chromatography oct 2014

Pharmaceu)calProcessDevelopment

Chromatography

DepartmentofChemicalEngineeringandChemicalTechnology

RichardEsco<,2014

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PharmaceuticalProcess

DevelopmentChromatography

Content– History&Background– Types

• HPLC&uHPLC– Normalphase– ReversedPhase– Prepara)ve– SimulatedMovingBed– IonExchange– SizeExclusion

• GC– Packed– Capillary

• SuperCri)calFluid• ThinLayer• CapillaryElectrophoresis• Chiral

• Toillustratetherangeandscopeofthetechniques,instrumenta)onandapplica)ons.• Provideanapprecia)onoftheadvantagesandlimita)onsforuse‐op)misa)on.• TounderstandthesynergieswithengineeringprinciplesforTheore)calPlatesandScale‐up.

LectureAims:

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PharmaceuticalProcess

DevelopmentChromatography‐HistoryTowritewithcolours‐literallytranslatedfromitsGreekrootschromaandgraphein.

1903‐ChromatographywasfirstdevelopedbytheRussianbotanistMikhailTswe<‐heproducedasepara)onofplantpigmentsusingacolumnofcalciumcarbonate

1927‐DrHeinrichWieland(NobelLaureate)“Uptonowwehavelearnedwithmuchefforttodis)ll,crystalliseandrecrystallise,andnowtheycomealongandjustpourthestuffthroughatube!”

1938‐IzmailovandShraiberimplementedTLC

1941‐ArcherJohnPorterMar)nandRichardLaurenceMillingtonSyngedevelopedliquid‐liquidpar))onchromatographysepara)ngvariousaminoacids

1944‐AJPMar)ncreatedpaperchromatography

1947‐FritzPriorandErikaCremerseparatedoxygen&carbondioxide‐gaschromatography

1952‐AJPMar)ndevelopedGas‐Liquidchromatography(NobelPrize)

1956‐J.J.VanDeemterintroducedtheequa)onwhichshowsthedependenceofthetheore)calplateheight(HETP)onthemobilephaselinearvelocity.OriginallyintroducedforGC,butithappensthatthesamephysicalprocessesoccursinHPLC.

1960‐JohnKnox(Edinburgh)introducedIon‐pairchromatography,reducedparametersandporousgraphi)ccarbon.

1963‐JCGiddingsusedsilicagelofsmallspecifiedpar)clestoachievehighresolu)onandspeed.

1966‐Hovarthcoined“HPLC”

1972‐Chemicallymodifiedsta)onaryphases(reversedphase)wereprepared

1973‐Merckintroducedsphericalpar)cles

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PharmaceuticalProcess

DevelopmentChromatography‐Types

Thebasisofalltypesofchromatographyisthepar))onofthesamplecompoundsbetweenasta)onaryphaseandamobilephasewhichflowsoverand/orthroughthesta)onaryphase.Differentmechanismsfromdifferentcombina)onsofgaseous,solidorliquidphasesgiverisetothemaintypesofchromatography

•Adsorp)on•Par))on•IonExchange•SizeExclusion

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PharmaceuticalProcess

DevelopmentChromatography‐Types

• Adsorp'onChromatographyusesasolidsta)onaryphaseeg,silicagel,ac)vatedcarbonandaliquidorgaseousmobilephase.Solutesareseparatedaccordingtotheirdifferentadsorp)oncharacteris)csontothesta)onaryphase.

• Par''onChromatographyisbasedonathinfilmformedonthesurfaceofasolidsupportbyaliquidsta)onaryphase.Solutesequilibratebetweenthemobilephaseandthesta)onaryliquidphase.

• IonExchangeChromatographyaresin(thesta)onarysolidphase)isusedtocovalentlya<achanionsorca)onsontoit.Soluteionsoftheoppositechargeinthemobileliquidphasearea<ractedtotheresinbyelectrosta)cforces.

• MolecularorSizeExclusionChromatographyalsoknownasgelpermea)onorgelfiltra)on,thistypeofchromatographylacksanya<rac)veinterac)onbetweenthesta)onaryphaseandsolute.Theliquidorgaseousphasepassesthroughaporousgelwhichseparatesthemoleculesaccordingtotheirsize.Thesmallporesexcludethelargersolutemolecules,butallowsmallermoleculestoenterthegel,causingthemtoberetained.Thiscausesthelargermoleculestopassthroughthecolumnatafasterratethanthesmallerones.

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PharmaceuticalProcess

DevelopmentChromatography‐Techniques

• HPLC&uHPLC– Normalphase(Polarsta)onaryphase&non‐polarmobilephase)

– ReversedPhase(Non‐polarsta)onaryphase&polarmobilephase)

– Prepara)ve(Columndiametersof2cm‐1m)

– SimulatedMovingBed(con)nuouscountercurrentchromatography)

– IonExchange(polymerphaseswhichexchangeionswithsolutes)

– SizeExclusion(eghighlycrosslinkedpolymethacrylatecolumns)

• GC– Packed(Carbowax‐PEG,Chromosorb‐diatomaceousearth)

– Capillary(CoatedegPEGorsiloxanes)• SuperCri)calFluid(Normalphasewithsupercri)calCO2+egMeOH)

• ThinLayer(sta)onaryphaseiscoatedonasolidsupportegglass)• CapillaryElectrophoresis(separatesionsbasedontheirelectrophore)cmobility

withtheuseofanappliedvoltage)

• Chiral(thesta)onaryphasecontainsasingleenan)omerofachiralcompound)

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PharmaceuticalProcess

DevelopmentReversedPhase

• Thetechniqueofusingalkylchainscovalentlybondedtothesolidsupportcreatesahydrophobicsta)onaryphase,whichhasastrongeraffinityforhydrophobiccompounds.Theuseofahydrophobicsta)onaryphasecanbeconsideredtheopposite,or"reverse",ofnormalphasechromatography‐hencetheterm"reversedphasechromatography”

• Alkyl(R)bondedphasesaresilicabasedandarepreparedbyreac)ngthehydroxylgroupsonthesurfaceofthesilicawithorganicsilylchloridesorsilylesters.Anyremainingunreactedsilanolgroupsareblockedbysubsequentmethyla)onwithegtrimethylsilazane.

• Themostpopularcolumnisanoctadecylcarbonchain(C18)bondedsilica.ThisisfollowedbyC8bondedsilica,puresilica,cyanobondedsilicaandphenylbondedsilica.

• NotethatnotallC18columnshaveiden)calreten)onproper)es.Surfacefunc)onalisa)onofsilicacanbeperformedinamonomericorapolymericreac)onwithdifferentshort‐chainorganosilanesusedinasecondsteptocoverremainingsilanolgroups(end‐capping).Whiletheoverallreten)onmechanismremainsthesame,subtledifferencesinthesurfacechemistriesofdifferentsta)onaryphaseswillleadtochangesinselec)vity.

• Commonapplica)onsinclude:Materialpurityassessment,reac)onmonitoring,purifica)on,iden)fica)onofimpuri)es,andqualitycontrol.

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PharmaceuticalProcess

DevelopmentInstrumenta)on‐HPLC

Agilent 1200

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PharmaceuticalProcess

DevelopmentInstrumenta)on‐GC

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PharmaceuticalProcess

DevelopmentExampleApplica)onGCSepara)onof3,4,5‐TrimethoxybenzaldehydeandImpuri)es

Column:25metres;,0.3mmIDfusedsilica,0.14umOV‐1701

Temperature:130oCisothermal

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PharmaceuticalProcess

DevelopmentExampleApplica)on

GCSepara)onStandardMixtureofReferenceMaterialsusingFIDdetec)on

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PharmaceuticalProcess

DevelopmentExampleApplica)onGCSepara)onofInsec)cidePermethrin

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PharmaceuticalProcess

DevelopmentExampleApplica)onGCSepara)onofPharmaceu)calRawMaterialandIntermediates

Column:OV‐7Detector:FIDTemperature:180oCto270oC

andE/Zisomersof

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PharmaceuticalProcess

DevelopmentExampleApplica)onGCSepara)onofOilofSassafras

Column:25meterby0.3mmIDfusedsilica, 0.14umpolyethyleneglycol20M

Temperature:60oCfor4mins,5OCperminto180oC

Detector:FID

Thisfragrantoilisdis)lledfromtherootbarkisextensivelyusedinthemanufactureofthecoarserkindsofperfume,andforscen)ngthecheapergradesofsoap.Theoilusedinperfumesisalsoextractedfromthefruits

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PharmaceuticalProcess

DevelopmentExampleApplica)onGCSepara)onPolychlorinatedBiphenyls‐PCBs

100ppmofAroclor1015and1260

Column:OV‐73

Temperature:60oCto275oC

Detector:ElectronCapture

PCBswerewidelyusedasdielectricandcoolantfluids,forexampleintransformers,capacitors,andelectricmotorss

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PharmaceuticalProcess

DevelopmentSimulatedMovingBed

Slide 17

PharmaceuticalProcess

DevelopmentCESimula)on

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PharmaceuticalProcess

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.

Instrumenta)on‐SFC

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PharmaceuticalProcess

Development

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PharmaceuticalProcess

Development

• Theore)calAspects

– Efficiency‐reducedparametersandVanDeemterplots

– Resolu)on‐Theore)calPlates

– ColumnLoadingsandcycle)mes.

• Detectors

– FID,TCD,UV‐DA,MS,ECD,Fluorescence

• Quan)ta)on/Calibra)ons– ResponseFactors

– Rela)veResponseFactors

– InternalStandards

– StandardAddi)on

– Prepara)veScale‐up

H = A + B/u + Cu

h = H

dp

v =udp

Dm

Theore)calAspects

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PharmaceuticalProcess

DevelopmentTheore)calAspectsReten)on

Compoundswillspendsome)meinthesta)onaryphase,andsome)meinthemobilephase.The)mespentbyanindividualmoleculeineachofthe2phasesiscalledthecapacityorreten)onfactork.Thera)oof)mespentinthe2phasesisequaltothera)oofthemassofthecompoundsinthe2phases.

k=)mespentinthesta)onaryphase=massinthesta)onaryphase)mespentinthemobilephase massinthemobilephase

Thera)ooftheconcentra)onofacompoundinthe2phasesiscalledthepar))oncoefficient(K)

K = molar concentration in the stationary phase

molar concentration in the mobile phase

ThefactorskandKarerelatedtoeachotherbasedonanotherparametercalledthephasera)oβ.

β = volumeofmobilephase volumeofsta)onaryphase k = K/βand

Whichshowsthatthemassra)oisafunc)onoftheconcentra)oninthetwophasesandtherela)vevolumeofthetwophases

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PharmaceuticalProcess

DevelopmentTheore)calAspectsReten)on

The)meittakesforacompoundtotravelthroughthecolumn(fromwhenananalyteisinjectedtowhenitreachesthedetector)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)vityα α =k2

k1

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PharmaceuticalProcess

Development

Alsoknownascolumnefficiency,thenumberoftheore)calplatesisamathema)calconceptanditisanindirectmeasureofpeakwidthforapeakataspecificreten)on)me.

Theore)calAspectsNumberofTheore)calPlates(N)

N = 5.54 tR

wh

2

N=numberoftheore)calplatestr=reten)on)mewh=peakwidthathalfheight()me)

Acolumnwithahighnumberoftheore)calplateswillhaveanarrowerpeakatagivenreten)on)methanacolumnwithalowerNnumber.

Highcolumnefficiencyisbeneficialsincelesspeaksepara)on(meaningloweralpha,α‐selec)vity)isrequiredtocompletelyresolvecomponents.Onsta)onaryphaseswherethealphas(α)aresmall,more efficient columns are needed. Column efficiency is a func)on of the column dimensions(diameter, length and film thickness), the type of carrier gas and its flow rate or average linearvelocity, and the compound and its reten)on. For column comparison purposes, the number oftheore)calplatespermeter(N/m)isouenused.

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PharmaceuticalProcess

Development

Anothermeasureofcolumnefficiencyistheheightequivalenttoatheore)calplate‐Handusuallyreportedinmillimeters.Theshortereachtheore)calplate,themoreplatesare"contained"inanylengthofcolumn.This,ofcourse,translatestomoreplatespermeterandahighercolumnefficiency.

Theore)calAspectsHeightEquivalenttoaTheore)calPlate(H)

H = L

N

N=numberoftheore)calplatesL =Lengthofcolumn(mm)

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PharmaceuticalProcess

Development

Thehighertheresolu)on,thelesstheoverlapbetweentwopeaks.Separa)onisonlythedistanceor)mebetweentwopeakmaxima(alpha,α).Resolu)ontakesintoconsidera)onbothalpha(α)andthewidthofthepeaks.Itiscalculatedusingeitheroftheequa)onsbelow.Baselineresolu)onusuallyoccursatresolu)onnumberof1.50;however,thereisnovisiblebaselinebetweenthetwopeaks.Numbersgreaterthan1.50indicatethereisbaselinebetweenthepeaksandnumberslessthan1.50indicatethereissomedegreeofco‐elu)on.

Theore)calAspectsResolu)on‐R

R = 1.18 t

R2 - t

R1

wh1

+ wh2

R = 2 t

R2 - t

R1

wb1

+ wb2

tR1 = retention time of first peaktR2 = retention time of second peakWh1 = 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)

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PharmaceuticalProcess

DevelopmentResolu)onandEfficiency

Resolu)onisrelatedtothesepara)onorcolumnefficiencyandalsotheselec)vity(α).

Theeffectofchangingtheseparameterscanbeeasilyassessedusingtheequa)on:

k2 = retention factor of second peak

Selec)vity α =k2

k1

R = N

4

- 1

k2

k2 + 1

α

α

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PharmaceuticalProcess

DevelopmentExample1

2 3

)me tR1 tR2 tR3

N = 5.54 tR

wh

2

t0

Wh1

Wh2

Wh3

Average N = 6931

(104) (146) (152)

(3.7)

(3.7)

(4.0)

(61.5)

N1 = 5.54

104

3.7

2

= 4377

N2 = 5.54

146

3.7

2

= 8626

N3 = 5.54

152

4.0

2

= 7791

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PharmaceuticalProcess

DevelopmentExample1

2 3

t0

)me tR1 tR2 tR3

Wh1

Wh2

Wh3

(104) (146) (152)

(3.7)

(3.7)

(4.0)

R = 1.18 t

R3 - t

R2

wh3

+ wh2

R = 1.18 152 - 146

4.0 + 3.7

=0.78

(61.5)

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PharmaceuticalProcess

DevelopmentExample1

2 3

t0

)me tR1 tR2 tR3

Wh1

Wh2

Wh3

(104) (146) (152)

(3.7)

(3.7)

(4.0)

(61.5)

α

1.47

1.37

= 1.07=

k2

= 152 - 61.5

61.5

= 1.47

R = 6921

4 1.07 - 1

1.07

1.47

1.47 + 1

=0.80

α =k2

k1

R = N

4

- 1

k2

k2 + 1

α

α

k1

= 146 - 61.5

61.5

= 1.37

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PharmaceuticalProcess

DevelopmentExample1

2 3

t0

)me tR1 tR2 tR3

Wh1

Wh2

Wh3

(104) (146) (152)

(3.7)

(3.7)

(4.0)

(61.5)

=1.5forbaselineresolu)on

1.5 = N

4 1.07 - 1

1.07

1.47

1.47 + 1

N = 24,068

R = N

4

- 1

k2

k2 + 1

α

α

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PharmaceuticalProcess

DevelopmentTheore)calAspectsVanDeemterrela)onship

H = L

N

ThevalueofHdependsprimarilyonfourfactors,1)thevelocityofthemobilephase,2)mul)pathdiffusion,3)thediffusionofthecompoundinthemobilephase,and4)thetransferofthecompoundbetweenthesta)onaryphaseandthemobilephase.Forcolumnspackedwithpar)cles(HPLCcolumns),thesefactorscanbeexpressedbythefollowingformula

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

u is the average linear mobile phase velocity,A is a constant expressing diffusion due to non uniformity of the packing.B is a constant expressing the longitudinal diffusion coefficient in the mobile phaseCs is the mass transfer term in the stationary phaseCm is the mass transfer term in the mobile phase

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PharmaceuticalProcess

Development

TheATerm

Inpackedcolumns,peak‐broadeningistheresultofanumberoffactors.Asmoleculesoftheanalytemovethroughthecolumn,theytakemanydifferentpathsaroundthepackedpar)cles.Someofthesepathsareundoubtedlylongerthanotherssoasthemoleculesmovethroughthecolumn,theytendtospreadout.Theamountofspreadingisaffectedbythenatureofthecolumnmaterialandhowwellthecolumnispacked.Thisfactorisgenerallypropor)onaltothepar)clesizeofthepackingmaterial.Thisfactormustbetakenintoaccountforpackedcolumns,butforcapillarycolumns,thistermisnotneededsincetherearenopar)cles

12

Flow

Direction

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.

Theore)calAspects

Slide 33

PharmaceuticalProcess

DevelopmentTheore)calAspects

TheLongitudinalDiffusionTermB/u

Longitudinaldiffusionalsocontributestopeakbroadening.Inthisprocess,analytesdiffusefromareasofhighconcentra)ontomorediluteareainfrontofandbehindthemovingband.Thedegreeoflongitudinaldiffusionisreducedtosomeextentbythepackingmaterial.Atlowveloci)eslongitudinaldiffusionhasanega)veeffectonresolu)on,butthiseffectisnegligibleathigherveloci)es.Thistermisveryimportantingaschromatographyasdiffusioncoefficientsingassesareordersofmagnitudehigherthaninliquids.Inliquidchromatography,thistermistypicallyclosetozerorela)vetotheotherterms.

TheMassTransferTermsCu.

Themasstransfertermrelatestothefactthatequilibriumbetweenthemobileandsta)onaryphasesisneverrealisedinachromatographycolumn.Ittakes)meforanalytesinthemobilephasetomoveintothesta)onaryphase.Becausenoequilibriumisreached,someoftheanalytesaresweptaheadoftheofthemainband.Italsotakes)meformoleculestomoveoutofthesta)onaryphase,andsomeoftheanalytesmoleculeswillbeleubehindbytherapidlymovingmobilephase.Likethelongitudinalterm,themasstransfertermisbasedondiffusion.However,longitudinaldiffusiontakesplaceparalleltothedirec)onofflow,andthereforeisinverselyrelatedtothemobilephaseflowrate,whilemasstransferdiffusiontakesplaceperpendiculartotheflowrate.Asaconsequence,thefasterthemobilephasemoves,theless)methereisforequilibriumbetweenthephasesandthemasstransfereffectonpeakbroadeningisdirectlyrelatedtomobilephasevelocity.

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PharmaceuticalProcess

Development

Forhighresolu)on,thesediffusionfactorsshouldbeminimized(plateheightshouldbesmall).Thereisamop)mumatminimumH.TheVanDeemterplotshowsthattheeffectofthevariousband‐broadeningparametersonplateheight.Peakbroadeningduetomul)pathdiffusionisrela)velyconstantoverthenormalrangeofmobilephaseveloci)es,whilepeakbroadeningduetomobilephasemasstransferincreaseswithmobilephasevelocity.Onthecontrary,peakbroadeningduetolongitudinaldiffusionishighatlowveloci)esandhasalessereffectasmobilephasevelocityincreases.Theoveralleffectisthatthereisanintermediatevelocitythatyieldsthesmallestplateheightandhencethehighestresolu)on.However,intheinterestofspeedofanalysis,recommendedveloci)esareouensetsomewhathigherthatthis.

Linear Velocity, u

Pla

te H

eig

ht,

H

Multipath Term, A

Mass Transfer (both), Cu

Longitudinal diffusion, B/u

A + B/u + Cu

Theore)calAspects

Slide 35

PharmaceuticalProcess

Development

Decreasingpar)clesizehasbeenobservedtolimittheeffectofflowrateonpeakefficiencysmallerpar)cleshaveshorterdiffusionpathlengths,allowingasolutetotravelinandoutofthepar)clefaster.Thereforetheanalytespendsless)meinsidethepar)clewherepeakdiffusioncanoccur.No)cethatasthepar)clesizedecreases,thecurvebecomesfla<er,orlessaffectedbyhighercolumnflowrates.Smallerpar)clesizesyieldbe<eroverallefficiencies,orlesspeakdispersion,acrossamuchwiderrangeofusableflowrates,butmuchhigherbackpressures

Theore)calAspects

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PharmaceuticalProcess

DevelopmentTheore)calAspectsGiddingsintroduceddimensionlessparametersforHandalsoforthelinearvelocityu.Dimensionlessparametersallowthedirectcomparisonoftheefficiencydifferentcolumnspackedwithdifferentpar)clesizepackingmaterials.Accordingtothetheory,awellpackedcolumnshouldhaveareducedplateheight(h)intherangeof2‐3atareducedvelocity(v)ofabout3.

ReducedmobilephasevelocityvAdimensionlessmeasureofthemobilephasevelocitycomparedtodiffusionintothepores.

h = H

dp

v =udp

Dm

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

u=linearvelocityofthemobilephasedp =par)clediameterDM=diffusioncoefficientofthesoluteinthemobilephase.

Withtheseparameters,anempiricalformoftheVanDeemterequa)onwasderivedbyProfJohnKnox(Edinburgh,1960)

h = B

v+ Av

1/ 3 + Cvh = 2

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PharmaceuticalProcess

Development

Slide 38

PharmaceuticalProcess

Development

GCThermalConduc/vityDetector(TCD)

TheTCDisatrulyuniversaldetector.Itconsistsofaheatedsensorinathermostatedchamber,throughwhichtheeffluentflows.Heliumisusuallyusedasacarriergas,asithasthehighestthermalconduc)vityofanygas,exceptforhydrogen.Asthepeakselute,thethermalconduc)vityofthegasinthechamberchanges.Thischangestheheatflowfromtheheatedsensor,throughthegas,tothewalls.Sincethesensorisbeingheatedataconstantrate,itbecomesho<erasthethermalconduc)vityoftheeffluentdrops.Thechangeintemperatureofthesensingwirefilamentorthermistorchangesitsresistance.

Thedetectorislimitedbyitsrela)velylowsensi)vity,comparedtootherdetectors,andusuallyhasafairlylargedeadvolume.Itis,therefore,notverysuitableforcapillarywork.

Detectors

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PharmaceuticalProcess

Development

GCFlameIoniza/onDetector(FID)

TheFIDisnearlyuniversallysensi)vetoorganiccompounds,andshowsgoodsensi)vityandexcellentlinearity.Thecolumneffluentisfedintoaflamefueledbyhydrogen,withaforcedairflow.Apoten)alofseveralhundredvoltsisimposedbetweenthe)poftheflameburnerandthecollectorwhichsurroundstheflame.Asthesamplecomponentsburn,theyproduceaburstofions.Theseproducea)nycurrentbetweentheflame)pandthecollector.

TheFIDdetectorhasanumberofadvantages.Theresponseisroughlypropor)onaltothenumberofcarbonatomsintheflameatany)me.Thedetectorisinsensi)vetoinorganicgases,water,carbondioxide,sulfurdioxide,nitrogenoxidesandothernon‐combus)blegases.Thedetectorhasaverywidelinearrange,overabout7ordersofmagnitude,hasalowdeadvolumeofabout1ml.

Detectors

Slide 40

PharmaceuticalProcess

Development

Ultraviolet(UV)detectorsarefairlygeneralinapplica)on,sincemostorganiccompoundsabsorbatsomewavelengthsintheUVspectrum.However,theuseofwavelengthsbelow210nmisusuallynotuseableforanalysisbecausemostsolventswhichwouldbeusedaseluentswouldalsoabsorbintheseareas.TheresponseofthisdetectordependsonBeer'sLaw,andthereforegivesalinearresponseover4‐5ordersofmagnitude.Thedetec)onlimitsvarywidely,dependingonthesamplecomponentanditsex)nc)oncoefficientatthewavelengthbeingused.Inthemostfavorablecases,1ngorlessofacompoundmaybedetected.

DetectorsHPLC

Othercommonlyuseddetectorsinclude

Refrac)veIndexFluorescenceElectrochemicalMassSpec

Slide 41

PharmaceuticalProcess

DevelopmentQuan)ta)onData from chromatograms may be used to obtain the relative or absolute concentration of components in amixture, providing good resolution is achieved. The Peak area, from integration of the detector signal duringelution of a component, is proportional to the amount of that component in the sample. However, the responseof a detector varies from one compound to another; for example, the HPLC ultraviolet detector depends onabsorption of electromagnetic radiation, the spectra of the components and the detection wavelength used.

There are four principal methods for obtaining quantitative information:

1. Normalising peak areas2. Internal standards3. External standards4. Standard addition methods.

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

Slide 42

PharmaceuticalProcess

DevelopmentQuan)ta)on2. 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 thechromatogram.

The procedure involves analysing a test mixture sample containing known amounts of each component plus apredetermined amount of the internal standard (I.S.) to calculate the Response factor RF.

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

Where:Ax=AreaofthecompoundofinterestCx=Concentra)onofthecompoundAis=AreaoftheinternalstandardCis=Concentra)onoftheinternalstandard

Oncetheresponsefactorisknown,analysisofanunknownmixtureisachievedbyaddinganaccuratelyknownamountofinternalstandardandthencarryingoutthechromatography.The concentration of each component iscalculated using the equation above, rearranged to give

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

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PharmaceuticalProcess

Development

3. External Standard Method

Automated sample injection systems and multiport injection valves(HPLC) have good reproducibility so that a series of injections can bemade with a variation in sample volume of < 1 %. A set of standardmixtures containing known concentrations of the analytes is analysedand their peak areas recorded. A calibration graph of area versusconcentration can be drawn for each analyte to confirm a linear detectorresponse and from which the amount of the analyte in a mixture can bedetermined

Quan)ta)on

Concentra)on

4.StandardAddi'on.Thestandardsolu)on(solu)onofknownconcentra)onofanalyte)isaddedtotheunknownsolu)on.Atypicalprocedureinvolvespreparingseveralsolu)onscontainingthesameamountofunknown,butdifferentamountsofstandard.Forexample,five25mLvolumetricflasksareeachfilledwith10mLoftheunknown.Thenthestandardisaddedindifferingamounts,suchas0,1,2,3,and4mL.Theflasksarethendilutedtothemarkandmixedwell.

Theideaofthisprocedureisthatthetotalconcentra)onoftheanalyteisthecombina)onoftheunknownandthestandard,andthatthetotalconcentra)onvarieslinearly.Ifthesignalresponseislinearinthisconcentra)onrange,thenaplotsimilartothatshownisgenerated

Slide 44

PharmaceuticalProcess

DevelopmentScale‐up

Onceadesiredanaly)calsepara)onhasbeenachieved,aloadingstudyisouenperformedtodeterminethecapacityofthepackingmaterialandthescale‐upfactorcalculated:

Scale - up factor = Diameter Prep( )2

X Length Prep

Diameter Analytical( )2 X Length Analytical

Thisfactorisusedtocalculatetheprepara)vecolumnloading,egforcolumnsofthesamelengthbutwith3.9and19mmIDa24mgloadingcanbeusedfroma1mganaly)calloading

Theequivalentflowraterequiredforthesamelinearvelocityiscalculatedfrom:

Flow Rate Prep( ) = Flow Rate Anal( ) X Diameter Prep( )2

Diameter Anal( )2

Slide 45

PharmaceuticalProcess

DevelopmentScale‐upPrepara)vechromatographyisgenerallycarriedoutundermassand/orvolumeoverloadedcondi)onsinordertoincreasetheproductthroughput.Involumeoverloading,thesampleconcentra)onismaintainedinthelinearregionoftheisothermandthevolumeisincreasedun)lthethroughputisop)mized.Inmassoverloading,thesampleconcentra)onisincreasedbeyondthelinearadsorp)onregion,resul)nginasymmetricbandprofiles,withself‐sharpeningfrontsandtailingrearboundaries.Acombina)onofvolumeandmassoverloadingiscommonlyusedtomaximisethroughputinprepara)veelu)onchromatography.

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PharmaceuticalProcess

DevelopmentChromatography

• Tounderstandthebasicprinciplesofthedifferentmajortypesofcommonlyusedchromatographictechniquesandhowtoassesschromatographicperformance.

• Tobeabletoapplytheore)calconsidera)onstoprac)calexamples,forexample– Useandselec)onofquan)ta)veprocedures.– Scale‐updecisionsandprocedures.

LearningOutcomes

ExampleQues)ons:

• ExplaintheA,BandCtermsoftheVanDeemterequa)onandhowyoucancomparetheperformanceofdifferentHPLCcolumnspackedwithdifferentpar)clesizematerialswiththeore)calvalues.

• Foragivensepara)oncharacterisethechromatographicperformanceintermsofN,Rsanda.

• Describehowyouwouldcalculatetherela)veresponsefactorforcomponentB.• Inscalingupthesepara)onfroman4.5mmidanaly)caltoa76.2mmid

prepara)vecolumn;fromthegivendatawhatflowratewouldyouuse;b)whatsamplemass/columnloadingwouldyouuse?

FurtherReadingsugges)ons:

PRINCIPLESANDPRACTICEOFCHROMATOGRAPHY,RaymondP.W.Sco<,Chrom‐EdBookSeries‐on‐lineIntroduc)ontoModernLiquidChromatography,LloydR.Snyder,JosephJ.KirklandandJohnW.Dolan

Slide 47

PharmaceuticalProcess

Development

• Semba BioSciences

Acknowledgement