Colunm chromatography

For B.Pharm, 5th Semester

1. COLUMN
CHROMATOGRAPHY

2. COLUMN CHROMATOGRAPHY
•Column chromatography inchemistry,analysis,
pharmacognosy,etcisamethodusedtoseparate/purify
individualchemicalcompoundsfrommixturesofcompounds.
•Itisoftenusedforpreparativeapplicationsonscalesfrom
microgramsuptokilograms.
•Thisisasolid-liquidtechniqueinwhichthestationaryphaseisa
solid&mobilephaseisaliquid.
•Columnchromatographyisanextremelyvaluabletechniquefor
purificationofsyntheticornaturalproducts.
•CompoundsareseparatedbyColumnChromatographythrough
thesamemechanismasTLC

3. COLUMN CHROMATOGRAPHY
•ColumnChromatographywasdevelopedbythe
Russian-Italianbotanis,MikhailSemyonovichTswett,
in1906usedadsorptioncolumnsinhisinvestigations
ofplantpigments.

4. PRINCIPLE
Adsorption
•Mixtureofcomponentsdissolvedinthe
MobilePhaseisintroducedintothecolumn.
•Componentsmovesdependingupontheir
relativeaffinities.
•Relativeaffinities:Itisdefinedasan
equilibriumconstantrepresentingtheratioof
theequilibriumactivitiesofacomponentin
twodifferentphases.

5. A chemist in the 1950s using column chromatography.
The Erlenmeyer receptacles are on the floor.

8. COLUMN CHROMATOGRAPHY
•Adsorptioncolumnchromatography,theadsorbent,
packedinaglasscolumn,andasolvent,themobile
phase,thatmovesslowlythroughthepackedcolumn.
Asolventusedasamobilephaseiscalledaneluent.

9. COLUMN CHROMATOGRAPHY
•Acompoundattractedmorestronglyby
themobilephasewillmoverapidly
throughthecolumn,andelutefrom,or
comeoff,thecolumndissolvedinthe
eluent.
•Incontrast,acompoundmorestrongly
attractedtothestationaryphasewill
moveslowlythroughthecolumn.

10. Experimental aspects of column
chromatography:
•Adsorbents:Theusualadsorbents
employed in column
chromatographyaresilica,alumina,
calciumcarbonate,calcium
phosphate,magnesia,starch,etc.,
•Aluminaisgenerallysuitablefor
chromatographyoflesspolar
compounds(Non-polarsolvents).
Silicagelgivesgoodresultswith
compoundscontainingpolar
functionalgroups.

11. COLUMN CHROMATOGRAPHY
•Adsorbent in C.C should meet following criteria
◘Particles should be spherical in shape & uniform in size
◘Mechanical stability must be high
◘They shouldn’t react chemically
◘It should be useful for separating for wide variety of
compounds
◘It should be freely available & inexpensive
(Theparticlesizeofthecommerciallyavailablegradeis
intherange50–200µm.)

12. Selection of Stationary Phase
•Successofchromatographydependsuponproperselection
ofStationaryPhase,itdependsonthefollowing:
1.Removalofimpurities
2.No.ofcomponentstobeseparated
3.Lengthofthecolumnused
4.Affinitydifferencesb/wcomponents
5.Quantityofadsorbentused

13. Mobile Phase
•Theyactassolvent,developer&eluent.
Thefunctionofamobilephaseare:
•Asdevelopingagent
•Tointroducethemixtureintothecolumn–assolvent
•Todevelopingagent
•Toremovepurecomponentsoutofthecolumn–as
eluent

14. •Thechoiceofthesolventisdependonthesolubility
characteristicsofthemixture.Thesolventsshouldalso
havesufficientlylowboilingpointstopermitready
recoveryofelutedmaterial.
•However,polarityasseenthemostimportantfactorin
adsorptionchromatography.
•Differentmobilephasesused:(inincreasingorderof
polarity)
•Petroleumether,carbontetrachloride,cyclohexane,
ether,acetone,benzene,toluene,esters,water,etc
•Itcanbeusedineitherpureformorasmixtureof
solvents

16. COLUMN CHARACTERISTICS
•Themainfunctionofallthecolumnsistosupport
thestationaryphase.
•Thematerialofthecolumnismostlygoodquality
neutralglasssinceitshouldn’tbeaffectedby
solvents.Anordinaryburettecanalsobeusedas
columnforseparation.
•Columndimensions-length&diameterratio
(10:1,30:1or100:1)
•Variousaccessoriesareattachedtothetopand
bottomofthecolumnformaintenanceofthe
elutionprocess.

17. PREPARATION OF THE COLUMN
•Itconsistsofaglasstubewithbottomportionofthe
column–packedwithglasswool/cottonwoolormay
containasbestospad,
»Abovewhichadsorbentispacked
»Afterpackingapaperdisckeptonthetop,sothatthe
adsorbentlayerisnotdisturbedduringtheintroductionof
sampleormobilephase.

18. Packing techniques in Column
Chromatography
•Therearetwotypesofpreparingthecolumn,theyare:
i.Drypacking/dryfilling
ii.Wetpacking/wetfilling
•Thecolumnshouldbefreefromimpurity,beforeusing
column,itshouldbewashedproperlyanddryit.
•Beforefillingcolumnwithstationaryphase,cotton/glass
wooliskept
•Itshouldbeuniformlyfilled

19. Dry Packing Technique
Adsorbentispackedinthecolumnindryform
Filledupto3/4
th
oftheactualheightofthecolumn
Fillthesolvent,tillequilibriumisreached
DEMERIT:Airbubblesareentrappedb/wMobilePhase&
StationaryPhase→cracksappearintheadsorbentlayer.
•Afterfillingtappingcanbedonetoremovevoidspaces.

20. COLUMN CHROMATOGRAPHY

21. COLUMN CHROMATOGRAPHY
WetPackingTechnique
»ideal&commontechnique
Thematerialisslurredwithsolventandgenerallyaddedto
thecolumninportions.
◊AllowtosettleS.Puniformly&avoidthecrackinginthe
columnofadsorbent.
»Solidsettledownwhilethesolventremainupward.
»Thissolventisremovedthenagaincottonplugisplaced.
it’susuallyfasterandseemstouseuplessliquid

22. Introduction of the Sample
•Thesamplewhichisusuallyamixtureofcomponentsis
dissolvedinminimumquantityofthemobilephase.
•Theentiresampleisintroducedintothecolumnatonce
andgetadsorbedonthetopportionofthecolumn.
•Fromthiszone,individualsamplecanbeseparatedbya
processofelution.

23. COLUMN CHROMATOGRAPHY

24. COLUMN CHROMATOGRAPHY

25. COLUMN CHROMATOGRAPHY
Developmenttechnique(Elution)
•Byelutiontechnique,theindividualcomponentsare
separatedoutfromthecolumn.
•Thetwotechniquesare:
(i)Isocraticelutiontechnique:Inthiselutiontechnique,same
solventcompositionorsolventofsamepolarityisused
throughouttheprocessofseparation.
•Example:chloroformonly

26. COLUMN CHROMATOGRAPHY
(ii) Gradient elution techniques:
(Gradient –Gradually)
Solvents of gradually ↑ polarity or ↑ elution strength are used
during the process of separation.
E.g. Initially benzene, then chloroform, then ethyl acetate then
methenol

27. COLUMN CHROMATOGRAPHY
DETECTIONOFCOMPONENTS
•Ifthecompoundsseparatedinacolumnchromatography
procedurearecolored,theprogressoftheseparationcan
simplybemonitoredvisually.
•Ifthecompoundstobeisolatedfromcolumnchromatography
arecolourless.Inthiscase,smallfractionsoftheeluentsare
collectedsequentiallyinlabelledtubesandthecomposition
ofeachfractionisanalyzedbyTLC.

28. COLUMN CHROMATOGRAPHY
•Elutingthesample:Componentsa,b,andcseparateas
columnprogresses.
•Fractionscanbecollectedintesttubes,vials,beakers,or
flasks.

29. COLUMN CHROMATOGRAPHY
Analyzingthefractions:
•Analyzethefractionsbythin-layerchromatography

30. FACTORS AFFECTING COLUMN EFFICIENCY
1.Dimensionofthecolumn:columnefficiencyhasbeen
improvedbyincreasinglength/widthratioofthecolumn.
2.Particlesizeofcolumnpacking:separationtobeimproved
bydecreasingtheparticlesizeoftheadsorbent.
3.Uniformityofpacking:Nonuniformpackingresultsin
irregularmovementofsolutesthroughcolumn&less
uniformzoneformation,(i.e.bandbroadningortailing)
4.Temperatureofthecolumn:Thespeedoftheelution
increasesathighertemperatures.
5.Qualityofsolvents:Solventsshouldbeoflowviscosity(to
giveefficientresolution)&highvolatility(togetrapid
recoveryofthesubstances)
6.Continuity of flow: Discontinuous flow disturbs resolution
7.Condition of adsorbent: Deactivation of adsorbent decreases
separation.

31. APPLICATIONS
►Separationofmixtureofcompounds
►Purificationprocess
►Isolationofactiveconstituents
►Estimationofdrugsinformulation
►Isolationofactiveconstituents
►Determinationofprimaryandsecondaryglycosidesin
digitalisleaf.
►separationofdiastereomers

32. COLUMN CHROMATOGRAPHY
AdvantagesofC.C
»Anytypeofmixturecanbeseparated
»Anyquantityofmixturecanbeseparated
»WiderchoiceofMobilePhase
»Automationispossible
DisadvantagesofC.C
»Timeconsuming
»moreamountofMobilePhasearerequired
»Automationmakesthetechniquesmorecomplicated&
expensive

33. TLC vs. CC
•Same type of separation technique (liquid-solid separations)
•TLCis muchsimpler(it doesn’t require as much preparation
asCCdoes with all of the packing material, solvent and eluent),
•Cheaper(even thoughCCis cheap as well when compared
toHPLC)
•Takes less time to finish.
•Columnrequires more amount of solvents
•Column Chromatography is that it’s not really reproducible.
•Column Chromatographyis better becausecolumns are
longerthan a TLC plate. Also, TLC (as opposed to column
chromatography)takes place in an open systemwhich means it
can be affected by humidity or temperature

34. References
1. Pharmaceutical Analysis-II, Instrumental
Methods by P.C. Kamboj.
2. Instrument methods of analysis by Scoog and
West.