column chromatography

COLUMN CHROMATOGRAPHY

Under the guidance of:Mr.Ch.Devadasu

Assistant ProfessorDept. of PA&QA

Submitted by:J . RameshRegd No:12AB1R0088IV/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: 5222133

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

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 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.


chromatography

Liquid chromatography

Gas chromatography

Liquid -solid chromatography

Liquid-Liquid chromatography

eg: GLC GSC

eg: column adsorptionTLC,HPTLC

HPLC

eg: column partition Paper partition

HPCPC

Mobile phase: GasMobile phase: liquid

Classification of Chromatography A/c to physical state of mobile phase


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

HPCPCGC(GLC)

Stationary phase: solid Stationary phase: liquid


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)


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


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.


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)


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

Hydrogenbonding

Vander wallsinteraction

Stationary phase silica gel Revese phase (ODS silica gel)

OH

O

Mechanism of adsorption:


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

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 13Fig . 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.


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)


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


Ion exchange resin-H + M+ Ion exchange resin-M+ + H+


-CH-

SO3- H+

strong acid cation exchange resin

( R-SO3H2O R-SO3-H+ )

Induction ofbasic groups

(Quartenaryammoniumhydroxide/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


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.

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 19Fig . 2:gel permeation chromatography

Affinity chromatography:

It is based on the Lock & key mechanism prevalent in biological

system.

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 20Fig . 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 componentsVignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 21

Materials of construction

Good quality neutral glass,plastic or

nylonAdsorbent(stationary phase)/adsorbate (mixture)

weight ratio30:1

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:


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 Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 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


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


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.


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.Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 27

Convex and concave adsorption isotherms:


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.


ELUENTS OR SOLVENTS OR MOBILE PHASES USED:


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.

Incr

easi

ng e

lutin

g po

wer

Light petroleum ether (petroleum ether,hexane,heptaneCarbon tetra chlorideCyclohexaneCarbon disulphideBenzeneTolueneChloroformEtherEthyl acetateAcetoneAlcoholsWaterPyridineOrganic acidsInorganic acids&bases

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 31Fig . 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.



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.


Fig 9:PROCESS OF DRY PACKINGVignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 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.



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.


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.


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.


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


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).


A plot of amount of substance per ml of eluate against volume of

eluate will gives the following chromatogram.

Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 43Fig13: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.



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


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


Inorganic ions Separeation of copper,cobalt,nickel etc Organic

Purification of dyes e.g.sudan red,methylene blue,malachite greenSeparation of diastereomersSeparation of tautomers and racematesSeparation of cis and trans-pyrazolines

Plant constituents

Separation of geometrical isomers of cis and trans isomers of bixin and crocetin dimethyl ether using aluminaPurification of steroids carotenoids,chlorophils and xanthophyllsSeparation of alkaloids and glycosides.

Drugs and pharmaceuticals

Purification of vitamin-B12Oxytetracyclin and oleandomycinSeparation of the urinary 17-ketosteroidsSeparation of cartisol from plasma

Separation of amino acids,proteins,phospholipids and triglycerides.

APPLICATIONS:


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.


Me

MeC8H17

HO

0

Me

MeC8H17

0

CholestenoneCholesterol

Examples:

1.Separation of cholestenone from cholesterol:

Stationary phase:copper (n) oxide

Mobile phase: Light petroleum

Temp. conditions:300-314°c


Stationary phase:Activated silica

Mobile phase:Light petroleum

Temp. conditions:40-60°c

2.Separation of (E)-isomer from the (Z)-azobenzene:



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.


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

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.


ACKNOWLEDGEMENT


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……..?


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column chromatography

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