column chromatography ppt

COLUMN CHROMATOGRAPHY
Under the guidance of:
Mr.Ch.Devadasu
Assistant Professor
Dept. of PA&QA
Submitted by:
J . Ramesh
Regd No:12AB1R0088
IV/IV B. Pharm.,
VIGNAN PHARMACY COLLEGE
(Approved by AICTE, PCI & affiliated to JNTU-K)
VADLAMUDI, GUNTUR DISTRICT – ANDHRA PRADESH, INDIA PIN NO: 522213

CONTENTS
 History & Introduction to chromatography
 Classification of chromatography
 Definition of chromatography
 Types of chromatography
 Requirements for column chromatography
 Factors affecting efficiency of column
 Applications
 Conclusion
2Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

 A group of techniques for the separation of the compounds of mixture.
The term chromatography was coined from two greek words
kromatos-colour
graphos-written
 In 1906 the Russian scientist M.Tswett separated different coloured
constituents of leaves by passing an extract of the leaves through a column
of calcium carbonate.
HISTORY
“Meaning Colour Writing”
Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213
3
INTRODUCTION

DEFINITION:
 M. Tswett (1906) defined chromatography as The method in which
the components of a mixture are separated on an adsorbent column in a
flowing system.
 The international union of pure& applied chemistry(IUPAC)definition of
chromatography:
“Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary, while the other(mobile phase)moves in a definite
direction”.
INTRODUCTION
4

 The stationary phase may be a solid or a liquid supported on a solid (or)
a gel
-May be packed in a column eg: column chromatography.
-Spread as a layer on a glass/aluminium plate eg: TLC, HPTLC.
-Distributed as a liquid film eg: GLC.
 The mobile phase may be a liquid/solvent/mixture of solvent or gas.
Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 5

chromatography
Liquid
chromatography
Gas
chromatography
Liquid -solid
chromatography
Liquid-Liquid
chromatography
eg: GLC
GSC
eg: column adsorption
TLC,HPTLC
HPLC
eg: column partition
Paper partition
HPCPC
Mobile phase: GasMobile phase: liquid
Classification of Chromatography A/c to physical state of mobile phase
6

Classification based up on physical nature of stationary phase
Adsorption chromatography Partition chromatography
eg: column adsorption
Ion exchange
Size exclusion
TLC,HPTLC,HPLC
eg: column partition
Paper partition
HPCPC
GC(GLC)
Stationary phase: solid Stationary phase: liquid
7

Classification based on polarity of stationary phase & mobile phase
Normal phase chromatography Reverse phase chromatography
Stationary phase: polar(silica gel)
Mobile phase: non-polar
(n-hexane)
Stationary phase: non polar
(Octadecylsilane or C18)
Mobile phase: polar (Acetonitrile,
water)
8Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213

COLUMN CHROMATOGRAPHY
Introduction:
 Column chromatography is a separation technique in which
components of mixture is separated by using a glass column packed
with stationary phase and the liquid mobile phase flowing
continuously through the column.
Principles of column chromatography:
Column adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography or Gel permeation chromatography:
Affinity chromatography
9

Adsorption chromatography:
 The principle underlying the separation of the compounds is
adsorption at the solid-liquid interphase.
 solid stationary phase like silica gel and liquid mobile phase is
used.
 In column chromatography the interaction between
adsorbent & component is usually reversible.
 The various components will move down the column
until, they are arranged in order of their affinity move down
the column at a faster rate than those with the greatest
affinity for the adsorbent.
10

The separation is based on differences in affinities towards the stationary
phase.
Those compounds which have greater affinity towards stationary phase
will move slower rate when compared to compound with less affinity.
 In straight-phase chromatography, four commonly used solvents are:
hexane; dichloromethane; isopropanol; methanol
(Increasing strength)
In reverse-phase chromatography, four commonly used solvents are:
water; methanol; acetonitrile; tetrahydrofuran
(Increasing strength)
11

CHCH3
COOH
H3CO
H3CO
CHCH3
HOOC
OH
Si O Si O Si O Si O Si O
OH OC18H37 OC18H37 OC18H37
O O O O O
Si Si Si Si SiO O O O OO
OSi
Si
Hydrogen
bonding
Vander walls
interaction
Stationary phase silica gel Revese phase (ODS silica gel)
OH
O
Mechanism of adsorption:
12
Normal phase chromatography Reverse phase chromatography

Partition chromatography:
Distribution of solutes between two immiscible liquids.The two immisible liquids are
liquid stationary phase, liquid mobile phase (Or) gaseous mobile phase.
In partition chromatography separation is based up on the differences in partition
coefficient of the individual components in a mixture.
According to Nernst distribution law,
The ratio of activities of the solute into two immiscible liquid phases is known as a
partition coefficient.
as1
k=
as2
where 1=organic phase
2=aqueous phase
13
Fig . 1:partition
chromatography

In dilute solutions the activities can be replaced by solubilities then k is written as kD.
kd = kso/ kaq
where so=solubility of solute in organic phase.
saq=solubility of solute in aqueous phase.
When solubilities are replaced by concentration of solute now the kD becomes D.
D=Distribution ratio.
D= C0/CW
where Co =concentration of solute in organic phase
Cw=concentration of solute in aqueous phase
 ‘D’ depends upon the nature of the both phases, nature of solute & temperature.
14

In case of gas chromatography the stationary phase is high boiling organic liquid
(polydimethyl siloxanes) where as the mobile phase is inert gases
(Helium,nitrogen,Argon)
Examples: column partition
Gas liquid chromatography
High performance centrifugal partition chromatography
Paper partition chromatography
counter current extraction chromatography (counter current distribution)
15
k= concentration of solute in stationary phase
concentration of solute in mobile phase

Ion-exchange chromatography:
Ion exchange chromatography is the process by a mixture of charged ions can be
separating using an ion exchange resin.
The principle of separation is by reversible exchange of ions between the ions present
in the solution.
Example:
solid matrix of solution cation retained solution
cation exchange resin containing by the solid matrix
cations of ion exchange resin
16
Ion exchange resin-H + M+ Ion exchange resin-M+ + H+

17

-CH-
SO3
-
H+
strong acid cation exchange resin
( R-SO3
H2O R-SO3-H+ )
Induction of
basic groups
(Quartenary
ammonium
hydroxide/
halide/nitrate
CH
CH-N+
(CH3)3 x-
Counter ion
Strong base anion exchange resin
X-=OH-, CL-, NO3-
counter ion
CH=CH2
+
CH=CH2
CH=CH2
Polymer matrix
sulfonated HSO3CL,H2SO4
18

Gel permeation chromatography:
 It is also called as gel filtration or size
exclusion chromatography.
 In size exclusion chromatography, the
stationary phase is a porous matrix
made up of compounds like cross linked
polystyrene, cross like dextrans , polyacrylamide
gels, agarose gels etc.
 The separation is based on their (analytes)
molecular sizes since the gel behaves like
a moleculae sieve.
 This technique is used for the separation of
proteins,polysaccharidees,enzymes and synthetic
polymers.
19
Fig . 2:gel permeation chromatography

Affinity chromatography:
 It is based on the Lock & key mechanism prevalent in biological
system.
20
Fig . 3:Affinity chromatography

Requirements for column chromatography:
1. Column characteristics & selection
2. Stationary phases
3. Mobile phases
4. Preparation of column
5. Introduction of sample
6. Development of column
7. Detection of components
8. Recovery of components

Materials of construction
Good quality neutral
glass,plastic or
nylon
Adsorbent(stationary phase)/adsorbate (mixture)
weight ratio
30:1
Column length to diameter ratio(cm) 10-15:1 or 30-100:1
Multi component system is present Long column is used
Components with similar affinities for adsorbent
are present
Long column is used
Components with different affinities for adsorbent
are present
Short column is
used
Column characteristics & selection:
22
Table:1: selecting a suitable column
dimension

Stationary phases in column chromatography:
 Stationary phases used in column adsorption chromatography are also
known as adsorbents.
Requirements of an ideal stationary phase:
1.They should be insoluble in solvents or mobile phases.
2.chemically inert.
3.colourless to facilitate observation of zones.
4.should have reproducible properties from batch to batch.
5.The particle should have uniform size distribution and have spherical
6. particle size :60-200µ
23

 The most commonly used chromatographic adsorbent is silica or
silisic acid or silica gel (80-100 mesh or 100-200 mesh size, which has a
particle size of 63-200µm) .
 They adsorbs polar and unsaturated substances by the formation of
hydrogen bonds with the hydroxyl groups on the silicon atom.
Type of mesh size in microns
60/120 mesh 120-250 micron
100/200mesh 75-150 micron
70/230 mesh 63-200 micron
230/400 mesh 37-63 micron
70/ 325 mesh 45-200 micron
24
Fig . 6: Silica gel and their
mesh

Table 2: Adsorbents used in column chromatography
Weak activity Medium activity Strong activity
Sucrose Calcium carbonate Activate magnesium
silicate
Starch Calcium phosphate Activated alumina
Inulin Magnesium
carbonate
Activated charcoal
Talc Magnesium oxide Activated magnesia
Sodium carbonate Calcium oxide Activated silica
25

Adsorption isotherms:
 The amount of a solute adsorbed from the solution by an
absorbent can be determined by shaking a known weight of
adsorbent with a known volume of solution at fixed temperature
until equilibrium is attained.
 The adsorbent is filtered off & the concentration of the
substance in the filtrate is determined by any suitable means.
 A plot of equilibrium concentration of species in a solution and
that of amount adsorbed is called adsorption isotherms.
26

Linear adsorption isotherms:
 These are obtained when the amount of substance absorbed per gram of
adsorbent is proportional to the concentration of solution.
 The adsorption coefficient(k=m/Cs) is a constant value, in this linear
adsorption isotherm.
Convex adsorption isotherms:
 These are obtained when adsorption from weak solution is
greater than from strong solutions.
Concave adsorption isotherms:
 These are obtained when adsorption from strong solution is greater than
from weak solutions.
27

Convex and concave adsorption isotherms:
28
 Tailing is particularly likely to occur where adsorption is involved in the
separation process.
 Both effects become more pronounced at high concentrations and are
therefore symptomatic of overloading the column sample.

 Adsorption isotherms and related elution patterns of substances
from a column of adsorbent.
m = weight of substance adsorbed per g of adsorbent.
Cs = Concentration of solution.
Cf = Concentration of each fraction.
f = Number of each fraction.
29

ELUENTS OR SOLVENTS OR MOBILE PHASES USED:
30
 Mobile phase act as a solvent to introduce the mixture into the column
as developer to develop the zones for separation and as an eluent to remove
the pure component out to the column.
 Strain(1942) has arranged the solvents in order of eluting power. A
grouping of solvents in order of chromatographic strength(polarity index)
is known as elutrophic series.

Increasingelutingpower
Light petroleum ether (petroleum
ether,hexane,heptane
Carbon tetra chloride
Cyclohexane
Carbon disulphide
Benzene
Toluene
Chloroform
Ether
Ethyl acetate
Acetone
Alcohols
Water
Pyridine
Organic acids
Inorganic acids&bases
31
Fig . 7:mobile phase

Preparation (packing) of the column:
a) Slurry packing (wet packing method):
The adsorbent is suspended in the mobile phase and stirred very
well to remove all air bubbles. The resulted slurry is then poured in
to the column.
After slurry application, the column must be allowed to settle
overnight. This is the ideal method of column packing.
At the bottom portion of the column a piece of glass wool or
cotton/whattman filter paper disc must be added before the slurry
application.
32

33
PROCESS OF WET PACKINGFig . 8

b) Dry packing:
In this method the dry adsorbent is poured to the column directly
Vibration is applied to get rid of air bubbles then the mobile phase is
passed through the adsorbent.
The demerit with this method is that air bubbles are entrapped between
the solvent and the stationary phase. This method cannot be applied in
gel chromatography.
34

Fig 9:PROCESS OF DRY PACKING
35

c)Sample introduction in column chromatography:
1) Wet application: Dissolve the sample in the initial mobile phase and
apply by pipette to the top of the column.
 This is very good method but in most of cases the samples are not soluble in
the initial mobile phase.
36

2) Dry loading:
 Dissolve sample in any volatile solvent.
 The sample solution is then adsorbed an small weight of adsorbent and the
solvent is allowed to evaporate.
 The dry adsorbent loaded with the sample is then applied to the column.
Fig . 10:sample adsorbed in silica gel Fig.11:column loaded with dry sample

Development of column:
 Removal of individual components from a column is called
development of column.
Frontal analysis: This technique was developed by Tiselius in 1940
In this method, the solution of sample mixture is added continuously
on the column. No mobile phase (solvent) is used for development of
column.
A mixture containing A,B,C is added on the column.
If component A is least adsorbed, component B is adsorbed to
intermediate extent and component C most strongly to the column
adsorbent material.
38

The mixture flows through the column, the least adsorbed A runs down
the column fast, component B to intermediate extent while ‘c’ is retained at
the top of the column.
 A plot of amount of substance against volume of eluate is gives a
chromatogram.
39

Displacemental Analysis:
 In this method, a small volume of mixture is added to the column and
elution is carried out by a solvent containing a solute which has adsorptivity
for column material.
 The adsorbed constituents of mixture are displayed by the solute from
mobile phase.
 Each solute in the mixture in turn displaces another substance solute which
is less firmly adsorbed.
 The least adsorbed constituent is pushed out of the column. The substance
used in mobile phase is called as displacer.
40

The plot of amount of substance (conc. In eluate) against volume of
eluate is given by:
 Displacement analysis technique is mainly used in preparative work
and is not suitable for analysis since some overlapping may occur
41
Fig.12:Displacement analysis

Elution analysis:
 It is a common method used in column chromatography.
 In this method a small volume of mixture to be separated is added on the
top of column& mobile phase is allowed to flow through the column.
 The mixture introduced on the column gets separated into individual as the
components of mixture are adsorbed to the column material to different
extent.
 on other phase of mobile phase, each component of mixture is eluted out as
separated components (called eluate).
42

A plot of amount of substance per ml of eluate against volume of
eluate will gives the following chromatogram.
43
Fig13:Elution analysis

a) Isocratic elution: (Iso means same or similar)
In this elution technique, the same solvent composition or
solvent of sample polarity is used throughout the process of
separation.e.g.(chloroform only as a solvent or CHCL3:MeOH=1:1)
b) Gradient elution:(gradient means gradual)
In this elution tecenique,solvents of gradually increasing
polarity or increasing elution length are used during the process of separation.
44

45
DETECTION & RECOVERY OF COMPONENTS:
Fractions are collected by elution analysis
Each fraction is examined by TLC using suitable experimental
conditions
Those fractions which give same Rf values in TLC are added as a
common fraction
From the column fraction, solvent is evaporated, dried & the
materialis collected in eppendorf container
After spectral analysis (NMR, MS, X-RD etc.)the compound is
identified

46

Table 3: Factors affecting column efficiency
Factor Effect
Particle size of solid
stationary phase
Decrease in size improves separation
Column dimensions Efficiency increases as ratio length
Column temperature Increase in column temperature results in speed
of elution but does not improve separation
Solvent It should be of low viscosity & high volatility
Solvent flow rate Uniform and low flow rate gives better resolution
Conduction of adsorbent Deactivation of adsorbent decreases separation
Concentration of solutes Substances of high concentration moves slowly
47

Inorganic ions Separeation of copper,cobalt,nickel etc
Organic
Purification of dyes e.g.sudan
red,methylene blue,malachite green
Separation of diastereomers
Separation of tautomers and racemates
Separation of cis and trans-pyrazolines
Plant constituents
Separation of geometrical isomers of cis
and trans isomers of bixin and crocetin
dimethyl ether using alumina
Purification of steroids
carotenoids,chlorophils and xanthophylls
Separation of alkaloids and glycosides.
Drugs and pharmaceuticals
Purification of vitamin-B12
Oxytetracyclin and oleandomycin
Separation of the urinary 17-ketosteroids
Separation of cartisol from plasma
Separation of amino
acids,proteins,phospholipids and
triglycerides.
APPLICATIONS:
48

2.column chromatography can be used for the estimation of drugs in
formulations or crude extracts:e.g.
1) Determination of % w/w of strychnine in syrup of ferrous phosphate with
quinine and strychinine.
2) Determination of primary and secondary glycosides in digitalis leaf.
3) Determination of phytomeandione in injection and tablets.
4) Determination of flucinnolone acetonide or betamethasone 17-valerate in
formulated products.
49

Me
Me
C8H17
HO
0
Me
Me
C8H17
0
Cholestenone
Cholesterol
Examples:
1.Separation of cholestenone from cholesterol:
Stationary phase:copper (n) oxide
Mobile phase: Light petroleum
Temp. conditions:300-314°c
50

Stationary phase:Activated silica
Mobile phase:Light petroleum
Temp. conditions:40-60°c
2.Separation of (E)-isomer from the (Z)-azobenzene:
51

52
CONCLUSION
 Column chromatography is a conventional tool for separation of
phytohemicals, removal of impurities and purification of drugs.
 Effective separation of constituents from different sources in
preparative scale (milligram to gram) can be achieved by column
chromatography.
 Availability of wide range of stationary phases makes the technique
to be used for different kinds of mechanisms.
 Understanding the basic principles of column chromatography
enables us to find solutions for current research problems.

53
REFERENCES:
 A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry, 4th edition,
part two, page no: 86-105.
ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181.
 DAVID G.WATSON, Pharmaceutical analysis, page no: 270 - 271.
 B.K. SHARMA, Instrumental methods of chemical analysis, page no : C-8 to C-15.
Dr. A.V. KASTURE, Dr. K.R. MAHADIK, Dr. S.G. WADODKAR, Dr. H.N. MORE,
Pharmaceutical analysis volume – II, page no: 10-17.
 VOGEL’S, Text book of quantitative analysis, page no:289- 314.
 G.DEVALA RAO, A text book of advanced pharmaceutical analysis, page no:332- 335.
54

54
 Mahfouz m abdel-gawad, maher a el-hashash, mortada m el-sayed..,
Chromatographic isolation of Allium cepa (ssp. Red onion) and its cytotoxic
activity against human liver carcinoma cell lines (HEPG2); International Journal
Of Pharmcy and Pharmaceutical Sciences, vol 6, Page no: 109-110.
 Rachana gohel, bhavna solanki, nilesh gurav.., Isolation and
characterization of shatavarin iv from root of asparagus racemosus willd ;
International Journal Of Pharmcy and Pharmaceutical Sciences, vol 7, page
no: 363-365.
ARTICLES:

• Thank you for paying attention.
•I sincerely thank my guide CH.DEVADASU sir for giving me the
valuable guidance.
•My honored thanks to principal DR.P.SRINIVASA BABU sir for giving
me this opportunity.
•A special thanks to seminar committee.
55
ACKNOWLEDGEMENT

56
QUESTIONS
1) what are the temperature conditions used In separation of (E)-isomer
from the (z)-azobenzene…………….?
2) silica gel is also called as…………….?
3) In reverse phase chromatography what is the polarity of stationary
phase…………..?
4) write the formula for Nernst distribution law…………..?
5) what is the stationary phase used in gel permeation
chromatography……..?

57

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