Agilent1200 Infinity系列可變波長探測器的性能特點VIP

PerformancecharacteristicsoftheAgilent1200InfinitySeriesVariableWavelengthDetectors

Fasterresults,improvedsensitivityandabsolutedatasecurity

TechnicalOverview

Introduction

TheAgilent1200InfinitySeriesVariableWavelengthDetectors(VWD)aredesignedforhighestopticalperformance,compliancewithGLPregulationsandeasymainte-nance.Twoversionsareavailable:theAgilent1260InfinityVWD(G1314F),andtheAgilent1290InfinityVWD(G1314E)withhighsamplingratesforultra-fastHPLCandUHPLC.Thesedetectorsofferthefollowingfeaturesandbenefits:

Highersamplingratesupto160Hzforultra-fastHPLC

(Agilent1290InfinityVWD)orupto80Hz(Agilent1260InfinityVWD)

Datarecoverycard(DRC)providesforunique"data-never-lostinsurance"(Agilent1290InfinityVWD)

Deuteriumlampforhighestintensityandlowestdetectionlimitoverawavelengthrangeof190to600nm

Optionalflow-cellcartridgessuchasstandard(10mmpathlength/14μLvolume),highpressure(10mm/14μL),micro(3mm/2μL)andsemi-micro

(6mm/5μL)areavailableandcanbedeployedaccordingtoapplicationneeds

Easyfrontaccesstoflowcellandlampforfastreplacement

FlowcellandlampwithRFIDtagforsafeidentification

Lampinformation,includingpartnumber,serialnumber,productiondate,numberofignitions,totalburntime

Cellinformation,includingpartnumber,serialnumber,productiondate,nominalpathlength,volume,maximumpressure

Built-inelectronictemperaturecontrol(ETC)forimprovedbaselinestability

Built-inholmiumoxidefilterforfastverificationofwavelengthaccuracy

PAGE

5

Opticaldesign

Figure1showstheopticalsystemoftheAgilent1260and1290InfinityVWD.Itsradiationsourceisadeuteri-um-arcdischargelampfortheultravio-let(UV)wavelengthrangefrom190to600nm.Thelightbeamfromthedeu-teriumlamppassesthroughalens,filterassembly(inopen,cut-offorholmiumoxideposition),entranceslit(1mmstan-dard),sphericalmirror(M1),grating,asecondsphericalmirror(M2),abeamsplitter,andfinallythroughtheflowcelltothesamplediode.Thebeamthroughtheflowcellisabsorbeddependingonthesolutionsinthecell,inwhichUVabsorptiontakesplace,andthesamplephotodiodeconvertstheintensitytoanelectricalsignal.Partofthelightisdirectedtothereferencephotodiodebythebeamsplittertoobtainareferencesignalforcompensationofintensityfluctuationofthelightsource.Aslitinfrontofthereferencephotodiodecutsoutlightofthesamplebandwidth.

Bandwidthistypically6.5nm.Wavelengthselectionismadebyrotat-ingthegrating,whichisdrivendirectlybyasteppermotor.Thisconfigurationallowsfastchangeofthewavelength.Thecut-offfilterismovedintothelightpathabove370nmtoreducehigherorderlight.

Datasamplingrates

Thereisamajortrendtoreduceanaly-sistimesinordertoincreasesamplethroughput.Analysistimesaslowas

0.6minandpeakwidthsof0.4sarenowachievablewithmodernLCequip-mentsuchastheAgilent1290InfinityLCsystem.Thishasplacedhighdemandsonthedatasamplingrate.Detectorsmustbefastenoughtopro-videsufficientdatapointsforthesesmallpeaks.Table1showsthedatasamplingratesettingsoftheAgilent1260and1290InfinityVWD.

Forultra-fastLCapplication,usetheAgilent1290InfinityVWD.Formoreconventionalapplications,theAgilent1260InfinityVWDoffersasufficientlyhighdatasamplingrate.

Thispublicationevaluatesandcom-parestheperformanceoftheAgilent1260InfinityVWDandtheAgilent1290InfinityVWDtotheperformanceofanearlierAgilent1200SeriesVWDmodel.Performancecharacteristicsevaluatedinclude:

Driftandnoise

Limitofdetection(LOD)foranthracene

Detectionofimpuritiesatlevelsbelow0.03%ofthemaincompound

Sensitivityimprovementsbetweenultra-fastLCandconventionalLC

Deuteriumlamp

Cut-offfilterHolmiumoxidefilter

Slit

Samplediode

Mirror1

Flowcell

Beamsplitter

Mirror2

Referencediode

Linearityofcaffeine

Figure1

Opticalsystemof1200SeriesVWDandVWDSLPlus.

Influenceofdatarateonresolution,peakwidthandpeakcapacity

Influenceofdifferentcellsonresolu-tion,noiseandsignal-to-noiseratio

Equipment

AnAgilent1260InfinityBinaryLCsystemwasusedfortheevaluation,comprisingthefollowingmoduleswithfirmwarerevisionsA.06.01orhigher:

Agilent1260InfinityBinaryPumpwithAgilent1260InfinityMicroDegasser

Agilent1260InfinitySeriesHighPerformanceAutosampler

Agilent1260InfinityThermostattedColumnCompartment

Agilent1260InfinityVWDandAgilent1290InfinityVWD

Agilent1200SeriesVWD(earliermodel)

AgilentZORBAXRRHT1.8μmcolumns

1260InfinityVWDPeak

width(min)

Responsetime(s)

Samplingrate(Hz)

1290InfinityVWDPeak

width(min)

Responsetime(s)

Samplingrate

<0.003125

<0.0625

80

<0.0012

<0.03

160

>0.003125

0.0625

80

>0.0012

0.03

160

>0.00625

0.125

80

>0.0025

0.06

160

>0.0125

0.25

40

>0.005

0.12

80

>0.025

0.5

20

>0.01

0.25

40

>0.05

1

10

>0.025

0.5

20

>0.1

2

5

>0.05

1

10

>0.2

4

2.5

>0.1

2

5

>0.4

8

1.25

>0.2

4

2.5

>0.4

8

1.25

Table1

Peakwidths,responsetimesandsamplingratesoftheAgilent1260and1290InfinityVWD.

Noiseanddrift

Absorbance[mAU]

Noiseanddriftarekeyparameterswhenevaluatingtheperformanceofdetectors.Figure2showsthenoiseanddriftbehavioroftheAgilent1260and1290InfinityVWDcomparedtothatoftheearlierAgilent1200SeriesVWDmodel.ThebaselinenoiseoftheAgilent1260and1290InfinityVWDistypicallyafactorofthreetofivetimesbetterthantheearliermodel.Theexperimentaldatapresentedhereshowsanimprovementinbaselinenoisebyafactorof3.4.

Thedriftbehavioralsodependsstronglyonhowsensitivethedetectorreactstochangesinambienttempera-ture.Thedatapresentedhereshowsa2.4-foldimprovementindriftfortheAgilent1260and1290InfinityVWD.BothdetectorswerewithinAgilent’sspecifications.

LimitofdetectionofanthraceneVariablewavelengthdetectorsarefrequentlyusedforqualitycontrolapplications.Onerequirementforensuringbestproductqualityistheabilitytodetectallimpuritiespresentinafinalproduct.Thiscanbedonereli-ably,ifthedetectorexhibitslownoisebehavior.Figure3showstheevaluationofthelimitofdetectionforthe1260and1290InfinityVWD,5pgofanthracenewereinjectedin3μLofacetonitrile.Thelimitofdetectionwas

0.228pgwherebythecalculationwasbasedonthe5pginjection.

0.1

0.08

Earliermodel

Noise:<0.44×10-5AUDrift:<0.99×10-4AU

0.06

0.04

0.02

1260and1290InfinityVWD

0 Noise:<0.126×10-5AU–3.5-timesbetter

Drift:<0.42×10-4AU–2.4-timesbetter

-0.02

-0.04

0

10

20

30

40

50

Time[min]

Figure2

NoiseanddriftofVWDvs.earliermodel.

ChromatographicconditionsColumn: RestrictioncapillaryMobilephase:Water

0.2

0.1

AnthraceneLOD:0.228pg

Noise:0.002mAU

0

-0.1

-0.2

-0.3

0.5

1

1.5

Time[min]

2

2.5

Absorbance[mAU]

Flowrate: 1mL/min

Figure3

Wavelength: 254nmResponsetime:2s

Peakwidth: >0.1minColumntemp.:36°C

Measurementofthelimitofdetectionofanthracene.

Chromatographicconditions

Sample: Anthracene,1.6668pg/μL,5.004pg/3μLColumn: AgilentZORBAXSBC18,2.1x50mm,

1.8μm

Mobilephase:Water/Acetonitrile,30/70Flowrate: 0.5mL/min

Detection:Wavelength251nmPeakwidth:>0.025min(20Hz)Cellvolume:14μL

Pathlength: 10mmInjectionvol.:3μLColumntemp.:36°C

Short0,12mmidcapillarieswereusedtoconnectinjectionvalve,columnanddetector.The1.6μLvolumeheatexchangerwasusedinthedetector.

Absorbance[mAU]

Figure4showsacomparisonoftheAgilent1260and1290InfinityVWDwithanearliermodelfortheevaluationofsignal-to-noiseratio.Anthracenewasusedassamplewherebythesameamountwasinjectedandthesamepeakwidthsettingsusedforbothdetectors.TheresultsshowthattheAgilent1260and1290InfinityVWDshowedbetternoisebehaviorandthesignal-to-noiseratiowasimprovedbyafactorof4.6.

Detectionofimpurities

Figure5showsacomparisonoftheAgilent1260and1290InfinityVWDwithanearliermodelfortheanalysisofimpuritiesatlevelslessthan0.03per-centofthemaincompound.TheimprovednoisebehavioroftheAgilent1260and1290InfinityVWDensuredbetteridentificationandquantitativeresults.Theincreaseinsignal-to-noiseratiowasbyafactorofabout3.

Performancecomparisonbetweenconventionalandultra-fastLC

Absorbance[mAU]

Anotherwaytoincreaseperformanceandsamplethroughputsimultaneouslyistousecolumnswith1.8μmparticlesinsteadof5μmparticle.Figure6showsacomparisonbetweenaconventionalLCapplicationandanultra-fastLCapplication.

A150×4.6mm,5μmcolumnwasusedfortheconventionalLCapplica-tionandtheruntimewas10min.Fortheultra-fastLCapplicationa50×

4.6mm,1.8μmcolumnwasusedand

1.75

1.50

1260and1290InfinityVWD

S/N:507.5

Noise:0.0026mAU

ImprovementFactor:4.6

1.25

1.00

0.75

0.1

0.05

0

-0.05

-0.1

-0.15

-0.2

-0.25

EarliermodelS/N:110.2

Noise:0.01mAU

0.50

0.25

1.41.61.82.02.22.42.62.8

0

-0.25

0

0.5

1

1.5

2

2.5 Time[min]

Figure4

Comparisonofsignal-to-noiseratiousinganthraceneassample.

Chromatographicconditions

Sample: 16.668pg/μL,50.04pg/3μL

Column: AgilentZORBAXSBC18,2.1×50mm,1.8μmMobilephase:Water/Acetonitrile,30/70

Flowrate: 0.5mL/min

Detection: (Oldermodel)251nm,peakwidth>0.025min(14Hz),14μLflowcellvolume,10mmpathlength

(1260and1290InfinityVWD)251nm,peakwidth>0.025min(20Hz),14μLflowcellvolume,10mmpathlength

Injectionvol.:3μLColumntemp.:36°C

1.0

0.8

0.6

0.4

0.2

0

-0.2

-0.4

Oldermodel

Peak1:S/N=3.1,peakwidth=0.0204minPeak5:S/N=4.1,peakwidth=0.0234minNoiseP-to-P:0.025mAU

2

4 5

1

1.0

0.8

0.6

0.4

0.2

0

-0.2

-0.4

2

1260and1290InfinityVWD

Peak1:S/N=8.6,peakwidth=0.0219minPeak5:S/N=12.4,peakwidth=0.0225minNoiseP-to-P:0.0071mAU

4 5

1

0 0.5 1.0 1.5 2.0 2.5 Time[min]

Short0.12mmIDcapillarieswereusedtoconnectinjectionvalve,columnanddetector.The1.6μLvolumeheatexchangerwasusedinthedetector.

Figure5

Analysisofimpuritiesatlevels<0.03%ofmaincomponent.

Chromatographicconditions

Sample: Tramadol,2.112mg/mL,containingfourimpurities(peaks1,2,3and4)Column: AgilentZORBAXSBC-18,4.6×50mm,1.8μm,for600baroperationMobilephase:SolventA:Water+0.2%TFA,SolventB:Acetonitrile+0.16%TFAGradient: 17to45%Bin2.8min,holdfor0.2min

Stoptime: 3min

Posttime: 1min

Flowrate: 2.2mL/min

Injectionvol.:3μL,10swashforexteriorofneedleColumntemp.:30°C

Detection: (1260and1290InfinityVWD)270nm,peakwidth=0.025min(20Hz),10mmpathlength(oldermodel)270nm,peakwidth=0.025min(14Hz),10mmpathlength

Absorbance[mAU]

theruntimewasdecreasedto0.6min.Forthisshortenedruntime,thedatasamplingrateoftheAgilent1290InfinityVWDwasincreasedfrom>0.05min(peakwidth)to>0.0025min.

Increasingthedataratecausesanincreaseinbaselinenoiseandanincreasebyafactoroftwowasobservedinthisexample.Increasingthedataratealsoinfluencesthesignal-to-noiseratio,whichwasmeasuredtobeabout1.5to2timesbetterfortheultra-fastLCapplication.Table2summarizestheresultsofbothapplications.

Atwofoldincreaseinthelimitofdetec-tionwasachievedforthelateelutingpeaks.TheAgilent1260and1290InfinityVWD,whichtypicallydeliverathreefoldincreaseinsignal-to-noiseratio,wereabletotakeadvantageofthecolumnswith1.8μmparticlesandfacilitateda10to20-foldincreaseinspeedandafivefoldincreasein

signal-to-noiseratio.

Detectionlinearity

ThedetectionlinearityoftheAgilent1260and1290InfinityVWDwasdeter-minedusingcaffeine.Elevendifferentconcentrationsfrom0.1to1000μg/mLwereanalyzed(Table3).Overthetest-edconcentrationrangethelinearitywaswithin±5%,(Figure7).

600

1

500

400

Ultra-fastLC

AgilentZORBAXSBC18,50×4.6mm,1.8μmRuntime=0.6min

300

200

5

100

9

0

0.1

250

0.2

1

0.3

0.4 0.5 0.6

200

ConventionalLC

AgilentZORBAXSBC18,150×4.6mm,5μmRuntime=10min

150

100

50

5

9

0

1

2

3

4

5

6

7

8

9Time[min]

Figure6

PerformancecomparisonbetweenconventionalLCandultra-fastLC.

Chromatographicconditionsforultra-fastLC

Sample: Phenonetestmix(orderno.5188-6529),diluted1:10Column: AgilentZORBAXSBC18,50×4.6mm,1.8μmGradient: 50-100%ACNin0.3min

Flowrate: 5mL/min

Stoptime: 0.6minTemperature:60°CInjectionvol.:3μL

Detection: Peakwidth>0.0025min,datarate160Hz,standardcellpathlength10mm

ChromatographicconditionsforconventionalLC

Sample: Phenonetestmix(orderno.5188-6529),diluted1:10Column: AgilentZORBAXSBC18,150×4.6mm,5μmGradient: 35to95%ACNin10min

Flowrate: 1.5mL/min

Stoptime: 10minTemperature:50°CInjectionvol.:3μL

Detection: Peakwidth>0.05min,datarate10Hz,standardcellpathlength10mm

Table2

PerformancecomparisonbetweenconventionalLCandultra-fastLC.

10 500.

Parameter

ConventionalLC

Ultra-fastLC

Calibrationlevel

Amount(μg/mL)

Runtime

10min

0.6min(17

1

0.977

timesfaster)

2

1.953

Peakwidth

3

3.906

peak2

0.0492min

0.00698min

4

7.812

S/Npeak2

1814.9

2001.0

5

15.625

S/Npeak5

1094.6

1618.7

6

31.25

Resolution

7

62.5

peak5

4.24

2.37

8

125.

S/Npeak9

55.8

124.2

9

250.

11 1000.

Table3

Concentrationsusedforlinearityevaluation.

Influenceofdatasamplingrateonresolution,peakwidthandpeakcapacity

Itisimportanttosetthedatasamplingratecorrectlytogeneratesufficientdatapoints.About20datapointsperpeakarerequiredtoobtaincorrect

Responsefactor[amount/area]

1.00E-01

9.00E-02

8.00E-02

7.00E-02

6.00E-02

5.00E-02

4.00E-02

0.00E+00 2.00E+02 4.00E+02 6.00E+02

±5%Error

8.00E+02 1.00E+03 1.20E+03

quantitativeresults.Ifthedatasam-plingrateistoolow,peaksseparatedinthecolumnbecomemergedinthe

Figure7

Amount[μg/mL]

detectorandleadtoincorrectquantifi-cation.Settingthedatasamplingratetoohighcausesanincreaseinbaselinenoise.Toselecttheoptimumdatasam-plingrate,thepeakwidthofthesmall-estpeakshouldbeusedasareferenceandtheratesetaccordingly.Figure8showsanexampleofanultra-fastanalysiswithdifferentdatasamplingratesettings.

InTable4,theresultsarecombinedandshowthatadatasamplingrateof

160Hzisoptimumforultra-fastapplications.

Influenceofdifferentflowcellsonresolution,noiseandsignal-to-noiseratio

AvarietyofflowcellsareavailablefortheAgilent1260and1290InfinityVWD,whichcanbeinstalledusingthesamequickandsimplemountingproce-dure.Theflowcellshaveintegratedradio-frequencyidentification(RFID)tagsthatholdspecificinformationaboutthecellsuchaspartnumber,cellvolume,pathlength,andsoon.AnIDtagreaderinthedetectorcollectsthisinformationandtransfersittothecon-trolsoftware.Table5showsthecellsthatareavailable.

InTable6,recommendationsaremadewhatflowcellmatchesthecolumnused.Ifmorethanoneselectionisappropriate,usethelargerflowcelltogetthebestdetectionlimit.Usethesmallerflowcellforbestpeakresolution.

LinearityofAgilent1260and1290InfinityVWDfrom1to1000μg/mL.

Chromatographicconditions

Sample: Caffeineat11concentrations

Column: AgilentZORBAXSBC18,50×4.6mm,1.8μmMobilephase:Water/Acetonitrile,85/15

Flowrate: 1mL/min

Detection: 272nm,response2s,peakwidth>0.1min,standardflowcellwith14μLvolumeand10mmpathlength

Responsetime=8s,1.25Hz

0

400

0

Responsetime=4s,2.5Hz

500

0

Responsetime=2s,5Hz

Responsetime=1s,10Hz

0

Responsetime=0.5s,20Hz

0

2000

0

Responsetime=0.25s,40Hz

Responsetime=0.12s,80Hz

0

Responsetime=0.06s,160Hz

0

1

2

3

45

6 78

9Responsetime=0.03s,160Hz

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 Time[min]

Absorbance[mAU]

Injectionvol.:10μLColumntemp.:36°C

Figure8

Influenceofdatasamplingrateonresolutionandpeakwidth.

Chromatographicconditions

Sample: Phenonetestmix(orderno.5188-6529)Column: AgilentZORBAXSBC18,50×4.6mm,1.8μmGradient: 50-100%ACNin0.3min

Flowrate: 5mL/min

Stoptime: 0.8min

Temperature:60°CInjectionvol.:2μL

Detection: Seefigureforresponsetimeanddatasamplingrate,standardflowcellwith14μLvolumeand10mmpathlength

Forultra-fastapplicationswithshortcolumnsandhigherflowrates,theparametersettingsshowninTable7arerecommend.

Flowcellpathlength

Lambert-Beer’slawdefinesalinearrelationshipbetweenabsorbanceandthepathlengthoftheflowcell.

Absorbance(A)=logI0/I=Cdwhere:

Aisdefinedasthequotientoftheintensityofthetransmittedlight,I0,dividedbytheintensityoftheincidentlight,I0.

istheextinctioncoefficient,which

isacharacteristicofagivensub-stanceunderaprecisely-definedsetofconditionsofwavelength,solvent,temperatureandotherparameters.

Cistheconcentrationoftheabsorb-ingspecies(usuallying/Lormg/L).

disthepathlengthofthecellusedforthemeasurement.

Asaresult,flowcellswithlongerpathlengthsyieldhighersignals.Althoughnoiseusuallyincreasesslightlywithincreasingpathlength,thereisagaininsignal-to-noiseratio.Whenincreas-ingthepathlength,thecellvolumeusuallyincreasesaswell.Typically,thiscausesmorepeakdispersion.Asa

rule-of-thumbtheflowcellvolumeshouldbeaboutonethirdofthepeakvolumeathalfheight.

Responsetime(s)

Resolutionpeak5

Peakwidthpeak2(min)

Peakwidthpeak6(min)

0.03(160Hz)

2.61

0.00635(0.381s)

0.0049

0.06(160Hz)

2.56

0.00656

0.005

0.12(80Hz)

2.48

0.00667

0.00521

0.25(40Hz)

2.11

0.00764

0.00642

0.5(20Hz)

1.45

0.0107

0.00976

1(10Hz)

0.89

0.0183

0.0172

2(5Hz)

n.a.

0.0343

4(2.5Hz)

n.a.

8(1.25Hz)

n.a.

Table4

Influenceofresponsetimeonresolutionandpeakwidth.

Celltypestandard

Semi-micro

Micro

Highpressure

Max.pressure[bar]

40

40

120

400

Pathlength[mm]

10

6

3

10

Volume[μL]

14

5

2

10

Table5

Flowcellsfor1260and1290InfinityVWD.

Columnlength

<=5cm

10cm

20cm

>=40cm

Typicalpeakwidth

0.025min

0.05min

min

min

Typicalflowrate

Microflowcell

Recommendedflowcell

Semimicroflowcell

Standardflowcell

0.05-0.2mL/min

Internalcolumndiameter 1.0mm

0.2-0.4mL/min

2.1mm

0.4-0.8mL/min1-5mL/min

3.0mm

4.6mm

Table6

Flowcellrecommendations.

Columninsidediameter

2.1mm

3.0mm

4.6mm

Practicalflowrate(ml/min)

0.4-5

1-5

2-5

Flowcellvolume,pathlength

2μL,3mm

5μL,6mm

14μL,10mm*

*Forultrafastanalysiswithstepgradientsthemicroflowcell(2μL3mm)givesthebestperformance.Iflongercolumns(>50mm)forhigherresolutionareused,thenthenextlargerflowcellisthepreferredchoiceforhighersensitivity.

Table7

Flowcellrecommendationsforultra-fastanalysis.

Parameter

14μLvolumeflowcell,10mmpathlength,

2mL/minflowrate

14μLvolumeflowcell,10mmpathlength,

1mL/minflowrate

μLvolumeflowcell,

mmpathlength,1mL/minflowrate

μLvolumeflowcell,

mmpathlength,1mL/minflowrate

Peakwidthofpeak2

0.0229min

0.0442min

0.0400min

0.0417min

Peakwidthofpeak5

0.0214min

0.0425min

0.0383min

0.0396min

Peakwidthofpeak9

0.0211min

0.0425min

0.0387min

0.0400min

Resolutionofpeak5

4.16

4.06

4.48

4.33

Signal-to-noiseofpeak2

6318.5

4760.5

3389.4

4926.9

Signal-to-noiseofpeak9

365.6

264.5

188.6

271.3

Table8

Influenceofdifferentcelldimensionsonpeakwidth,resolutionandsignal-to-noiseratio.

Figure9andTable8showtheinfluenceofdifferentflowcellsonpeakwidth,signal-to-noiseratio,resolutionandnoise.The14μLvolumeflowcellwasevaluatedusingtwodifferentflowrates.Withaflowrateof2mL/minanda3minutegradient,theperformanceofcolumnswith1.8μmparticleswassig-nificantlybettercomparedtoaflowrateof1mL/minanda6minutegradi-ent.Ataflowrateof1mL/min,the

2μLvolumeflowcellgavesmallerpeakwidthsduetolesspeakdispersionafterthecolumn.Theperformanceofthe

5μLvolumeflowcellwasbetweenthe14and2μLvolumeflowcells.Thedif-ferentpathlengthsyieldedsignificantlydifferentsignal-t