HPLC STUDY

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY(HPLC)
BY-SUBHASMITH PRADHAN
Chromatographyisa technique toseparate mixturesof substancesintotheircomponentsonthe
basisof theirmolecularstructure andmolecularcomposition.
Thisinvolvesastationaryphase (asolid,ora liquidsupportedona solid) andamobile phase (aliquid
or a gas).The mobile phase flowsthroughthe stationaryphase andcarriesthe componentsof the
mixture withit.Sample componentsthatdisplaystrongerinteractionswiththe stationaryphase will
move more slowlythroughthe columnthancomponentswithweakerinteractions.
Thisdifference inratescause the separationof variuoscomponents.Chromatographicseparations
can be carriedoutusinga varietyof stationaryphases,includingimmobilizedsilicaonglassplates
(thin-layerchromatography),volatilegases(gaschromatography),paper(paperchromatography)
and liquids(liquidchromatography).
High performance Liquid Chromatography
Highperformance liquidchromatography(HPLC) is basicallyahighlyimprovedformof columnliquid
chromatography.
Insteadof a solventbeingallowedtodripthroughacolumnundergravity,itis forcedthroughunder
highpressuresof upto 400 atmospheres.Thatmakesitmuchfaster.
All chromatographicseparations,includingHPLCoperate underthe same basicprinciple;separation
of a sample intoitsconstituentpartsbecause of the difference inthe relativeaffinitiesof different
moleculesforthe mobilephase andthe stationaryphase usedinthe separation.
Types ofHPLC
There are followingvariantsof HPLC,dependinguponthe phase system(stationary) inthe process:
1. Normal Phase HPLC
Thismethodseparatesanalytesonthe basisof polarity.NP-HPLCusespolarstationaryphase and
non-polarmobilephase.Therefore,the stationaryphase isusuallysilicaandtypical mobilephases
are hexane,methylenechloride,chloroform, diethyl ether,andmixturesof these.
Polarsamplesare thusretainedonthe polarsurface of the columnpackinglongerthanlesspolar
materials.

2. 2. Reverse Phase HPLC
The stationaryphase isnonpolar(hydrophobic) innature,while the mobile phase isapolarliquid,
such as mixturesof waterandmethanol oracetonitrile.Itworksonthe principle of hydrophobic
interactionshence the more nonpolarthe material is,the longeritwill be retained.
3. Size-exclusion HPLC
The columnis filledwithmaterial havingpreciselycontrolledpore sizes,andthe particlesare
separatedaccordingtoits theirmolecularsize.Largermolecules are rapidlywashedthroughthe
column;smallermoleculespenetrate inside the porousof the packingparticlesandelutelater.
4. Ion-Exchange HPLC
The stationaryphase has an ionicallychargedsurface of opposite charge tothe sample ions.This
technique isusedalmostexclusivelywithionicorionizable samples.
The strongerthe charge on the sample,the strongeritwill be attractedtothe ionicsurface andthus,
the longeritwill take toelute.The mobile phase isanaqueousbuffer,where bothpHand ionic
strengthare usedto control elutiontime.
Instrumentation of HPLC
High-performance-liquid-chromatography-hplc

3. As showninthe schematicdiagraminFigure above,HPLCinstrumentationincludesapump,injector,
column,detectorandintegratoror acquisitionanddisplaysystem.The heartof the systemisthe
columnwhere separationoccurs.
Solvent delivery system(mobile phase)
 The mobile phase inHPLCrefersto the solventbeing continuouslyappliedtothe columnor
stationaryphase
 The mobile phase actsas a carrier to the sample solution
 A sample solutionisinjectedintothe mobilephase of anassay throughthe injectorport
 As a sample solutionflowsthroughacolumnwiththe mobile phase, the componentsof that
solutionmigrate accordingto the non-covalentinteractionof the compoundwiththe
column
 The chemical interactionof the mobile phaseandsample ,withthe column,determine the
degree of migrationandseparationof componentscontainedinthe sample
 The solventsormobile phasesusedmustbe passedthroughthe column athighpressure at
about1000 to 3000 psi.thisisbecause asthe particle size of stationaryphase isaround5-
10µ, so the resistance tothe flow of solventishigh.
1. Solvent Reservoir
Mobile phase contentsare containedinaglassreservoir.The mobile phase,orsolvent,inHPLCis
usuallyamixture of polarand non-polarliquidcomponentswhoserespective concentrationsare
varieddependingonthe compositionof the sample.
2. Pump
A pumpaspiratesthe mobile phase fromthe solventreservoirandforcesitthrough the system’s
columnand detector.Dependingonanumberof factors includingcolumndimensions,particlesize
of the stationaryphase,the flowrate andcompositionof the mobilephase,operatingpressuresof
up to 42000 kPa (about6000 psi) can be generated.
The role of the pumpis to force a liquid(calledthe mobilephase)throughthe liquidchromatograph
at a specificflow rate,expressedin millilitrespermin(mL/min).
 Normal flowratesinHPLC are inthe 1-to 2-mL/minrange.
 Typical pumpscan reach pressuresinthe range of 6000-9000 psi (400-to 600-bar).
 Duringthe chromatographicexperiment,apumpcandeliveraconstant mobile phase
composition(isocratic) oranincreasingmobile phase composition(gradient).

4. Types of HPLC pumps
There are several typesof pumpsusedforHPLCanalysis,most commonlyusedare reciprocating
pistonpump, syringe pumpand constantpressure pump
1. Reciprocating piston pumps:
 Consistsof a small motordrivenpiston whichmovesrapidlybackand forthin a hydraulic
chamberthat may varyfrom 35-400µL involume
 On the back stroke , the separationcolumnvalve isclosed,andthe pistonpullsinsolvent
fromthe mobile phase reservoir
 On the forwardstroke, the pumppushessolventoutof the columnfrom the reservoir
 A wide range of flowratescan be attainedbyalteringthe pistonstroke volume duringeach
cycle , or by alteringthe stroke frequency.
 Dual and triple headpumpconsistsof identical piston chamberunitswhichoperateat180
or 120 degreesoutof phase(thissystemissignificantlysmootherbecause one pumpisfilling
while the otherisinthe deliverycycle.

5. 2.Syringe type pump
 These are most suitable forsmall bore columnsbecausethispump deliversonlyafinite
volume of mobile phase before ithastobe refilled. These pumpshave avolume between
250 to 500mL
 The pump operatesbya motorizedleadscrew thatdeliversmobile phase tothe columnata
constantrate .The rate of solventdeliveryis controlledbychangingthe voltage onthe
motor.

6. 3.Constant pressure pump
 In these typesof pumps,the mobile phase isdriventhroughthe column withthe use of
pressure fromthe gas cylinder
 A low-pressure gassource isneededtogenerate highliquid pressures
 The valvingarrangementallowsthe rapidrefillof the solventchamber whosecapacityis
about70mL
 Thisprovidescontinuousphase flow rates
3. Sample Injector
The injectorcan be a single injectionoran automatedinjectionsystem.AninjectorforanHPLC
systemshouldprovide injectionof the liquidsamplewithinthe range of 0.1-100 mL of volume with
highreproducibilityandunderhighpressure(upto4000 psi). The injectorservestointroduce the
liquidsample intothe flowstreamof the mobile phase foranalysis.
 It isequippedwithsix portvalvessothata sample canbe injectedintothe flow pathat
continuouspressure
 For a manual injector,the knobismanuallyoperatedtodeliverthe sample tothe column
 The knob issetto LOAD positionforsample injectionusingasyringe,the sample isinjected
intothe sample loop,whichis separatedfromthe flow path
 The knob isturnedto INJECTpositionandthe eluenttravelsthroughthe loopfromthe
pumpand deliversthe sampletothe column
 Typical sample volumesformanual injectorare 5-to20-microliters (μL).
 The injectormustalsobe able towithstandthe highpressuresof the liquidsystem.
 An autosampleristhe automaticversionforwhenthe userhasmany samplestoanalyze or
whenmanual injectionisnotpractical.Itcan continuouslyInjectvariablevolume aof 1 μL –
1 mL

7. 4. Columns
Columnsare usuallymade of polishedstainlesssteel,are between50and 300 mmlongand have an
internal diameterof between2and 5 mm. Theyare commonlyfilledwithastationaryphase witha
particle size of 3–10 µm.
 It isusuallymade of stainlesssteel towithstandhighpressure causedby the pumptomove
the mobile phase throughthe columnpackingother material include PEEKandglass
 The small particlesinside the columnare calledthe “packing”what cause the highback
pressure atnormal flowrates.
 Columnpackingisusuallysilicagel because of itsparticle shape,surface properties,and
pore structure give us a goodseparation
 Othermaterial usedinclude alumina,apolystyrene-divinyl benzenesyntheticoran ion-
exchange resin–Pellicularparticle:original,Spherical,nonporousbeads, proteinsandlarge
biomoleculesseparation(dp:5μm)– Porousparticle:commonused,dp:3 ~ 10 μm. Narrow
size distribution,porousmicro particle coatedwiththinorganicfilm
 The dimensionsof the analytical columnare usually-straight,Length(5~25 cm),diameterof
column(3~ 5 mm),diameterof particle(35μm).Number(40k ~ 70 k plates/m)

8.  Guard columnisusedto remove particularmatterand contamination,itprotectthe
analytical columnandcontainssimilar packingitstemperature iscontrolledat< 150 °C, 0.1
°CAsmentionbefore ,columnsare dividedintodifferenttypes accordingtotheirfunctions
(see typesof HPLC)
5. Detector
A detectorservestomeasure the amountof those molecules
 The detectorprovidesanoutputto a recorderor computerthat resultsin the liquid
chromatogram
 Detectorisselectedbasedonthe analyte orthe sample underdetection
 CommonlyuseddetectorsinHPLCis-
Ultraviolet(UV)
 Thistype of detectorrespondstosubstancesthatabsorblight.
 The UV detectorismainlytoseparate andidentifythe principal active componentsof a
mixture.
 UV detectorsare the most versatile,havingthe best sensitivityand linearity.
 UV detectorscannotbe usedfor testingsubstancesthatare low in chromophores (colorless
or virtuallycolorless) astheycannotabsorb lightat low range.
 Theyare cost-effectiveandpopularandare widelyusedinindustry.

9. Fluorescence
 Thisis a specificdetectorthatsensesonlythosesubstancesthatemit light.Thisdetectoris
popularfortrace analysisinenvironmentalscience.
 As itis verysensitive,itsresponse isonlylinearoverarelatively limitedconcentrationrange.
As there are not manyelementsthat fluoresce ,samplesmustbe syntesizedtomake them
detectable.
MassSpectrometry
 The mass spectrometrydetectorcoupledwithHPLCiscalledHPLC-MS.HPLC-MSis the most
powerful detector, widelyusedin pharmaceutical laboratoriesandresearchand
development.
 The principal benefitof HPLC-MSisthat it iscapable of analyzingand providingmolecular
identityof awide range of components.

10. RefractiveIndex(RI)Detection
 The refractive index (RI) detector usesamono chromatorand is one of the leastsensitive
LCdetectors.
 Thisdetectorisextremelyusefulfordetectingthose compoundsthatare non-ionic,donot
absorbultravioletlightanddonot fluoresce.
 e.g.sugar,alcohol,fattyacidand polymers.
6. Data Collection Devices
Data processing unit (Computer)
 Frequentlycalledthe datasystem, the computernotonlycontrolsall the modulesof the
HPLC instrumentbutittakesthe signal fromthe detector andusesit to determinethe time
of elution(retentiontime) of the sample components(qualitative analysis) andthe amount
of sample (quantitative analysis).
 The concentrationof each detectedcomponentiscalculatedfromthe areaorheightof the
correspondingpeakandreported.
OPERATION Switchoninstrument-ChecksystemsetupPrime the pumpPrepare the column
Set-upsoftware(forsystemflushing)Software setup(forsample run) Sampleinjection
Chromatographdata acquisition.

11. High-performance-liquid-chromatography-hplc
Applications of HPLC
The informationthatcan be obtainedbyHPLC includesresolution,identificationandquantification
of a compound.Italsoaidsin chemical separationandpurification.The otherapplicationsof HPLC
include :
PharmaceuticalApplications
 To control drug stability.
 Tabletdissolutionstudyof pharmaceutical dosagesform.
 Pharmaceutical qualitycontrol.
EnvironmentalApplications
 Detectionof phenoliccompoundsindrinkingwater.
 Bio-monitoringof pollutants.
Applicationsin Forensics
 Quantificationof drugsinbiological samples.
 Identificationof steroidsinblood,urineetc.
 Forensicanalysisof textile dyes.
 Determinationof cocaine andotherdrugsof abuse inblood,urine etc.
Food and Flavour
 Measurementof Qualityof softdrinksandwater.
 Sugar analysisinfruitjuices.
 Analysisof polycycliccompoundsinvegetables.
 Preservativeanalysis.
Applicationsin Clinical Tests
 Urine analysis,antibioticsanalysisinblood.
 Analysisof bilirubin, biliverdininhepaticdisorders.

12.  Detectionof endogenous Neuropeptidesinextracellularfluidof brainetc.