Chromatography

Unit operations of Bioseparations

UnitoperationsofBioseparations
Solidliquidseparations

Centrifugation
Sedimentation
Filtration
Microfiltration

Solidsconcentration
S lid
t ti
crystallization,precipitationandextraction

Concentrationofsolubleproducts
Concentration of soluble products
adsorptionandaffinitychromatography

Purification
chromatography

What is Chromatography?
WhatisChromatography?
Ability
Abilitytoseparatemoleculesusing
to separate molecules using
partitioningcharacteristicsofmoleculeto
remain in a stationary phase versus a mobile
remaininastationaryphaseversusamobile
phase
Onceamoleculeisseparatedfromthemixture,it
Once a molecule is separated from the mixture it
canbeisolatedandquantified

Canchromatographyidentifycomponents?
Can chromatography identify components?
Notwithoutthedetector chromatographyisthe
process of separation!
processofseparation!

Chromatography: Introduction
Chromatography:Introduction
Dynamicseparationprocessbasedoninterphase
y
p
p
p
masstransferinvolving
Stationaryphasewhichisgenerallyarrangedintheform
ofapackedbed
Mobilephasethatflowsthroughthestationaryphase

SSubstancestobeseparatedarecarriedalongthrough
bstan es to be separated are arried alon thro h
thepackedbedbythemobilephase
These
Thesesubstancesselectivelybindreversiblyonto(or
substances selectively bind reversibly onto (or
partitioninto)thestationaryphasetodifferentextents
Basedonthisthesesubstancesmovethroughthecolumnat
differentvelocities

ChromatographicSeparation

Chromatography: Basic components

Chromatography:Basiccomponents
Majorcomponents:
Mobilephaseflowsthroughcolumn,carriesanalyte
Gas=GasChromatography(GC)
Liquid=LiquidChromatography(LC),ThinLayer
Li id Li id Ch
h (LC) Thi L
Chromatography(TLC)
Supercriticalfluid
Supercritical fluid =SupercriticalFluidChromatography(SFC)
Supercritical Fluid Chromatography (SFC)

Stationaryphasestaysinaplace,doesnotmove
GC,LCplacedinsideofthecolumn
, p
TLC layerofasorbentontheplate

TheSEPARATIONisbasedonthepartitioningbetween
p
g
themobileandstationaryphase

Basic Chromatographic Terminology

BasicChromatographicTerminology

Chromatograph:Instrumentemployedforachromatography.
St ti
Stationaryphase:Phasethatstaysinplaceinsidethecolumn.Can
h
Ph
th t t
i l
i id th
l
C
beaparticularsolidorgelbasedpacking(LC)orahighlyviscous
liquidcoatedontheinsideofthecolumn(GC).
M bil h
Mobilephase:Solventmovingthroughthecolumn,eitheraliquid
S l t
i th
h th
l
ith
li id
inLCorgasinGC.
Eluent:Fluidenteringacolumn.
Eluate:Fluidexitingthecolumn.
Elution:Theprocessofpassingthemobilephasethroughthe
column.
Chromatogram:Graphshowingdetectorresponseasafunctionof
atime.
Flowrate:Howmuchmobilephasepassed/minute(ml/min).
Linearvelocity:Distancepassedbymobilephaseper1mininthe
column(cm/min).

Chromatographic separation
Chromatographicseparation
Mobile phase
t0
t1

t2

St ti
Stationary
phase
h

t3

Chromatography equipment
Chromatographyequipment
Sample

Column

Mobilephase
reservoir

Pump

Detector

Chromatograms
Mobile
phase

Mobile
phase

Mobile
phase

Mobile
phase

Mobile
phase

Sample

Concentration

Migrating
bands
Time

Peaks

Resolutionofseparation
p
Resolutionoftwopeaksfromoneanother=tr/wav
WeWantResolution>1.5

Theseparationisworsewiththeincreasingpeakwidth

Chromatographic elution
Chromatographicelution
Isocratic
i elution
l i

Gradient elution

A
B
A

Gradients
B

Step
Linear

Non-linear

Chromatographic columns
Chromatographiccolumns

Packed
bed

Packed Open
capillary tubular

Membrane

Monolith

Separation mechanisms
Separationmechanisms
Adsorption
Adsorption
Ion-exchange
Reverse phase
Hydrophobic
H d h bi interaction
i t
ti
Size exclusion
Supercritical fluid
Affinity

Separation Mechanisms
SeparationMechanisms
Ionexchange
Basedonelectrostaticinteractionsbetweenthe
moleculesandtheadsorbent

Affinitybinding
Basedonstereospecific
Based on stereospecific recognitionoftarget
recognition of target
moleculesbyligands
theshapeoftheligand
p
g
iscomplimentarytothe
p
y
shapeoftheentiretargetmoleculeoratleasta
portionofthemolecule

Separation Mechanisms
SeparationMechanisms
Reversedphase
Reversed phase
Partitiontypebehaviour
Usedforadsorptionofnonpolarmolecules
Used for adsorption of non polar molecules

Hydrophobicinteraction
Basedoninteractionbetweenhydrophobic
patchesonmoleculesandthoseontheadsorbent
Mainlyusedforproteinseparations
M i l
df
t i
ti

Separationprinciplesinprotein
chromatographicpurification
h
hi
ifi i

Adsorptionchromatography
p
g p y

Adsorption chromatography
Adsorptionchromatography
Biologicalmoleculeshavevaryingtendenciesto
adsorbontopolaradsorbents
silicagel,alumina,diatomaceousearth,charcoal

Performanceoftheadsorbentreliesstronglyon
thechemicalcompositionofthesurface,i.e.thetypesand
concentrations of exposed atoms or groups
concentrationsofexposedatomsorgroups.

Theorderofelutionofsamplecomponentsdepends
primarily on molecule polarity
primarilyonmoleculepolarity.
Becausethemobilephaseisincompetitionwithsolutefor
adsorptionsites,solventpropertiesarealsoimportant.
Polarityscalesforsolventsareavailabletoaidmobilephase
selection

Ion exchange
Ionexchange
Ionexchangeadsorbents
charged
chargedgroupsattachedonto
groups attached onto
insolublesupportmaterial

Examples
Cellulose,Cellulosederivatives
Cellulose Cellulose derivatives
Agarose,Acrylicresins
Crosslinkeddextrans

Cation
exchange
h

Chargedgroupsforcationicresins
Ch
d
f
ti i
i
Carboxylic,carboxymethyl,
sulphopropyl

Chargedgroupsforanionicresins
Ch
d
f
i i
i
Diethylamino ethyl(DEAE)
Quarternary aminoethyl (QAE)

Anion
exchnge

Functional Groups Used in IEC

FunctionalGroupsUsedinIEC

Ionexchange
Ion
exchange
chromatographyof
proteins

Cation ExchangeMedia
Exchange Media

Anion Exchange Media

AnionExchangeMedia

Parameters influencing ionexhange

Parametersinfluencingion
exhange
pH
ForbindingproteinsonanionicadsorbentspHis
maintainedabovepI;oncationicadsorbentspHiskept
below pI
belowpI

Ionicstrength
Greatertheionicstrengththelessertheamountbound
Greater the ionic strength the lesser the amount bound

Buffertype
Th
Thechargedligands
h
d li d presentpresent
t
t ontheadsorbent
th d b t
areassociatedwithmobileexchangeablecounterions
Somecounterions aremoreexchangeableandhence
g
theiruseresultinhigherbindingoftargetmolecules

Elutionofmoleculesboundonion
exchangeadsorbents
h
d b
Solution
Solutioncontaininghighconcentrationofneutralsalt
containing high concentration of neutral salt
e.g NaCl
Athighconcentrationthesaltshieldstheelectrostatic
interactionsbetweenthetargetmoleculesfromthe
adsorbentresultingindesorption
Thechargedelectrolytesalsocompetewiththetarget
The charged electrolytes also compete with the target
moleculesforbindingandhencedislodgethetarget
molecules

Boundmoleculescanalsobeelutedbychangingthe
solutionpH

Protein

Isoelectric pH

L
Lysozyme

11 0
11.0

Ovalbumin

4.6

Conalbumin

6.1

Eggwhiteproteins

Separationoflysozyme fromhen
eggwhiteusingcation
hi
i
i exchange
h

Othereggwhite Lysozyme +NaCl

proteins

Cation
exchange
adsorbent+
N Cl
NaCl

+NaCl
Cation
exchange
adsorbent+
lysozyme

Cation
exchange
adsorbent+
NaCl

SeparationofhumanimmunoglobulinGfromhuman
serum albumin using anion exchange
serumalbuminusinganionexchange
Protein

Isoelectric
pH

HSA

4.9

HIgG

7.0

HSA+NaCl
HI G
HIgG

HASandHIgG

+NaCl

Anionexchange
adsorbent

Anionexchange
adsorbent+HSA

Anionexchange
adsorbent+
NaCl

Affinitybinding

Basedonstereospecific recognitionof
targetmoleculesbyligands

Affi it bi di
Affinitybinding

Affinity ligands andpurifiedproteins

Affinityligands
and purified proteins
Immobilizedligand

Purifiedprotein

Divalentandtrivalentmetal Proteinswithanabundance
ion
ofHis,Tryp andCys
residues
id
Lectins
Glycoproteins,cells
Carbohydrates

Lectins

Reactivedyese.g Cibacon
bl P
blue,Proscion
i blue
bl

Mostproteins,particularly
nucleotidebindingproteins
l id bi di
i

Nucleicacids

Exo andendonucleases,
polymerases,othernucleic
l
th
l i
acidbindingproteins

Affinity ligands andpurifiedproteins

Affinityligands
and purified proteins
Immobilizedligand

Purifiedprotein

Aminoacids(e.g.Lys,Arg)

Proteases

Nucleotides,cofactors
,
substratesandinhibitors

Enzymes
y

Proteins A and G
ProteinsAandG

Immunoglobulins

Hormones,drugs

Receptors

Antibodies

Antigens

Antigens

Antibodies

Supportmatricesforaffinityadsorption

Affinitypurificationofamonoclonalantibody
frommammaliancellculturesupernatant

Impurities

Monoclonalantibody

Cellculture
supernatant

+
LowpH
buffer
Protein A affinity
ProteinAaffinity
adsorbent

ProteinAaffinity
adsorbent+
d b
Monoclonal
antibody

ProteinAaffinity
ff
adsorbent

Reverse phase adsorption

Reversephaseadsorption
Basedonpartitioning
p
g
Partitioningtakesplaceintoaverythin
immobilised layerofhydrocarbonandnot
layer of hydrocarbon and not
withinabodyofextractingsolvent

H
Hydrophoic
d h i lowpolaritysubstancesC4to
l
l it
bt
C4 t
C18areusedforsolutebinding
chemicallyboundtosolidsupportmaterial
suchassilica

Reversedphaseadsorption
p
p
Polarmedia

Adsorption
Immobilised hydrocarbonlayer

NonPolarmedia

Desorption

Reverse phase separation of insulin

Reversephaseseparationofinsulin
Filtered
fermentation
media +
media
Acetonitrile

Impurities

Insulin

+
80%
acetonitrile
+20%IPA
C 18 reverse
C18reverse
phaseadsorbent

Adsorbent+
d b
Insulin

C18reverse
phaseadsorbent

Hydrophobic interaction based adsorption

Hydrophobicinteractionbasedadsorption
Based
Basedontheinteractionbetweenthehydrophobic
on the interaction between the hydrophobic
patchesonmoleculesandontheadsorbent
Mainlyusedforproteinseparation
y
p
p
Inaqueoussolutionshydrophobicaminoacids are
shieldedbyastructuredlayerofwatermolecules
Removalofwatermoleculesbytheadditionofanti
chaotropic agents(NH4)2SO4,Na2SO4)facilitates
interaction of the exposed hydrophobic patches with
interactionoftheexposedhydrophobicpatcheswith
theadsorbent

Agarose isthemostwidelyusedsupportmaterial

Ligands coupledtoachromatographicmatrix
b l id l ether
byglycidyl
h
Hydrophobicpatches
Hydrophobic
patches
createdbygraftingalkyl
andaromatic
hydrocarbongroupson
agarose

Alkyl

Butyl

Phenyl

Octyl

Hydrophobicinteractionbasedadsorption

Molecule

Structuredwater
Structured
water
inbulksolution

Adsorption
Hydrophobiclayer

Lowsalt
concentration

Desorption

Hydrophobicinteraction
Twoaliphaticcarbonchains
(blackcircles)areimmersedinwater
Whentwochainsareincontact,
thesurfaceareathatiscoveredwith
orderedwatermoleculesis
decreasedandsomewaterisina
state of less order in the bulk of the
stateoflessorderinthebulkofthe
water.
Thefreeenergyofthesystem
decreases,andthusthebinding
g
togetherofthetwomoleculesis
favored.

Hydrophobicinteractionofaprotein
Twooctyl chainsarecoupledtoa
chromatographicmedium
Anenergygainisachievedbythe
interactionoftheoctyl chainswith
nonpolar moietiesonaprotein
Thecarbonatomsofthehydrophobicpartsofthe
h
b
f h h d h bi
f h
proteinareindicatedwithblackcircles
Theoxygenatomsofthewaterwith
whitecircles
hi i l
Chargedatomsorgroupsoftheproteinwithwhite
circlescontainingplusorminussigns.
Thehatchedareaindicatestheinteriorpartofthe
protein

Hydrophobicinteraction:SeparationofrEGF
y p
p
Filtered
Fil
d
fermentation
media+
Ammonium
Ammonium
sulphate

Impurities

rEGF

Hydrophobic
interaction
adsorbent

Adsorbent+
d b
rEGF

Ammonium
sulphate
free
solution

Hydrophobic
Hydrophobic
interaction
adsorbent

Partition chromatography
Partitionchromatography

Partition chromatography
Partitionchromatography
Normalphasechromatography
Normalphase chromatography
Whenthestationaryphaseismorepolar
th th
thanthemobilephase,
bil h

Reversephasechromatography
Whennonpolarcompoundsarebeing
p
y
separateditisusualtouseastationary
phasewhichislesspolarthanthemobile
p
phase

Reversedphase
Reversed
phasechromatography
chromatography
A
Acommonstationaryphaseforreverse
common stationary phase for reversephase
phase
chromatographyishydrocarbonwith8or18
carbonsbondedtosilicagel
Solventsystemsmostfrequentlyusedare
wateracetonitrile andwatermethanol
Aqueousbuffersarealsoemployedtosuppress
ionisationofsamplecomponents.

Elutionisgenerallyinorderofincreasing
solutehydrophobicity.

Ionexchangechromatography
g
g p y

Si e exclusion chromatography
Sizeexclusionchromatography

Affinity chromatography
Affinitychromatography

Applications

PharmaceuticalsandFinechemicals
Biotechnology
y
Environmentalanalysis
Foodsandnutraceuticals
Watertreatment
Analysisofchemicals
g
Diagnostics
Processcontrol

Chromatography:Theoryofretention

Free

y
Diffusion

Bound

Diffusion
z

Factorsinfluencingthe
transformationofshape
f
f h

Interactionwiththestationaryphase
Interaction
with the stationary phase
Nonidealinletdistribution
Radialdispersion
di l di
i
Axialdispersion
Golay Taylordispersion

Theoryofretention
The association of a component with the stationary phase is
quantified in terms of the capacity factor (k):

nS
k =
nM
VS c S
VS
k =
=K
VM c M
VM
1
k = K

nS andn
d M arethenumberofmolesofa
h
b
f
l
f
componentboundtothestationaryphaseand
thatpresentinthemobilephaserespectivelyin
any element of column volume
anyelementofcolumnvolume
VS andVM arethevolumeofstationaryphase
andmobilephaserespectivelyinanyelementof
columnvolume
cS andcM arethecorrespondingvaluesfor
concentrationofthecomponentinthese
phasesrespectively,andK isadistribution
coefficient

isthevoidage fraction.k'shouldideally
haveavaluebetween1and10

TheoryofRetention
Theretentiontimeofthemobilephase
(tM)withinacolumnwouldbebased
purelyonhydrodynamicconsiderations
y t
M

t R = (t R t M )
'

tR

tR '
k =
tM

tM
t

1
t R = t M 1 +
K

VC
=
Q

VC
( + K K )
tR =
Q
Q=mobilephaseflowrate
Q=
mobile phase flow rate
K=distributioncoefficient
Vc =columnvolume

Resolution and selectivity

Resolutionandselectivity
t R2
tR1

tM

Concentration

w1

w2

Time

Resolution

t R 2 t R1
R=
0.5(w1 + w2 )

Selectivityparameter()
K 2 k2 t R 2 tM
=
= =
K1 k1 t R1 tM

Chromatography:
Plateconcept
Achromatographiccolumnisassumedtobe
A h
t
hi
l
i
dt b
madeupofalargenumberofhypotheticalplates
Eachoftheseplatesisequivalenttoone
Each
of these plates is equivalent to one
equilibriumstage
The
Theplateconceptisborrowedfromdistillation
plate concept is borrowed from distillation
Thegreaterthenumberofplates,thebetterthe
separationislikelytobe
p
y
Theheightequivalenttoatheoreticalplate
(HETP orsimplyH)shouldbeassmallaspossible

l
N

tR
N = 16
w

Definition of Plate Height

DefinitionofPlateHeight
ThebreadthofaGaussiancurveis
relatedtothevariance2.
Therefore,theplateheightcanbe
d f d
definedasthevarianceperunit
h
lengthofthecolumn:

H=2/L
Plateheightcanbedefinedas
columnlengthincmwhichcontains
34% of the solute at the end of the
34%ofthesoluteattheendofthe
column(asthesoluteelutes)
Thepeakwidthcanalsoberepresentedintermsoftime,,where:

=/v
/
=/(L/tR)

Band Broadening
BandBroadening
Apartfromspecific
p
p
characteristicsofsolutesthat
causedifferentialmigration
Averagemigrationratesfor
A
i ti
t f
moleculesofthesamesolute
arenotidentical
Threemainfactorscontributeto
thisbehavior
LongitudinalDiffusion
L
it di l Diff i
ResistancetoMassTransfer
StationaryPhaseMassTransfer

Longitudinal Diffusion
LongitudinalDiffusion
Molecules
Moleculestendtodiffuseinalldirections
tend to diffuse in all directions
becausethesearealwayspresentina
concentration zone as compared to the other
concentrationzoneascomparedtotheother
partsofthecolumn
H =K D /V
L

1 M

DM=thediffusionofsoluteinthemobilephase

Notveryimportantinliquidchromatographyexceptatlowflowrates

Resistance to Mass Transfer

ResistancetoMassTransfer
StationaryPhaseMassTransfer
Stationary Phase Mass Transfer
MobilePhaseMassTransfer
MultiplePathEffects
li l
h ff

Stationary Phase Mass Transfer

StationaryPhaseMassTransfer
Notallmoleculespenetrateto
p
thesameextentintothe
stationaryphase
Some
Somemoleculesofthesame
molecules of the same
solutetendtostaylongerinthe
stationaryphasethanother
molecules

Hs=K2 ds2V/Ds
ds =thethicknessofstationaryphase
the thickness of stationary phase
Ds=thediffusioncoefficientofsolutein
thestationaryphase

Mobile Phase Mass Transfer

MobilePhaseMassTransfer

Solutemoleculeswhichhappentopass
through some stagnant mobile phase
throughsomestagnantmobilephase
regionsspendlongertimesbeforethey
canleave
Moleculeswhichdonotencountersuch
stagnantmobilephaseregionsmove
bl h
faster
Othersolutemoleculeswhichare
g
locatedclosetocolumntubingsurface
willalsomoveslowerthanothers
locatedatthecenter(hydrodynamic
chromatography)
Some solutes which encounter a
Somesoluteswhichencountera
channelthroughthepackingmaterial
willmovemuchfasterthanothers

HM =K3dp2V/D
/ M

Multiple Path Effects

MultiplePathEffects
Multiple
Multiplepathswhichcan
paths which can
befollowedbydifferent
moleculescontributeto
bandbroadening
HE =K4dp

Theoverallcontributionstoband
b d
broadening
Ht =H
= HL +H
+ HS +H
+ HM +H
+ HE +H
+ HV
Ht istheoverallheightequivalenttoatheoretical
plate resulting from the contributions of the
plateresultingfromthecontributionsofthe
differentfactorscontributingtobandbroadening

Ht =kk1dp +k
+ k2DM/V+K
/V + K3ds2V/Ds +K
+ K4dp2V/DM
Ht =A+B/V+CSV+CMV

Practical Implications
PracticalImplications
It
Itturnedoutthatresistancetomasstransfer
turned out that resistance to mass transfer
terms(K3ds2V/Ds andK4dp2V/DM)aremost
importantinliquidchromatography
Shouldbeminimizedby
a.Decreasingparticlesize
gp
b.Decreasingthethicknessofstationaryphase
c.Workingatlowflowrates
d.IncreaseDM byusingmobilephasesoflow
viscosities

Practical Implications
PracticalImplications
The
Thelongitudinaldiffusionterm(k
longitudinal diffusion term (k2DM/V)isthe
/V) is the
mostimportantoneingaschromatography
Reducingthisterminvolves:
Reducing this term involves:
a.Workingathigherflowrates
b.DecreasingD
b D
i DMbyusingcarriergasesofhigher
b
i
i
f hi h
viscosities

vanDeemter Equation
q
B
H = A + + Cu
u

A,B andC aresystemandoperating

conditiondependentconstants

u istheaveragevelocityof
mobilephasewithinthe
column
column
A representseddydiffusion
andisduetothevariability
ofpathlengthfollowedby
f th l th f ll
db
fluidstreams
(B/u)representsaxialdiffusion
ofthecomponentinthemobile
f h
i h
bil
phase.
(Cu)representstherateof
materialtransferbetween
mobilephaseandstationary
phase.

H
B/u opt

H min

Cu opt

u opt

vanDeemter Equation
Theoptimumflow
ratecanbefoundby
takingthefirst
derivativeofequation
dH /dV =O B/V2 +C
Visoptimumwhen
V
is optimum when
dH /dV=O,therefore,
C=B/V2optimum
Voptimum ={B/C}0.5

Theoreticallycalculatedvelocityisalwayssmalland
inpracticealmosttwiceasmuchasitsvalueisused
inordertosavetime

Problem
Eggwhiteproteinsarebeingseparatedbyisocraticchromatography
using a 10 cm long column having 250 theoretical plates The
usinga10cmlongcolumnhaving250theoreticalplates.The
distributioncoefficientsfortheproteinsareasfollows
Protein
Ovalbumin
Conalbumin
Lysozyme

Distributioncoefficient
0
1
5

Ifthevoidagefractionofthecolumnis0.45andthemobilephase
If
the voidage fraction of the column is 0 45 and the mobile phase
retentiontimeis10minutes,predicttheretentiontimeofthethree
proteins.Commentontheselectivityandresolutionsofseparations

Solution
Theresidencetimesofthethreeproteinscanbeobtainedusingthe
equation
ti
1

t R ,ovalb

t R = t M 1 +
K

1 0.45
= 10 1 +
0 = 10 min
0.45

t R ,conalb

1 0.45
= 10 1 +
1 = 22.22 min
0.45

t R ,lys

1 0.45
= 10 1 +
5 = 71.11min
0.45

Theselectivityofseparationcanbeobtainedusingequation
K 2 k 2 t R 2 t M
=
=
=
K 1 k1 t R1 t M

Theselectivityofthethreeproteins
Ovalbumin

Lysozyme

Conalbumin

conalb / ovalb

1
= = undefined
0

lys / conalb

5
= =5
1

Thepeakwidthcanbedeterminedusingtheequation

tR
N = 16
w

wovalb

10
=
= 2.52 min
250
16

wconalb

22.22
=
= 5.62 min
250
16

71.11
wlys =
= 2.52
2 52 min
250
16

Theresolutioncanbedeterminedusing

t R 2 t R1
R=
0.5(w1 + w2 )

Rconalb / ovalb

22.22 10
=
= 2.99
2 99
0.5 ( 2.52 + 5.62 )

Rconalb / ovalb

71.11
71
11 22.22
22 22
=
= 4.14
0.5 ( 5.62 + 17.99 )

Feasibility:
The three proteins are obtained as separate resolved peaks
Thethreeproteinsareobtainedasseparate,resolvedpeaks

Problem
Aplasmidwasfoundtohavearetentiontimeof10minutesina
chromatographic column of 0 01 m3 andavoidagefraction0.3.
chromatographiccolumnof0.01m
and a voidage fraction 0 3
Thedistributioncoefficientoftheplasmidisknowntobeequalto
2.
p
Calculatethemobilephaseretentiontimeandflowrateatwhich
theabovesepartion wascarriedoutwewouldliketousethesame
columnmobilephasesystemtoseparatetheplasmidfromRNA
(which has a capacity factor of 4 66)
(whichhasacapacityfactorof4.66).
Commentonthefeasibility

Solution
Themobilephaseretentiontimecanbecalculatedusingthe
equation

1 0.3
10 = tM 1 +
2
0.3

tM = 1.76
.76 min

1
t R = t M 1 +
K

Thecapacityfactorofplasmidcanbedeterminedusing

k = K

1 0.3

k = 2
4 66
= 4.66
0.3

ThecapacityfactorofRNAis4.66too.Therefore
4.66
=1
4.66
ThereforeRNAandplasmidcannotbeseparatedbythis
chromatographicseparationprocess

Problem
AlbuminisbeingseparatedfromIgG byisocraticchromatography
usinga50cmcolumnhavingavoidagefractionof0.25anda
diameter of 10 mm at a mobile phase flow rate of 10 ml/min
diameterof10mmatamobilephaseflowrateof10ml/min.
ThedistributioncoefficientsforIgG andalbuminare1and0.1
respectively.
Ifthealbuminpeakhascharacteristicpeakwidthof0.52minutes,
p
p
,
predicttheselectivityandresolution.
Whenthemobilephaseflowratewasincreasedto20ml/minthe
When
the mobile phase flow rate was increased to 20 ml/min the
HETPwasfoundtoincreaseby80%.
Predicttheselectivityandresolutionatthehigherflowrate

TotalcolumnvolumeV=Lxd2/4=39.25~39ml
Themobilephaseretentiontimecanebecalculated

VC
=
Q

39 0.25
tM =
min = 0.975 min
tM
10
1
K
Theretentiontimescanbecalculatedusing t R = t M 1 +

1 0.25

t R ,alb = 0.975 1 +
0.1 min = 1.27 min
0.25

t R , IgG

1 0.25
= 0.975 1 +
1 min = 3.9 min
0.25

Thenumberoftheoreticalplatesinthecolumncanbecalculated
fromalbuminretentiontimedateusingequation
tR
N = 16
w

1.27
N = 16
= 95
0.52

ThepeakwidthofIgG canbecalculatedusingequation

tR
N = 16
w

w IgG =

t R , IgG

3.9
=
= 1.6 min
N
95
16
16

Theresolutionofseparationcanbecalculatedusingtheequation

t R 2 t R1
R=
0.5(w1 + w2 )

33.99 1.27
1 27
R=
= 2.58
0.5 (1.6 + 0.52 )

The selectivity can be calculated using equation

Theselectivitycanbecalculatedusingequation

K2
1
=
=
= 10
K1
0 .1
1
Theheightofatheoreticalplatecanbecalculatedusingequation

l
50
H=
=
= 0.526cm
N 95

Atthehigherflowrate,theheightofthetheoreticalplateis
increasedby80%.
y
Therefore

H=1.8x0.526cm=0.95cm

Thenumberoftheoreticalplateswillbereducedto N=50/0.95=

52

VC 39 0.25
tM =
=
min = 0.4875 min
Q
20

The mobile phase retention time at higher flow rate is

Themobilephaseretentiontimeathigherflowrateis

t R ,alb

1 0.25

= 0.4875 1 +
0.1 min = 0.633min
0.25

Thenewretentiontimesare

t R , IgG

0 25
1 0.25
= 0.4875 1 +
1 min = 1.95 min
0.25

Thepeakwidthsare

w IgG =

walb

t R , IgG

0.633
=
min = 0.35 min
N
52
16
16

t R ,alb

1.95
1
95
=
=
min = 1.08 min
N
52
16
16

Theselectivityisindependentoftheflowrate,i.e still10
Theresolutionis

t R 2 t R1
1.95 0.633
=
= 1.84
R=
0 ( w1 + w2 ) 00.5 ( 00.35
0.5
3 + 11.08
08 )
Hence the proteins can still be separated at the higher flow rate
Hencetheproteinscanstillbeseparatedatthehigherflowrate


1 k ave

R = 0.5 N

+ 1 1 + k ave

Resolution
= 0.5(k1 + k 2 )
k ave

(( t / t 0 ) 1) 2
C = C 0 exp
2
Shapeandyieldofpeak
2

Chromatographicpeak
g p p

C0 =Maximumconcentration
M i
t ti
t0=Timeatwhichmaximum
concentrationisreached
2=Varianceofthepeak
= Variance of the peak

12

Concentration

10

C0

Thetotalamountofa
component eluted (Mtotal)
componenteluted(M
)
fromacolumn

M Total = Q C dt

t0
0
0

w =4

10

Time

12

14

16

18

20

Theyieldofacomponentintheeffluentcollectedinthetime
The
yield of a component in the effluent collected in the time
periodt1 tot2
% yield

t2

t1

C dt

100
C dt

ThepurityofacomponentAinbinaryseparation(i.e.separation
of A and B) in effluent collected in the time period t1 tot
ofAandB)ineffluentcollectedinthetimeperiodt
to t2
t2

C A dt

t1

% purity A = t2
t2
100
C A dt + C B dt
t
t1

Chromatogram for A
6
5

Conc. (g/l)

Thetwochromatogramswere
obtainedfortwodifferent
compoundsAandBbyinjecting
puresamplesofthesesubstances
into a 30 cm long column
intoa30cmlongcolumn

4
3
2
1

WewouldliketoseparateAandB
fromamixturecontainingthe
sameamountsofthese
substancesaspresentinthepure
p
p
samplesusedforobtainingthe
chromatograms.

10

Time (min)

Chromatogram for B
12
10

Conc(g//l)

Themobilephaseresidencetime
ofthecolumnwasfoundtobe2
minutesandthevoidage
d h
d
f
fraction
wasdeterminedtobe0.3.

8
6
4
2
0
0

10

12

Time (min)

14

16

18

20

Calculate:
1.Theselectivity
2.Theresolution
3 The respective capacity factors
3.Therespectivecapacityfactors
4.Theplateheightofthechromatographiccolumn
5.Therespectivedistributioncoefficients
Ifwecollectthecolumneffluentfromthestartuntil7minutes,
calculate:
1.ThepurityofAinthecollectedmaterial
2.ThepercentyieldofAinthecollectedsample

tR1 =5min

Chromatogram for A

W1 =4min

Conc. (g/l)
)

5
4
3
2
1
0
0

Time (min)

10

Chromatogram for B

W2 =8min

tR2 =10min
12

Conc(g/l)

10
8
6
4
2
0
0

10

12

Ti
Time
(min)
( i )

14

16

18

20

Theselectivity(using1ascomponentAand2ascomponentB)
From chromatograms: tR1 =5minandt
Fromchromatograms:t
5 min and tR2 =10min;givent
10 min; given tM =2min
2 min
Selectivity

Resolution

Capacityfactors

K 2 k 2 t R 2 t M (10 2 ) min
=
=
=
=
= 2.67
(5 2) min
K1 k1 t R1 t M
R=

t R 2 t R1
10 5
=
= 0.833
0.5(w1 + w2 ) 0.5(4 + 8)

k1 =

t R1 ' 3 min
=
= 1.5
tM
2 min

k 2' =

t R 2 ' 8 min
=
= 4 .0
tM
2 min
i

Theplateheightofthechromatographiccolumn
2

2
t R1
5
= 16 = 25
N1 = number of plates for 1 = 16
4
w1
2

2
t R2
10

= 16 = 25
N 2 = number of plates for 2 = 16
8
w2

l
0 .3 m
H = HETP = =
= 0.012 m
25
N

Distributioncoefficients

K1 =

k1'

K2 =

k 2'

0.3
= 1 .5
= 0.643

1 0 .3

0 .3
= 1.71

= 4 .0
1
1 0.3

ThepurityofAinthecollectedmaterial

C A dt

t1

% purity A = t2
t2
100
C A dt + C B dt
t

t1
1
t2

(( t / t 0 ) 1) 2
C = C 0 exp
2
2

 t 5 2
0 C0, A exp 2 A dt

%A =
7
7
t 5 2
t 10 2
0 C0, A exp 2 A dt + 0 C0, A exp 2 B dt

Bysolvingthedefiniteintegralsintheaboveequationweget

7 5
5
2 AC0, A Erf
+ Erf

2
2

A
A

%A =

7 5
5
7 10
2 AC0, A Erf
+ Erf
+ 2 B C0, B Erf

2 A
2 A
2 B

w
=
4

10
+ Erf

2 B

Th f
Therefore
1 d B =2
2
A =1and

100

Fromthechromatograms,C0,A =5g/landC0,B =10g/l

Substitutingthevaluesintheaboveequationweget
%A=78.5%
TheyieldofAinsamplecollectedfrom0to7minutesisgivenby
7

C
% y ie ld =

0,A

10

C
0

0,A

exp

exp

t 5

dt
2 A
100
2
t 5

dt
2 A
2

SolvingthedefiniteintegralsaspreviouslywegetyieldofA=97.7%

Differential migration of two solutes A and B

DifferentialmigrationoftwosolutesAandB

Parametersusedforthecharacterisation of
differential migration
differentialmigration
Ve
Volumeofelutingsolventrequiredtocarrythe
solutethroughthecolumnuntilitemergesatits
l t th
h th
l
til it
t it
maximumconcentration

Capacityfactor,k
Selectivityorrelativeretention,
VT
Th
Thetotalvolumeofagelcolumnforgel
l l
f
l l
f
l
chromatography

Gelpartitioncoefficient,Kp
Vi

Ve=Vo+KpVi

(Ve Vo )
k=
Vo

K 2 k2 t R 2 tM
=
= =
K1 k1 t R1 tM
VT=Vo+Vi+Vs
Vi =aWr

internalvolumeofliquidintheporesoftheparticles
which is difficult to measure accurately
whichisdifficulttomeasureaccurately
Wr g
(VT V0 )
Viisusuallycalculatedusingaismassofdrygel(a) Vi =
1 + Wr w
andthewaterregainvalue (Wr)

Problem:Gelpermeationchromatography
Apilotscalegelchromatographycolumnpackedwith
Sephacryl resinisusedtoseparatetwohormonesAandB.
Thecolumnis5cmindiameterand0.3mhigh;thevoid
volume is 1 9x104 m3.
volumeis1.9x10
Thewaterregainvalueofthegelis3x103 m3 kg 1 dry
Sephacryl;thedensityofwetgelis1.25x103 kgm3.
ThepartitioncoefficientforhormoneAis0.38;thepartition
The
partition coefficient for hormone A is 0.38; the partition
coefficientforhormoneBis0.15.
Iftheeluant
If
th l
t flowrateis0.7lh
fl
t i 0 7 l h11,whatistheretentiontime
h t i th
t ti ti
foreachhormone?

Thetotalcolumnvolumeis:VT=V0 +Vi +Vs

VT = r 2 h = (2.5 10-2 m) 2 (0.3m) = 5.89 10-4 m3

VoidvolumeV0 =1.9x104 m3;w =1000kgm3

InternalvolumeVi =aWr

Vi =

Wr g
1 + Wr g

(VT V0 )

a ismassofdrygelandWr isthewaterregainvalue
g isthedensityofwetgeland
is the density of wet gel and w isthedensityofwater
is the density of water
( 103 m3 kg
(3
g 1 )(
)(1.25 103 kgm
g 3
4 3
4 3
Vi =
(5
(5.89
89

10
m

1.9
1
9

10
m)
3 3
1
3
1 + (3 10 m kg )(1000kgm )
= 3.74 104 m3

VolumeofelutingsolventVe =Vo+KpVi
KpA =0.38andKpB =0.15
VeA =1.9x104 m3+0.38(3.74x104m3)=3.32x104 m3
VeB =1.9x104 m3+0.15(3.74x104m3)=3.32x104m3
Retentiontimeassociatedwiththeseelutionvolumes

3 .3 2 1 0 4 m 3
tA =
= 2 8 m in
i
3
1m
1h o u r
0 .7 lh 1
.
1 0 0 0 l 6 0 m in
2 .4 6 1 0 4 m 3
tB =
= 2 1 m in
3
1m
1h o u r
1
0 .7
7 lh
.
1 0 0 0 l 6 0 m in

Selectionoftheseparation
l
f h
mechanisminLCbasedonthe
p
criteriaofsamplemolecular
weight,solubilityand
conductivity

Highperformanceliquid
chromatography(HPLC)
h
h (
)
HPLC
HPLCusesveryhighpressures(upto4000psi)
uses very high pressures (up to 4000 psi)
andverysmallparticlesize(downto3m)
Fourmainchromatographictechniquesthat
Four main chromatographic techniques that
usealiquidmobilephase
PartitionChromatography(mostwidelyused)
P titi Ch
t
h (
t id l
d)
LiquidSolidChromatography
IonExchangeChromatography
h
h
h
SizeExclusion(GelPermeation)Chromatography

FactorsInfluencingtheColumnEfficiency
i i id h
inLiquidChromatography
h

Particlesize
Particle
size
Flowrate
Thickness of stationary phase
Thicknessofstationaryphase
Mobilephaseviscosity
Diffusion of solute in mobile and stationary phases
Diffusionofsoluteinmobileandstationaryphases
Howwellacolumnispacked
Sample size (g sample/gpacking)
Samplesize(g
sample/g packing)
InRPLCefficiencyalwaysdecreasesasthesamplesizeis
increased

ExtraColumn
Extra
ColumnBandBroadening
Band Broadening
Extracolumnbandbroadeningbecomesveryimportantfor
smallborecolumns
Majorcontributors
Multiplepathseffects
Multiple paths effects
Longitudinaldiffusion
Masstransferinstationaryandmobilephases

Othersourcesofbandbroadeningunrelatedtocolumn
materialsandoccuroutsidethecolumn

Fittingsdeadvolume
Fittings
dead volume
Tubinglengthanddiameter
Detectorvolume
I j ti
Injectionvolume
l

InstrumentsforLiquid
Chromatography:Pumps
h
h
Reciprocatingpumps
amotordrivenreciprocatingpistoncontrolstheflowofmobilephase
withthehelpoftwoballcheckvalvesthatopensandcloseswiththe
pistonmovement.
Theflowisthusnotcontinuousanddampingofflowisnecessary.
The flow is thus not continuous and damping of flow is necessary.
Thisisaccomplishedusingpulsedamperswhicharealongcoiled
capillarytube

DisplacementPumps
composedofaonedirectionalmotordrivenplungerthatpushesthe
mobilephasepresentinasyringelikechamber
Thevolumeofdisplacementpumpsislimitedwhichlacksconvenience.A
constant flow rate is usually obtained with syringe like pumps
constantflowrateisusuallyobtainedwithsyringelikepumps

Pneumaticpumps
simplestwhereathemobilephaseispushedoutofthemobilephase
containerbythepressureofapressurizedgas.
Theflowisdependentonthebackpressureofthecolumnandusually
theflowislimitedtopressuresbelow2000psi.

Sample Injection Valves

SampleInjectionValves

Columns
Columnsarealmostalwaysmadefrom
stainlesssteel
withmostcommondimensionsintherangefrom
g
25cmlongandabout4.6mminternaldiameter

Pellicular orporouspackingmaterialsare
p
p
g
usuallyused
Pellicular packings arenonporousglassorpolymer
are nonporous glass or polymer
beadsrangingfrom30to40m
Porouspackings
p
g aremostlysilicabasedwith
y
particlediametersfrom310m

Column type
Columntype
Analytical
Analytical2.0
2 0 3
3
Microbore 2 0.5
Capillary<0.5
C ill
0

Fingertightendfittingcolumn
showingfritcap

Removableendfittingcolumn

Radialcompressioncolumncartridge

PEEKcolumn
PEEK
column
(poly(etherether
ketone)

PEEKoborecolumn Integralguardcolumn

Standaloneguardcolumn

Guardcolumnsareusedtoprotectthe
Guard
columns are used to protect the
mainanalyticalcolumn

S
Somecommerciallyavailablebondedphases
i ll
il bl b d d h

Fluorescence
UVVIS

Detectors

RI

Theoreticalproteintitrationcurves,showing
hownetsurfacechargevarieswithpH

Negatively
charged
proteins
Neutralorpositively
charged proteins
chargedproteins

Typicalaffinitypurification
Affinitymediumisequilibratedinbindingbuffer
SSampleisappliedunderconditionsthatfavorspecific
l i
li d d
di i
h f
ifi
bindingofthetargetmolecule(s)toacomplementary
bindingsubstance(theligand).Targetsubstances
bind specifically but reversibly to the ligand and
bindspecifically,butreversibly,totheligand
and
unboundmaterialwashesthroughthecolumn
Targetproteinisrecoveredbychangingconditionsto
Target
protein is recovered by changing conditions to
favorelutionoftheboundmolecules.Elutionis
performedspecifically,usingacompetitiveligand,or
nonspecifically,bychangingthepH,ionicstrengthor
p
y, y
g g
p ,
g
polarity.Targetproteiniscollectedinapurified,
concentratedform.
Affinitymediumisreequilibratedwithbinding
buffer

Typical affinity chromatogram

Typicalaffinitychromatogram

Commontermsinaffinitychromatography
Matrix:forligand attachment.
Matrixshouldbechemicallyandphysicallyinert.
Spacerarm:usedtoimprovebindingbetween
ligand andtargetmoleculebyovercomingany
effectsofsteric hindrance.
Ligand:moleculethatbindsreversiblytoaspecific
target molecule or group of target molecules
targetmoleculeorgroupoftargetmolecules
Binding:
bufferconditionsareoptimizedtoensurethatthetargetmoleculesinteract
buffer
conditions are optimized to ensure that the target molecules interact
effectivelywiththeligand andareretainedbytheaffinitymediumasallother
moleculeswashthroughthecolumn.

Elution:
Bufferconditionsarechangedtoreverse(weaken)theinteractionbetweenthetarget
moleculesandtheligand sothatthetargetmoleculescanbeelutedfromthecolumn.

TerminologyusedinAffinity
Chromatography
h
h
Wash
bufferconditionsthatwashunboundsubstancesfromthecolumn
withoutelutingthetargetmoleculesorthatreequilibratethecolumn
back to the starting conditions (in most cases the binding buffer is
backtothestartingconditions(inmostcasesthebindingbufferis
usedasawashbuffer).

Ligand coupling
covalentattachmentofaligand toasuitablepreactivatedmatrixto
createanaffinitymedium.

Pre
Preactivated
activatedmatrices
matrices
matriceswhichhavebeenchemicallymodifiedtofacilitatethe
couplingofspecifictypesofligand.

Capacity of ion exchanger

Capacityofionexchanger
Quantitativemeasureofitsabilitytotakeup
y
p
exchangeablecounterions
Thetotalioniccapacity
thenumberofchargedsubstituentgroupspergramdry
ionexchangerorpermlswollengel
Measuredbytitrationwithastrongacidorbase.
y
g

Availablecapacityforthegel
Theactualamountofproteinwhichcanbeboundtoan
i
ionexchanger,underdefinedexperimentalconditions
h
d d fi d
i
l
di i
dynamiccapacityfortheionexchangerunderdefinedflow
rates

Schematicrepresentationofthe
methodology based on average surface
methodologybasedonaveragesurface
hydrophobicity ( surface)

Schemefortherefoldingofdenatured
proteinwithHPHIC.

A,adsorption;
D d
D,desorption;
ti
DH,dehydration;
H,hydration;
MP mobile phase;
MP,mobilephase;
ST,stationaryphase.

Gas chromatography
Gaschromatography
Gassolidchromatography
Gasliquid
Gas
liquidchromatography
chromatography

Gas chromatography detectors:

TCD FID andd ECD
TCD,