

2 Up to pH 10, pM increases with pl. The colour of the indicator-metal
ton complex depends upon the pM, which in tum depends on ph.
‘THFES OF DETERMINING END POINT IN COMPLEXOMETRIC TITRATIONS

1. Potentiometric method: Using ‘
Poesie method can be appled for determining the end pot
SE Titration of tron and copper can be done by this

i
E
i

pM ls the negative logarithm of metal ion concentration.
pM log IM
‘The value of pM can be derived from the equation for stability constant,

x Oe

IM.
then, ow = BR
or Tog) = tog PATE — tog

and ph © log gly wi

ben DO = 1400, Le when there le equal activity of metal lan ang 2, Xylenol orange: It has yellow colour in acid solution and red colour

complex ts present, pM = «pl or pM = pK in alkaline solution. The metal complexes are red in colour, I ls
is used tn the estimation of Aluminium hysroxide gel Abumintum
pat INDICATORS USED IN COMPLEXOMETRIC fulphate, Akiminium hydroude, Titanium diaxde and Zinc
‘The mest commonly used indicalora are: undecylenate.
Mcedant Ertochrome T or Solochrome Black 3. Murexide: (Ammontum purpurate] IL is used tn the estimation of
3 noi ENT, ig calcium in the presence of mngnestum, This is because magnesium
2 Xylenol orange - murexide complex ls less stable than calcium - muredée complex.
3. Morexide 5

4. Calcon mixture }

Structure el indicators
1. Mordant Black Ih: Free indicator is blue in colour (pH 10). On
complexation with metal lona, a pink colour is formed, Below pit
6.3 apd above 11.5, M ts reddish in colour. Hence it in used at
about ph 10. 1 hs used in the estimation of metal tons like Calcium,

Magnesium, Zinc, Cadmium, Manganese, Lead and Mercury.

4, Caicon mixture: It is Sodium 2-hydrexy-1-{2-hydrexy-1- napkıtlylazo)
It cannot be used either with enidising lores lke Ferrie, Cerrte and naphthalene-4-sulphonate, IL is otherwise called as Solochrome Dark
Vanadate tons or with reducing ona like Stannous and Titanous, It blue, Mt is used tn the assay of Caletum carbonate and Calcium

| DAS

Qe

Musa peto at pH 12)

chloride.
Nan
occasionally used for specific Utratiens or
| other applications, are Catechol violet, Methyl thymel blue, Alizarine
Prk ott 0) | Bourne blue, Sodium alizarín sulpbonate, Diphenyl carbazone, Tiron
i 1 2-dityydrosypbenyt-3,S-disutpbonate) and PAN [Pyridyl Azo

How does an Indicator in Complexometste Titration work?
In any Utration,
‘Stability of indiestor-Metal complex < Stability of Chelate-Metal complex,

IM the beginning of a titration, when Indicator ts added to metal tony
to be determined, the colour of the contents of the flank is that of the
dicatee-metal complex. At the end point, when all the metal tons have
been complexed by the chelating agent, free Indienter is Mberated, which
has a different colour. This polit only ls called as the end point.

TTPES OF COMPLEXOMETRIC TITRATIONS
1. Direct titration

2. Back titration

3. Replacement of one complex ty another
4, Alkallmetrie titration of metals

1. Direct titration

To & welghed amount of the substance to be estimated. measured
lume of sable bulfer schulen and few drops of indicator ts added.
The contents are tried agunst standardised disodium edetate solution
‘the end point altem by the colour change. A blank titration ls carried
eut omitting the substance to be determined, but contains all the other
élites like buffer & indicator. The volume ol cdetate consumed in blank
tration la subtracted from that obiadned In the original estimation,

‘Examples of such estimation:
Bismuth + Nitrate, carbonate, oxynitrate, subnitrate
Calehum - Chloride, gluconate. lnctate
Magnesium - Carbonate. oxide, stearate, sulphate, trisilicate
Zinc - Sulphate, oxide, undreenoate

2, Back titration
Back tration la necessary in the following cases:
- metals which are precipitated as hydroxides In the pH used
+ for insoluble substances like Lead sulphate, Calcium oxalate
- those react slowly with disodium acetate
+ those farm stable metal chelate complex than with indicator

3. Replacement of one complex by another

When sharp end polnt ls not obtained by direct or back titration,
this method is followed. In this method, the metal ton Is determined by
displacement of an equivalent amount of magnesiue ar zinc from less
sable edetate complex, The liberated ions are titrated against edetate,

Ma + Zax + MX? zo
Example: Cadmium, lead and mercury can be determined by this

4. Alkallmetrió titration of metals
Mnt + Ho > Mx Oe 2H

When a polyvalent ton in added to a complex, protons are liberated.
‘This acidity ls quantitatively titrated with an alkall but tn an unbuffered

solution. An acid-base Indicator itself ls sufetent, instead of pM ing

erauve polentlmetric method of determining end Point can aly gy

‘Masking and demasking agents are those which
Jus she, tn order to eskmaie a specie ion Me & Comp

‘Need for masking or demasking agents

L EDTA os well us its salt, disodium ede
sel mel los. During estimation af forms compleces with

Apte ao are cimas. Te gies false al ne ee

2 Sometimes when two or more
iad a des te site

estimated in a mixture,

2 Addition of complexing agents: Addition ef these complexing agents
causes formation of complexes with tnterfertng lons. These complexes
are more stable than the edetate complexes and thus eltmination of
tmpurtuies and selective titration can be done easily,

Int fons
unintusa, Iron, Titandum

lascorbie acid + Perrosyanide
Mercury, Cadmium, Zine, Arsenic,[Dimercaprol in alkaline medium
|antumony, Tin, Lead, Bisruth

jum Mouride

Mercury. (Potassium ledide
lAemintum. Tran [ren
(Aluminturm, tro

Silver, Copper. Mercury. Iron, Zune, Potaastum cyanide In alkaline

(Cadmium. Cobalt, Nickel

3. pH control: The edetale complexes with alkaline earth metals are
not stable below pH 7. But in this pH region and upto pH 3,
complexes with Tin (Sn), Iran (Pe), Cobalt (Co) and Thortum
(th) are stable and can be selectively titrated by varying pH.

Preparation of 0.05M disodium cdctate

Dissolve 18.6g of disodium ethylene diamine tetetracetate in. sulfictent
water lo produce [000ml and standardise the sobution.

Standardisation of 0,05M disodium edetate
Method 1

Weigh accurately 12514, of ealctum carbonate and transfer into a
450m! standard Mask. Add minimum quantity of dilute hydrochlorte acid
lo dissolve the calcium carbonate, by balling off the carbondioxide. Coal
and make upta volume with distilled water, Pipette out 20ml of this solution
la to a conical flask, add Sel of Ammania - Ammontum chloride buffer
and few drops of Caleon mixture as indicator (alternatively, Modernt Black
Ht mixture or Exiochrome Black T or Solochrome Black T can be used}.
‘The contents of the flask are Utruted against 0.05M disodium edetate (TV).
until pink colour changes to full blue colour of the indicator, A blank
the volume of

„ Wt of Calcium carbonate «
Strength of 0.05M disodium edetate gg

|

23. ERRORS, PRECISION AND ACCURACY

‘of results, Reproducibility of
the 7
cen e seme nee vale to ar ae re
Measurement. in other wards, I. the agreement between matasratients
et have been made tn exactly the same way. The thet terms which are
tuned to describe the precision of a set of replicate data.

1. Standard devation - It ts the root mean square devintion
from

erors: The error of a measurement in an inverse measure of Ita necuracy.
‘The smaller, the error, greater la the accuracy of the analysis.

Errors can be expressed as either Absolute error or Relative error
Absolute error = Observed value = True value

Ghecrved value — True value
Relative error = ale Joue x 100%

Errors cant be broadly classified into 2 types:
L Random or Indeterminäte errors
I. Systermatic or determinate errors

L Random or Indeterminate errors: are those which
Laso possess
and follow po Le and invariably have fluctuating values, This 1yP*

systematic or determinate errors: Syslematic errors have a definite
assignable cause or reason. ‘Ths type of error can be avoided,
Tinimiscd or corrected, Different types of errors are

a. Methodic errors; These are Inherent to the analytical method. These
are often introduced from non-ideal chemécal or physical behaviour
‘of reagents and reactions upon which a method ls based. Some of
the sources of this type of error are slowness or incompleteness of
chemical reactions, loss by volatility, adsorption of analyte on solid
precipitates, tnstabllity of reagents, contaminants and chemical
interferences,

b. Personal error: Are those introduced Into a measurement by
Judgement of a person or analyst. Examples of such errors are -
estimating the position of a pointer between 2 divisions of a scale,
colour of the solution at the end point of a titration, level af liquid
with respect to a graduation in a pipette, ete. These types of errors
can be minimised or avoided by care and self discipline.

Es
B

1. Mat ln Beers law?

2 What in Lambert's da?

3, What ts the principle invubred in Colorimetry?

À Wht ts the suvelengh range of visible ren
5. Which is the source of light tn colortmetry?
What da the diference between doux ad Een
7. Which detector la used in Colorimetey?

A What da the sample cell made of, In Coborimetry?
0, What are the applications of Coloctmetsy?

10. Name the ressens for deviations from Beer's lam?
AL. What ds a flier? Name the different typen

12. Explalo few to select Miter?

A What is a monochromatic ligne?

14. What in a Polyehromatic Wht?

15. Name a source of Monschromatic Wight

16. Name a source of Polyehromatte light

17. What are the types of Monschromatora?

18. What 18 the principle involved la a prism monochromator?

19, What is he principle Involved in a grating rmenochromatcr?

20. Name the different types of grating manochromatarı?

23. Ge the equation for different types of ating monochromator,
22. What ts Absorbance LA

2%. What is molecular extinction co-efficient?
77. WX da changed, what will happen to Absorbance LA?
28, Y Concentration ls changed, what will happen to Absorbance (AI?
29, 16% ts changed what will happen to E?
30. What in Iscabestic paint?
31. What are the different methods of Quantitative anabysts?
32 What is spectrophotametsic Utratsoer? Give comple
3%. What la Une difference between Calorimeter and Spectrophotometer?
34. What are the diferencen between Single beast and Double beam Instruments?
35, What ts band pass?
2. ULTRA VIOLET SPECTROSCOPY

a
1, What ta the wavelengih of UV region’?

2. Of 200mm and 400m, wich one has tae energy?

A Name the diferent types of electrons? Where are Uhey present?
4, Name the diferent types of electronic transitions.

5, Which transition requires lowest energy?

E Which transition requires highest energy?

2

can we ps po regen fe ot We?
A yw caro har UV spect «20007

9, What ts o sue of ght In UV?

10. What ln desi?

12. What i the normal pathlength of seit se)
13. Wa che sample cell made up ef in UV?
14. Weich detector ts med In UV? &

15. Which Mer or emoncchramatee u used In UV?
O

17. What ds an munnchreme?

18, Wat I Buihochromse stat?

MD What ts Hypsochromie abet?

20. What bs Myperehromis flee?

21. What ts Hypochromse effect?

22. What ta the elect of conjugation on dama
23. What in the eect of cross conjugation am hea?

24. What 2 the effect of geoenetrcal tsemeriam (cintre) on Da

3. What in the effect of alkyl minditudin Gn Aes?
26, What ln spectrophetemetric Uria?

20. What ace the fees methods of Quantitatiee anal?
28. How 16 analyse mixtures of substances by UV (Quantitative)?

2. What are Le qualitative applications of UV?
30, Whack solvents ca be wed tn UV? Why?
31. How hs the abnarbed UY edits energy lot?

À. What la singlet state?
2 What in doublet state?
3, Wheat lo triplet state?

6, at are We way In which the abe UV energy can Le I?

9.1 rene, wal tbe were oe eal dl ne uP
ta that of absorbed Might?

a. ny the wae of he etd Bt iran fom ao rel
ue?

a. me eit ght of age march than abr WO?

IR wnt cect oot conn ran cx gt wae tl ee?
11, What ts Flourescence? {in eleetronie terms)
12 What ls Phesphorescence? lin eleetronie termal

BEE
É
:
:
i
Ä

‘What in static quenching? Grre example.

7. OF the follwing, which fector increases Rourescence MERAY:
rigid molecule / Mexsble molecule
b. elretren donating group / électron withdrawing roy

38. Which ts the source off ght In flourimeter? |
‘37, Wat are the sivantages of fourmmetry? |
38. Can you analyse a noefourescent substance by flourimetry? How?

4, NEFILO-TURNDIMETAY
N What lo Nepas?
2 What in Turbitimeto?

3. What da Tyra eier?
4 What da the wavelength of the
ar Mg scattered when compared to incident

Hat in |
© in whic ange la ls menue in Repeat? |
7, ln wich ange L ts measured in Turbidimetry? |
Se mere ae mar ty Mehmet whee
9 Compare Nephlometry aad Fiourkmetiy.

10, Contrast Nephlometry und Pleurknetry.

5 What ts the wavelength el the Dot transratied neem
‘when compared lo

12. Contrast Turbidinetry and Colorimetry.
13. What should be the concentration of a mpenalon to be anuiwed dy

¥ What #6 the source of light tn Nephlörsetry, Turbidimety?
15, Which elector hu used in Nephibenetry, Turbidimetry?
18, Which factors affect scattering of light ty suspensions?
17. What ts Tusbidimetric (tration?

18. Which ef the following can be used ax Nephiometer, How?
a Flourmeter b, Coleimeter

10. We of the following can be used as Turbídimetes, How?
a Flourkneter b. Colorimeter

20. How can you do quantiiatie analysis using Nephloturbidimeter?

E INFUARED SPECTROSCOPY UM
1. What ts Che wavelenjih of IR reggee?
2, Which segon of IR bs used in Pharmaceutical Analyste?
3 What ds ware number?
4. Why ls wave fsumber scale used instead of wavelength scale?
5. What de you mean by resiecular vibration’?
& What are the different types of energy for a coslecule
7. What is Natural frequency of vibrasen?
A What is the relationship between wavelengths aed ware number?
9. What ln the relationship between ware mumber and energy?
10. What are the criteria for a molecule to absorb IR radiation?
AL. Mist IR regen ts divided into tien, Wat are they?
12 How well you convert warthength to wave number?
13, What ts dipole moment?
IM. What are the different types of vibration?
15, What are stretching vibrations?
1. What are bending veto?

si

17. How wi you express the tla mamber of ae a

present lo a
18: Whats the source GR radio?
10, How 1 sapling of salda done In IR APR
20. How la samp ol de done o Rape?
M. wich ype of Mes and roenchromern are used in IR?
2. Mame the detectors sed Im IR spectrophotomeler?
2. wi ds PTR?
34. What are the advantages of FT-IR?
25. What de you mean by Group Frequency regen?
28 What do you mean by Pinger print region’?
77. How wi you Mestily m fonctions group by IR?
38 Mow wl you Sent a drug substance by IR?
28. How wil you Mens the Impursties to a drug by IR?
30. How wit you performs quamtitalive analysis in IR?
6, NUCLEAR MAGNETIC RESONANCE
1, Wheat da MM?
2 Wat ds Pa
3 What i Che erence between KM and PM
4, Wich compounds gre HMR Spectra?
5. Why do compounds with edd mass number only we NMR spectra?
6. What la stone ener
7, What la musa mumber?
What are queetum numbers?
9 What In spin quantum fahr?
10. What ln precessicaal order?
i rene am an
Peinciple in NMR
YAM me eld men ancrensed, what happens to precesstnal

ja Wi a compound absorb rathowave Ärgueney, af magnetic Meld de not
applied, Whey?

15. What ls relaxation process?

16, What are the types of relaxation process in NMR?

17. What are the components of NMR spectrophotometer?

18. What strength of magnetic Held in commonly used in NAM?
10. Which radiation energy la commonly used in NMR. AL what frequency?
20. What are the requirements for a solvent to be used in NMR?
What are deuterated scbrenta. Where are they used?

Give examples of solvents used in NMR.

What ts Shen?

‘What is Desbieiding?

‘What is chemical alu?

How in chemical alt measured?

What in internal reference or reference standard?

Wal are the requirements of reference standard?

Give examples for reference standard?

What are the applications of NMR?

Prem NMR spectra, bow will you Mentily
a types of protons bo Environment of protons
© No. of protons of each type d. No. of adjacent protons

How will you perform quantitative analysis bn NMR?

LL HIECTHON sr RERORANCE (mal
1, What ta ESR and EPR?
2, What ts studied Im ESR?
3. Which types of cempounds give ESR spectra?
4. Where are unpaired electrons present?
5S. Which type of radiation 19 used in EMR?
8, What in spin quantuen number?
7, What is the strength of magnetite field used?

E

EEBBENBBERB

®

AL Which microwave frequency In sed In ESR?
What is he source of acre?

10, Which ts the detector und In ESR?

11, Name the reference standards used tn ESR?
12 What is 16 value?

13. How la JE value calculated?

14. What are the application» of ESR?

8. MASS SPECTROMETRY (MS)
ee

L What la masa spectra?

2 How is mans spectra obtained?

2 How mass and nus of ton path related?

A What are the diferent types of mass spectrometers?

5. What la We principle in EIMS (Electron Impact Mass Spectrometer!?
6, What la resstution?

7, What is the difference between singe focussing and double focussing musa

B. What ds the priociple in CIMS (Chemical fonination Mass Spectrometer!?
9. What are the different typen of peaks obtained?

10. What in Mass peak (Parent peak?

2. What 1 Dane peak?

12. What in Mel peak?

13, What in M2 peak?

M4. What are the applications of mann spectroscopy?

9._POTENTIOMETRY
— —
1, What la Potentiometry?
2 How in potential fem?) of a solution enensared?
3 What ds indicator electrode?
4. Give examples for indicator electrode.
5, What in reference electrode?

5. One examples for reference electrode.
7 vna pair of electrodes lo ment commonly used In pit meter or

ig What ds Nernst equation?

10. How is pH measured?

1, What in the relasoaship between emf and pit?

Ma What are the admis of gas eee or ether ican dec?

13, What are the advantages of mures camel electrodo rer other reference
lectrodes?

A What ty the difference between pH meter and potetiometer?
18. What are the components of a pH meter?

16. What do you mean by cardos of pH meter?

17. How is canton of pH meter dene?

18 Name the factors whieh affect potectial ef a selben.

18. Why la temperature Knob necessary in pH meter?

A What ie te il
23. What im dead atop cod polo technique?

determinan of
30. How in dead stp end pot age spp In 08
water?

4, What Es the selationship between resistance und. conductivity?

5. What ase the factors afleting conductivity?

6 What is apeciie resistance?

7. Wat in the relationship between resistance and specific resistance?
4 What la the relationship between conductivity and specific condactmiy?
9, What is cell constant?

10. How is conductivity of a solution measured?

1. Which types el cells are used to measure conductivity?

12. What ave tht components of a conductivity meter?

13, What la the init of conductivity?

14. What is equivalent conductirey

15. What ls molar conductivity?

16. What la jante conduct

17, What ls the effect of tien on apeeifle conductivity, equivalent conductidiy
and molar conduetity?

18 How la cell constant measured?

10, Why 1 cell constant determined?

20. What ts the elect of temperatura on conductivity?

20. What ls a conductometric titration?

22. What are the advantages of conductometric titrations?

23. What are the precautions to be observed in conductometrie titrations?
24. How ha a conductometrse ration performed?

25, la specific conductrity measurement required in conductomnetrie titration?
26. What la conductivity seater?

77. Where ln conductinty water used?

28. Which cation bas highest tonic conducta?

st.

a ES e nnpon»r

Which anton has highest tome conductivity?

How ts the end point determined in conductometric ttmtion?
What are the applications of conductivity measurements?
What are the applications of condurtometric ttmtians?

11. POLAROGRAPHY

What la oxidation En electronic terms?

‘what ls reduction tn electronic terms?

‘What ia electrochemical reduction?

‘What in the principle in polarographic anabysis?
‘What is Eu (Half ware potential?

‘What in diffusion current?

‘What is residual current?

‘What is migration current?

What in polarographic sardina?

What is the use of marina suppressor?

Give examples of maxime suppressors,

What bo the use off supporting electrolyte? Give example,
Why ls nitrogen purging required tn Polarography?
What ts the alternative to nitrogen pare?
How ts electrochemical cxidaon/reductlon dene?
‘What is a polarisable electrode? Give example,
What is a non-polarisable electrode? Give example.
Why ts DME used? What are the advantages?
What la Movie equation?

|. What are the factors which affect diffunion current?
L How does capillary eharmcteristics affect difusion current?

What are the advantages of polarography?
How is qualitative analysis performed?

|. How is quanistative analysis done?

35. What are the various quite methods used in polarography?

2% Mow in drogue shared? -
27. How is mercury dupe altered?

28 What le direct comparison method?

20 What In calibration curve mxthod?

20 What da pot ton method?

31. What 15 standard addition method?

30 Mow ie mesure of lens arulysed qualitatively and quantitatively?

22 What are the applications of polarography?

12, AMPEROMETRIC TITRATIONS

‘What In Aanperemetry?

What in che priscile in Amperumetre trio

How 1 he potential selected in amperometric trations?

What are the compocenta of apparatus used in amperometric trations?
Mame the electrodes used in amperometric utrations?

What are the advantages of ampercenetite (trations?

Ce examples of nét-mducitle ton.

Mow wil you analyse u non-rrducible ton by amperometry?

Che cammples of electroreducitle ton.

What are the advaninges of HOMES

AL How ln the end poo determined ln amperemetri: trations?

12 What are he applications of amperometric Lradina?

What are the advantages of amperometric tration ever polarography?
a Ihe tags of amperometne trations ever potentcmetr /

|
Hi Sole cimowATooRAPITY (oc)
L Wal de chromatogeasiy?

2 What are the principles d seperaticn

3, What ie adeerpuon diremategrshy) Nr

Beene ee eee

4 Give examples of technique for adsorption chrocualogtuphhy?
5 What ts Parution chromatogragtey?
6 Gree example where partite ts the principle of separation.

7, Is the interacióón between components and stationary phase In adsorption
chromatography - reveraiBile for) irreversible?

8 ls adsorption phenomena, temperature dependent (or) independent?

9. Is partition phenomens, temperature dependent for) independent?

10. What ls partition coefficient?

11, What ds the relationship between partition coefficient and R value of a
component,

12. What are the general requirements for deing a separation by column
ehromatngraphy?

13 Name some weak adsorbents,
14 Name some strong adsorbents,

16. What are the requirements for a good adsorbent?

16. What are the functions of a mobde phase?

17. What da an eluent?

‘What is an eluate?

‘What ts chaton?

Give examples of sclventa with low ehaling power

Give examples of solvents with high eluting power.

Mame the diferent elution techniques.

‘What la tocratle technique?

‘What ts gradient technique?

ln which of the chromatngraphic technique, gradient elution should mot be
used. Why?

(Give an ideal column Jengih : diameter ratio.
Give the ideal adsorbent : adsorbate ratio.
‘What should be the lenglh of (be column for separating componente

SR RERBBRBE BEE

3 What ts ution volume?

35. lo reverse phase mode, which type of stationary phase and mobile phase
28 used: Polar / Non pola.

36. in normal phase mode, which (ype of stationary phase and mobile phase
in ved: Pelar / Noo-polat

3 ln normal phase mode. the following ds eluted fat:
n Polar component D, Nem polar component
38 la reverse phase mode, the folowing component ts eluted frat:
a Polar substance b Non polar substance
14, THIN LATER CHROMATOGRAFHY (TLC)

1, What ts thio, layer chromatography?

2 What ds dhe principe of separation in TLC?

3. What are the adeantages of TLC?

4 Wink ace the general requirements In TLC Lechaique?
5. Mame the stationary phases used in TLC?

6, What in the diference between Silica gel H, O, GF?
7. Name the diferent grades ef Alumna.

8, For Siienge! O, in what ratio st ts mined À
Gare ne wich water for making shotty.

0, Give the standard dimensisem of TLC plates
10. Name the callerent methods of preparing TLC plates

11, What in the thicisess of adserben
pt ee her ia

How ts activation of TLC platen done?

Why ls activation of TLC plates necessary?

How are the activated TLC plates stored. Why?

How is a sample prepared for spotting in TLC plaie?
Which concentration of sample is used for spotting. Why?
Which aspect of sampling in necessary in quastitaive TLC?
What ts development of TLC plate?

What in edge feet in TLC?

Te avoid edge effect, what must be dane, in TLC,
Name the mobile phases used for development ln TLC?
Name a low polar solvent?

Name a high polar solvent?

What are the methods of detection in TLC?

Name a non-specific spray reagent.

Name a non destructive detecting technique.

Name a specific spray reagent.

Explain quantitative techniques in. TLC.

‘What is the use of denstiometer?

What in HPTLC?

Wat ls Rr, Ra, Ru?

‘What ts the use of determaning Re fe and Ru?
‘What lo bo dimensional TLC? When st in used?

13, PAPER. CHROMATggB FT 29
1, What is the principle In Paper eheomatograptey?
2, What de you mean by paper adsorption chromatngrapy/?
3, What de you mean by paper partition efromategraphy?
4. What la ie paper made up of in paper chromatograpty’?
5 Give examples of modified papers.
& Which type of paper ts normally used: hydrophilic hydrophobic,

BEES SE

ESS:

BREEEBESEREEBR

16. Ge a mpecilo spray reagent to detect
a abs ‘cardiac ghromside
& akdehde ar hetones € phenols
vun h proteins

17. How win you perform quantitative analysis in paper chromalograpty?

€ amine acid
L tasnins

16. ION EXCHANGE CHROMATOGRAPHY.

1. What de you mean ty lon exchange chromatagraphy?
2 What la the principe of separation?
3 Which plays a rele im separation
a Pheystcal/ehemical forces b, rewerntble/Irrevernible reaction,
4. What in an kan exchange resin?
Bo What da the nature of polymeric rmsin mari?
6, Gwe un example of nature resin à Cation b, anton
7. Whieh portan of men conmins exchangeable mes?
8. Which part of the reto ts responde for strength?
‘9, Which functional group can be present in
alone exchange rein
eat exchange resin
exchange tee
jong, anionic exchange resin.

i

aaa ARS EEE RTC PORT ON

11. Which physical form of ton exchange resin has greater eficheney?

14. How is the efficiency of an lon exchange resin measured?

15. Which packing method in used tn Jon exchange chromatagraphy?

16, Which are the developing soivents used tn Jon exchange chrumatagrapliy?
17, Wiseh techniques are used to analyse ehients?

10, What is regeneratien of a resin?

20. How ‘will you regenernte eatlon exchange resto?

AL. How will you regenerate anton exchange rest’?

2. What are the applications of lon exchange chromatogmpiy

17, GAS CHROMATOGRAPHY (GC)
1. Which do used as mobde phase in Gas chromatograpty?
2 What ts the principle of separation in Oss Salad chromatagraphey?
3. What ts the principle of separation is Gas Liquid chromatography?
4 Name the statlooary phases used in Cas meld chromatograety.
5 Name the stationary phases used in Gas Liquid chromatrgraphy,
6. What are the requirements fer m compound to be analysed by Cas Liquid
chromatography?

7. Under what conditions Cas Solid chromaingraphy la preferred over Cas
Liquid chromatograpty?

& Which instrumental components are required in Cas Liquid curesauogmptey?

D, Give examples of carter gases weed in Gas Liquid chromatagrapti/?

10. Mase the flow meters used?

13, re de dimensions of preparative can In Cas Liquid chromatography?
14. Way cohumn temperature sainenanct I necesa?

15. What ts tsothermal peogrsemening?

16. What ds temperature progressing?

m la partion coefchnt, dependent / independent of temperature?

IB. Way prcheaters are present along with lefecting devices?

19. What are the end charmcteritics of a detector?

24, ln Kethoremeter. a gas with lower thermal conductivity should pot be used.

25. What is the principle and which carrier gas is sustable for use Im
a Katharneneter
la Pene Hasta detector
©. Amon ionisation detector
4 Electron capture detector

20, Which detector gres the best response?

27, Whal is the functcn of an integrator?

28, What is the reason and remedy foc a forward tailed peak? rooting)
20. What la the reason and remedy for a tailed peak?

30, What la Retention time?

3. What la Retention volume?

32. What ts specie retention volume?

DD What te separation factor?

34. If the separation factor bas a higher value, what Inder regarding
= Peter Mme Parton cell.

36. What ts resobutioo? What should be the optimus value!
36. What is METF How ho measured?
3. What eleseney? How la it measured?

39, Fer a column lo be efficient, what should be the value
we + more or less
b, of HETP + more or less.

‘What ls Ihe material of column used in Gas Liquid chromatography?
Give examples of stalienary phases In Gas Liquid chromatography?
‘What sould be the characteristics of a solid support?

What ts support pretreatment?

‘What are Bonded phases in Gas Liquid Chromatography?

What la Dertrattsation?

Why dertvatisalion Is dene?

Give examples for reagents. used in derrratiation’?

‘What is Preceluimn dertatisation?

What is Posttohsma denvattsation?

What is Ube principle sed in quabraive ana?

What prince is used tn quantitative analysis?

What are the injection devices available in Gas Liquid chremaingraphy

8. HO! FERTORMACE LIQUID CHOMATOORAPEY MILE)

1. What are the differences between column chromaingraphy and HPLC, with

reference to
fa Particle Size A, Column ste € Premure d. Sample hood.
e Com E Choice of stalienery phase LE Seale of operation.

2. What are the techniques of separation in HPLC, based on
à Modes of chrocntapmaphy Principe of separation
€. elution technique 4. Scale of opera e Type of analysts

& What ts Normal phase chromatography?

4. What is Reverse phase chrematagrapty?

5, What are the components of HPLC (in generall?
6. What is Asgeratie elution technique?

7. What ls gradient elution technique?

lig tal A La bo Reverse phase chremaiagraply

€ lon exchange chromatography lon pur chromatography

BLEBRARESARES

Sue ection chromaingraphy {.Alinky chromatagrapkıy
À Gil phase chroxangrapy
8, What da Cia or ODS? What lo tx use in Chrematograpiiy?

10. What are the methods of solvent degaasing?

11. What are the diferent infecting devices im HPLC?

12 Wat is the use of guard colucan in HPLC?

13. Clay analytical columos based on particle sise and functional group.
14. What are the detectors used in HPLC?

15. What is Retention time and Retention vob?

10, What is rome

17, What in a theseetiel_ plate?

16. What 1 HEAT

19. What ts eflicieney of a columa?

20. What ls the ise of calculating asymmetry factor?

‘21, What are the applications of HILO?

22. What ds the bests of qualitative analysts in HPLC?

23. What is the basis of quantitative analysis in HPLC?
24. How will you check the presence of impurities in HPLC?
2, What is internal standart?

19. US _TITRATIONS
1, What ds an acid?
2 What da à base?

3 What in noo-aquesus tration?

4 Way la non-aqueous Brut done for meale acts aid weak bases?
5, How are scvents clase

& What de you mess by Aprotic solvent? Give example,

7, Whit de you mean by Prutageaie solvent? Give expe.

& What do you mean by Protophitic schent? Cine: cxausple.

9. What de you mean by Aciphipretie aarent?
10, What la leveling edfece? =e

11. Name a levelling solvent for weak and strong base

12 Name a levelling solvent for weak and strong ación.

18. What is differentiating effect?

Name a diferentiating solvent for weak and strong base.

Name m differentiating solvent for weal and strom acid.

What 1s ontum ton?

Whey ls titration of weak bases done with perehlarie acid in acetic acid?
How ts 0.1N perchloric acid prepared?

How will you standardise 0.18 perchloric acid?

How will you determine end potot la non-aqueous Utration?

Nowe the electrodes uned lo detect end point in Non-aquenus trations?
Give examples for indicators used in Non-aqueous trations.

Name the class of compounds analysed by Noevaqueous trations?
How will you Utrate halogen ack! salts of weak bases?

25, Why is mercurio acetate added in Non-aqueces titrations of halogen acid
salts of weak bases?

PBBEBERER FRE

29, HEDOX Taro
1, ‘What ds oxidation?
2 What in reduction?
3 Name some oxidising agents,
4. Give examples of reducing agents
5, What la redox potential?
4. How well you standardise ceric amencntum sulphate?
7. What are redox indicators?
B Give examples of redox indicator?
9. How dees a redax indicator work?
10. How wll you select mm indicator für a redox ttratio!?
11, What are the different methods of determining end palet?
12. Give examples of drugs which can be titrated agua cermk amo
‘sulphate.

13. Espia how Fern stn a cedar tnt:
Y Wome, De ciated ened to tt at ak af ro tion by
cames meihad.

15. He wal you standart tant hote?
comica of drag which can be Utrnted agatoat Utanous Chone,
an, pusomsanon TITRATIONS

1, Wha ts a diastisation utmtion?

2 Which dus of drugs can be cure hy discusion reaction?

& What are the methods ef determiniag end point An dimmotisatinn titration?

A. What ls external indicator meibed. Give example.

A. Mow will you stasdardise DIM sodium nitrite?

& Explain the procedure for carrying out Diazotiaation titrations?

T. One examples for compounds directly titrated with NaNOa.

A. Mn drag does mot contain free amino group, how wll you cary out
Austin Vera?

16, Gm

© Give examples of drugs coowerted to free amino group.

10. How ta starch dde paper prepared?

XL, How does a starch iodide paper werk as indicator?
22, COMPLEXOMETRIC TITRATIONS

PSE
1. What in a comphong agent / chelating agent?

2 What in and

3 Whut is a coordination compound?

4, What ms a sequestering agent?

3. What ls the difference between unidentate and polylentate son’?
4 What is Werners coordination number? What la ls slgnieance?
7. Name the functional groups persent in complentos agents,

B Name the fusion groups present in sequestering agente.
4. What da EDTA

10. Why da discdium echte used instemd of EDTA

11, ln what ratio EDTA forma complex with pobrralent tens?

12 How la stabAity content expressed?

13. Name the factors which influence stabiity constant?

14, Why polyenlent ions are trated with EDTA using allaline butler?
16, Name the buffers used tn complexcenetric titrations?

16; What are the methods of determining end paint In ceemplexsmetne utrations?
17. What i pM Indicator?

la How does n pM Indicator work in complexometrio titration?

19. What in the relationship between pa and pH?

20, Give examples of pM indicators.

31. Way different pM indicators are used lo different trations?

2. What are the types of complenometnie trations?

28. Why back tration ls adrocaled ln some estimations?

‘What do you mean by replacement utration?

Explain alkalimetnic utration of metals.

Why buffers are not used in alkalıeine ration of metals?
What ts masking agent?

‘What ts demasking agent?

What la the need for masking and demusking agents?

How will you standardise 0.05M disodium edetate solution?
Eagle a ee e lee,

SBRBRS REE

Give examples of compounds analysed hy direct uration.
Give examples ef compounds analysed hy back Ueration.

‘What is the difference between Meedant black 1 mod Merdant black II
mixture?

35, What in be difference between Calcoo and Caleon mixture?

FER

25. UNITS, DILUTIONS & FORMULAE

ums
‘The discussion will pertain to few units only.
Ter Werelengits [For Prequeney

1 metre = 100 cestimebre or 1 Kits or 1 Kilo Herta = 10° Ma
1000 etmetre fe
À srflimetre = 1000 microns Lam 1 Mi or 1 Mega Hertz = 10" He

dam = 1000 masometre (mm 1 Ola or Giga Herte 10% He

1 em = 1000 picometre (pod [1 THe or Term Mertz 107 tte
Lens 10% For Weight
1 um = 104m 1 Ridogram = 1000 gram
Imm = 10% 1 gram = 1000 milligram
a= 10m 1 milligrasn = 1000 microgram ad
pm = 10 7m Tmicrogram (a) = 1000 nanagraen ing
Percentage eehitions & conversions 1 mg = 10%
1% w/v = Igram in 10000 of solvent Lg = 10%
A w/w gram les LOOgrmms of sobrent 1 ng = 10%

Normally %w/v solutisos are used
Conversions:

1. To convert rf to mg/m: Multiply by 10
Mu x 10.0 © mg/ml

Problem: Convert 2w/v to mg/ml

Formula; Zw /w x 10.0 = 20my/mi
2. The answer la 2000/00

Checking: ZU w/v means 2gram/1000l

2 To convert mg/l to % w/v: Divide by 10

= ey,

problem: Convert, Somg/m to 9% w/v

Le. 3¢/100mi = 3 wiv

3. To convert Mw/v to ‚g/m: Multiply by 10.000
ww x 10,000 = pg/ml

problem : Convert w/v to pg/ml
yormula: 2 w/v x 10,000 = 20,000%¢/enl

Checking: 2% w/v means Zgrum/100ml
Le. 200mg in 100m! « 20mg/eml « 20,000n6/m!

4 To convert ja/m to % w/v: Divide by 10,000 (Checking + 29 above

For preparing calibration curves, ditions have Lo be prepared fem
stock solutions. In dilutions, the folowing must be observed:

1, Mintmum quantity of steek solution la to be used,

2 AL any step, dilution should not be more Wan 5 or 10 times,
[This ia to miniaise error In dilution)
D ko em er La o 10mi can be done, but nat Iml a 100
The total volume of solution should be aufficient for
subsequent ditutions,

4. In order to avoid

following, format
Ina volume + final velume (Coneentnation/ ml

in calculations, It la better 16 use the

Keeping the condicos ln mind, et us werk out an example

‘Problem 1
Prepare a ealbration curve from 10-SOyg/mi, The given stock sahitog
ds mg/m (tmg/mi = 1000k/ rl

I

10m» L00p/ rail
Amt + 10m (1092/01
Ami + 100 209g/m0)
ami > 1000 (3094/00
oak + 10001 (Oil
Sl + 10m (5081)

Now the above 5 solutions are ready for calibration curve
Problem 2

Prepare a calibration curve fram 0.Ipg/anl to O.5pg/ml using 1eng/rl
stock solution

Stock solution = Imgfml = 1000pg/m

ps

LE q (100yg/ mt)

LE LL.
+ LL]

Ant + 10081 (0. Hg/ en)
Am) > 10ml (0.2 en)
‘Stal > 10ml (0.3 uni)
ml + 10nd (0.4yg/ral)
Seal + 1Oml (0.5 yp/m)

These 5 solutions ase ready for calibration curve

FORMULA (FOR TITRATIONS)
1. FOR STANDARDISATION OF TITRANT
1. When primary or secondary standard solution Is available
VN, = Val

Vi & Ni = Volume & Normality of solution 1
va & Na = Volume & Normality of solution 2

W any 3 of these values are known, the 4th one can be calculated

2, When primary standard solution is not prepared, but substance
An the form of powder ls available

„ esp mean tkm.
Shengh of dant ‘Titre vadar + Ey mi. factor

Wh of Potamabc blog phate
(eg Strength of IN Sodium hydroxide = LAA A

IL FOR ESTIMATION AND ASSAY
1, For estimation of a substance present In the given container
Transfer the contents quantitatively to a contcal flask.
Perform the titration and find out the Utre value.

‘Amd, present in given container = Titre value « Strength of tra « Eq. wt. factor

2. For estimation of u substance present in 100ml of the
given solution

Pipetie out 10ml of the given solution to a conical flask, perform
the tration and And out the titre value.

ahr» Seng tr + wt tor +
Ama. prevent tn Wm of che grin atten = TEESE ug ef wees Bvt eter ÉD

3. To find thespercentage purity of a zum material

of penedes, transfer to a conical flask, add indicator
pio phere as per requirements and ttrale against a dirant

Find out the titre value.

Perentnge purty of ven substance « Bes BSS Ese tn

4. To find out the amount of drug present in each tablet
‘The following steps are followed:
Weight accurately 20 tablets

Find the average weight of one tnblet
(Total weight of tablets divided by 20)

Fihely powder all the 20 tablets.

‘Weigh a quantity of powder as specified in monograph.
Perform the titration and find out the titre value.

The amount of drug in each tablet is calculated ns follows:
à ut

Asount of drug in each Be

5. To fied the % purity of tablets
Follow the steps as given in Formula 4 (as above).

. . + pu ER!
© pry of tablets Bl à rt eS

26. FLAME PHOTOMETRY
+ Principle
7 Components of a Flame Photometer
% Burner (with fuel and oxidant)
* Filter / Monochromator
# Detector
# Readout Device
Schematic dingram of
À Flame Photometer
Flame Speetrophotometer
* Double beam Flame Spectrophotometer
Applications
% Qualitative analysis
À Quantitative analysis
Methods: Direct Comparison Method
Calibration Curve Method
Standard Addition Method
Internal Standard Method
Interferences and Methods to overcome

* Preparation of Standard Stock solutions

FLAME PHOTOMETRY

metry ia alo called as Flame Emission Spectroscopy,
ne ara) atom are involved in the emission of radiation when

introduced into the laste.

Principle

When a solution of metallic salt is sprayed on to a flame, fine droplets
ase (armed. Due to the thermal energy of the flame, the solvent in the
droplets evaporate, leaving behind fine residue, which are converted to
neutral atoms, These neutral atoms are converted to extted state atoms
by the thermal energy of the Game. As the exited state is not stable,
these exited atoms return to ground state, with the emission of radiation
al specific wavelength. The wavelength of the radiation emitted is
characteriatic of the element and is used to identify the element
(Qualitative Analysis); The intensity of the radiation emitted depends
upon the concentration of the element analysed [Quantiative Analysis).

liquid formation fine formation of
sample ofdroplets residue neutral ators
A & intensity Emission of Excitation
‘of exiitted radiation — Radiation of — of atoms by
measured

Specific wavelength Thermal energy
Although it is thearetically possible to analyse all the elements by
Mame photometry, the availability of turner, fuel and oxidant combinations
and technical reasons make it practically possible 10 analyse Group IA
‚elements (like Li, Na, K) and Group [LA elements like Ca, Mg). The intensity
of the radintion emitted depends upon the proportion ef thermally exited
‘Moms, which in turn depends upon the temperature ef the fame,

Pruction of free storms thermally exited = Le Ae ttre

where N* = po. of atoms in exited state

Me » of atom in ground state
À + Constant for element

AE = difference in energy level of exited and araucd state

k = Bolteman constant
‘T= Flame temperature
‘The wavelength of the Ught emitted depends upon the difference in
the energy levels of atoms in the exited and ground state, Since atoms of
each element han specific exited and ground state energy levels, the
wavelength of radiation emitted in different for different elements, For
example, sodium emits yellow radiation ($89nm), potassium emita orange
(767nm), calcium emita brick red colour (422, 554 and 626020] & Lithium
at 670nm. Some of the elements emit at a single wavelength (primary
emission), whereas other elements emits secondary emissions at different
wavelengths.

The wavelength of the radiation emitted in given by the following
‘equation:

Wacko of geminal (A) ¿PE

where h = Planks constant
€ = Velocity of light
Ea, Es = energy levels of exited and ground state respectively
The intensity of the radiation emitted dependa upon the concentracion
of the element present in the solution. Higher the concentration, more, ia
the flame intensity and lower the concentration, less is the flame intensity.
The intensity of the spectral emission line is given by the following
equation:

Intensity of spectral emission line (y) = MS catar

Where E = Energy of exited state
absolute temperature h = Plank’s constant
= freq. of radiation Y = flame volume (aperture ratio)
Ar = no. of transitions each exited atom undergoes / sec,

No = no. of free metal atoms in ground state per unit volume
Bs = statistical wt. of exited atomic state
BT) = partial function of the atom overall states

k = Bolteman constant

Instrumentation / Components af u Flame Photsaneter
1. Burner (with fuel and oxidant)
2. Piter / Monochremator
3. Detector
4. Read out device

1. BURNERS

‘There are different burners available, which are used to spray the
sample solution into fine droplets, mix with fuel and oxidunt, so that à
homogenous Bare of stable intensity is obtained. The most common ones
are Mecker burner, Total Consumption burner and Laminar flow (premix)
burner,

‘Total Consumption Burner

The construction of a Total
Consumption Burner is simple and is
sown in Fig-26.1. tn thin, the sample
solution is aspirated through a
capillary by the high pressure of fuel
and oxidant and burnt at the tip of
the bumer. The advantage is that, the
design is simple and the entire
sample is consumed. But the demerit
ls that, an uniform and homogenous
flame is not obtained, since droplet
sizes vary, Thin leads to Quctuations
in the Mame intensity.

This bumer is the most widely used, because of its merits lke
Srl in the Dane intensity. In this type, the sample solution, fol
exit are mixed before reach the bi k fow
= sie prep adit dei alert dropless

an outlet at the botters. The diagram of the Laminur flow burner in shown
in Fig 26.2,

ine

as 1
in

(aL Len or en u
Fuel and Oxidants

If the temperature of the flame in too low, it may not cause exitation
al neutral atoma. If the temperature is too high, it may cause ionisation
of atoms and thus sufficient population of stems in exited state may not
secur. Hence the temperature of the flame ía critical. This makes it
hecessary to select ideal combination of oxidant and fuel which given the
desired temperature in flame photometry.

The following table shows the different combinations of fuel and
axidant to get desired dame temperature, In most cases, a conventional
flame photometer uses compressed air as oxidant and liquified petroleum
Gas (LPC) as fuel

Flame temperature
Fuel ‘Oxidant
| Air On |
Propane 200% 200°C
Hydrogen 19000 | 2300c
Acetylene move | more

2 FILTER / MONOCHROMATOR

In flame photometry, the wavelength as well as intensity of the
An RU he eee fines en ee

monochromator is to be used. A detailed description of filters and
monochromators is presented in Chapter 1 (Page 1-14 to 1-20). Typically,
a simple flame photometer contains a filter whee! (containing several filters
for either Ca, U, Na or K) and when a particular element has to be
analysed, the specific filter ix selected,

2 DETECTOR

‘The radiation emitted by the elements is mostly in the visible region,
Hence conventional detectors like photo voltaic cell or photo tubes can be
used. In a flame opectrophotometer, phatomultiplier tube in used as
detector. A detailed descriptian of these detectors, is presented in Chapter.)
(Page 1-21 10 1-23).

4 READ OUT DEVICE

The signal from the detector is shawn as a response in the Dí
Read Out drin. The rad ae played in an army sola Ph line
Intensity),

‘SCHEMATIC DIAGRAM OF A FLAME FHOTOMETER
‘The schematic diagram of a flame photometer is given in Fig 26.3.

4 u Detect
+ mur.
re
u nk
a | Cost
HINTER

Tie. 26.3, Sehematis dlagram of a Flame Paotemater
‘The components were

‘Typically, a Mame

solution to fine droplets. When the sprayed droplets are ignited, the solvent
is evaporated by the thermal energy of the flame, leaving behind fine
residue. This fine residue is converted to neutral atoms, The neutral atoms
ase exited by the thermal energy of the flame to exited ators. These exited
atoms are unstable and hence loose energy in the form of radiation and
returns to ground state. The wavelength as well as the intensity of the
ight radiation emitted is detected by using a filter and a photometric
detector,

A concave mirror is used to focus the light onto a filter wheel
(containing Li, Na, K and Ca filters), in which the desired position can be
selected based upon the element to be measured. Thin Light passes through
a alit and falls on the detector. The detector response is shown in the
digital display device (% Flame Intensity).

FLAME SPECTROPHOTOMETER

A Mame spectrophotometer consists of similar components of a Name
photometer, in which the filter is replaced by a prism or monochromator
and the photoveltaic cell is replaced by a phototube or photamultiptier
tube.

DOUBLE BEAM
FLAME SPECTROFHOTOMETER

A schematic diagram of m
double beam spectrophotometer la
abr in the following Figure 26.4.

In this, the radiation from the
flame passes through two different
filtern or monochromators and the
resulting beams fall onto two
= different detectors. The double beam
configuration is used to moniter à

=
=
sample element and an internal
an standard element (a second
múbstance used in quantitative
is by

analysis internal standard
method, detailed in the next section).
For example, Uthium is used as

=

|
|

internal standard in the estimation of either calcium, sodium or potassium,

Double beats flame ia used when internal standard
method of quantitative analysis in used, to avoid certain errors tie
fluctuations in Mame intensity, errors in atomising the sample due to high
‘viscosity al solutions, etc.

PHARMACEUTICAL APPLICATIONS OF FLAME FHOTOMETRY

1. Qualitative analysis

Flame photometry in used to identify the elements in a given
sample solution. This is done by a technique called ux Peak matching,
where at lenst 3 peaks of emission spectrum should match when
sample and standard spectra ure recorded, For example, Calcium
emits radiation at 4220m, 354nm and 626nm. If the spectrum of the
sample shows emission maximum at these wavelengths, then the
sample can be identified by using the standard.

2. Quantitative analysis

Flame photometry ia mostly used to determine the concentration of
the dements belonging to Group IA and Group DA elements. Most often,
the concentration or the amount of elements like lithium, calcium, sodium
es potassium in samples can be determined.

The following are some of the quantitative applications:
a. Concentration of ealelai én serum.
b, Concentration of calcium, sodium, potassium in urine,

© Amount of sodium, potassium, calcium and magnesium in
intravenous Muids, oral rehydration salts,

4. Assay of potassium chloride in syrup,
©, Concentration of Lithium in serum for therapeutic drug monitoring.

The concentratisa or the amount of elem
any one of the following four methods, can be determined ly

a Direct comparison method

b. Calibration curve method

e Standard addition method

d. Internal standard method
a Direct comparison method

In this method, the % flame intensity (% FI) of a standard solution
and sample solution is compared. The merita of this method are, less time
is required for the estimation and more standards need not be prepared.
‘The demerit is that, it is subject to more errors, especially when the

concentration of the sample and the standard are in the non-linear region
of calibration range.

Cane. of fon in sample solution MEL SSA Cone, of jon in Std.

À. Calihration curve method
In this method, a series of standard solutions of the element to be
estimated, is prepared and a calibration curve of Concentration (Vs) %
Flame intensity is made. From thin cabration curve, the concentration of
sample solution is determined from itn % flame Intensity. The detailed
procedure is as follows: (for determination of sodium}.
i Prepare a stock solution of Sodium chloride solution (1000pg/eni).
fi. Prom the stock solution, prepare a series of standard solutions of
Sodium (10, 20, 30, 40 and S0pg/ ml}.
Hi, Select the sodium filter in the instrument.
he. Set the air pressure of 0.4 - 0.5 kg/oqem and set the fame in
the burner.
Y. Une distilled water (or demineralized water) and set 10 0% flame
vi Set 100% flame intensity (or full scale in the instrument] by using
the maximum concentration of standard solution in the calibration
region (Söpg/ mil:

vi Use the other standard solutions (10-A0pg/mi] as well as sample
solutien and determine the % flame intensity,

vi Construct a calibration curve of concentration of standard (Vo) %
flame intensity.

ix. From the % flame intensity of sumple solution, the concentration
of jon in the sample solution can be determined.

Note: If the % flame intensity of sample solution is more than 100%
‘or full seale, it can be duted accordingly, #0 that the % flame intensity
lies within the calibration region.

€ Standard addition method

ln this method, the given sample solution ia divided into aliquots and
to each, increasing concentrations of standard solution af the nase element
in added. The % flame intensity of sample solution us weil as those of
susnple solutions to which standard solutions were added were determined.
A calibration curve as shown in Fig, 26.5. in constructed, with negative
ass, The line when intrapolated, meets at negative x-axis, which is the
‘concentration of the jon present in the given sample solution.

This method is used, when the interfering elements cannot be removed.
dificult to be removed from the sample matrix Standard addition
hei enreumes physical ar annie interference, but cannot avoid
‘atioeie interference. (eq). Standard addition method is used in the
estimation of Calcium in Magnesium Chloride far diatyais,

The following detailed dl
‘standard addition method. procadise wil erplain the methodology for

|
!

Er

Let us assume that the concentration of enleium in the given sample
ja x ng/ml. Pipette out Sml aliquots of sample solution into each of four
Tal standard flanks. To the flasks, add respectively Sm! of 1Ovg/ml,
20ug/tsl, 3044/ml and 40pa) nl of standard solution of calcium ions,

Determine the % Name intensity of sample solution (xag/ml) as well
as those of (x + ‘LOpigy/ eal), 6e + 2019/mul), (x + 3044/0001 and (x + 4Qug/ml).

Plot a calibration curve ax shown in Fig 26,5. Intrapolate the line to
meet negative x axis image of x axis) which ia the concentration
‘of the sample ion in the given sample solution,

de Internal standard method

Internal standard method is used to avold or minimise the errors due
to uctuations in the flame intensity, errors in atomising the sample
solution due to high viscosity, ete..

ln this method, a known concentration of a different element is added
to standard solutions as well as to sample solution. The following example
will dustrate the procedure adopted in internal standard method.

(eg) Estimation of Ca in unknown sample

1. Add kmowm concentrations of strontium to various standard
solutions of Ca and to sample solution,

2. Measure the fame intensity of standard and sample at 227nm for
Ca & 260nm for strontium, simultaneously (since double beam
flame photometer in used}.

3, Plot a graph of Cone. af Ca (Va) Ir / loso for standard solution.

4. Concentration corresponding to Isar / liso of sample is read from
the graph

Interferences

Although flame photometry ia simple in operation, it has some
demerits due to various types of interferences. The following are the types
of interferences seen commonly in flame photometry and the means to
overcome the same are discussed,

|. Background absorption: This occurs due to the sample matrix,

flame iteelf, scattering, absorption by similar allali halides, ete,
‘Remedy, Use of gratings vill avoid or minimise there interferences,
‘Spectral line Interference: = Atomic line Interference, occurs due
to the presence of other cations, which can emit radiation in the
same region of emission by that of the element under analysis,
‘This is also called ax Cation-Cation Interference or molecular
spectral Interference. Por example,

a. Orange band of Ca - 543 - 622nm interference with Na doublet
589 & 589,6nm and Ba line at 553.60
b. Iron 324.7nm interferes with Copper 324.Bam
Iron 285.2nm interferes with My 285.2nm
© Aluminium interferes with emission lines of Ca and Mg
de Ma and X mixturen interfere with each other,
Remedy 1. Extraction of interfering material
2. Calibration. curve of interfering material
3, Use of gratings instead of prisma / filters
. Vapoarisation interference:
Chemical type, which occurs due to presence of ‘
ali the diaocition with ether male a M

in pila to high viscosity (eq) dextrose, sucrose (interferes

Overcome by.
1. Choice of flames, burners, atomisera & adit
Use of Acetylene / Nitrous oxide r thermally
Phosphates, sulphates, añicates, retratado =
2. Additives: ‘
Releasing agents: Add few ppm of Lanthalum/Stronitiuen as
chalet fe mme interference due to POs,
af POs (cation anion » interference Fe re
Tonisaticn interference
More ionisation depopulates neuf
o ral atom both i
exited a
mute. Hence It decreases the sensitivity tha netted =

of
‘iy ioalable lona lice K, Ca, arontium

exited state, Hence it decreases the sensitivity of the method.
Remedy: Add excess of easily ionisable ions like K, Cs, strontium
as suppressants to the sample and standard solution
(100-10004g/mi). These have lower ionisation potential (<7.5 ev),
and hence they are preferentially ionised over the elements to be
analysed.

Preparation of Standard stock solutions of Sodium, Potassium, Calcium
and Lithium

1. Sodium ion solution (FP) - Dissolve 2,4978 of sodium chloride in
1000m1 of demineralined water to give 1000pg/ml of Nat.

2. Potassium ion solution (FP) + Dissolve 1.908 of potassium chloride
in 100ml of demineralised water to give 1000yg/ml of K+.

3. Calcium ion solution (FP) - To 2.503g of calcium carbonate, add
25ml af IM HCI (to dissolve the carbonate), make upto 1000m!
with demincralised water to give 1000yg/ml of Cat,

4. Lithium fon solution (FF) - Ta 5.32g of Lithium carbonate, add
SOml of HCI and make upto 1000ml with demineralised water to
give 1000)g/ral of Lit

Prom the standard stock solutions, working standard solutions for

calibration curve can be prepared by suitable dilutions with demineralised
water.

27, ATOMIC ABSORPTION SPECTROSCOPY
Introduetion
© Principle
+ Components af Atomic Absorption Spectrophotometer (AAS)
% Hollow Cathode Lamp (HCL)
% Burner (with fuel and oxidant)

% Chopper

# Detector

Ÿ Readout Device
* ‘Schematic Diagram of Atomic Absorption Spectrophotometer
+ Applications + Quantitative analysis

+ Comparison of Atomic Absorption Spectrosc
‘vith Flame Photometry (FP) a

Interferences in AAS.

. INTRODUCTION

Atomic absorption spectroscopy deals with the absorption of specific
wavelength of radiation by neutral atoms in the ground state, (The
henomenon is similar to UV spectroscopy, where absorption ef
radiation by molecules acct).

Neutral atoms are obtained by spraying the sample solution of element
sing a burner (like in flame photometer). Specific wavelength of radiation
fs generated by using a hollow cathode lamp. For determination of every
dement, separate hollow cathode lamp is required.

Principle

When a solution of metallic salt is sprayed on ta a flame, fine droplets
fire formed. Due to the thermal energy of the Mame, the solvent in the
droplets evaporate, leaving a fine residue, which are converted to neutral
atoms.

These neutral atoms absorb radiation of specific wavelength, emitted
by hollow cathode lamp (HCL). Hallow cathode lump is filled with the
Vapour of clement, which gives specific wavelength of radiation. For the
determination of every element, hallow cathode lamp is selected, which
fentains vapour of the element to be analysed. Although this appears to
be the demerit of AAS, specificity can be achieved only by the une af HCL.

The intensity of light absorbed by the neutral atoms in directly
Proportional to the concentration of the element and obeys Beer's law over
A wide concentration range, The intensity of radiation absorbed by neutral
Moms in measured using photometric detectors (Photo tube or Photo
maliglier tube),

The schematic diagram of the principle of AAS is given us follows:

Liquid formation of fine
Sample * “droplets = residue

Neutral atoms: |
Measurement of Abo epecko
Intensity of radiation ¢ wavelongthol + formation of
‘absorbed by using wavolangt of neutral atoms.

In AAS, the temperature of the Oume is mot critical, since the
thermal energy of fame is used just to atomise the sample solution to
fine droplets, to form a fine residue and later to neutral atoms. The
exitation of neutral atoms is brought about only by radiation from hollow
cathode lamp and mot by the thermal energy of the fame,

‘The number of elements which can be determined by AAS in far more
when compared to those that can be analysed by flame photometry,

Components of Atomic Absorption Spectrophotometer
N Hollow Cathode Lamp

The las er the source of light in AAS in a hallow eathade lamp, The
cathode, is made up al specie element or alloys of elements or canting
of elements on catbode. When current is applied between anode and
cathode, metal atems emerge from hallow cup and collides with filler gas,
which in normally argon or neon, Due to these collisions, number of metal
atoms are excited and emit their charucteristic radiation. Thi
Characteristic radiation in abworhed by neutral atoms af the same element
in ground state, which occur in the flame, when sample solution is sprayed.

‘The dingram of hollow cathode lamp is given below:

Argon or
Neon gas rio

Sica
> window

Fig 274. Diagram of Hollow Cathode Lamp
In ad do! poso ta vos m rn of Ket wth à mouschromator (ike
* üther apectiophetometers) because this ent gives a radintion

‘with a band width of Inn, wher
Width of 0.001 te DOI, nn 8 the hollow cathode lamp gives a band

„ , the light source should provide a line width less than the
jon line width of the element to be determined.

k
y

The demerit in AAS is that, for determination of every element,

HCL has to be used. Thin can be overcome by using multi-element

amps. (og) Two element lamps like Na/K, Ca/Mg, Cu/Za and three element
amps like Ca/Mg/Zn are available.

Barnes (with fuel and oxidant)
As discussed earlier, the temperature of the flame is not critical, but

“atemisation of sample solution, like in flame photometry is required.

Bumera, with fuel and oxidant as specified in flame photometry are used

“ter Chapter 26).

por

Earlier, the instruments had choppers, which rotate like a fan, allows
alternatively radiation from flame alone or the radiation from HCL and
flame. This produces a pulsating current (signal), which is used to measure
the intensity of light absorbed by elements, without interference by
radiation from the fame itself

In modern instruments, where flameless techniques (electrothermal
technique) are used, the lamp itself is modulated at a frequency, The
‘=xplifier is switched in synchronisation with the lamp and hence acts as
A phase sensitive detector. This eliminates all errors in signals (other than
HCL), hence only the intensity of absorption by the element is measured.

Monschremator

Some elements have a single absorption line (Principal Hine}. But
several elements have more than one absorption line (secondary lines).
Hence it is necessary to select the spectral line for absorption
Measurements. Moreover, it is necessary to isolate the line spectrum of
“ment from that of the emission by the gas in the lamp, or from the
background signal of the flame. Hence a monochrorater which can provide
fed resolution of Inm or less is required, A detailed description of
Monochromatora is presented in Chapter 1 (Page 1-14 to 1-20).

ana

ensity of radiation abserbed by elements, in the UV or vinible
ree 190400} ean be detected using Photometric detectors (Photo
Tube or Photo multiplier tube}. A detailed description of detectors in
presented in Chapter 1 Page 1:21 ta 1-23)

Readout Device

‘The readout device is capable of displaying the absorption spectrum
as well as the absorbance at a specified wavelength (similar to UV
spectroscopy). Beer's law is obeyed over a wide concentration range.

Schematie Diagram of AAS [Single beam)

Fig 27.2, Single beam AAS.

A single beam AAS consists of the components described in detail [as
above) and the schematic diagram is given in Fig 27.2.

‘The sample solution is sprayed onto a flame, with the use of a fuel
and oxidant, using an efficient mixer/burner (as used in a flame
photometer). The neutral atams present in the flame absorb light radiation
emitted by the hollow cathode Lamp (of same element to be analysed) and
passes on through a monochromator {to isolate the required radiation) and
the intensity of radiation absorbed js measured by using a photometric
detector. Thus the intensity of radiation absorbed by neutral atoms is

To eliminate the detector response due to radiation (by the flame
| choppers were used in olden days. In modern instruments, electronic
jces ase used which eliminates such background signal from the Mame.

Fig 27.3. Schematic Diagram of Double beam AAS

‘The two channel double beam AAS consista of two hollow cathode
lamps, for the determination of 2 elements. The schematic diagram is given
la Fig 27.3, Thin type of instrument has the advantages like, measurement
of concentration of two different elements simultaneously and use of
interna] standard in one of the channels, This double beam design
diminates the errors due to fluctuations in the conditions of the Mame,
tke viscosity of sample, temperature of sample solution, speaying rate, etc,
Than double beam AAS offers the above advantages, when compared 16 &
tingle bear AAS.

(terferences

AAS is less Sable to be affected by interferences, when compared to
Flame Photometry. The technique of AAS is especially free from Cationte
interferences. This is because of the absorption of sharp resonance lines
kom halls nda dama However, AAS has the following interferences.

3, Estimation of Magnesium, Zinc, etc in blood.
4, Estimation of Zinc in Zine Insulin injection.
5. Estimation of Mercury in thiomersal solution.
6. Estimation of lead in Calcium carbonate, petrol, etc.

7. Estimation of elements in soil samples, water supply, effluents,
ceramica, ete

‘The following table shows some of the elements estimated by AAS,

‚wurelength of principal absorption line and the lower detection límit,

ppm | | Element | à (om) | ppm |

F
I

Physical interfereneee: as described in Flame Photometry (refer
® 5:36.12). The methods to overcome this type of interference is also

th Anicale Interferences: as described in Flame Photometry (refer
P.26-12}. The methods to overcome this type of interference is also
ren in flame photometry.

€ Scattering effects: This occura due to the presence of high
concentrations of interfering clement. This can be climinated by
tuning a continum ght source (deuterium lamp), in addition to
hollow cathode Lan.

A Tonic Interferences: Such interferences are seen in hot Mames,
‘when the thermal energy of the Mame is aufficient for fonisation,
‘This decreases the proportion of neutral atoms in the ground state. Í y
Hence easily ionizable elements Uke Potassium (2000)q/ml) is Tod
added to the sample solution, which is preferentially ionized than +
the elements of interest,

a

Applicaticas of AAS

AAS de mainly used for quantitative analysis of various elements
present indifferent samples. It ls net used for qualitative analysts, since =
unless separate lamps are used, it is not possible to (deatify various (Parameter / | + | AB '
elements present in the given sample. |_ Characteristics —

7 radiation emitted: > eadlation absorbed

Calibration curve method in used in quantitative analysis, where pmp pra isa EE
various standard solutions of the element to be determined are prepared r en ee |
and the absortiance of each solution is determined. A calibration curve of 5
concentration of elements (Vo) absorbance la made, from which the
concentration of the element in the sample solution is determined. Beer's
law is obeyed aver a wide range of concentration, Low levels of detection
Tr a eee ae sees

‘The following are some of the applications of AAS;
L ru ECS A EE ES

2, Estimation of elements like Copper, Nickel and Zine in food

28, PAPER ELECTROPHORESIS

Introduction
Principle
Components of Paper Electrophoresis
2 Paper (Type & dimensions)
#+_ Electrodes & voltage applied
# Electrolytes / Buffers used
+ Typos of Paper Electrophoresis & Procedure
À Low voltage / High voltage
% Horizontal / Vertical / Continuous

” Factors governing migration of ions
Advantages & Disadvantages
Applications of Paper Electrophoresis

14 INTROPUGTION 59
| paper electrophoresis is a separation technique, where lona af diferent
pris on a medium of paper (moistened with a buffer],
‘he application of a voltage between two electrodes, which are in contact
the paper.
paper electrophoresis ía primarily used to separate mixture of amino
proteins and poptides, although it can be uned for other substances,

Bs À mixture of ions or ioniaable substances is applied on the centre of
previously immersed in a buffer of known ionic strength. This
paper is placed across two trays, Glled with buffer, into which two
des re immeraed. When a voltage is applied across these electrodes
foe loma or foninable substances migrate towards anode or cathode, based
on their charge and other factors, Neutral oc noncionisable substances do
M get migrate. Anionic substances move towards ‘anode and cations move
towards cathode, Ultimately, there is separation of anionic, cationic and
Rondon / zwitterionic substances. The spots / banda which migrate can
be detected by using appropriate spray reagents or visualiing agente ee
in paper chromatography and can be quantiied using densitomet=r Both
Goabtacive analysis ond quantitative analysis can be performed in paper
eectrophoresix.

‘the dectrophoretic mobility, Le. migration of ions / loniasbls
tetes are based primarily os. charge of particles, size and shape of
molecules, the voltage applied, pH and lonic strength of butler used
¡encantos of electrolyte), dilectrc constant and viscosity el medium,
nettes of the paper used in separation and environmental factors
auch as temperature.

ir the charge on the molecule is mare, the migration is faster. I the
change on the molecule is less, then the migration in slower. Bieger
molccules migrate slowty, where as amaller molecules move faster

Based on Stoke's law, the electrophoretic mobil of a molecule can
‘be given by the following equation:

Paper

In Papes electrophoresis, paper la used as the supporting medium.
But in other types, agar gels, polyacrylamide gels (PAGE), starch blocks
er cellulose acetate strips are used as medium for separation. Normally
Whatman® filter paper (Grade 3MM or No.1) of suitable dim Sem
to Sem wiäth) with a length so that both ends of the atrip of paper touches
the buffer solution, kept in the electrode vessels, The paper to be used
de washed with distilled water followed by 0.1M HCl or 0.01M EDTA to
remove impurities,
i) Electrodes & voltage to be applied

The electrodo in the form of a thin wire, in made up of carbon or
platinum. A DC voltage of about 8-15V/cm length of paper is normally
applied, ln Low voltage esis, the voltage acroan two electrodes

is about 100-200V, with m current of OAmAmp per em width or
1.SeaAmp/ strip.

In High voltage electrophoresis, a potential of about 50-215V/em
(Total 10,000V/ strip) la applied across the electrodes,

(li) Electrolytes / buffers uned
Buffers of different pH and onic strength are used in separation

procesa. The pH of buffer to be used. the of de
uk dependa upon the types of compound

‘The ionic strength (15) of the buffer used in paper electrophoresis
alfects the migraticn velocity of compounds. Migration of compounds is
inversely proportional to the joule strength. At law one strength, migration

is faster, but the tanda a
(adhe on tek and appear sed. Urualy onic strengths

‘The following are some of the bulfers used:
1, Barbitone buffer (Veronal buffer) (0.07 mole/Htre, pH 8.6), IS - 0.05
3. Tris-acetate buffer (0.07 mole/litre, pH 7.6]

3. Citrate buffer (0.07 mole/litre, pH 3.0 or pH 6.8)

© Other buffers of different pH and ionic strength can also be used for
feparation, based on the type of compounds.

ype of Paper Electrophoreal (PE)
“© ‘There ure two typen of Paper electrophoresis based on the voltage
Le. Low voltage or High voltage Paper electrophoresis. The following
lable gives the details:
N [ T Voltage/em | Woltage/ trip
Low voltage PE | 8-15V/em 100-300
High voltage FE | 50-215V/em | 10,000V
High Voltage Paper Electrophoresis has the following advantages:
1. Separation is faster, Henee less time is required for separation.
2 Sharp bands are obtained, since there is lens diffusion af banda.
3. As sharp bands are obtained, séparation of closely related
compounds can be achieved.
4. More number of samples can be analyned simultaneously.
However, the technique of High Voltage Paper Electrophoresis has the
fetewing demerits:
1. It ls dangerous to the operator, since high voltage is applied.
2. More heating effects are seen because of high voltage and the paper
becomes dry, '
In most laboratories, only Low Voltage Paper Electrophoresis in being
tried out.
Morizontal / Vertical / Coatinwous Electrophoresis

There ase different types of electrophoresis instruments based on the
design of the instrument. The dingram of these 3 types of instruments are

Horizontal and Vertical modes are used in analytical scale, where an
continuous electrophoresis is used en a preparative scale (Le. A large
quantity of sample mixture is separated into individual compounds and
they are used for different purposes).

‘The principle in all the modes are same, but the design of each

instrument varies,

In Vertical / Horizontal type (Fig 28.1 & Fig 28.2), buffer solution of
known pH and fonde strength is filed in two beakers / troughs. Whatman
fer paper of suitable grade and convenient dimensions are cut (2.5-Sem
‘width und suitable length) and immersed in buffer solution, 10.204] of
sample solution is applied at the centre of the paper and fixed in position.

transparent lid is closed for safety as well as to prevent evaporation
Auffer / solvent, A suitable potential (100-300V, also refer p-28-3) is
sed across two electrodes dipped in buffer solution.

When such potential is applied across the electrodes, migration of

jons and anions take place towards cathode and anode respectively.

jonisable / neutral substances do not move, Hence separation of
unds from the mixture takes place,

N ju this vertical mode, the migration of ions are assisted by gravity
‘ed hence a typical separation takes place in about 6-8 hours, After
jeficient migration, the paper in taken out and dried, to fix the spots /
hands. Then the compounds / bands / spots can be visualised like in
per chromatography. The quantitation of spots can also be carried out
using densitometer.

‘The horizontal mode is similar to the vertical mode, in principle.
However, the paper is placed on a flat bed, as ahown in Fig 28.2, The
we

procedure to be followed is same us that of vertical type, In horizontal
mode, it takes about 12-14 hours for separation.

tinuous eleetrophoresis (Fig 28.3) is meant far preparative
sansa Gene posta mth vola ran € ms oi 5
applied continuously aa the centre of a paper. The application of voltage
eauses migration of samples and hence compounds are separated as
‘banda. Thus each band is made to fall down and pure compounds are
collected in separate containers. The solvent is evaporated and pure
fractions are reused.

Factors governing migration of lona.

The following are the factors affecting migration of ions in Paper
electrophoresis.

1. Charge of less The electrophoretic mobility is directly
proportional to the charge of the molecules (refer equation on
928-2}, which means that mobility of molecules in higher when
its charge is higher, Hence when a mixture is ions with X" and

X" are separated, the band of X?" moves faster than X".

2. Size of the lens The electrophoretic mobility is inversely
Proportional to its size, Le. the mobility in more, when the size is
less and vice versa, The mobility also depends on the shape of the
molecule,

3, Viscosity of the medium: The clectrophoretic mobility is inversely

proportional to the viscosity of the medium (buffers or other
substances used}.

# Voltage applied: Higher the voltage applied, faster the separation
and sharp bands are obtained. However steps have to be taken to
Prevent evaporation of the solvent / buffer, due to the heat
rnerated by high voltage.

5. pH of buffer and Tonic strength; The ionic strength (15) of the
bulles used in paper electrophoresis alfects the migration velocity
al compounds, Migration of compounda in inversely proportional to
‘the jonl strength. At low ionic strength, anigration is faster, but
the separated bands appear diffused. Usually ionic strengths (18)
of 0.05 - 0.5 in used in most separations.

287

| The technique in easy to follow.
“The cost of the instrument is low.

4, Operational costs are less - only paper and bufferle] are used.
4, Number of samples can be separated on a single paper, at a time,

i i eins
Wide variety of ionisable substances such as amino acids, prot
5 nd peptides, antibities, allaloids, ce, can be separated.

The time required for separation is more, Le. 6-8 hours in vertical
mode and 12-14 hours in horizontal mode.

Use of high voltage may bo dangerous, unless precautions are

taken.

of Paper electrophoresis '
Paper is is used mainly for the separation of ionizable
pS , by using tillers of different pit und ion strength, The
y i are some of the pharmaceutical applications Paper
aeetrephoresia, |
1 Separation of amino acids (into acidic or basic or witterionic type):
4 a

2 Separation eins in werum (into albumin, 04 ag. P an
Qe great the percentage of each

component can be estimated using densitometer.

3. Separation of Lipoproteins in serum (in case of hyperlipidemia).

4. Separation af enzymes in blood.

& Separation of allalaids and antibioticn in different samples can be
carried out.

»

29. HIGH PERFORMANCE
THIN LAYER CHROMATOGRAPHY (HPTLC)

+ Instrumentation
+ Stationary phase - types el material - sizes - activation
+ Mobile phases
+ Application of sample
+ Pre-conditiening of chamber
+ Development chamber - Twin trough
+ Development f chromatogra
* Densitormeters - Detectors - UV - Visible - Fluorescence
* Destvatisation techniques
* Automated Method Development
+ Photograph of HPTLC instrument
© Applications
+ Orbers
Comparison of HPTLC with TLC

* Comparison of HPTLC with HPLC

F

INTRODUCTION | |; | +!

High performance thin layer chromatography is an improved,
and an automated form of thin layer chromatography. lt is also
‘pied an flat bed chromatography. It offers high performance separations
à comparison to TLC (Thin layer chromatography). Although the principle
mais similar to TLC, there Es difference ut each level of operation, such
‘a wide variety of pre-conted stationary phases, application of samples on to
HITLC plates using auto applicator, detection of compounds by using various
destometric detectors (UV / visible / fluorescence] and use of software for
ta processing. All of these avances make HPTLC, a superior, expensive
sed high performance technique when compared to TLC,

‘The principle in HPTLC ls adsorptton, where the stationary phase ls a
seid and the mobile phase ie liquid. The stationary phase ts coated either
te aluminium Coll or glass plates of various sizes Before the application of
pie, the plates are activated (nurface), if they are not freshly opened) A
nal) amount fyi) of sample or multiple samples Is applied on bottom edge
the plate uning an auto sampler (applicator).'Thix plate is developed by
Pacing in a HPTLC development chamber made up of glass (eg. Twin trough
chamber), and the mobile phase (a solvent or mixture of solvents) to be used
{a placed in the development chamber. When mobile phase moves up the layer
stationary phase, against gravity, separation of compounds takes place due
lo diference in the affinity of various compounds towards stationary phase. As
Be 3 compounds have the same affinity towards stationary phase, separation
takes place, The time required for this separation is called as development
time, Normally a distance of about 5-15em la allowed for better separation.
The separated spots can be visualized using densitometste scanners - UV /
tibio J fluorescente mode based on the property of the components, The
Analysis can be done qualitatively and quantitatively, using software,

Stationary phase: Frecosted HPTLC plates are available in various
‘sizes, mostly in aluminium foils and sometimes in glass. Sines such as 20 x
‘Mem, 10 x 2bcm, 5 x 20cm or 10 x 10cm are available, A plate of specific
dimension 10cm x Sem can also be cut from larger site plates. These plates
are conind with stationary phases which can work in normal phase ot reverse
phase modes of chromatography.

Types of stationary phase material: Pistes for normal / reversed
phase mode arc available, Mates coated with Silica gel 60 F254, Amina /
Dial / Nude / Cellulose / Reversed Phase materials such as C2, C8, C18 are
available, Depending on the nature of compounds to be separated, stationary
phase can be chosen,

Particle sise: The particle size is typically S-7um, which offers high
‘surface area leading to high performance separations due ta more number
4 theoretical plates when compared to thin layer chromatography, where the
particle aie is about 10-15 ym.

Beale: Analytical / preparative: Analytical scale ls uted for the
‘separation of nanogrum quantities. It la preferable to apply small amount
of sample as a spot for better separation either manually or using auto
applicatos. For analytical separations, the thickness of the adsorbant layer
ls O.1mm-0.25mm. When the quantity of sample to be separated is more,
Separation is difficult. In such case, preparative scale in used. Preparative
‘scale la sed for the separation of larger quantities of sample like 10mg to

ig The adsorbant layer in this technique can vary from O.Senm do 2m
‘thickness,

‘Activation of HPTLC plates: Freshly cut plates from packed anes, do
pot require any activation. But when plates are exposed to atmosphere for
longer periods, moisture and other volatile components are being adsorbed
(on the surface. Hence these plates require activation, Activation la n process
of beating the plates to about 110-120°C for about 30 minutes, when the
‘Adectbed components are remaved Em the epee, a 5

y of separation, Without activation, better separations cannot be

Moblle phases : A wide variety of mobile phases are used, based on the
jure of sample (number and type of samples to be separated), the nature
phase and the complexity of separation. The mobile phase can
be a pure solvent of à tnixture of solvents. Sometimes they are used with or
out addition of small amount of acids / alkalis / salts ax bulfers. When
trough chamber (for diagram, please refer to Fig 14.3 on page 14:6] ía
for development, a volume of 10-18 of mobile phase la sufficient for
slopment. It is preferable to prepare the mobile phase freshly and avoid
the sume repeatedly, an composition of mobile phase can change an
rage, 3-4 component mobile phase is to be avoided, an the composition
be altered when more volatile component evaporates quickly, leading ta
“change in the composition of mobile phase. In case of normal phase mode,
| the solvents are of non-polar in nature. In case of reversed phase mode, the
obents are polar in nature, (For more details an mobile phase, please refer
lo Chapter 14 on TLC),

Application of sample: The sample solution which contains the
npounda to be separated in applied as a narrow band using automatic
epticators, to get better separations Typically solutions of 0.1-1 up/anl
‘Concentrations are applied in analytical scale separations. After the application
ef sample, the solvent ls allowed to evaporate, by keeping the plate in a
À hamber at about 60°C.

b Preconditioning of chamber: Preconditioning of HPTLC chamber la
Required for efficient separations, It la the process in which the chamber
‘ie saturnted with the mobile phase components used for development. This
» Sruares equilibrium is established kn the atmosphere within the chamber
| "ith the mobile phase components, A good approach will be to line the
| hunber with filter paper moistened with the mobile phase before keeping the
“HPTLC plate, Alternatively pour mobde phuse ln the twin trough chamber,
‘Set anide for 30 minutes, place the HPTLC plate quickly and close with the
D 0 00 Kae ale sos Tease A ee

Development tray/chamber: The developinent chamber is made of
ass, like in the case of TLC, But unlike TLC, small chambers are sufficient,
as better separation takes place over shorter distances. A classic example
for the development chamber la twin trough chamber, where 2 plates can
be placed slemultancously in a single chamber and the mobile phase can be
reed, because of unique design. (For diagram, please refer to chapter on
TL, page 14-6),

Development af chromatogram: After keeping the HPTLC plate la ane
of the trough of twin trough chamber, migration of mobile phase takes place.
‘The distance travelled by the solvent front can be shorter or longer. When the
migration distance la longer, there is better separation for similar conditions,
when compared to shorter migration distance, Hence longer migration distance
is preferred. A migration distance of about 5-7 em, with an average of Gem is
preferred in a plate length of 10 cm, Developanent time is typically 1Ominutes
fin contrast to about 30 minutes in TLC). It ts the time required for the
‘sebvent front to mave from the paint of sample application to the lending edge
‘of the plate (almost close to the top edge of the plate}, When the solvent front
reaches the leading edge of the plate, remove the plate from the chamber and
allow the mobile phase to evaporate in an open atmosphere.

Densltometer : Detectors + UV / Visible / Fluorescence - After the
chromatogram la developed and the mobile phase gets evaporated, scan
the plate using densitometer which uses UV/Visible/ Fluorescence mode
depending upon the property of the mixture of compounds, The densitometer
scans the entire chromatogram qualitatively and quantitatively nt the desired.
Wavelengih settings Most commonly, m wavelength of 254nm is chosen, as
majority of compounds have good absorbance at this wavelength,

Dectvatisation techniques (Post chromatographic): Sometimes when
the compound cannot be detected, a spray reagent in used which reacts with
the separated compounds and can be easily detected by using denaitometric

‘The purpose of derivatisation is ax follows:

To make non detectable substances to detectable ones

To increase detection limits

To selectively detect a few components in a mixture

‘To convert non fluorescent compounda to fluorescent compounds

nee»

Automated method development:

During method development, different combinations af stationary and
mobile phase have to be tried In order to optimise the separation of mixture
ol compounds. Nowadays it la possible to have automated ayatersa for
method development using sofware and programming, with inputs based on
a fewer separations.

Two dimensional chromatography: This is applied to separate a
complex mixture, lke in TLC [please refer te the chapter on TLC},

PHOTOGRAPH OF HPTLC

Computer controlled Densitometer/Scanner

‘Sample applicatar (Linomat 5 |

Photographs : Courtesy: ANACHROM (CAMAG)

APPLICATIONS:

‘The applications of HPTLC ix very vast, since whatever be the compounds
‘which can be analysed dy TLC, be analysed using HPTLC technique. Moreover,
ta the technique is much sensitive and email sample volume is sunt for
detection and estimation, various applications are possible In several fields.

The following are some of the categories of applications.

Pharmaceutical Applications:
> Quantitative analysin of drug substances in formulations and biological
fluids
+ Assay of active components and multicomponent analysis
+ Content uniformity in dosage formulations
Presence of impurities in drugs
> Stabilliy tenting and forced degradation studies

> Preservatives » eg, Butplated Hydroxy Anisole, Butylated Hyroxy
Toluene, parabens In formulations

à Phytocenatitvents in plant extracts: Emumples are presented in
Indian Herbal Pharmacopoeia and US Herbal Pharmacoposis,
where estimation of | phytoconstituents In various herbal
products are made. Eg, Sion Wort, Piperine in Piper Nigrum,
Caffiene in Tea/Coffee, Nicotine in Tobacco, ete.

3 Adulteration of plant extracts

> Quality control / authentication / E
extracts (eg) Gartlc extract (Atlin,
ginkgo, ginseng, etc.

nger printing /sereerdng of plant
allicin), valerian, askwagandha,

> Phytochermical analysis af volatile ois 1e. Eugenel in Clove oil

Clinical applications

> ted In pharmncokinetes and metabolism to detect ngs and

metabolites, screening for drugs of abuse.

Forensic applications
Detection and estimation of traces of drugs or poisonous compounds

Conmetle analysis
+ Presence of colouring agente, preservatives, trace materials and
prohibited substances,
Food Analysis

+ Estimation of vitamins, pesticides and quality control of food
substances

> Aflatoxin, Mycotoxins, Pesticide residues in food articles
+ Curcumin - curcumincida in Turmeric powder
+ Sudan - 1,11, 11, IV ete in chills powder / whole chilli

> Other food colours: Erythroaine, Ponceau AR, Carmoisine, Tartrazine,
Sunset yellow FCF, ete at PPh level can be detected.

+ Cholesterol in edibie el
> Safran in Food
Environmental Analyala
++ Detection of polluenta and residues in air/water.
Industrial Applications
+ Process development, optimisation and monitors of chemical reactions
and cleaning validation.
Miscellaneous

+ Coupling of HPLC with HPTLC cas also be done Sor the automatic check
‘of purißied fractions fresm HPLC.

> Coupling of HPTLC with masa spectrometer : HPTLC is coupled with MS

for te detection ef after
Li nel compounds after separation of compounds, leading

wherein a mixture of substances la separated based on their molecular sien,
using a porous matrix of stationary phase and a buffer as mobile phase, The
el oF porous matrix in a cross linked bead that forms a three dimensional
netwark of u polymer, egdextran, agarose, polyucrylamide, ete.

Principle

A group of compounds with different molecular sites (or weights) pass
through a porous matrix of stationary phase, with buffer as mobile phase.
Large molecular size compounds cannot pass through the pores and are
‘excluded from the column faster along with veld volume. Small molecular
weight compounds clute later from the column, as they all have to pass
through the porous matrix. Molecules with partial access to pores ehute from
the column in the order of decreasing size. The amaller the partictes, Inter
they elute. Due to this, separation of compounds with varying motecular
‘ites take place, When the eluents pass through a detector feg. Absorbance),
A chromatogram is obtained, like in HPLC, The following diagram Ihustraten
the mechanism of separation in gel Altration chromatography.

Le he

fi

‘The foigwing la a typical ebromatograra obtained in gel fltraton
In
ah
tou
4
’

Advantages of gel filtration technique

A wide range of porous matrices are available to separate varying
molecular sises

Separations are carried out at room temperature oF colder. The
‘separations are independent of temperature, jonic strength & pH.

‘The technique la useful to estimate molecular alze.
Useful for desalting from large molecules as well ax for separation of
‘coenpaunds

5. By the use of suitable matrix, high resolution separations can be
achieved,

6. Wide range of buffers can be used to stabilise macromolecules
proteins] and also to do isocratic separation.

Inatrumentation

The following are the components required to perform gel filtration

technique:

+ Stationary phase : Gels auch as Dextran, agarose or polyacrylamide,
are used based on the nature of sample.
Eg. Sephadex G-75 (dextran based) in used to separate molecular
‘weights from 3,000 to 70,000 M.W. Components with M.W. above
70,000 are excluded rom the column.
‘Bio-Gel P-150 (polyacrylamide based) ia used for fractionation in the
range of 15,000-1,50,000 MW.
Sepharase 6B -(agarose based) is used for fractionation in the range of
10,000-4,00,000 M.W.
Due to advances in technology, stationasy phase media are available
‘which can separate compounds with MW ranging from 100 to
8,00,00,000.

+ Mobile phase: Butlers, eg, phosphate buffer pH 7, sodium
chloride solutions, ammonium acetate, ammoniue bicarbonate,
ethylenediamine acetate
‘Separations can also be carried out in the presence of ions or co-
factors, detergents, urea, guanidine HCL,

* Elution : Collected as fractions like in column chromatography for
‘preparative separations

* Detection ; On line detection using UV absorbance detector is done
{eepecially 280nm or 21400) and a graph of Elution Volume (ml) Va
Molecular Weight in semi log. paper ía made. For calibration of the
gel in the column, calibrators (proteins of known molecular weight)
like Yeast alcohol dehydrogenase (1,50,000 Dalton»), Bovine serum
albumin (66,000 Daltons), Carbonic anhydrase (29,000 Daltons), and

119,400 daltons) are used and a graph of MW vn elution
ee ered below la platted, For unknown. compounds,
Separation carried out ning the same column of gel, elution volume la
determined and rem the graph, the molecular weight ofeach campound

la determined.
Figure cl calibration curve
1000000:
0000!
MW
40000.
3
Lau y à
Eton vota {mi}
‘Procedure for gel filtration technique

‘Step:

‘Preparation of colema: The required stationary phase porous beads
des the form of powder are made into a alurry with mobile phase and allowed
to become A gel, Sometimes heating may be required, depending upon the
material to form a gel After auffcient time, the column made of glans or
Inest material bs filed to get a packed bed of gel. Typically the column has a
length of about 10cm to 60cm depending upon various factors,

Washing of the column: The packed bed is equilibrated with buller,
‘which (lle the pores la the beads ax well as the space between the beads.
Now the packed bed of column is ready for separation. Although buffer is
‘ot required to lemprove resolution, buffer in sed at the end of separations
fo remove any molecules which ase present in, the column to facilitate next

separation.
na

Loading of the sample: The compounds to be separated, in the form of
solution. la changed into the column, followed by flow of mobile phase with a
specific low rate under gravity (atmospheric pressure),

Etution using mobile phase buffers}: Elution is carried out with
buffer at optimsisn flow rate (eg. 0.25-Sml/min) to give maximum resolution
of peaka and small volume fractions are collected for detection of individual
component. Depending upon the separations, a pressure of up to 350pai
(pounds. per square inch) are used,

Detection of compounds: Detection of compounds can be carried out
by connecting the outlet of column to detector, Usually UV absorbance is
monitored at 2L4rum or 2800m and a chromatogram is obtained.

Collection of fractions, in case of preparative separations (may not be
required in analytical separations}: When preparative separations are carried
eut, based on the response of detector, individual compounds can be collected
separately.

Factors affecting resolution in gel filtration
1, Sample volume
2, Ratio of sample volume to column volume
3, Flow rate of mobile phase
4, Column dimensions
5, Particle sise and its distribution
6, Packing density
7. Pate size of the particles
8. Viscosity of the sample and buffer

Note: Usually buffer composition does not allect resolution but are
required to maintain the nature of compounds during separation.

om) «31. COUNTER CURRENT EXTRACTION "HR"

m of protatna I mixture As tbe molecular wright of each

Ta dlerent, ges of required apecifcation la used lo separate | + Introduction
e pot int pre enpanet. This technique i most rule |
M separation o anal protein, ls proteins, plynucleaides, MA = Principle
perme, small ius parce, peude, alar protein, peptide |
hormenes, monoclonal antibodies, etc.

© Instrumentation

+ Determination of approximate molecular weight : Procedure an
red in detection ia followed. The ge cohumn is caltrated, folomed + Factors alfecting extraction
ty separate of unknown compounds in the same columo. From the Solvent selection,
ak volume, and the calibration graph, the approximate molecular Operating Conditions,

: Mode of Operation
weight I determina. Extractor Type
+ Puriicatios of macromolecules, proteins, ensymen, amino acids, Design Criteria

; When impurities are present, they can also be

Separated, as the moleculas alses of iupurities are diftrent when © Applications
‘eanpared to components of interest. Each fraction a collected separately
= re + Pharmaceutical & others

+ Deasiting of proteins: When salts are used in the separation of proteins
in to fractions, to remove these salta for purification of proteins, pel
tration la uned. Salta travel alower in the gel and these large molecules
are exchuded frors the column faster,

Couster current extraction a method of multiple liquid iquld extraction
technique where separation of components having variable solubility in two
dmiscille quid phases ds achseved,

ln a conventional liquid liquid extraction, 2 components (eg, “a? and "b")
are distributed between 2 immiscible liquids, according to their partition co-
ficients, Sul pure “a” and pure “b" are nat present ln these 2 líquida, even

after reaching equifbrium,

An the counter current extraction, 2 immiscible solvents Bow in an
opposite direction, in multiple stages, equilibrium is established and after
several stages, pure “a” and "U" can be obtained,

In counter current extraction, when 2 components *a* and “b*, having
varying affinity or partition coretBcient, la distributed between 2 immiscfhle
solvente (eg, X and Y) which are allowed to flow in opposite direction,
‘separation of pure “a” and °° takes place, in multiple stages, us described
le the figure I, in the next page,

In the frst stage, when equilirisim is achieved, In container 1, selvent X
Gighter or upper phase) and solvent Y (heavier or lower phase) will have both
components “a” and "b", Of course, based on their distribution coefficient,
let us say, present more in X and "b" la present more in Y, The upper
‘phase (solvent X) la transferred to next container 2, with similar composition
of solventa. Fresh solvent X is added to container 1.

‘Aber achieversent of equilfirium In container 2, now the upper phase will
‘contain less of “4”, due to its low sctubility In X and more of “a”, This upper
Phase then tranaferred to container 3 with similar compasltken of solvents.
Now, the Upper layer of eattainer 1 fs then tranaferred to container 2 and Fresh

‘The lower phase (solvent Y) of container | contains the pure component of “b*.

Fig 1. Diagramatie representation of counter current extraction

else

0

Sen

E

is

The value of “e dependa upon various factors described later In this
chapter, Also, the number of steps required to separate “a* and “t", depends
upon the difference in their distribution coefficient, When the difference
thateieon them fe mae. fier bises bre nt Bit when the difivenre kn’

is lesa, then more steps are

the distribution coefficient between “a” ara!

required. +
Instrumentation

A simple type of apparatus on laboratory scale ls a Craig apparatus
(Fig 2}. This tube has provision for separating upper layer and transferring
to next tube, where heavier schvent ony ts placed, Fresh solvent da added ta
tube 1

‘Stan of Cycle

Fig 2 Craig Apparatus

Models are available to contain about 20-25 tubes, which can be
connected in sequence and used for demo purpose fas given below),

On industrial scale, the design of extractora ía different and depends
upon no, of factors described below:

Factors affecting extraction

* Solvent selection

+ Operating Conditions

+ Mode of Operation

+ Extractor Type and

Design Criteria

Solvent selection, depends upon the type of solute mixtures, Other

factors affecting solvent selection are beiling point, density, interfacial tension,

viscosity, corrosiveness, Anmmability. texicity, stability, compatibility with
product, availability and cost

Applications
+ Separation of components from mvnthetic mixtures,
+ Separation of components from plant extracts,
+ Purlfcation af compounds (removal of lepuritles}

32. ULTRACENTRIFUGATION

Introduction
+ Principle
+ Instramentation
+ Ultracentrifuge - Rotors & Cell
Applications
+ Examination of Sample Purity
+ Molecular Weight Determination
+ Analysis of Associating Systems

+ Sedimentation and Diffusion Coefficients - Detection
of conformational changes

+ Ligand Binding

INTRODUCTION. .

Ultracentrifugation, commonly used to denote Analytical
Ultra Centrifugation (AUC) in uned to analyse biological molecules
(macromolecules such as proteins) in solutions, using specially
designed centrifuges, which can separate Into multimeric staten based
on the sedimentation rate, This produces a molecular weight profile
of compounds, by which multimeric state(s) of that molecule can be
known. (Monomer: small molecular weight compound; Dimer - double
the monomer; tetramer = multiple of 4 monomers, Multimer; multiple
times the monomer). Equilibrium analysis can also be performed, to
provide dynamic equilibrium information Le, association/dissociation
rates of the multimeric states.

‘There are 2 types of analytical ultracentrifugation, le,, Sedimentation
velocity AUC and sedimentation equilibrium AUC.

Sedimentation Velocity analytical ultracentrifugation (SV-AUC} is
performed to obtain sedimentation coefficient values and the relative
percentages of manemer/multimer components and aggregates observed
in the given samples,

Sedimentation Equilibrium analytical ultracentrifugation (SE-AUC)
in used to asseas the interaction and dissociation between sample

components,

Principle

When a mixture of proteins (macromolecules) in solution is
centrifuged under high speed (40,000 - 60,000 rpm, rotations per
minute), a centrifugal force of 2,50,000g (g=gravity) is achieved, Due
to this, sedimentation and separation in to multimeric states occur. Ig
masa representa a weight of 250kg under this high rpm conditions, A
profile of molecular weight of compounds takes place and they can be

analysed in transparent cell (online) which is present in rotor, based
‘on the measurement of absorption or refraction.

‘The advantage is that, this method in capable of separating wide
molecular weight ranging from several hundreds (eg. Sucrose) to
several millions (eg. Proteins, carbohydrates, nucleic acids and several
large molecules), The substances ahould have different absorbance
or refractive index compared to that of solvent. Sedimentation
equílibrium methods require only small sample sizes (20-120 iL} and
low concentrations (0.01-1 g/L}, Sedimentation depends upon size
and shape of the protein. The sedimentation process is governed by 3
factors = gravitational force, the buoyancy and hydrodynamic friction,

Instrumentation

‘A typical analytical ultracentrifuge (Beckman-Coulter) is a specially
designed equipment which has a rotor which can rotate up to 40,000-
60.000rpm (rotations per minute) which produces up to 2,50,000g
Ig=gravity). The cell can hold a sample volume of 100-4004, between
transparent windows, used to pass UV / visible light for detection,
based on the property of compounds, For example, for aromatic amino
acids, a wavelength of 280nm is used and for peptide backbone, a
wavelength of 230nm can be used. Based on the compounds to be
analysed, detection also can be done using laser interferometer based
on refractive index difference between solvent and that of compounds.

A schematie dingram of analytical ultracentrifuge is shown in next
page:

Figure 1 Schematics ol he anaiytica utracentitugation detection

The below mentioned diagram shows a plot of different zones va
absorbance,

«Examination of sample = Sedimentation methods
allow determination of purity, assessment of large and small
‘contaminants, size distribution in poly disperse samples, integrity
of native structure, ete.

+ Molecular weight determination: For proteins, nucleic acid and
carbohydrates in solution, molecular weight can be determined at
very low concentrations using small volumes of samples.

+ Analysis of associating systems Sedimentation is valuable
in studies of the changes in molecular weight when molecules
associate to form more complex structures,

+ Sedimentation and diffusion coefficients Detection of
conformational changes : The overall size and shape of a
macromolecule or complex in solution can be determined.

+ Ligand Binding: Absorbance optics are particularly well suited
to studies of ligand binding, because of the ability to distinguish
between ligand and acceptor.

33. RADIOIMMUNOASSAY (RIA) .

+ Introduction

© Principle

© Merits and demerits of RIA
© Steps followed in RIA

+ Detailed Procedure in RIA
+ Applications of RIA

ELISA

* Advantages of ELISA

The following dingram thows the relationship between the
concentration
— of unlabeled antigen and radicar ofthe complex,

RIA (Rediolmmuncassay) in the technique used for the estimation of
any nd T ng 1a BERGE D engendre

amd moanuring the Tadioactivity of the resultant labeled antigen-antibody

complex. In this reaction, Inbeled aa weil as unlabeled antigen competes
‘with antibodies of apecific quantity. The free antigen (unlabeled) and antigen
‘antibody complexes ure separated out before the estimation of radioactivity.

‘Principle

N he principle of RIA in based on the antigen-antibody reaction, In which

radio labeled antigen (exogenous) competes with endogenous antigen for the
limited sites of the specific antibody against the same antigen, The radio |
labeled antigen should have wimilar biological activity and immunogenicity
ike that of native antigen.

L Migh Specificity
Antibody + Antigen + Labeled Antigen + Antigen -Antibody + rar > mad PER there la no interference from other
Labeled Antiges-Antíbody + 2
j SEE up 10-1
Antigen + Labeled Antigen de Leido en ed
The free antigen and labeled antigen are separated and washed out, a High Precialon (repeatability of the
The radioactivity (typically “9 or MC or 7H) of the labeled antigen-antibody e eck eran
comple in meaauced.uning scintillation counter or farsa counter and the es ele) variety of compounds in various Beide —
‘measured radioactivity is inversely proportional to the concentration of ligand immunology, " aneslogy, epidemiology and clínicas
(antigen). More is the concentration of antigen, lesser is the radioactivity of |
the comples and vice versa. Desserits of RIA
|
Diapramatically, the above reaction is represented as follows: | 1 Special expertise and safety precautions are required,
(Wb = antibody: Ag = antigen to be measured: * = radio labeled) 4 materials are used and dispose, M radioactive

2
Expentve compared to other methods, at radiación =

4 Ada Node!
¿=D + D I Diet X Di 3 Development of sei antibodies othe agen,

‘Steps followed im RIAs

1." Preparation and characterization of antigen fused as calibrator)
The antigen whose concentration ia to be meanized, should be
available in pure form to be used as calibrator. Also itis required to
be sed to create antibodies, which are used in the assay process, to

2. Radio labeling of antigen: This fs also called as tagging where the
antigen fs tagged with radioactive element like 11 oF *C or 1, to be
detected usleg gamena counter or scintillation counter,

3. Preparation of specific antibody: Specific antibodies are produced by
injecting the antigen in mouse, rabbit or guinea pig and collecting
and purifying the antibodies. In case of drugs or some compounds
eg Morphine, steroids, hormones}, which are not antigenic, they
are combined with albumin, to become antigenic, which leads to the
production of antibodies in serum.

4. Development of the assay methodology. (described under detailed
procedure)

5. Validation of method using parameters like senaltivity, specificity,
precision, recovery, Bneurity and dilution,

Detailed procedure in RIA *

‘The assay is curried out usually in weil plates [typically 96 wells plate),
‘where the inner surface of well is coated with antibodies. Radioactive antigen
is added to all these wells, which forms a complex with antibodies. The
calibrator (unlabeled antigen of known concentration) is added to multiple
‘wells. Antigen of unknown concentration (ample) is also adéed to one of the
wells, Competition between labeled and unlabeled antigen takes place, due
lo listed binding sites present in antibodies. Radio-lnbeled antigen bound
to antibody is separated from unbound mdio-labeled antigen by precipitation
| chromatography / gel fltralion, etc, This leads to displacement of radio
labeled antigen by unlabeled antigen from the antibody complex formed,
Añer incubation time, the plates are removed and washed, This remaves
Ina mil and unbound entices. The well pieten ore then masse Dr

———

of free antigen, lesser is the radioactivity in the comple. rom tha graphy af
radioactivity vs concentration, the unknown concentration of antigen can be
determined.

Applications of RIA

> RIA has been used to may plasma even of
Most of hormones: insulin in human plasma, --HCG in females,
‘vasopressin;

> Digitoxin or digoxin in patients receiving these drugs;
+ Certain abused drugs, eg morphine;

> Presence of hepatitis B surface antigen (HBsAg) in donated blood, HIV
162, etc.

> AnthDNA antibodies in systemic lupus erythematosus (SLE)
ELISA (Ensyme Linked Immuno Sorbent Assay)

As maioactivity materials are difficult to be handled and expensive, the
RIA ls replaced by ELISA technique, where specie ensyme is used. Hence

there la no requirement of radioactivity measurement and detection ln by
colorimetric method.

Advantages of ELISA technique:
1. No tadionetivity in used
2. Detection can be done In nanamoles (highly sensitive),
Method description:
‘The ELISA technique Is used to identify a protein as well determine the

concentration of proteln. The ELISA technique combines the specificity of
antigen-antibody reaction as well as the sensitivity of enzyme assays,

‘An ELISA de a 6 atep-pencedure:

4. Micre titre plate wells are coated with antigen,
All unbound sites are blocked to preverst false positive resulta;
‘The antibodies, specific to the antigen are added to the weils;
‘Anti-mouse IgO conjugated is added to an enayme;

Reaction of a substrate with the crayme to produce a coloured product,
thus indicating a positive reaction.

peep

Sebematie Diagram of ELIBA test

os
a md y eras
mn
‘seme
‘Types of ELISA essaye

Direct method, Indirset method and sandwich method

‘There are 2 variations in the ELISA method. The method can be used
to determine the antigen for a specific antibody or to measure antibodies for
à apecife antigen,

‘Applications of ELISA.

2 ALIEN has eneofthe moet important analytical ol, for the determination
of protein an well aa its concentration.

+ ELISA la commonly used for the following serology assays like Hepatitis
Rene AE

B &C,RIVIGT, VORE (called as RPR-Rapid Plasma
—

Other applications are listed below; > oor mas

+ Most of hormenes: eg. Testosterone, progesterone,
‘Insulin in Seman plasma, -B-HCG in females, vasopressin;

> Digitoxin or digonin in patients receiving these drugs;
+ Certain abused drugs, eg, morphine;

+ Presence of hepatitis B surface antigen (HBsAg) in donated
Hepatitis A & C virus; HIV 1 & 2, ete. ig ra

> AnthDNA antibodies in systemic bupus erythematon
Immurioglobulins IgG, IgA, IgM, etc. en

+ Pharmaceutical and other related Applications

The 6 Parties {voting members) are the founder members of ICH
which represent the 3 regulatory bodies and 3 rescarch-based induntey in
the European Union, Japan and the USA. The 3 regulatory bodies are EU
(European Union), MHLW (Ministry of Health, Labour and Welfare, Japan) and
USFDA (United States Food and Drugs Administration), The 3 research based

Association], and PHRMA (Pharmaceutical Research and Manufacturers of
America).
PROA

/=\
SES

==

£ &
‘The 3 observers (non voting members) are WHO (World Health Organisation),
EFTA (European Free Trade Association), and Canada (represented by Health

Canada}, This important group of non-voting members mets as a link between
the ICH and non-ICH countries and regions.

[merma]
[Secretariat]

EN
€

ICH ls operated via the ICH Steering Committee, which is supported by
ICH Co-coordinatorn und the ICH Secretariat, The ICH Secretariat operates

Grup) and Discunion Group mectingn. The ICH Secretariat alo pts
kamistative support for the GCO (Olebal Co-ordination Grup) and Med DRA

(Medical Dictionary far Regulatory Activities)

At the time of ICH Conferences, the Secretariat is responsible for the
technical documentation and for Maison with the speakers for the Conference.
COnpeniatonal aspects of the Confereoces are handled by the industry and
regulatory partes in the country where the Conference takes place.

There ls a ICH steering committee which decides the policies and
procedures of CH, selecta topiea for harmonisation and moniters the progress
ef harmonisation initiatives, The Steering Committee meets at least twice a
year with the location rotating between the 3 regions.

Since the beginning, euch of the 6 co-sponsors has 2 seats on the ICH
Steering, Committee (8C) which oversees the harmonisation activities. IFPMA
provides the Secretariat and participates aa a non-voting member of the
Steering Committee.

Fundamental to the senooth running of ICH has been the designation, by
‘tach of the 6 co-sponsors, of an ICH Coordinator to act as the main contact
peist with the ICH Secretariat and ensure that ICH documents are distributed
to the appropriate persona within the area of their resporsibiity.

Each party has also established a Contact Network of experts within
their own organisation or region in onder to ensure that, in the discussions,
they reflect the views and policies of the componsor they represent. The
way in which this network operates diera according to the administrative
structure of the party concerned.

Due to the structural diferences within the EU and MHLW, ICH Technical
Coordisatars alto designated from the EMEA (European Medicines

respectively, They support the ICH Coordinator and facilitate every action
of the Steering Committee members i the region, mainly by applying their
scientife knowledge. Their roles include acting as a contact point between:
the experts within EMEA and PMDA and the ICH Coordinator at the main
regulatory body, and as a contact point with the ICH Secretariat.

Expert Working Group (EWO)

la a group that discusses the need for harmonisation within

. specific

eisentiße domain. The outcome of brainstorming sessions la recornmendations
for topics for ICH harmonisation, for Steering Committee consideration.

After consideration of a Concept Paper, the Steering Committee
sequent the development ola Business Pan, cuinng the cota and benefits
‘of harmonising the tople proposed by the Concept Paper. The Business Plan la

complementary to the Concept Paper and focuses in
: particular on regulatory

‘The 5 Steps in the development
= and implementation of ICH guidelines

Step 1. Building scientific consensus

‘Step 2. Agreeing on a draft text

Step 3. Consulting regional regulatory agencies
Step 4. Adopting, harmonized guidelines
Step 5. Implementing guidelines in ICH regions

‘ QUE Evaluation and recemmendation of pharmacopoeial von

ICH Guidelines: testa for use in the ICH regions Now.2007
guidelines developed in 4 area (as of nom): QSARI) Viral safety evaluation of biotechnology products
The following ase the derived from cell lines Truman or anal orig Sep.1999
Topic Document Code 958 Quality of biotechnological products: Analysis
of the expression construet in cella used for
Lo Quality A Qi to Q10 Production of r-DNA derived protein products Now.1995,
. Qe Quality of biotechrabogica) producta: Stabalty
2: 8 + 831089 testing of biotechnological bislogieal products Nov.1998
3 Bir 2 ELwELS Q5D Derivation and charactesiaation of cell substrates
2 used for production of biotecnología /biologícal products Jul,1997
4, Mulídisciplinary =: MI to M4 OSE Cocparabôlty of bicerchnotogieal/bislogieal products
subject to changes in their manufacturing process Nov.2008
For each of these 4 areas, the guidelines developed and reached step 4 ds = vet
guidelines added to this Bat in future) ‘Specifications: test procedures and acceptance
tees un (leu (more oe ‘teria for new deu substances and new
drug products ; Chemical substances 001999
QUALITY que Specifications: test procedures and acceptance
Document Tide pag certerin for biotechslagical/ biological products Mar. 1999
. ar Good Manufacturing Practice Quide for active
QUAI Stability tating of new drug mbstances prarmacentical Ner:2000
ac products (2% Revision) am
| SRI) ‘Pharmaceutical development Nov 2008
QUE Suit test Photostablty testing of
‘pew drug substances and producto Rov 1906 Li Quality Risk Management Nev.2008
QC Baby testing for new domage forma Nov 1996 0 Punmaceuties] Quality Systens Jun 2008
OID Bracketing ard matrbelng designs for stability testing 1]
of new drag substances and products paa ner
Fa SIA Guidéline sa the Need for Cascinogeskeitg
QUE Evaluation for stability data ied stadies of Pharmaceuticals Nor. 1995
AN O Validation of analytical procedures: Tent and Método — Nov | m veis tr Carcinageniity of Farmer ul 1997
SS SR i ng ect jé | SIE Dose section for Carcinogenicity Seudien of
BRD Impurities in mew drug products Jus.2006 Pharmaceuticals Mar 2008

RA Impruritien: Guideline for residual solvents Teb.2009 | SA Guidance on specific aspects of regulatory

sm

STA

Ost 194

Sep 1998

Now 2008

Jul 1997

Nov 2000

May 2008
Sep 2008
Get 2008

Get 1994

Det 1994

Feb.2001

Nov 2005

a
ul

SESELSCE

eu

an

E

Hs

mé

Pharmacoriglance planning +
Structure & content of enical study reports
Dose-Htesponse information to support drug registration
Eine dnctuen in the acceptability of Sure clinical data
‘Ocod Clinica! Practice: Consolidated pullin:

‘Studies In wupport of special populations: Geriatrics
General considerations fer clinical trials
Statistical princspies for chaical triala

Choice of control group and related lasues
An clinical triada

‘Clinical investigation of medicinal product
in the pediatric population

Principles for clinical evaluation of new
draps

‘The Clinical evaluation af QT/QTe interval
probeta

for non-antéarrhythmie
Definitions foe Genomic Blomaricers,
Wharmacugenomics, Pharmacogenetics,
Genomic Data and Sample Coding Categories
Genomic Biomarkers Related to Drug Response:
‘Content, Structure and Fermat el Qualification
‘Submissions

MA ISR (RAI Riectronie trunamdasion of individual case

Nov 2004
Now 1995
Mar 1994
Mar 1998
May 1996
Jun 1993
dul 1997
Feb 1998

Jui 2000

Maz 2000

May 2005

Now 2007

Fev 2001

MA Cr in ceca ma ut Mor 2000
Mail el ow 2000
Mn ne urn
wane me ne

‘be downloaded
‘The guideline document on each of the above topic can

from the ICH website (reference given at the end of thin chapter). As these

documents are veluminor», they are nat Ineluded bere, and readers are

ugpeied wo reer individual guideline for further rending, a al the guidlines

may not be applicable to all the readers, Same of the more usefil guidelines
from a atudent perspective are as follows:
QU) Vaisation of Analytical Procedures:

‘Test acc Meibodelogy How 2005

o Good Mamufacturing Practice Guide for

tive Pharmaceutical Ingredient We

Qi Pharmaceutical Development ov 2008
09 Quai Wink Manage Now 2005
QUO Phermaceutienl Quality Syrie Jun 2008
May 1996

RG (NI) Good Clinical Practice Consolidated Cuuidelime

‘The ICH process has achieved success because it In based an scientiße
consensus developed between industry and regulatory experta and because
of the comesdtment of the regulatory parties to implement the ICH tripartite,
harmonired guidelines and recommendations,

Carrest status on harmonisation activities

‘The ICH Steering Committee and other interested parties have agreed 10
continue in their comsnitment to purme future harmonisation activities.

ICH han been ‘wuccessful la Achleving Harmonisation, initially of
technical guidelines and more recently on the format and content al
registration applications, All parties agree that there is a need to maintain
this harmonisation in the interest of the patient and public health to prevent
unnecessary duplication without compromising the regulatory obligations
cof safety, quality and efficacy. New approaches to the maintenance of the
products of harmonization are needed. In addition, further harmontsation
activiica should be continued in a focused manner.

» Implementation and monitoring: The current magnitude of successful
harmonisation actions and the need for these to remain current in a
rapidly changing international environment calls for focusing more effort
‘on the implementation and monitoring of ICH commitments. These will
he key to the success, in particular, of the Corman Technical Document
(ETD), in which the creation of implementation groups or task forces.
‘will be important.

+ Selection of new topics: The Steering Committee agrees that it is
important to continue with the work of harmoëlestion after ICH 5,
provided that the selection of possible new topics continues to be
carried out in a systematic and focused manner. Experience with recent
harmonisation activities have identified a need for an even more careful
advance examination of a potential topic far future harmonisation in
order to ensure that it is feasible, able to be implemented tn a timely
manner, nnd provides a high added value for all of the ICH partners, New
activities will moat Weely be in arcas where there are mew technological
advances, new innovative medicines, or in post marketing arcas, Tapie
aciection should be preceded, where necessary, ty a feasibility stay
and/or a seientihe pre-screening.

+ Recent interactions between the regulators involved in ICH have identified
post marketing activities ax a future area where increased regulatory co-
operation can help to contribute to the enhancement of the protection
of the health af citizens on a more international basis.

timely introduction of new medicinal products, and their availability
to patients;

à. To contribute to the protection of public health from an International
‘perspective faded upon revision in 2000};

A To monitor and update harısonlsed technical requirements leading
ko a greater mutual acceptance of research and developisent data;

4. To avoid divergent future requirements through harmonisation of
selected topics needed as a result of therapeutic advances and the
development of new technologies for the production of medicinal

6. To facilitate the dissemination and communication af information
en harmoniscd guidelines and thelr use such as to encourage the
implementation and integration of common standards

Reference for further reading:

e Ju og

‘Absorption Filters, 1-14
Absorption spectroscopy, 1-5
Accuracy, 23-1
Activation of TLC plates, 14-4
‘Adsorption chreseatography, 13-1
Amperomnetrie titrations, 12-1
Asphiprotic solvents, 19-3
Anion exchange, 16-2
‘Soden

oF
Anti-stokes ftuorescence, 3-4
Apretic solvents, 19-2
Argon ionisation detecter, 17-10
Ascending development, 15-4
Asymmeiry factor, 17-17
Atomic absorption
spectrophotometer, 27-5
Atomic spectroscopy, 1-4
Auzschsome, 1-27
Barrer Layer Celis, 1-25
Base peak, 5-5
Baihorhreeic ahift, 1-28
Beers law, 1-10
Bending vibrations, 53
Eiamprrometay, 9-17
farmers in flame photometera, 264
Caleen mixture, 22-7

Calibratian curvo method, 1-31,
113, 26-9

Capillary column in GLC, 17-6
‚Carrier gan, 17-3

MGM coman, 10-5

Cerric ammonieis sulphate, 20-2
Chelating agent, 22-2
Chemical quenching, 3-10
Chemical abit, 6-7

Chiral chromatography, 12-3
Chromatography, 13-1
Chromogenic agent, 1:28
Chromephore, 1-27

Colsional deactivation, 3-2
Collislonal quenching, 3-10
Colorimeters, 1-13

Colorimetry, 1-8

Calama chrumatagraphy, 13-4
ohms lo OLE, 17:5
Columns in HPLC, 18-9
Commercial potentiometer, 9-11
Comparison ef AAS with PP, 27-8
Compleig agente, 22-2
Complesermeteie rations, 22-1
‘Conductametric titrations, 10-8
Conductometry, 10-1
‘Conjugation, 2-4, 35

Counter current extraction, 13-2
Cross conjugation, 29

Current voltage curve, 11-2

Dead stop end technique,
CA mr

Demasking agents, 23-10
Demineralisation el water, 16-9

[Derivatisation La GLC, 17-19
Descending development, 154
Deshielling, 6-6

Detecting agent, 148, 155
Detector de HPLC, 18-10
Develepinent tank, 14:5
evista from Beer's law, 1-26
Diameistien titrations, 21-1
Difirenalating elect, 10-3
Difracten Grating, 1-18

Deuble beum filter fuorimeter, 3:13
Double beats Name
mpectropbatemeter, 26-7

Double bean spectrephatometer,
125, 27 ö

Dosbiet slate, 32
Dropging mercury electrode, 11-3

E (0% tom), 1-2
Edge effect, 14-4
EDTA, 22-3

Efficiency al column, 17-17

Electromagnetic Radiation, 1-3
ee ee ee

Electron impact Mass Spectrometer, |
a '

Electron Spin Resonance, 7-1
Electronic: spectroscopy, 1-5
Electronic transitions, 2-3
Biectrophoretic mobility, 28:2
Etuste, 13411

Ehuent, 13-11

lution, 13-11

Emission spectroscopy, 1-5
End abserption, 2-5

Energy levels, 23

Energy of radiation, 1-3
Equivalent conductivity, 10-3
Ervers, 23-1

Excitation process, 1-6
Extended conjugation, 2-4
Fabry-Perot Filters, 1-15
Filters, 1-14

Pinger print region, 57
Flame lonisation detector, 17-10
Plaine phetometer, 20-6
Flame photometry, 261
Plame spectrophotorseter, 267
Phusreseest indicators, 3-18
Fluorescence types, 34
Fluorescence, 3-2
Pluorimetry, del

Plow meter, 17-4

Premeaney, 1-4

of values, 74
Gas ehresatography, 17-1

Gas liquid chrematogeaphy, 17-1
Gas solid chrematography, 17-2

PURE SE

Gel permeation chromatography, 13-3

Glass Cells,

Glass electrode, 9-1

Glass plates,

Gradient bation technique, 13-10

144

Gratings, 1-18

Group frequency regios, $7
Half serve potential, 11-2

ETF, 17-16

Hollow cathode lamp, 27-3
Horisnntal devebopenenit, 15-5

HPLC pump,
MALE, 184

187

HPTLG, 14-12
Hydrogen Electrodes, 9-4

‘Hydrophilic mobde phases, 153
Hydrophobic mobile phases, 154
Hyperchromic effect, 1-28

Hypectsromie effect, 1:28
Hypuschressie ahif, 1:28
llkcovic equation, 11:8
indicator electrode, 9-7

Infrared Spectroscopy, 5-1
Injections devices in OLE, 17-5

Injreraen ln

HPLC, 16-8

Anterferentes; 26-11, 27-6 °°
Interferenee Filters, 1-18

Interna) standard

11-8, 18-13, 26-11
fon exchange capacity, 167
Yon exchange chromatography, 16-1
Jon selective electrodo, 9-9
onic product al water, 10-6
Joue strength, 28-3
Incas point, 1-28
Inocratie ehition technique, 13-10
tsothermal pengramzning, 17-7
Katharecieter, 17-5
‘Meto-enal tautomerism, 2-12
Kiystreo, 7-3
Lamberto Lane, 1411
Laminar flaw burner, 26-4
Leveling effect, 19
Youd, 222
Limiting current, 115
Lithium methoxide, 19-11
Mel prak, 895
M+2 peak, 8-5
Magnate spectroscopy, 1-5
Maskdeg agents, 22-10
Masa peak, 85
Masa Spectroscopy, 8-1
Mass Spectrometers, 84
Membrane selective elsetruden, 9-9
Microwave ‘onidgr, 7-3

Migration current, 11-514! mont
Miscallasioun techniques, 1-2
Malar conductivity, 10-3
Malecular spectroscopy, 1-4
Manschrumatern, 1:14

Mordant black 11, 22-6

Murexite, 227

‘electrons, 2-2

Natural Sequency of vibration, 5-2
Mephelometes, 46

Nephelametry, 4-1
Mephelecurbidimeter, 4-7

Mermat equation, 9-4

MR spectrophetosseter, 6-5

MR Spectroscepy, 6-1
Yoo-Aqvewus izations, 19-4
Normal phase, 18-3
Octadecyisilane (ODS) column, 14-15
Onis ins, 194

Paper chromatography, 15-1
Paper electrephoresis, 28-1
Partition chromatography, 13-2
Partition coefficient, 17-2

Parier column chromatography,
1313 il

Perchlerie acid, 10:5
PL meter, 941
Phosphotescenet, 3-2
Phot multiplier tubes, 1-22
Paste tubes, 1-22

Phase Cells 1-29
Photoreeteie Detectors, 1-21
Physical methods, 1:2

PA fe electrons, 2-2

pka determination, 2-9
Platinum electrode, 103
‘pM indicators, 225
Polarographée maxima, 11:3
Polarography, 11-1
Patyilentate, 22-2
Polystyrene Cela, 1:21
Potassium methexide, 19-11
Potentiometric titrations, 9-12
Potenticmetry, 9-3
Frecesalonal frequency, 6-2
Precision, 23-1

Preparative TLC, 14-12
Pretreatment of solid support, 17-13
Principle in Colorianetry, 1-9
Prism monochromator, 1:17
Protegenic solrenta, 19-3
Protopiilie solvents, 19-3
Pump, 16-7

Quarts Cells, 1-21
Quenching, 3-9

Radial development, 15-5
Radioactive metheds, 1-1
Redox indicators, 20-3

Reference standard dí ESR, at
Reference standard in NMR, 6-7
‘Reflective type prisme, 1-18
Relractive type Prism, 1:17

Regering af in chan rl,

Reinforcesnent, 1-18
Relaxation Process, 1:7, 6-4
Residual current, 11-5
Resins, 16-3

Resolution, 17:15
Resonance fhuorescence, 3-4
Retastion time, 17:14
Retention vobame, 17-14
Reversed phase, 18-4

Rf value, 149, 18-7
Hheodyne injector, 18:9

Rm value, 149, 157
Rotameter, 174

ag pairs ice decode,

RP-14 column, 18-15

RPA colin 18-15

Rx value, 149, 15-7

Sigma (e) electrons, 22

Sample cells, 1-20

Saturated Calomel Electrode, 9-6
‘Seartered light, 4-2

‘SCOT columns, 17-6

‘Self quenching, 3-9

r

Separation facter, 17-8 0
Sequesteriag agent. 222

‘Shielding, 6-6

Silver-Silver chloride electrode, 96
Simple potentiometer, 9:9

‘Siigle bean colorimeter, 1-24
Single beam filter usrimeter, 3-12
Single bras UV, 27

Singlet excited state, 32

Singlet ground state, 3-2

dise exclusion chromatography, 13-3
Soap bubble meter, 17-4

‘Sedium nitrite, 21-3

Sobres degassing in HPLC, 168
Specific conduetivity, 102

‘Specific resistance, 10-2

Spectral matehiog, 5-7

aditition method,
11-9, 26-10

‘Static quenching, 3-10
‘Stepwise Fluorescence, 3-5
Stoker Quorescence, 34 "
Stretching vibrations, $3
‘Talling of peaks, 17:14, 17-17
Temperature programming, 17-7

‘Visualising agent, 148, 15-5
Wavelength, 14

Ware number, 14, 53

‘Werner's co-erdination mumber, 22-2

438 of nahe!

ur berm

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