Lecture 9 Introduction to Chromatography “The Science of 9讲介绍色谱学”.ppt

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Objectives
DefineHighPerformanceLiquidChromatography(HPLC).
Describeindetailtheset-upofanHPLCsystem.
Determineequivalenceinpolarityamongdifferentmobilephasecompositions.
Lecture7–HPLC,23-10-2019
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RealisticSet-UpofyourHPLC(location:instrumentalanalysislaboratory,B7-303)
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Lecture7–HPLC,23-10-2019
?
HPLCisonemodeofchromatography,oneofthemostusedanalyticaltechniques.
,,themixtureseparatesintoitscomponents.
studyhplc/
Theinteractionofthesolutewithmobileandstationaryphasescanbemanipulatedthroughdifferentchoicesofbothsolventsandstationaryphases.
Asaresult,HPLCacquiresahighdegreeofversatilitynotfoundinotherchromatographicsystemsandithastheabilitytoeasilyseparateawidevarietyofchemicalmixtures.
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Lecture7–HPLC,23-10-2019
HPLCinpharmaceuticalanalysisfromdrugdiscoverytoqualitycontrol
HPLCisthedominanttechniqueforpharmaceuticalanalysisusedinresearch,development,andqualitycontrolwiththefollowingfunctions:
Drugdiscovery:findingnewchemicalentities(NCE)foradoptionasnewdrugdevelopmentcandidates.
Chemicaldevelopment:developingviablesyntheticroutesandscale-upprocessesforsynthesizingactivepharmaceuticalIngredients(API).
Pharmaceuticaldevelopment:developingdosageformswithoptimizeddeliveryandstabilityprofilesforclinicalsuppliesandfinalproducts.
Drugmetabolism/pharmacokinetics(DMPK):evaluatingthemetabolicandpharmacokineticprofilesofthedrugcandidatesinanimalmodelsandhumanclinicalstudies.
Qualitycontrol(QC):assessingthequalityofthefinalmanufacturedproductsagainstpublishedspecificationforproductrelease.
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Lecture7–HPLC,23-10-2019
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Highresolution().
Smalldiameter()stainlesssteel,glassortitaniumcolumns.
Columnpackingofstationaryphasewithverysmall(3,5and10μm)particles.
Relativelyhighinletpressuresandcontrolledflowofthemobilephase.
Continuousflowdetectorscapableofhandlingsmallflowratesanddetectingverysmallamounts.
Rapidanalysis.
ThesecharactersreferofcoursetoHPLC,canyoucomparethesecharacterstotheonesrelatedtoclassiccolumnchromatographythatyouperformedinthelab.?
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Lecture7–HPLC,23-10-2019
-UpofHPLC
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Lecture7–HPLC,23-10-2019

.
Gradientconstruction:
High-pressuregradient
Low-pressuregradient(morecommon)wherepro-portioningofthesolvents(upto4)throughafour-wayvalveatlowpressuretakesplace,thenpumpingthemixtureathighpressureintothecolumnisperformed.
Requirementsofpumpingsystem:
Pulsefreeoutput.
-10mL/min.
%.
Corrosionresistant(stainlesssteel&Teflon).
Generationofpressureupto6000psi(400Bar).
ModernHPLCsystemshavebeenimprovedtoworkatmuchhigherpressures,andthereforeareabletousemuchsmallerparticlesizesinthecolumns(<2 μm).These"UltraHighPerformanceLiquidChromatographyUPLC"systemsworkatupto100 MPa(15,000 lbf/in²),orabout1000 Bar.
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Lecture7–HPLC,23-10-2019
DoeshighpressureinHPLCconstituteanexplosionhazard?Why?
tocolumn
solvent
ballcheck
valves
topulse
damper
(in90%ofsystems)
Disadvantage: pulsedflow,thusdampingnecessary
Advantages: smallinternalvolume(35–400L),highoutputpressure(10,000psi)adaptabilityforgradientelution,constantflowrates(largelyindependentofcolumnback-pressureandsolventviscosity).
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Lecture7–HPLC,23-10-2019

:usingaflowcell,mostcommontype(why?).Employdeuterium,tungstenorxenonlampswithmonochromatorstochoosetheoptimumλ.
AspecialtypeofUVdetectors:thephotodiodearraydetectors(PDA).
:
Fluorescenceismeasuredafterexcitationoftheeluatewithalaser.
Adv.:verysensitiveDisadv.:-fluorescinganalytesbycovalentattachmentoffluorophorstoanalyteseitherpriortochromatographicseparation,orbyaddingderivatizationreagentstotheeluatebetweencolumnanddetector.
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Lecture7–HPLC,23-10-2019
Theflowcell