High performance liquid chromatography.pdf

HPLC AND IT'S ALL COMPONENTS, WORKING

1. HPLC
MUTHYLA BALA KRISHNA,
Asst.Professor,
Pharmaceutical Analysis
VIPW

2. INTRODUCTION
•HPLC stands for“High-performance liquid
chromatography”(sometimesreferredtoas High-
pressureliquidchromatography).
•High performance liquid chromatography is a
powerfultoolinanalysis,ityieldshighperformance
and high speed compared to traditional columns
chromatography because of the forcibly pumped
mobilephase.
•HPLCisachromatographictechniquethatcan
separatea mixtureofcompounds
•Itisused in biochemistryandanalyticalchemistryto
identify, quantify and purify the individual
componentsofa mixture.

3. •Chromatography:physicalmethodin
which separation of components takes
place between two phases-a stationary
phaseandamobilephase
•Stationary phase : The substance on
which adsorption of the analyte (the
substance to be separated during
chromatography)takesplace.Itcanbea
solid,agel,or asolidliquidcombination
•Mobilephase:solventwhichcarriesthe
analyte(aliquidor agas)

4. Chromatographictechniquesaredividedintodifferent
typesbasedon :
Thetypeofchromatographicbedused
i.e.columnchromatography(gaschromatography)and
planarchromatography(paperandthinlayer)
Thephysicalstateofmobilephase
i.e.gaschromatographyandliquidchromatography
Theseparationmechanism
i.e.ion-exchangeandsizeexclusion
HPLC is a type of liquid chromatography where the
sampleis forcedthrougha columnthatis packedwitha
stationary phase composed of irregularly or spherically
shaped particles, a porous monolithic layer , or a porous
membranebyaliquid(mobilephase)athighpressure.

5. COLUMNCHROMATOGRAPHY
Column chromatographyinvolvesthefollowing:
1.Adsorption/retentionofsubstanceon stationaryphase
2.Separationofadsorbedsubstanceusingmobilephase
3.Recoveryofindividualcomponentsbycontinuousflowofmobile
phase
4.Quantitativeandqualitativeanalysisofsoluteandthe
componentswhicharerecovered

6. •Whendistributionofsoluteisequalbetweentwophases,then
effectivedistributionKd =1
•Individual zones adjacent to each other in the column where
there is sufficient space for equal distribution of solutes in
betweenstationaryandmobilephaseare calledtheoreticalplates
•Lengthoftheoreticalplateinthecolumniscalledplateheight
•Morethetheoreticalplates, moreefficienttheseparation
•2000ormore theoreticalplatesareidealforacolumn
•Componentswhichhavemoreaffinitytowardsmobilephase
elutefirst

7. PRINCILPE
TounderstandtheprincipleofHPLC ,wemust firstlookatthe
principlebehind liquidchromatography
Liquidchromatographyisaseparationtechniquethatinvolves:
•theplacement(injection)ofasmallvolume of liquidsample
•intoatubepackedwithporousparticles(stationaryphase)
•whereindividualcomponentsofthesamplearetransportedalongthe
packedtube(column)by aliquidmovedby gravity.
Themainprincipleofseparationisadsorption.

8. •Whena mixtureof componentsareintroducedintothecolumn. various
chemical and/or physical interactions take place between the sample
moleculesand theparticlesofthecolumn packing.
•Theytravelaccordingtotheirrelativeaffinitiestowardsthestationary
phase. The component which has more affinity towards the
adsorbent,travelsslower.
Thecomponentwhichhaslessaffinitytowardsthestationaryphase
travelsfaster.
•Sinceno twocomponentshavethesameaffinity towardsthestationary
phase,thecomponentsareseparated

10. •HPLCisaseparationtechniquethatinvolves:
•theinjectionofasmallvolumeofliquidsampleintoatubepackedwith
tinyparticles(3to5micron(μm)indiametercalledthestationaryphase)
•whereindividualcomponentsofthesamplearemoveddownthepacked
tube(column)withaliquid(mobilephase)forcedthroughthecolumnby
highpressuredeliveredbyapump.

11. •These components are separated from one another by the column
packingthatinvolvesvariouschemicaland/orphysicalinteractions
betweentheirmoleculesandthepackingparticles.
•These separated components are detected at the exit of this tube
(column) by a flow-through device (detector) that measures their
amount.Theoutputfromthedetectoriscalleda liquidchromatogram
Inprinciple,LCandHPLC workthesameway exceptthespeed,
efficiency,sensitivityand easeofoperationofHPLC is vastlysuperior.

12. TYPESOFHPLC
I.BASEDONMODEOFSEPERATION
1.Normalphasechromatography-stationaryphaseispolar
(hydrophilic)andmobilefaceis non-polar(hydrophobic).
2.Reversephase chromatography-stationaryfaceis non-polar
(hydrophobic)andmobilefaceis
Polar(hydrophilic).
•Polar-PolarbondsandNonPolar-NonPolarbondshavemore
affinitythanPolar-NonPolarbonds.
•Reversephasechromatographyismore commonlyusedasdrugs
areusuallyhydrophilic

13. II.BASEDONPRINCIPLEOFSEPERATION
1.AbsorptionChromatography
•IntheAbsorption Chromatographysolutemoleculesbonddirectlytothe
surfaceof thestationaryphase
•the component which has more affinity towards mobile phase elutes
first&thecomponentwhichhaslessaffinitytowards stationaryphase
eluteslater.
No two components have same affinity towards mobile phase &
stationaryphase.

14. 2.Ion-exchangechromatography
•Ion exchangechromatographyisaprocess thatallowstheseparationof
ionsand polarmoleculesbasedon theircharge.
•Itcan beusedforalmostanykindof charged moleculeincludinglarge
proteins,smallnucleotidesand amino acids.
•Retention is based on the attraction between solute ions and charged
sitesboundtothestationaryphase.Ions ofthesamechargeareexcluded.
•The use of a resin (the stationary solid phase) is used to covalently
attachanionsorcationsontoit.Soluteionsoftheoppositechargeinthe
mobileliquidphaseareattractedtotheresinby electrostaticforces.
.

16. 3.Ion-pairchromatography
•Itisaform of chromatographyin whichionsin solutioncanbe "paired"
orneutralizedand separatedasanionpair on areversed-phasecolumn.
•Ion-pairing agents are usually ionic compounds that contain a
hydrocarbonchainthatimpartsacertainhydrophobacityso thattheion
paircan be retainedonareversed-phasecolumn.
4.gelpermeationchromatography
•Thistypeofchromatographylacksanattractiveinteractionbetweenthe
stationaryphaseandsolute.
•Theliquidor gaseousphasepasses throughaporous gelwhich
separatesthemoleculesaccordingtoitssize.

17. •Theporesarenormallysmalland excludethelargersolute
molecules,but allowssmallermoleculesto enterthegel,causingthemto
flow through a larger volume. This causes the larger molecules to pass
throughthecolumnata faster ratethanthesmallerones.

18. 5.AffinityChromatography
•Thisisthemostselectivetypeofchromatographyemployed.Itutilizes
the specific interaction between one kind of solute molecule and a
secondmoleculethatisimmobilizedon a stationaryphase.
For example, the immobilized molecule may be an antibody to some
specificprotein.Whensolutecontainingamixtureofproteinsarepassed
by this molecule, only the specific protein is reacted to this antibody,
binding it to the stationary phase. This protein is later extracted by
changingtheionicstrengthorpH.

19. 6.Chiralchromatography
•Itinvolvestheseparationofstereoisomers.Inthecaseof
enantiomers,thesehavenochemicalorphysicaldifferencesapartfrom
beingthree-dimensionalmirrorimages.Conventionalchromatographyor
other separation processes are incapable of separating them. To enable
chiral separations to take place, either the mobile phase or the stationary
phasemustthemselvesbemadechiral,givingdifferingaffinities
betweentheanalytes.

20. III.BASEDONELUTIONTECHNIQUE
1.Isocraticelution
•Aseparationinwhichthemobilephasecomposition
remainsconstantthroughouttheprocedureistermed
isocraticelution
•In isocratic elution, peak width increases with
retention time linearly with the number of theoretical
plates.Thisleadstothedisadvantagethatlate-eluting
peaksgetveryflatandbroad.
•Bestforsimpleseparations
•Often used in quality control
applicationsthatsupportandarein
closeproximitytoamanufacturing
process

21. 2.Gradientelution
•A separation in which the mobile phase
compositionischangedduringtheseparation
processis describedas agradientelution
•Gradientelutiondecreasestheretentionofthe
later-elutingcomponents so thattheyelute
faster,givingnarrower peaks .This alsoimproves
thepeakshapeandthepeakheight
•Bestfortheanalysisofcomplex
samples
•Often used in method
developmentforunknownmixtures
•Lineargradientsaremostpopular

22. IV.BASEDON SCALEOFOPERATION
1.AnalyticalHPLC
Norecoveryofindividualcomponentsofsubstance
2.PreparativeHPLC
Individualcomponentsof substancecanberecovered

23. V.BASEDONTYPE OFANALYSIS
1.Qualitativeanalysis
Analysisofasubstanceinordertoascertainthenatureofits
chemicalconstituents
Wecanseparateindividualcomponentsbutcannotassessthe
quantityinthisanalysis
2.Quantitaiveanalysis
Determining the amounts and proportions of its chemical
constituents.
Quantityoftheimpurityandindividualcomponentscanbe
assessed

24. INSTRUMENTATION

29. A. Solventdeliverysystem(mobilephase)
•Themobile phaseinHPLCreferstothesolventbeing
continuouslyappliedtothecolumnorstationaryphase
•Themobile phaseactsas acarriertothesample solution
•Asamplesolutionis injectedintothemobilephaseof anassay
throughtheinjectorport
•Asasamplesolutionflowsthroughacolumnwiththemobile
phase,thecomponentsofthatsolutionmigrateaccordingtothe
non-covalentinteractionofthecompoundwiththecolumn

30. •Thechemicalinteractionof themobilephaseandsample, withthe
column , determine the degree of migration and separation of
componentscontainedinthesample
•Thesolventsormobilephasesusedmustbepassedthroughthecolumn
athighpressureatabout1000to3000psi.thisisbecauseastheparticle
sizeofstationaryphaseisaround5-10µ,sotheresistancetotheflowof
solventishigh.

31. B.Pumps
•The role of the pump is to force a liquid (called the mobile phase)
throughtheliquidchromatographata specificflowrate,expressedin
milliliterspermin(mL/min).
•Normal flow ratesinHPLCareinthe1-to2-mL/minrange.
•Typicalpumpscan reachpressuresintherangeof 6000-9000 psi(400-
to600-bar).
•Duringthechromatographicexperiment,apumpcandelivera constant
mobile phase composition (isocratic) or an increasing mobile phase
composition(gradient).

32. TypesofHPLCpumps
There are several types of pumps used for HPLC analysis, most
commonly used arereciprocatingpistonpump,syringepumpand
constantpressurepump
1.Reciprocatingpistonpumps:
•Consistsofasmall motordrivenpistonwhichmovesrapidlybackand
forthinahydraulicchamberthatmayvaryfrom35-400µLinvolume
•Onthebackstroke, theseparationcolumnvalveis closed,andthe
pistonpullsinsolventfromthemobilephasereservoir
•On theforward stroke,thepumppushessolventoutofthecolumnfrom
thereservoir
•Awiderangeofflowratescanbeattainedby alteringthepistonstroke
volumeduringeachcycle, or by alteringthestrokefrequency.

33. •Dual and triple head pump consists of identical piston
chamberunitswhich operateat180 or 120 degreesoutof
phase(this system is significantly smoother because one
pump is fillingwhiletheotheris inthedeliverycycle.

34. 2.Syringetypepump
•Thesearemostsuitableforsmall bore columnsbecausethis pump
deliversonlyafinitevolume ofmobilephasebeforeithas toberefilled.
Thesepumpshave avolumebetween250to 500mL
•Thepumpoperatesbyamotorizedleadscrewthatdeliversmobile
phasetothecolumnataconstantrate.Therateofsolventdeliveryis
controlledbychangingthevoltageonthemotor.

35. 3.Constantpressure pump
•Inthesetypesof pumps,themobilephaseis driventhroughthecolumn
withtheuseof pressurefrom thegascylinder
•.Alow-pressuregassourceis neededtogeneratehighliquidpressures
•Thevalvingarrangementallowstherapidrefillof thesolventchamber
whosecapacityisabout70mL
•Thisprovidescontinuousphaseflowrates

36. C.Injector:
•Theinjectorservestointroducetheliquidsampleintotheflow streamof
themobilephasefor analysis.
•Itisequippedwith sixportvalvessothata sample canbeinjectedintoth
flowpathatcontinuouspressure
•Fora manual injector,theknob ismanuallyoperatedtodeliverthesampl
tothecolumn
•Theknob is set toLOADpositionforsampleinjectionusinga syringe
,thesampleisinjectedintothesampleloop, whichisseparatedfromthe
flowpath
•Theknob is turnedtoINJECTpositionand theeluenttravelsthroughthe
loopfromthepumpanddeliversthesample tothecolumn

37. •Typical sample volumes for manual injector are 5-to 20-microliters
(μL).
•Theinjectormust alsobeabletowithstandthehighpressuresofthe
liquidsystem.
•An autos ampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical. It can
continuouslyInjectvariablevolume a of 1μL–1mL

40. D.Column
•Considered the “heart of the chromatograph” the column’s stationary
phaseseparatesthesamplecomponents ofinterestusingvariousphysical
andchemicalparameters.
•Itisusuallymadeofstainlesssteeltowithstandhighpressurecausedby
the pump to move the mobile phase through the column packing other
materialincludePEEKandglass
•Thesmall particlesinsidethecolumnarecalledthe“packing”what
causethehighbackpressureatnormalflowrates.
•Column packingisusuallysilicagelbecauseof itsparticleshape
,surfaceproperties,and porestructuregiveus agood separation

41. •Othermaterialusedincludealumina,a polystyrene-divinylbenzene
syntheticoran ion-exchangeresin
–Pellicularparticle:original,Spherical,nonporousbeads,
proteinsandlarge biomoleculesseparation(dp:5 μm)
–Porous particle: common used, dp: 3 ~ 10 μm. Narrow size
distribution,porousmicroparticlecoatedwiththinorganicfilm
•Thedimensions of theanalyticalcolumnareusually
-straight,Length(5~25 cm),diameter of column(3~5mm),diameterof particle(35
μm).Number(40k~ 70kplates/m)

42. Guard column is used to remove particular matter and
contamination,itprotecttheanalyticalcolumnandcontainssimilar
packingits temperatureiscontrolledat<150°C,0.1°C
As mentionbefore , columnsaredividedintodifferent types
accordingto theirfunctions(seetypesof HPLC)

43. E.Detector:
•Thedetectorcandetecttheindividualmolecules thatelutefromthe
columnandconvertthedataintoanelectricalsignal
•Adetectorservestomeasuretheamountofthose molecules
•Thedetectorprovidesanoutputtoarecorderorcomputerthatresultsin
theliquidchromatogram
•Detectoris selectedbasedon the analyteorthesample under detection

44. CommonlyuseddetectorsinHPLC
Ultraviolet(UV)
•Thistypeof detectorrespondstosubstancesthatabsorb light.
•TheUVdetectorismainly toseparateand identifytheprincipalactive
componentsofa mixture.
•UVdetectorsarethemostversatile,having thebestsensitivityand
linearity.
•UV detectors cannot be used for testing substances that are low in
chromophores(colorlessor virtuallycolorless)as theycannotabsorb
lightatlowrange.
•Theyarecost-effectiveandpopularandarewidelyused inindustry

45. Fluorescence
•This is a specific detector that senses only those substances that emit
light.Thisdetectorispopular fortraceanalysisinenvironmentalscience.
•Asitisvery sensitive,itsresponseisonlylinearovera relatively
limited concentration range. As there are not many elements that
fluoresce, samplesmustbesyntesizedto makethemdetectable.
MassSpectrometry
•ThemassspectrometrydetectorcoupledwithHPLCiscalledHPLC-
MS.HPLC-MSisthemostpowerfuldetector,widelyusedin
pharmaceuticallaboratoriesand researchanddevelopment.
•Theprincipalbenefitof HPLC-MSisthatitis capableofanalyzingand
providingmolecularidentityof a wide rangeof components.

46. RefractiveIndex(RI)Detection
Therefractiveindex(RI)detectorusesamonochromatorandisoneof
theleastsensitiveLCdetectors.
•Thisdetectorisextremelyusefulfor detectingthosecompoundsthatare
non-ionic,do not absorbultravioletlightanddonot fluoresce.
•e.g.sugar,alcohol,fattyacidandpolymers.

47. F.Dataprocessingunit(Computer)
•Frequentlycalledthedatasystem,thecomputernotonlycontrolsallthe
modulesoftheHPLCinstrumentbutittakesthesignalfromthedetector
andusesittodeterminethetimeofelution(retentiontime)ofthesample
components(qualitativeanalysis)andtheamountofsample(quantitative
analysis).
•Theconcentrationofeachdetectedcomponentiscalculatedfromthe
areaorheightofthecorrespondingpeakandreported.

48. Switch oninstrument
Checksystem setup
Primethepump
Preparethecolumn
OPERATION

49. Set-upsoftware(forsystem
flushing)
Softwaresetup(forsamplerun)
Sampleinjection
Chromatographdataacquisition

50. STANDARDTESTPROCEDURE
BasedontheSTPthemethodof operationisdetermined
EXAMPLE:
Drugname:Pantoprazole(USP)
Description:whitetooff-whitecrystallinepowder
Solubility: freely soluble in water, very slightly soluble in phosphate
bufferatpH7.4, and practicallyinsolubleinn-hexane
Molecularformula:C
16H
15F
2N
3O
4S

51. Molecularweight:383.37
Halflife:1hour
Meltingpoint:Becauseofgradualdegradationof pantoprazolesodium
duringheating,themeltingpointcannotbe determined.

52. %purityofstandard
ASSAY
Assay isdoneto determinethemaincomponent inthedrug
StandardWt.inmg/dilution
Dilution/sampleWt.
inMg
%purityofstandard
Assay=samplearea/standardarea
Avgwt./labelvol.claim
rangeofassayis99%-101%
Forapowdersolution
Assay=samplearea/standardarea StandardWt.inmg/dilution
Dilution/sampleWt.
inMg
Foratablet

53. ChromatographicConditions
•MobilePhaseMethanol:buffer ina50:50ratio
InstrumentationConditions
•Column-C18,12.3x 4.0mm,5µm (silicagelparticlesize).
•InjectorVolume-20µm
•Flowrate-1.5ml/min
•Column OvenTemperature-RoomTemperature
•DetectorWaveLength-260nm
•RunTime-10minutes

54. 1.Preparationofsolutions:
1.Preparation of buffer : dissolve 1.32gm of diammonium hydrogen
orthophosphatein500ml of distilledwaterandadjustthepH to3with
orthophospharicacid(OPA)
2.Prepartationofmobilephase:
Add 500mlof preparedbuffersolutionto500mlofMeOH and mix
properly.
Filterthrough0.45µfilterpaper
Sonicatefordegassing
3.Preparationofstandardsolution
AddPantoprazolesesquihydratetoa20mlflaskandaddmethanol

55. Operation:
•Equilibratethecolumn with mobile phasetillthebaselineisstabilized
•Inject20µl ofmobilephaseintothesystemandrecordthe
chromatogram(blank)
voltage
time
•Inject20µlofstandardsolutionintothesystemandrecordthe
chromatogram
•Injectthe20µlofsample solutionintothesystemandrecordthe
chromatogram
•Washthecolumnwithdistilledwaterfor15 minutesandwithMeOH
for15 minutes

56. Washing
Washing-Methanol(anysolvent)for10-30minutes
-H20(10-30minutes)
Analysis
-MobilePhase
-Standard
-Sample
-H20
-MeOH
Preparationofstandardand&samplesisdone byPPM calculations
(Partspermillion)
PPM=wt.inmg/Vol.inml.1000(1PPM is1mgin1 ltsolution)
Washagain

57. Retentiontime(RT)
Inachromatogram,differentpeakscorrespondtodifferentcomponents
oftheseparatedmixture.
Timeelapsedbetweensample introductionand maximumofresponse, it
is the characteristic time it takes for a particular analyte to pass through
thesystem
Time takenfortheanalyte
to travel from the column
inlet to the point of
detection(maximumpeak)
PARAMETERS

59. •Forassaythemainpeak(maincomponent)isconsidered
•Forpuritytestallthepeaksareconsidered
AreaofpeakA=heightbase/2
Peakareagivesus theconcentrationof theanalyte

60. 2.Retentionvolume:
Retentionvolumeisthevolumeofcarriergasrequiredtoelute
50%ofthecomponentfromthecolumn.Itistheproductofretention
timeandflowrate.
Retentionvolume=Retentiontimeflowrate
3.Seperationfactor:
Separationfactoristheratioofpartitioncoefficientofthetwo
componentstobeseparated.
S=Ka/Kb=(tb-to)/(ta-to)

61. Where
to=Retentiontimeof unretainedsubstance
Ka, Kb =Partitioncoefficientsofa,b
ta,tb=Retentiontimeofsubstancea,b
Ifthereisabigdifferenceinpartitioncoefficientbetween2
compounds,thepeaksarefarapartandtheseparationfactorismore.
Ifthepartitioncoefficientof2compoundsaresimilar,thenthepeaks
arecloserandtheseparationfactorisless.

62. ofseparationof2
4.Resolution:
Resolutionisthemeasureofextent
componentsandthebaselineseparationachieved.
Rs=2(Rt
1-Rt
2)/w
1+w
2
5.HeightEquivalenttoaTheoreticalPlate(HETP):
Atheoreticalplateisanimaginaryorhypotheticalunitofa
columnwheredistributionofsolutebetweenstationaryphaseandmobile
phasehasattainedequilibrium.Itcanalsobecalledasafunctionalunit
ofthecolumn.
Atheoreticalplatecanbeofanyheight,whichdescribestheefficiencyof
eparation.IfHETPisless,thecolumnismoreefficient.IfHETPis
more,thecolumnislessefficient.
.

63. HETP=lengthofthecolumn/no.oftheoreticalplates
HETPisgivenbyVandeemter equation
HETP=A+B/u+Cu
WhereA=Eddydiffusiontermormultiplepathdiffusionwhich
arisesdueto
minimizedbyuniformityof
thepackingofthecolumn.Thiscanbe
packing.
B =Longitudinaldiffusiontermormoleculardiffusion.
C= Effectofmasstransfer.
u=flowrateorvelocityofthemobilephase.

64. 6.Efficiency:
Efficiencyofacolumnisexpressedby thetheoreticalplates.
n=16 Rt
2/w
2
Wheren=nooftheoreticalplates
Rt=retentiontime
w=peakwidthatbase
Rtandwaremeasuredincommonunits(cmormm,minorsec).Noof
theoreticalplates
Ishigh,thecolumnissaidtobehighlyefficient.ForGLC,avalueof
600/metreissufficient.ButinHPLC,highvalueslike40,000to70,000/
meterarerecommended.

65. 7.Asymmetryfactor:
Achromatographicpeakshouldbesymmetricalaboutitscentre
andsaidtofollowGaussiandistribution.Butinpracticeduetosome
factors,thepeakisnotsymmetricalandshowstailingorfronting.
Frontingisduetosaturationofstationaryphaseandcanbe
avoidedbyusinglessquantityofsample.Tailingisduetomoreactive
adsorptionsitesandcanbeeliminatedbysupportpretreatment.
Asymmetryfactor(0.95to1.05)canbecalculatedbyAF=b/a(b,a
calculatedby 5% or10%ofthepeakheight).
Broadpeaksoccurduetothemoreconc.ofsample,largeinjection
volume,columndeterioration.
Ghostpeaksoccurduetothecontaminationofthe
column,compoundfrom earlierinjections.

66. Negativepeaksoccurifmobilephaseabsorbanceislargerthan
sampleabsorbance.
Peakdoublingoccursduetotheco-elutionofinterfering
compound, column overload,channelingincolumn.
Baselinespikesoccurduetotheairbubblesinthemobilephase
and/ordetector,columndeterioration.

67. 1.Calculationofthenumber of TheoreticalPlates(half-height
method)
Where:
N=Numberoftheoreticalplates
V
e= elution volume or retention time (mL, sec, or cm)
h=peak height
w
1/2=widthofthepeakathalfpeakheight(mL, sec,or
cm)
SYSTEMSUITABILITY

68. 2.Calculationofthe number of TheoreticalPlates(USPmethod)
Where:
N=Numberoftheoreticalplates
V
e= elution volume, retention time or retention distance (mL, sec, or cm)
h=peak height
w
b=widthofthepeakatthebaseline(mL, sec,orcm)

69. 3.CalculationofpeakAsymmetryFactor(usedbyTosoh)
Where:
A
s=peakasymmetry factor
b=distancefromthepointat peakmidpointtothetrailingedge
(measuredat10%ofpeakheight)
a=distancefromtheleadingedgeofofpeaktothemidpoint
(measuredat10% ofpeakheight)

70. 3.CalculationofpeakAsymmetryFactor(usedbyTosoh)
Where:
A
s=peakasymmetry factor
b=distancefromthepointat peakmidpointtothetrailingedge
(measuredat10%ofpeakheight)
a=distancefromtheleadingedgeofofpeaktothemidpoint
(measuredat10% ofpeakheight)

71. Where:
T=tailingfactor(measuredat5%ofpeakheight)
b=distancefromthepointatpeakmidpointtothetrailingedge
a=distancefromtheleadingedgeofthepeaktothemidpoint
4.CalculationofTailingFactor(USPmethod)

72. 5.CalculationoftheHeightEquivalenttoa TheoreticalPlate
(HETP)
Where:
H= Heightequivalentof atheoreticalplate
L= Lengthofthecolumn
N= Numberoftheoreticalplates
Dimensions:whenusingHPLCorUHPLCcolumns,His
usuallyexpressedinµm.

73. 6.CalculationofReduced PlateHeight(h)
Giddings introduced dimensionless parameters for H and also for the
linearvelocity.Dimensionlessparametersallowthedirectcomparisonof
the efficiency of two or more columns packed with different particle size
packingmaterials.Accordingtothetheory,awellpackedcolumn should
haveareducedplateheight(h) intherangeof2-3atareducedvelocity
(v)ofabout3.Hereweonlyprovidetheformulafor h.
Where:
h=reducedplateheight(sometimes referredtoasthenumber of
bandwidths)
H=heightequivalentofatheoreticalplate(µm)
d
p=meanparticlesize(µm)

74. 7.CalculationofchromatographicResolution

75. CALIBRATION
Calibrationof HPLCisdone tochecktheperformanceof itsinstrument.
1.flowrate(pumpcalibration)
2.Detectorandinjectorlinearity
3.Systemprecision
4.Columnoventemperature
5.Detectorwavelengthaccuracy

76. 1.Pumpcalibration
•Disconnectthecolumnandconnecttheinletandoutlet
tubing’swith a union.
•Primeallthelinesat5 ml/minflowratewithwaterand
ensurethatflowlineis freefrom airbubbles.
•Set the flow rate at 1ml / min and collect the mobile
phase(water)inadrypreweighedbeakerandcollectthe
mobile phase for 10 min. weigh the beaker to get the
weightofmobilephase.
•Calculatetheflowratebydividingtheweightobtained
withweightpermland10 (runtime).
•Calculatethecorrespondingflowrate.Carryoutthe
experimentinduplicate.

77. Acceptancecriteria
0.05mlforsmallquantities
0.1mlforlargerquantities
2.DetectorandInjectorlinearity
Column:ODSorC18
Mobilephase:milliQwaterandacetonitrile(80:20)
Flowrate:1ml/min
Temperature:40Centigrade
Detectorwavelength:272nm
Runtime:10min

78. Preparationofstocksolution:
•Take 50mg of caffeine and transfer into 50ml of flask and make it up
tothemarkwith thediluent
•Caffeineisused a itgivesa multiwavelengthresponse and isstable
•PreparesolutionsofdifferentPPM(100-600)
•Injectthemandcalculatethearea
•Correlationcoefficientris usedtocheckthedetectorlinearityand
cannotbe lessthan.999
•r=NƩXY-ƩXƩY/√[NƩX²-(Ʃx)²][NƩY²-(ƩY)²]
•Thegraphobtainedbetweenconcentrationandareaislinear

79. 3.Systemprecision
•From the stock solution pipette out 1ml of stock solution and
transferinto10ml flask andmakeituptothemark withdiluentfor
100PPM conc.
•CalculatethepercentageofRSDofareasof5different injections
•%RSD =standarddeviation/averagevalueofabove5injection
areas100
•WhereSD=√Ʃ(X-M)²/n-1
N=#ofinjections
M=average area
X=area
Forsystemprecision%ofRSDis notmorethan1

80. 4.Columnoventemperature
1.Set thecolumnoventemperatureto30andleaveitfor30minutes
2.Openthedoorofthecolumn ovenandkeepthethermometerand
leaveitfor30 min
3.nownotedownthereadinginthethermometer
4.Similarlychangethecolumn oven temperatureto40C and50C and
repeattheaboveprocedure
Acceptancecriteria
2c

81. 5.Detectorwavelengthaccuracy
Column:C18
Mobile phase: HPLC grade methanal
Flowrate:1ml/min
Retentiontime:5minutes
Wavelength:272nm
1.Injectthemethanolandrecordblank
2.Inject20mlofstandardsolutionat 268nm
3.Similarlyinjectthestandardsolutionbyincreasingthenmupto
278nm(increasingit1 atatime)
Acceptancecriteria
273nm1

82. MAINTAINANCE
1.RESERVOIR
PossibleCause PreventiveMaintenance
1.Blockedinletfrit
2.Gasbubbles
1.a.Replace(3–6months)
b.Filtermobilephasewith
0.4-0.5µmfilter
2.Degasmobilephase,
sonification

83. 2.PUMP
PossibleCause PreventiveMaintenance
1.Airbubbles
2.Pump sealfailure
3.Checkvalvefailure
1.Degas mobile phase , do not
changemobilephaseduringrun
2.Replace(3months),cleanwith
1Nacid
3.Filtermobilephase;useinlet-
linefrit;keepspare
4.Impropercleaning 4.CleanwithIsopropylalcohol,
mobile phase container must be
cleaned with mobile phase and
othersectionswithsolvent

84. 3.INJECTOR
PossibleCause PreventiveMaintenance
1.Washing 1.washbeforeandafteruse
2.Rotorsealwear 2.
a.Donotovertighten
3.Syringe
b.Filtersamples
3.Sterilizewhenfreshsampleis
used

85. 4.Column
PossibleCause
1.Numberofinjections
2.Blockedfrit
3.Voidatheadofcolumn
PreventiveMaintenance
1.2000orless
2..a.Filtermobilephase
b.Filtersamples
c.Usein-linefilterand/orguard
column
3..a.AvoidmobilephasepH>8
b.Useguardcolumn
c.Useprecolumn(saturatorcolumn

86. 5.Detector
PossibleCause
1. Lamp failure;
decreaseddetector
2.Bubblesincell
PreventiveMaintenance
1.Replace(6months)or
keepsparelamp
response;increased
detectornoise
2.a.Keepcellclean
b.Userestrictoraftercell
c.Degasmobilephase

87. 6.Software
Updatefrequently,around6-12months

88. TROUBLESHOOTING
Identifyingproblemusingachromatogram
1.Theabovepatternoccurswhenthemobilephaseisnot
suitable

89. 2.Baselinenoise
I.Blankbase(normal)
II.Noisy:
•occurs when there is
contamination of
mobilephase
•impropercleaning

90. 3.Drift
•Temperatureis
unstable
•Contaminationof
column

91. 4.Peaktailing
•Impropermixingofmobilephase(90%ofthe time)
•Problemswithcolumn(30%of thetime)

92. AfteradjustingPH withbuffer
Goodresolution

93. 5.Peakfronting
•Lowtemperature
•Overloadingofsample
•Flowrate
•Overfillingofcolumn
•Insufficientmixing ofmobilephase

94. 6.Highconcentrationofsample

95. RT=4.0
7.Retentiontimevariation
Occursduetoproblemswithmobile
phase
RT=3.0
RT=3.5

96. 8.Communicationerror
Due toproblemincommunicationbetweendetectorandthe software

97. 9.Peaksplitting
Occurswhencolumnlifetimeis diminished

98. 10.Powerfluctuation

99. 11.Variationinarea
•Problemwith injector
•Fluctuationinflow rate
•Errorindetection
A=3010
A=3215
A=3516

100. 12.Ghostpeaks
•Contaminationofmobilephase
•Upsetequilibrium
•Production notfinished

101. 13.Detectorlifetimediminished

102. APPLICATIONS
HPLCisoneof themostwidelyappliedanalyticalseparationtechniques.
Pharmaceutical:
•Tabletdissolutionofpharmaceuticaldosages.
•Shelflifedeterminationsofpharmaceuticalproducts.
•Identificationofcounterfeitdrugproducts.
•Pharmaceuticalqualitycontrol.

103. Environmental
•PhenolsinDrinkingWater.
•Identificationofdiphenhydraminein sedimentsamples.
•BiomoniteringofPAHpollutioninhigh-altitudemountain lakesthrough
theanalysisoffishbile.
•Estrogensincoastalwaters-Thesewagesource.
•Toxicityoftetracyclinesandtetracyclinedegradationproductsto
•environmentallyrelevantbacteria.
•AssessmentofTNTtoxicityinsediment..

104. Forensics
•AmobileHPLC apparatusatdanceparties-on-siteidentificationand
quantificationof thedrugEcstasy.
•Identificationof anabolicsteroidsinserum,urine,sweatandhair.
•Forensicanalysisoftextiledyes.
•Determinationofcocaineandmetabolitesinmeconium.
•Simultaneousquantificationof psychotherapeuticdrugsin human
plasma.

105. Clinical
•QuantificationofDEETinHumanUrine.
•Analysisofantibiotics.
•Increasedurinaryexcretionofaquaporin2inpatientswithliver
cirrhosis.
•Detectionofendogenousneuropeptidesinbrainextracellularfluids.

106. FoodandFlavor
•Ensuringsoftdrinkconsistencyandquality.
•Analysisofvicinaldiketonesinbeer.
•Sugaranalysisinfruitjuices.
•PolycyclicaromatichydrocarbonsinBrazilianvegetablesandfruits.
•Traceanalysisof militaryhighexplosivesinagriculturalcrops.
Stabilityof aspartameinthepresenceofglucoseand vanillin

107. ADVANTAGESOFHPLC:
1.Separationsfast andefficient(highresolutionpower)
2.Continuousmonitoringof thecolumn effluent
3.Itcanbeappliedtotheseparationandanalysisofverycomplex
mixtures
4.Accuratequantitativemeasurements.
5.Repetitiveandreproducibleanalysisusingthesamecolumn.
6.Adsorption,partition,ionexchangeandexclusioncolumn
separationsareexcellentlymade.
7.HPLC ismoreversatilethanGLCinsomerespects,becauseithasthe
advantage of not being restricted to volatile and thermally stable
solute and the choice of mobile and stationary phases is much wider
inHPLC

108. 8.Bothaqueousandnonaqueoussamplescanbeanalyzedwithlittleor
nosamplepretreatment
9.Avarietyofsolventsandcolumnpackingareavailable,providinga
highdegreeof selectivityforspecificanalyses.
10.Itprovidesameansfordeterminationofmultiplecomponentsina
singleanalysis.