Column chromatography

Column chromatography

1. Column Chromatography
MahparaGondal
Rashid Latif College of Pharmacy

2. Content
Definition
Theoretical Aspects
Factors Affecting column Chromatography
Types of column
Packing of column
Development
Sample Application

3. Definition
“Columnchromatographyisamethodusedtopurify
individualchemicalcompoundsfrommixturesof
compounds.”
Itisoftenusedforpreparativeapplicationsonscalesfrom
microgramsuptokilograms.
Themainadvantageofcolumnchromatographyisthe
relativelylowcostanddisposabilityofthestationary
phaseusedintheprocess,BecauseothersystemslikeHPLC
nothavethiskindofadvantage.

4. CC vs TLC
Column chromatography (CC) works on the same principle as
thin layer chromatography (TLC).The main difference is that
TLC separates small amounts of materials and CC can be used
to separate large amounts (Even many kilograms) of material.
TLC has a thin layer of sheet of the stationary phase while cc
has a glass of teflontube packed with stationary phase. The
cross sectional area of the stationary phase in TLC is tiny, but
in CC it depends on diameter of the column.
Since the cross sectional area determines how much
compound can be loaded onto the TLC plate or CC column.

5. Theoretical concepts
1-Differential migration:
Differentcompoundsmovethroughthesystem
withdifferentratesofmovementthisiscalled
"differentialmigration".Thespeedofany
compoundinthemixtureisdeterminedbythe
numberofmoleculesofthatcompoundinthe
mobilephase.

6. Supposewehavemixtureofmaterials“A”and“B”:
A:Havemoreaffinitytomobilephaseeilargenumberof
moleculesarepresentinthemobilephase.
B:Havemoreaffinitytostationaryphaseeismallnumberof
moleculesarepresentinthemobilephase.Stationary Phase
Mobile Phase
A B
B
A
Bs
Bm
As
Am

7. Mathematical prsentationof differential
migration:
U: velocity of solvent (mobile phase).
Ux: velocity of material X.
R: fraction of material X in the mobile phase.
Ux= UR

8. IfR=1ieallthecompoundmoleculesarepresentinthe
mobilephase.
Ux= U X 1
Ux= u
:. Material X will move with the solvent velocity.
if R = 0.0 ieall the compound molecules are present in the
stationary phase.
Ux= u x 0.0
:. Ux= 0.0
:.MaterialXwillnotmoveatall.Foranymaterialtobe
separatedthroughthesystemitmustbedistributedbetween
themobilephaseandstationaryphase.

9. Capacity Factor:
Thefactorthatcontrolthedistributionofanymaterial
betweenthetwocompetitivephasesiscalled
“Distributioncoefficient”or“Capacityfactor”or
“Massdistributionratio”K’
(n)s
K’ =
(n)m
(n)s:Isthetotalnumberofmolesofthecompoundinthe
stationaryphase.
(n)m:Isthetotalnumberofmolesofthecompoundinthe
mobilephase.

10. t
r–t
0
K’ =
t
0
t
r:Timerequiredforthesampletocrossthe
column(retentiontime).
t
0:Timerequiredforthesolventmoleculestocross
thecolumn.
BiggerK’meansthatthematerialretainedmore
timeonthecolumneimoveslowly.SmallerK’
meansfastermovement.

11. 2-Movement of materials through the chromatographic system in
the form of “zones” or “bands”:
Itwasassumedthatthechromatographicsystemcomposedof
numberof“distributionsystems”or“equilibrations”called
“TheoreticalPlates”.Eachtheoreticalplateiscomposedof
stationaryphaseandmobilephase.Theheightofeachplateiscalled
“HeightequivalenttoTheoreticalPlate”(HETP).
Thenumberoftheoreticalplates“N”isimportantforseparation.
Increasing“N”resultedinnarrowerbandsandbetterseparation.

12. L
N=
HETP
L: Column length
“N” can be increased by:
1-Increase the length of the column (impractical).
2-Decrease the HETP.
How to decrease HETP:
Decrease the particle size of the stationary phase.
Proper selection of good mobile phase.

13. Materials move through the column as bands or zones and
velocity is controlled by K'.
Material with K' = 1 (64 molecules)
Thematerialwillbepresentinthemiddleofthesysteminthe
formofband.Ifweincrease“N”thematerialwillfrom
narrower.Narrowbandsallowbetterseparationofmixtures.32
32
2
2
16
16
16
16
16
16
12
12
12
12
12
12
8
8
8
8
8
8
8
8
4
4
4
4
2
2

14. 2
2
12
12
8
8
8
8
2
2
N= 5 N= 25 N= 150
N= 5 N= 25 N= 150
A
B
B
A
A
B

15. Factors affecting separation:

16. Factors due to Stationary Phase:
1-Particlesizeofthestationaryphase:
Reducingtheparticlesizeincreasesthesurfaceareaand
improveseparation.However,reductionoftheparticle
sizewilldecreasetheflowrateofthemobilephase.
InHPLCweuseveryfineparticlestogetverygood
separation.Theflowrateproblemissolvedbytheuse
highpressurepumpstopushthemobilephasethrough
thestationaryphase.Columnsaremadeofstainlesssteel
towithstandthehighpressure.

17. 2-Adsorbentactivity:Thechoiceofthesuitable
adsorbentisveryimportant.
3-Uniformityinpackingofthecolumn:Ifthestationary
phaseisnotpackeduniformlythenthebandswillbe
irregularandlessuniformresultinginpoor
separation.
4-Concentrationofthemixture:theproperratio
betweensampletobeseparatedandtheamountof
stationaryphaseisveryimportanttoomuch
samplesresultedinbadseparation.

18. Factors due to Mobile Phase:
1-Selectionofthepropermobilephase:Verypolarmobilephasewill
washoutallcomponentswithoutanyseparation.Ontheother
handverynonpolarmobilephasewillresultinbroadbandand
poorseparation.
2-Rateofflow:Slowerflowrateusuallyresultedinabetterseparation
andnarrowerbands.
3-Consistencyofflow:Thecontinuousflowofthemobilephaseduring
thewholeexperimentgivesbetterseparationthaninterruptingthe
flowthencontinueitlater.

19. Factors due to Columns:
Columndimensions:Increasingthelengthofthe
columnimproveseparation.However,thatusually
leadstoslowerflowrate.Alsoincreasingthecolumn
lengthsometimesisimpractical.
Columntemperature:Increasingthetemperature
usuallyreducestheadsorptionpowerofthestationary
phaseandincreaseelutionspeed.Thismayleadsto
decreaseintheefficiencyseparation.

20. Pressure:
High pressure above the column and low
pressure below the column increase the
efficiency of separation.
High pressure can be achieve by reservoir or
by using pressure devices (pumps) and
pressure below the column can be reduce by
using vacuum pump.

21. Types of columns:
1-Gravity Columns:
Themobilephasemovethroughthestationaryphasebygravityforce.
2-FlashColumns(Airornitrogenpressure):
Themobilephaseispushedbystreamofairornitrogenusingspecial
valves(Adaptors).

22. 3-LowandMediumPressureColumns(pumped):
Themovementofmobilephaseisacceleratedbyusingpumps
thatgeneratelowormediumpressure.Theincreaseinthe
flowrateshortenthetimeofseparation.

23. 4-Vacuum Columns [Vacuum liquid chromatography (VLC)]:
Theadsorbentisapplieddryintoasinteredglassfunnel.Thesampleis
appliedbydrymethodorassolution.Thenthemobilephaseisaddedportion
byportionandvacuumisappliedaftereachportiontocollecteachfraction.

24. 5-HighpressureColumns(HPLC):
Inthiscolumnsweuseveryfinesilicagelsogreat
increaserinseparationpower.However,theflowrateof
themobilephaseisseverelydecreased.Highpressure
pumpsareusedtopushthesolventthroughthecolumn
whichinthiscasemustbemadeofstainlesssteel.

25. Experimental Requirements
Stationary phase
Mobile phase
Packing the column
Sample application
Monitoring the column
Development technique
Isolation of the separated compounds
Detection of compounds

26. Stationary Phase
Alumina and silica are the two most popular
stationary phases in column chromatography.
These adsorbents are available in different
mesh size as indicated by number like silica
gel 60 or 230-400.
Smaller particle size use for flash
chromatography and larger particle size use for
gravity chromatography.

27. Mobile Phase
It is a mixture of organic solvents (unusually one
solvent only)
The most widely used solvents are cyclohexane,
benzene, chloroform, acetone, ether and acetic acid.
Solvents used in CC have three functions
1. They serve to introduce the mixture to the column
2. They effect the process of development.
3. They are used to remove require content.

28. Mobile phase:
Thechoiceofthecolumnmobilephaseisachieved
afterTLCstudyindifferentsolventsystems.
SystemsthatdonotmovespotsatallonTLCare
notgoodforcolumnseparation.
GoodsolventsystemmustproduceR
fvaluelessthan
0.6forallmaterialstobeseparatedbythe
column.Ifthesystemmovesthemmoreand
produceshigherR
fnoseparationwilloccur.

29. Packing of Columns:
The adsorbent is applied to the Column in two ways:
Slurry packing (Wet method):
Theadsorbentissuspendedinthemobilephaseand
stirredverywelltodriveoffallairbubbles.The
resultedslurryisthenpouredintothecolumn.Atthe
tapendofthecolumnapieceofglasswoolorcotton
mustbeaddedbeforetheslurryapplication.Sandmay
beaddedaftertheslurry.Afterslurryapplicationthe
columnmustbeallowedtosettleovernight.
Ingelchromatographytheadsorbentmustbesoakedin
themobilephaseovernighttoabsorbthemobilephase
andswell.

31. 2-Dry Packing:
Inthismethodthedryadsorbentispouredtothe
columndirectly.Vibrationistheappliedtogetridof
airbubblesthenthemobilephaseaspassedthrough
theadsorbent.Thismethodcannotbeappliedgel
Chromatography.

33. Sample Application
1-Wetapplication:Dissolvethesampleinthe
initialmobilephaseandapplybypipettetothe
topofthecolumn.Thisisverygoodmethodbut
inmostofcasesthesamplesarenotsolubleinthe
initialmobilephase.
2-Dryloading:Dissolvesampleinanyvolatile
solvent.Thesamplesolutionisthenadsorbedon
smallweightofadsorbentandthesolventis
allowedtoevaporate.Thedryadsorbentloaded
withthesampleisthenappliedtothecolumn.

35. Monitoring the column:
If the mixture to be separated contains colored
compounds then monitoring the column is very
simple. The color bands will move down the
column along with the solvent and as they
approach end of the column, so fractions will
collect order wise.

37. Development techniques
Isocratic
Gradient

38. Isocratic system:
Meansusingthesamemobilephasefromthebeginning
totheendoftheseparation.
Gradient:
Thepolarityofthesystemincreasedgraduallyduring
separationbyincreasingtheproportionofthemorepolar
solvent.AtypicalgradientmaybestartwithCHCl
3,
followedbyCHCl
3/MeOHmixtureswithgradual
increasein%ofMeOHtillallspotsareelutedfromthe
system.

39. Isolation
Whenallthematerialshavebeenremoved
fromthecolumn,thecolorsofthematerials
indicatewhichfractionscontainthe
compound.
Combinethelikeorsamefractionsandthen
evaporatethesolvents.

41. Detection of the compounds
Thedetectionofcoloredcompoundscanbe
donevisually.Butforcolorlesscompoundsthe
techniquedependsuponthepropertiesofthe
components.
E.g.UV-VISdetector,fluorescencedetector.

43. Application
1.Separationofmixtureofcompounds
e.galkaloids,glycosides,aminoacids,plant
extractsandotherformulations.
2.Removalofimpuritiesorpurificationprocess.
3.Isolationofactiveconstituents.
4.Isolationofmetabolitesfrombiologicalfluids.
Likeblood,plasmaorserumcanbeisolated.
5.Estimationofdrugsinformulationorcrude
extracts.

44. Reverse phase chromatography
Reversed-phasechromatography(RPC)includesany
chromatographicmethodthatusesanon-polarstationary
phase.
Thename"reversedphase"hasahistoricalbackground.
Inthe1970smostliquidchromatographywasdoneon
non-modifiedsilicaoraluminawithahydrophilic
surfacechemistryandastrongeraffinityforpolar
compounds-henceitwasconsidered"normal".

46. Cont…
Theintroductionofalkylchainsbondedcovalentlyto
thesupportsurfacereversedtheelutionorder.
Nowpolarcompoundsareelutedfirstwhilenon-
polarcompoundsareretained-hence"reversed
phase".
Allofthemathematicalandexperimental
considerationsusedinotherchromatographic
methodsapply(i.e.separationresolutionproportional
tothecolumnlength).

47. Silica Based Stationary Phases
Anyinertnon-polarsubstancethatachievessufficientpacking
canbeusedforreversed-phasechromatography.
Themostpopularcolumnisaoctadecylcarbonchain(C18)
bondedsilica(USPclassificationL1)with297columns
commerciallyavailable.ThisisfollowedbyC8bondedsilica
(L7-166columns),puresilica(L3-88columns),cyano
bondedsilica(L10-73columns)andphenylbondedsilica
(L11-72columns).
NotethatC18,C8andphenylarededicatedreversedphase
packingswhilecyanocolumnscanbeusedinareversedphase
modedependingonanalyteandmobilephaseconditions.It
shouldbenotedatthispointthatnotallC18columnshave
identicalretentionproperties

48. Mobile Phase Considerations
Mixturesofwateroraqueousbuffersandorganic
solventsareusedtoeluteanalytesfromareversed
phasecolumn.Thesolventshavetobemisciblewith
waterandthemostcommonorganicsolventsusedare
acetonitrile,methanolortetrahydrofuran(THF).
Othersolventscanbeusedsuchasethanol,2-
propanol(iso-propylalcohol).Elutioncanbe
performedisocratic(thewater-solventcomposition
doesnotchangeduringtheseparationprocess)orby
usingagradient(thewater-solventcompositiondoes
changeduringtheseparationprocess)

49. Affinity column chromatography
Affinitychromatographyisanapplicabletechniqueusedto
purifyproteins.Itisperformeddependingontheadvantageof
thehighaffinityofproteinsforspecificchemicalgroups.
Ingeneral,theinitialmixturethatcontaineddesiredproteins
wasaddedthroughthecolumntoallowbindingtooccur.A
washbufferwasgraduallyaddedtotheadditionofmixture.
Theelutionbuffersubsequentlyremovesunboundedprotein
fromthecolumnandcollected.

51. Utilization
Affinity chromatography is mainly used in biochemistry to
• Purify certain proteins from a mixture
• Reduce the amount of a certain protein molecule in a mixture of
multiple proteins
• Discover the affinity of substances to biological compounds, in
this case protein

52. Chiral column chromatographyis a variant ofcolumn
chromatographyin which thestationary phasecontains a
singleenantiomerof achiralcompound rather than being
achiral.
The two enantiomers of the same analyte compound differ
inaffinityto the single-enantiomer stationary phase and
therefore they exit the column at different times.
Conventional chromatography or other separation processes are
incapable of separating them.
Chiral Column chromatography

56. The chiral stationary phase can be prepared by attaching a
suitable chiral compound to the surface of an achiral support
such assilica gel, which creates a Chiral Stationary Phase
(CSP). Many common chiral stationary phases are based on
oligosaccharides such ascelluloseorcyclodextrin(in
particular with β-cyclodextrin, a seven sugar ring molecule).
As with all chromatographic methods, various stationary
phases are particularly suited to specific types of analytes.
Chiral Stationary Phases are much more expensive than
comparable achiral stationary phases.

57. Application
Thalidomideadrugadministeredinthe60’shastwo
enantiomers.Thisdrugwasusedassedativeandan
antidepressant,butwasfoundtocauseabnormalities
inthefetusofpregnantwomen.Althoughthisdrug
waspulledfromthemarketduetotheresultingbirth
defects.Thereisrecentliteraturethatsuggestthat
onlyoneoftheenantiomerscausedthedefects.Then
chiralchromatographyusetoseparatetheseisomers.

58. Flash column chromatography
Flashcolumnchromatographyisaquickand(usually)easy
waytoseparatecomplexmixturesofcompounds.
FlashColumnchromatographyusesthesameprinciples
discussedintheTLC.Wearerunningflashcolumnssincewe
willusecompressedairtopushthesolventthroughthe
column.Thisnotonlyhelpsprovidebetterseparation,butalso
cutsdowntheamountoftimerequiredtorunacolumn.

60. Preparing a flash column
1) Determine the dry, solvent-free weight of the
mixture that you want to separate.
2) Determine the solvent system for the column by
using TLC
3) Determine the method of application to the column.
You have three choices: neat, in solution, or deposited
on silica.

61. Cont…
4)Determinethesilicageltocompoundratio.
5)Picktheappropriatecolumn.Theamountofsilicagel
youaregoingtousedeterminesthesizeofyour
column.
6)Pickappropriatecollectiontesttubes.
7)Onceyouhaveselectedacolumn,youneedtoplug
thestopcockendsothatthesilicawillnotdrainout.
8)Mountthecolumninyourhood-duetothelarge
volumesofvolatilesolventsusedandthehealthrisks
associatedwithdrysilicagel,youshouldneverruna
columnoutsideofthehood.

62. Cont…
9)Closethestopcockandaddafewinchesofyour
solvent(eluent).
10)Addsand(driedandwashed)tothecolumnusinga
funnel,Orcanmakeslurryfirst.
11)Measureoutthecorrectvolumeofsilica.
12)Makeaslurryofthesilicabyaddingatleast1.5
timesthevolumeofsolventassilicayoujustmeasured
out.
13)Usingapowderfunnel,carefullyandslowlypour
theslurryintothecolumnmakingsurenottodisturbthe
layerofsand.

63. Cont…
14)Usingapipetteandyoursolventsystem,rinseanysilica
stucktothesidesofthetopofthecolumnintothesolvent
layer.
15)Onceallofthesilicaisoffthesidesofthecolumn,open
thestopcockandusethecompressedairtopackthecolumn.
16)Oncethecolumnispacked,addaprotectivelayerofsand
tothetopofthesilica.Thisshouldberelativelyleveland
about2cmthick.

64. Cont…
17)Usingthecompressedair,lowerthesolventlevel
untilitisevenwiththeheightofthesand.
18)Closethestopcockandputyourfirsttesttube
underthecolumnoutlet.
19)Carefullyaddyourcompoundtothecolumn-
whenaddingliquidsbesuretodripthemdownthe
sidesoftheglass,notdirectlyontothetopofthe
column.

65. Cont…
20)Carefullyfillthecolumnwithyoureluent.
21)Onceyouhavefilledthecolumnwithyoureluent,youare
readyto"run"thecolumn.Rememberthataquickflowrate
helpstogivegoodseparation.Adjusttheairpressuretogivea
swiftflowrate,Sokeepthepressureonandchangethetest
tubesoncetheybecomefilled.Remembertoreplenishthe
solventinthecolumnfrequently.
22)Monitorthecolumn'sprogressbyTLC—

66. Cont..
24)Onceyouhavedeterminedthatallofthe
compoundsyouareinterestedinhaveelutedfromthe
column,youarereadytowrapeverythingup.First,
putalargeconicalflaskunderneaththecolumn,and
useagreenKeckcliptoattachedyourcompressedair
sourcetothecolumn.
25)Whilethecolumnisdrying,starttocombine
fractions.UsingTLC,determinewhichtesttubes
containyourpurecompound(s).
26)Oncethesolventiscompletelyremoved,analyze
thecompoundsbyNMR.