DepartmentofChemicalEngineering

andChemicalTechnology

Pharmaceu)calProcessDevelopment
Chromatography
RichardEsco<,2014

Chromatography

Pharmaceutical
Process
Development

LectureAims:

Toillustratetherangeandscopeofthetechniques,instrumenta)onandapplica)ons.
Provideanapprecia)onoftheadvantagesandlimita)onsforuseop)misa)on.
TounderstandthesynergieswithengineeringprinciplesforTheore)calPlatesandScaleup.

Content

History&Background
Types
HPLC&uHPLC
Normalphase
ReversedPhase
Prepara)ve
SimulatedMovingBed
IonExchange
SizeExclusion
GC
Packed
Capillary
SuperCri)calFluid
ThinLayer
CapillaryElectrophoresis
Chiral
Slide 2

ChromatographyHistory

Pharmaceutical
Process
Development

TowritewithcoloursliterallytranslatedfromitsGreekrootschromaandgraphein.
1903ChromatographywasrstdevelopedbytheRussianbotanistMikhailTswe<heproduceda
separa)onofplantpigmentsusingacolumnofcalciumcarbonate
1927DrHeinrichWieland(NobelLaureate)Uptonowwehavelearnedwithmucheorttodis)ll,
crystalliseandrecrystallise,andnowtheycomealongandjustpourthestuthroughatube!
1938IzmailovandShraiberimplementedTLC
1941ArcherJohnPorterMar)nandRichardLaurenceMillingtonSyngedevelopedliquidliquidpar))on
chromatographysepara)ngvariousaminoacids
1944AJPMar)ncreatedpaperchromatography
1947FritzPriorandErikaCremerseparatedoxygen&carbondioxidegaschromatography
1952AJPMar)ndevelopedGasLiquidchromatography(NobelPrize)
1956J.J.VanDeemterintroducedtheequa)onwhichshowsthedependenceofthetheore)calplate
height(HETP)onthemobilephaselinearvelocity.OriginallyintroducedforGC,butithappensthat
thesamephysicalprocessesoccursinHPLC.
1960JohnKnox(Edinburgh)introducedIonpairchromatography,reducedparametersandporous
graphi)ccarbon.
1963JCGiddingsusedsilicagelofsmallspeciedpar)clestoachievehighresolu)onandspeed.
1966HovarthcoinedHPLC
1972Chemicallymodiedsta)onaryphases(reversedphase)wereprepared
1973Merckintroducedsphericalpar)cles

Slide 3

ChromatographyTypes

Pharmaceutical
Process
Development

Thebasisofalltypesofchromatographyisthepar))onofthesample
compoundsbetweenasta)onaryphaseandamobilephasewhichows
overand/orthroughthesta)onaryphase.Dierentmechanismsfrom
dierentcombina)onsofgaseous,solidorliquidphasesgiverisetothe
maintypesofchromatography
Adsorp)on
Par))on
IonExchange
SizeExclusion

Slide 4

ChromatographyTypes

Adsorp'onChromatographyusesasolidsta)onaryphaseeg,silicagel,ac)vated
carbonandaliquidorgaseousmobilephase.Solutesareseparatedaccordingtotheir
dierentadsorp)oncharacteris)csontothesta)onaryphase.

Par''onChromatographyisbasedonathinlmformedonthesurfaceofasolid
supportbyaliquidsta)onaryphase.Solutesequilibratebetweenthemobilephaseand
thesta)onaryliquidphase.

IonExchangeChromatographyaresin(thesta)onarysolidphase)isusedtocovalently
a<achanionsorca)onsontoit.Soluteionsoftheoppositechargeinthemobileliquid
phasearea<ractedtotheresinbyelectrosta)cforces.

MolecularorSizeExclusionChromatographyalsoknownasgelpermea)onorgel
ltra)on,thistypeofchromatographylacksanya<rac)veinterac)onbetweenthe
sta)onaryphaseandsolute.Theliquidorgaseousphasepassesthroughaporousgel
whichseparatesthemoleculesaccordingtotheirsize.Thesmallporesexcludethe
largersolutemolecules,butallowsmallermoleculestoenterthegel,causingthemtobe
retained.Thiscausesthelargermoleculestopassthroughthecolumnatafasterrate
thanthesmallerones.

Pharmaceutical
Process
Development

Slide 5

ChromatographyTechniques

Pharmaceutical
Process
Development

HPLC&uHPLC
Normalphase(Polarsta)onaryphase&nonpolarmobilephase)
ReversedPhase(Nonpolarsta)onaryphase&polarmobilephase)
Prepara)ve(Columndiametersof2cm1m)
SimulatedMovingBed(con)nuouscountercurrentchromatography)
IonExchange(polymerphaseswhichexchangeionswithsolutes)
SizeExclusion(eghighlycrosslinkedpolymethacrylatecolumns)
GC
Packed(CarbowaxPEG,Chromosorbdiatomaceousearth)
Capillary(CoatedegPEGorsiloxanes)
SuperCri)calFluid(Normalphasewithsupercri)calCO2+egMeOH)
ThinLayer(sta)onaryphaseiscoatedonasolidsupportegglass)
CapillaryElectrophoresis(separatesionsbasedontheirelectrophore)cmobility

withtheuseofanappliedvoltage)
Chiral(thesta)onaryphasecontainsasingleenan)omerofachiralcompound)

Slide 6

ReversedPhase

Pharmaceutical
Process
Development

Thetechniqueofusingalkylchainscovalentlybondedtothesolidsupportcreatesahydrophobic
sta)onaryphase,whichhasastrongeranityforhydrophobiccompounds.Theuseofa
hydrophobicsta)onaryphasecanbeconsideredtheopposite,or"reverse",ofnormalphase
chromatographyhencetheterm"reversedphasechromatography
Alkyl(R)bondedphasesaresilicabasedandarepreparedbyreac)ngthehydroxylgroupsonthe
surfaceofthesilicawithorganicsilylchloridesorsilylesters.Anyremainingunreactedsilanol
groupsareblockedbysubsequentmethyla)onwithegtrimethylsilazane.

Themostpopularcolumnisanoctadecylcarbonchain(C18)bonded
silica.ThisisfollowedbyC8bondedsilica,puresilica,cyanobonded
silicaandphenylbondedsilica.
NotethatnotallC18columnshaveiden)calreten)onproper)es.
Surfacefunc)onalisa)onofsilicacanbeperformedinamonomericor
apolymericreac)onwithdierentshortchainorganosilanesusedina
secondsteptocoverremainingsilanolgroups(endcapping).Whilethe
overallreten)onmechanismremainsthesame,subtledierencesin
thesurfacechemistriesofdierentsta)onaryphaseswillleadto
changesinselec)vity.
Commonapplica)onsinclude:Materialpurityassessment,reac)on
monitoring,purica)on,iden)ca)onofimpuri)es,andquality
control.

Slide 7

Instrumenta)onHPLC

Pharmaceutical
Process
Development

Agilent 1200

Slide 8

Instrumenta)onGC

Pharmaceutical
Process
Development

Slide 9

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onof3,4,5TrimethoxybenzaldehydeandImpuri)es

Column:25metres;,0.3mmIDfusedsilica,
0.14umOV1701
Temperature:130oCisothermal

Slide 10

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onStandardMixtureofReferenceMaterialsusingFIDdetec)on

Slide 11

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onofInsec)cidePermethrin

Slide 12

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onofPharmaceu)calRawMaterialandIntermediates

Column:OV7
Detector:FID
Temperature:180oCto270oC

andE/Zisomersof

Slide 13

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onofOilofSassafras
Thisfragrantoilisdis)lledfromtherootbarkis
extensivelyusedinthemanufactureofthe
coarserkindsofperfume,andforscen)ngthe
cheapergradesofsoap.Theoilusedinperfumes
isalsoextractedfromthefruits

Column:25meterby0.3mmIDfused
silica,0.14umpolyethyleneglycol20M
Temperature:60oCfor4mins,5OCper
minto180oC
Detector:FID

Slide 14

ExampleApplica)on

Pharmaceutical
Process
Development

GCSepara)onPolychlorinatedBiphenylsPCBs
100ppmofAroclor1015and1260
PCBswerewidelyusedasdielectricandcoolantuids,forexampleintransformers,capacitors,andelectricmotorss

Column:OV73
Temperature:60oCto275oC
Detector:ElectronCapture

Slide 15

SimulatedMovingBed

Pharmaceutical
Process
Development

Slide 16

CESimula)on

Pharmaceutical
Process
Development

Slide 17

Instrumenta)onSFC

Pharmaceutical
Process
Development

Many commercial instruments now perform both supercritical fluid chromatography and ultra high-performance/
pressure liquid chromatography (uHPLC).
This provides two orthogonal techniques and higher component selectivity.
With operating pressures up to 600 bar (9000 psi) capability small particle HPLC and SFC can be used.
Using gaseous CO2 as the mobile phase makes interfacing with mass spectrometers relatively straight forward.
Slide 18

Pharmaceutical
Process
Development

Slide 19

Pharmaceutical
Process
Development

Theore)calAspects

Theore)calAspects
EciencyreducedparametersandVanDeemterplots
Resolu)onTheore)calPlates
ColumnLoadingsandcycle)mes.
Detectors
FID,TCD,UVDA,MS,ECD,Fluorescence

h=

H
dp

v=

udp
Dm

H = A + B/u + Cu

Quan)ta)on/Calibra)ons

ResponseFactors
Rela)veResponseFactors
InternalStandards
StandardAddi)on

Prepara)veScaleup

Slide 20

Theore)calAspects

Pharmaceutical
Process
Development

Reten)on
Compoundswillspendsome)meinthesta)onaryphase,andsome)meinthemobilephase.The)me
spentbyanindividualmoleculeineachofthe2phasesiscalledthecapacityorreten)onfactork.The
ra)oof)mespentinthe2phasesisequaltothera)oofthemassofthecompoundsinthe2phases.
k=)mespentinthesta)onaryphase=massinthesta)onaryphase
)mespentinthemobilephase massinthemobilephase
Thera)ooftheconcentra)onofacompoundinthe2phasesiscalledthepar))oncoecient(K)
K =

molar concentration in the stationary phase

molar concentration in the mobile phase

ThefactorskandKarerelatedtoeachotherbasedonanotherparametercalledthephasera)o.

volumeofmobilephase
volumeofsta)onaryphase

and

k = K/

Whichshowsthatthemassra)oisafunc)onoftheconcentra)oninthetwophasesandtherela)ve
volumeofthetwophases
Slide 21

Theore)calAspects

Pharmaceutical
Process
Development

Reten)on
The)meittakesforacompoundtotravelthroughthecolumn(fromwhenananalyteisinjectedtowhenit
reachesthedetector)isknownasthereten'on'me(tr).Ifacompoundisnotretainedatall,itwills)lltake
)metotravelthroughthecolumn.Therefore,inordertomakearela)onshipbetweenkandreten)on)me,
anadjustmentmustbemadeforthistravel)me.
The)meittakesforanunretainedcompoundtotravelthroughthecolumnisouenknownasthedead
'me(to).
Usingthedead)me,thereten)onfactor(k)foracompoundcanberelatedtothereten)on)me.

k = (tr to)/ to
Wheretr=thereten)on)meofthecompound,andto=thedead)me

Selec)vity

k2
k1

Slide 22

Theore)calAspects

Pharmaceutical
Process
Development

NumberofTheore)calPlates(N)
Alsoknownascolumneciency,thenumberoftheore)calplatesisamathema)calconceptanditisan
indirectmeasureofpeakwidthforapeakataspecicreten)on)me.

tR
N = 5.54
wh

2
N=numberoftheore)calplates
tr=reten)on)me
wh=peakwidthathalfheight()me)

Acolumnwithahighnumberoftheore)calplateswillhaveanarrowerpeakatagivenreten)on
)methanacolumnwithalowerNnumber.
Highcolumneciencyisbenecialsincelesspeaksepara)on(meaningloweralpha,selec)vity)
isrequiredtocompletelyresolvecomponents.Onsta)onaryphaseswherethealphas()aresmall,
more ecient columns are needed. Column eciency is a func)on of the column dimensions
(diameter, length and lm thickness), the type of carrier gas and its ow rate or average linear
velocity, and the compound and its reten)on. For column comparison purposes, the number of
theore)calplatespermeter(N/m)isouenused.

Slide 23

Theore)calAspects

Pharmaceutical
Process
Development

HeightEquivalenttoaTheore)calPlate(H)

Anothermeasureofcolumneciencyistheheightequivalenttoatheore)calplateH
andusuallyreportedinmillimeters.Theshortereachtheore)calplate,themoreplatesare
"contained"inanylengthofcolumn.This,ofcourse,translatestomoreplatespermeter
andahighercolumneciency.

L
H =
N

N=numberoftheore)calplates
L =Lengthofcolumn(mm)

Slide 24

Theore)calAspects

Pharmaceutical
Process
Development

Resolu)onR
Thehighertheresolu)on,thelesstheoverlapbetweentwopeaks.Separa)onisonlythe
distanceor)mebetweentwopeakmaxima(alpha,).Resolu)ontakesintoconsidera)on
bothalpha()andthewidthofthepeaks.Itiscalculatedusingeitheroftheequa)ons
below.Baselineresolu)onusuallyoccursatresolu)onnumberof1.50;however,thereis
novisiblebaselinebetweenthetwopeaks.Numbersgreaterthan1.50indicatethereis
baselinebetweenthepeaksandnumberslessthan1.50indicatethereissomedegreeof
coelu)on.

t R2 - t R1
R = 1.18
w h1 + w h2

R = 2

t R2 - t R1
w b1 + w b2

tR1 = retention time of first peak

tR2 = retention time of second peak
Wh1 = peak width at half height of first peak (time)
Wh2 = peak width at half height of second peak (time)
Wb1 = peak width at base pf first peak (time)
Wb2 = peak width at base of second peak (time)

Slide 25

Resolu)onandEciency

Pharmaceutical
Process
Development

Resolu)onisrelatedtothesepara)onorcolumneciencyandalsotheselec)vity().
Theeectofchangingtheseparameterscanbeeasilyassessedusingtheequa)on:

R =

N
4

- 1

k2
k2 + 1

k2 = retention factor of second peak

Selec)vity

k2
k1

Slide 26

Pharmaceutical
Process
Development

Example
t
N = 5.54 R
wh

1
2

N1

104
= 5.54
3.7

N2

146
= 5.54
3.7

N3

152
= 5.54
4.0

= 4377
2

= 8626
2

= 7791

Wh1
t0

Wh2

(3.7)

(61.5)

(3.7)

Wh3
(4.0)

Average N = 6931

)me

tR1
(104)

tR2 tR3
(146)

(152)
Slide 27

Pharmaceutical
Process
Development

Example
1

t - t R2
R = 1.18 R3
w h3 + w h2

R = 1.18

152 - 146
=0.78
4.0 + 3.7

Wh1
t0

Wh2

(3.7)

(61.5)

(3.7)

Wh3
(4.0)

)me

tR1
(104)

tR2 tR3
(146)

(152)
Slide 28

Pharmaceutical
Process
Development

Example
R =

- 1

N
4

k2
k2 + 1

k2 =

152 - 61.5
61.5

= 1.47

k1 =

146 - 61.5
61.5

= 1.37

R =

6921 1.07 - 1
4
1.07

1.47
1.47 + 1

=
=0.80

k2
k1

1
2

1.47
= 1.07
1.37

Wh1
t0

Wh2

(3.7)

(61.5)

(3.7)

Wh3
(4.0)

)me

tR1
(104)

tR2 tR3
(146)

(152)
Slide 29

Pharmaceutical
Process
Development

Example
R =

1.5 =

N
4

k2
- 1
k2 + 1

N 1.07 - 1
4
1.07

=1.5forbaselineresolu)on

1
2

1.47
1.47 + 1

Wh1

N = 24,068
t0

Wh2

(3.7)

(61.5)

(3.7)

Wh3
(4.0)

)me

tR1
(104)

tR2 tR3
(146)

(152)
Slide 30

Theore)calAspects
VanDeemterrela)onship

H =

Pharmaceutical
Process
Development

L
N

ThevalueofHdependsprimarilyonfourfactors,1)thevelocityofthemobilephase,2)mul)pathdiusion,3)
thediusionofthecompoundinthemobilephase,and4)thetransferofthecompoundbetweenthesta)onary
phaseandthemobilephase.Forcolumnspackedwithpar)cles(HPLCcolumns),thesefactorscanbeexpressed
bythefollowingformula

H = A + B/u + (Cs + Cm) u

u
A
B
Cs
Cm

is the average linear mobile phase velocity,

is a constant expressing diffusion due to non uniformity of the packing.
is a constant expressing the longitudinal diffusion coefficient in the mobile phase
is the mass transfer term in the stationary phase
is the mass transfer term in the mobile phase

Slide 31

Theore)calAspects

Pharmaceutical
Process
Development

TheATerm
Inpackedcolumns,peakbroadeningistheresultofanumberoffactors.Asmoleculesoftheanalyte
movethroughthecolumn,theytakemanydierentpathsaroundthepackedpar)cles.Someof
thesepathsareundoubtedlylongerthanotherssoasthemoleculesmovethroughthecolumn,they
tendtospreadout.Theamountofspreadingisaectedbythenatureofthecolumnmaterialand
howwellthecolumnispacked.Thisfactorisgenerallypropor)onaltothepar)clesizeofthe
packingmaterial.Thisfactormustbetakenintoaccountforpackedcolumns,butforcapillary
columns,thistermisnotneededsincetherearenopar)cles
Flow
Direction
1

2
Pathways of two molecules
during elution. Distance traveled
by molecule 1 is longer than
that traveled by molecule 2, thus
molecule 1 will take longer to
elute.

Slide 32

Theore)calAspects

Pharmaceutical
Process
Development

TheLongitudinalDiusionTermB/u
Longitudinaldiusionalsocontributestopeakbroadening.Inthisprocess,analytesdiusefromareasofhigh
concentra)ontomorediluteareainfrontofandbehindthemovingband.Thedegreeoflongitudinaldiusion
isreducedtosomeextentbythepackingmaterial.Atlowveloci)eslongitudinaldiusionhasanega)veeect
onresolu)on,butthiseectisnegligibleathigherveloci)es.Thistermisveryimportantingaschromatography
asdiusioncoecientsingassesareordersofmagnitudehigherthaninliquids.Inliquidchromatography,this
termistypicallyclosetozerorela)vetotheotherterms.
TheMassTransferTermsCu.
Themasstransfertermrelatestothefactthatequilibriumbetweenthemobileandsta)onaryphasesis
neverrealisedinachromatographycolumn.Ittakes)meforanalytesinthemobilephasetomoveintothe
sta)onaryphase.Becausenoequilibriumisreached,someoftheanalytesaresweptaheadoftheofthe
mainband.Italsotakes)meformoleculestomoveoutofthesta)onaryphase,andsomeoftheanalytes
moleculeswillbeleubehindbytherapidlymovingmobilephase.Likethelongitudinalterm,themass
transfertermisbasedondiusion.However,longitudinaldiusiontakesplaceparalleltothedirec)onof
ow,andthereforeisinverselyrelatedtothemobilephaseowrate,whilemasstransferdiusiontakes
placeperpendiculartotheowrate.Asaconsequence,thefasterthemobilephasemoves,theless)me
thereisforequilibriumbetweenthephasesandthemasstransfereectonpeakbroadeningisdirectly
relatedtomobilephasevelocity.

Slide 33

Theore)calAspects

Pharmaceutical
Process
Development

Plate Height, H

Forhighresolu)on,thesediusionfactorsshouldbeminimized(plateheightshouldbesmall).Thereis
amop)mumatminimumH.TheVanDeemterplotshowsthattheeectofthevariousbandbroadening
parametersonplateheight.Peakbroadeningduetomul)pathdiusionisrela)velyconstantoverthe
normalrangeofmobilephaseveloci)es,whilepeakbroadeningduetomobilephasemasstransfer
increaseswithmobilephasevelocity.Onthecontrary,peakbroadeningduetolongitudinaldiusionis
highatlowveloci)esandhasalessereectasmobilephasevelocityincreases.Theoveralleectisthat
thereisanintermediatevelocitythatyieldsthesmallestplateheightandhencethehighestresolu)on.
However,intheinterestofspeedofanalysis,recommendedveloci)esareouensetsomewhathigher
thatthis.

A + B/u + Cu
Mass Transfer (both), Cu
Multipath Term, A
Longitudinal diffusion, B/u

Linear Velocity, u
Slide 34

Theore)calAspects

Pharmaceutical
Process
Development

Decreasingpar)clesizehasbeenobservedtolimittheeectofowrateonpeakeciencysmaller
par)cleshaveshorterdiusionpathlengths,allowingasolutetotravelinandoutofthepar)clefaster.
Thereforetheanalytespendsless)meinsidethepar)clewherepeakdiusioncanoccur.No)cethatasthe
par)clesizedecreases,thecurvebecomesa<er,orlessaectedbyhighercolumnowrates.Smaller
par)clesizesyieldbe<eroveralleciencies,orlesspeakdispersion,acrossamuchwiderrangeofusable
owrates,butmuchhigherbackpressures

Slide 35

Theore)calAspects

Pharmaceutical
Process
Development

GiddingsintroduceddimensionlessparametersforHandalsoforthelinearvelocityu.Dimensionless
parametersallowthedirectcomparisonoftheeciencydierentcolumnspackedwithdierentpar)cle
sizepackingmaterials.Accordingtothetheory,awellpackedcolumnshouldhaveareducedplateheight
(h)intherangeof23atareducedvelocity(v)ofabout3.

H
h=
dp

H=heightequivalentofatheore)calplate(m)
dp=meanpar)clesize(m)

Reducedmobilephasevelocityv
Adimensionlessmeasureofthemobilephasevelocitycomparedtodiusionintothepores.

udp
v=
Dm

u=linearvelocityofthemobilephase
dp =par)clediameter
DM=diusioncoecientofthesoluteinthemobilephase.

Withtheseparameters,anempiricalformoftheVanDeemterequa)onwasderivedbyProfJohnKnox
(Edinburgh,1960)

B
h =
+ Av1/ 3 + Cv
v

h=2
Slide 36

Pharmaceutical
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Slide 37

Detectors

Pharmaceutical
Process
Development

GC
ThermalConduc/vityDetector(TCD)
TheTCDisatrulyuniversaldetector.Itconsistsofaheatedsensorinathermostatedchamber,throughwhich
theeuentows.Heliumisusuallyusedasacarriergas,asithasthehighestthermalconduc)vityofanygas,
exceptforhydrogen.Asthepeakselute,thethermalconduc)vityofthegasinthechamberchanges.This
changestheheatowfromtheheatedsensor,throughthegas,tothewalls.Sincethesensorisbeingheated
ataconstantrate,itbecomesho<erasthethermalconduc)vityoftheeuentdrops.Thechangein
temperatureofthesensingwirelamentorthermistorchangesitsresistance.
Thedetectorislimitedbyitsrela)velylowsensi)vity,comparedtootherdetectors,andusuallyhasafairly
largedeadvolume.Itis,therefore,notverysuitableforcapillarywork.

Slide 38

Detectors

Pharmaceutical
Process
Development

GC
FlameIoniza/onDetector(FID)
TheFIDisnearlyuniversallysensi)vetoorganiccompounds,andshowsgoodsensi)vityandexcellent
linearity.Thecolumneuentisfedintoaamefueledbyhydrogen,withaforcedairow.Apoten)alof
severalhundredvoltsisimposedbetweenthe)poftheameburnerandthecollectorwhichsurrounds
theame.Asthesamplecomponentsburn,theyproduceaburstofions.Theseproducea)nycurrent
betweentheame)pandthecollector.
TheFIDdetectorhasanumberofadvantages.Theresponseisroughlypropor)onaltothenumberof
carbonatomsintheameatany)me.Thedetectorisinsensi)vetoinorganicgases,water,carbon
dioxide,sulfurdioxide,nitrogenoxidesandothernoncombus)blegases.Thedetectorhasaverywide
linearrange,overabout7ordersofmagnitude,hasalowdeadvolumeofabout1ml.

Slide 39

Detectors
HPLC

Pharmaceutical
Process
Development

Ultraviolet(UV)detectorsarefairlygeneralinapplica)on,since
mostorganiccompoundsabsorbatsomewavelengthsintheUV
spectrum.However,theuseofwavelengthsbelow210nmis
usuallynotuseableforanalysisbecausemostsolventswhich
wouldbeusedaseluentswouldalsoabsorbintheseareas.The
responseofthisdetectordependsonBeer'sLaw,andtherefore
givesalinearresponseover45ordersofmagnitude.The
detec)onlimitsvarywidely,dependingonthesample
componentanditsex)nc)oncoecientatthewavelength
beingused.Inthemostfavorablecases,1ngorlessofa
compoundmaybedetected.

Othercommonlyuseddetectorsinclude
Refrac)veIndex
Fluorescence
Electrochemical
MassSpec

Slide 40

Quan)ta)on

Pharmaceutical
Process
Development

Data from chromatograms may be used to obtain the relative or absolute concentration of components in a
mixture, providing good resolution is achieved. The Peak area, from integration of the detector signal during
elution of a component, is proportional to the amount of that component in the sample. However, the response
of a detector varies from one compound to another; for example, the HPLC ultraviolet detector depends on
absorption of electromagnetic radiation, the spectra of the components and the detection wavelength used.
There are four principal methods for obtaining quantitative information:

1.
2.
3.
4.

Normalising peak areas

Internal standards
External standards
Standard addition methods.

1. Normalising peak areas is simply the area of an individual peak calculated as a percentage of the total areas
recorded for all peaks in the chromatogram.

Slide 41

Quan)ta)on

Pharmaceutical
Process
Development

2. The internal standard method is a variation on the above, and is recommended for accurate quantitative work.
It eliminates the need for accurate injections since a reference standard is included in each sample analysed.
An internal standard is selected which has a retention time such that it is eluted in a suitable 'gap' in the
chromatogram.
The procedure involves analysing a test mixture sample containing known amounts of each component plus a
predetermined amount of the internal standard (I.S.) to calculate the Response factor RF.

F=((Ax)(Cis))/((Ais)(Cx))

Where:
Ax=Areaofthecompoundofinterest
Cx=Concentra)onofthecompound
Ais=Areaoftheinternalstandard
Cis=Concentra)onoftheinternalstandard
Oncetheresponsefactorisknown,analysisofanunknownmixtureisachievedbyaddinganaccuratelyknown
amountofinternalstandardandthencarryingoutthechromatography.The concentration of each component is
calculated using the equation above, rearranged to give

Cx=(Ax/AIS)x(CIS/RF)
Slide 42

Quan)ta)on

Pharmaceutical
Process
Development

3. External Standard Method

Automated sample injection systems and multiport injection valves

(HPLC) have good reproducibility so that a series of injections can be
made with a variation in sample volume of < 1 %. A set of standard
mixtures containing known concentrations of the analytes is analysed
and their peak areas recorded. A calibration graph of area versus
concentration can be drawn for each analyte to confirm a linear detector
response and from which the amount of the analyte in a mixture can be
determined
4.StandardAddi'on.Thestandardsolu)on(solu)onofknown
concentra)onofanalyte)isaddedtotheunknownsolu)on.Atypical
procedureinvolvespreparingseveralsolu)onscontainingthesame
amountofunknown,butdierentamountsofstandard.Forexample,ve
25mLvolumetricasksareeachlledwith10mLoftheunknown.Then
thestandardisaddedindieringamounts,suchas0,1,2,3,and4mL.
Theasksarethendilutedtothemarkandmixedwell.
Theideaofthisprocedureisthatthetotalconcentra)onoftheanalyteis
thecombina)onoftheunknownandthestandard,andthatthetotal
Concentra)on
concentra)onvarieslinearly.Ifthesignalresponseislinearinthis
concentra)onrange,thenaplotsimilartothatshownisgenerated
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Pharmaceutical
Process
Development

Scaleup

Onceadesiredanaly)calsepara)onhasbeenachieved,aloadingstudyisouenperformedtodeterminethe
capacityofthepackingmaterialandthescaleupfactorcalculated:

(Diameter Prep) X
(Diameter Analytical) 2 X
2

Scale - up factor =

Length Prep
Length Analytical

Thisfactorisusedtocalculatetheprepara)vecolumnloading,egforcolumnsofthesamelength
butwith3.9and19mmIDa24mgloadingcanbeusedfroma1mganaly)calloading
Theequivalentowraterequiredforthesamelinearvelocityiscalculatedfrom:
Flow Rate (Prep) = Flow Rate ( Anal) X

(Diameter Prep)
(Diameter Anal) 2
2

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Scaleup

Pharmaceutical
Process
Development

Prepara)vechromatographyisgenerallycarriedoutundermassand/orvolumeoverloadedcondi)onsinorderto
increasetheproductthroughput.Involumeoverloading,thesampleconcentra)onismaintainedinthelinear
regionoftheisothermandthevolumeisincreasedun)lthethroughputisop)mized.Inmassoverloading,the
sampleconcentra)onisincreasedbeyondthelinearadsorp)onregion,resul)nginasymmetricbandproles,with
selfsharpeningfrontsandtailingrearboundaries.Acombina)onofvolumeandmassoverloadingiscommonly
usedtomaximisethroughputinprepara)veelu)onchromatography.

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Chromatography

Pharmaceutical
Process
Development

LearningOutcomes

Tounderstandthebasicprinciplesofthedierentmajortypesofcommonlyusedchromatographictechniquesand
howtoassesschromatographicperformance.

Tobeabletoapplytheore)calconsidera)onstoprac)calexamples,forexample
Useandselec)onofquan)ta)veprocedures.
Scaleupdecisionsandprocedures.
ExampleQues)ons:

ExplaintheA,BandCtermsoftheVanDeemterequa)onandhowyoucan
comparetheperformanceofdierentHPLCcolumnspackedwithdierentpar)cle
sizematerialswiththeore)calvalues.
Foragivensepara)oncharacterisethechromatographicperformanceintermsof
N,Rsanda.
Describehowyouwouldcalculatetherela)veresponsefactorforcomponentB.
Inscalingupthesepara)onfroman4.5mmidanaly)caltoa76.2mmid
prepara)vecolumn;fromthegivendatawhatowratewouldyouuse;b)what
samplemass/columnloadingwouldyouuse?
FurtherReadingsugges)ons:
PRINCIPLESANDPRACTICEOFCHROMATOGRAPHY,RaymondP.W.Sco<,ChromEdBookSeriesonline
Introduc)ontoModernLiquidChromatography,LloydR.Snyder,JosephJ.KirklandandJohnW.Dolan

Slide 46

Acknowledgement

Pharmaceutical
Process
Development

Semba BioSciences

Slide 47