ThelargestportfolioofFastLCcolumns, andabroadfamilyofphasesacrossallparticlesizes forexceptionalflexibilityandscalability • ZoRBaX Rapid Resolution High definition RRHd columns –1.8µmcol
Trang 1Your Essential Resource for Columns & Supplies
Scan the QR code
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for more information
This information is subject to change without notice.
© Agilent Technologies, Inc 2012
Printed in Canada October 31, 2012
5991-1059EN
Trang 2Whetheryouareperformingconventionalorultra-fastchromatography,separatingbiomolecules, oranalyzingcomplexbasiccompounds,youcantrustAgilentfortheindustry’shighest-performing columnsthatdeliverthefast,reproducibleresultsyouneed–allengineeredwithAgilent’sunparalleled qualityandreliability.
• ZoRBaX Eclipse Plus columns –C18andC8columnsdeliversuperiorpeakshape,whilethe
phenyl-hexylbondedphaseandC18bondedphaseforPAHseparationsexpandselectivityoptionsfor moreapplications.AllEclipsePlusphasesareavailableinFastLC/UHPLCRRHDandRRHTcolumns, 1.8µm.Forscalability,theEclipsePlusC18phaseisverysimilartothePoroshell120EC-C18phase.
• InadditiontoPoroshell120andRRHDcolumns,ZoRBaX Rapid Resolution High Throughput
(RRHT) columns areathirdFastLCoptionwithover1401.8µmcolumnschoices.RRHTcolumns
areavailablein2.1,3.0and4.6mmids,allwith600barstability.
Andremember,whenyouchooseAgilentZORBAXLCcolumns,yougetmorethanjustadependable product.Youalsogetover40yearsofexpertise–alongwithunmatchedtechnicalsupport–fromthe world’slargestchromatographysupplier.Ontheweb,byphoneorinperson,Agilenthelpsyousolve theproblemsthatcanslowyoudownandgetinthewayofyourresults.
ThelargestportfolioofFastLCcolumns,
andabroadfamilyofphasesacrossallparticlesizes forexceptionalflexibilityandscalability
• ZoRBaX Rapid Resolution High definition (RRHd) columns –1.8µmcolumnsfeatureimproved
packingprocessestoachievestabilityupto1200barforusewiththeAgilent1290InfinityLCand otherUHPLCinstrumentsandareavailableinmorethan14phases,plusHILIC.
LC and LC/MS Columns
• Poroshell 120 columns –highefficiencyandhighresolutionwithupto50%lesspressurethan
sub-2µmcolumns.
Trang 3LC and LC/MS Columns for Small Molecule Separations
HPLC Column Selection 206
Fast Columns for Reversed-Phase HPLC/uHPLC 227
Poroshell 120 228
ZORBAX Rapid Resolution High Definition 1.8 µm 233
ZORBAX Rapid Resolution High Throughput 1.8 µm 239
Agilent UHPLC Guards 246
Columns for Reversed-Phase analytical HPLC 247
ZORBAX Eclipse Plus 248
ZORBAX Eclipse PAH 254
ZORBAX Eclipse XDB 256
ZORBAX 80Å StableBond 264
ZORBAX Rx 272
ZORBAX 80Å Extend-C18 274
ZORBAX Bonus-RP 278
ZORBAX Original Reversed-Phase Columns 283
Kits for Analytical HPLC 284
Pursuit 287
Polaris 298
TC-C18(2) and HC-C18(2) 304
PLRP-S 306
Preparative HPLC Columns 311
Agilent Prep LC Columns 311
ZORBAX PrepHT 314
Pursuit and Pursuit XRs Prep 319
Polaris Prep Columns 321
Load & Lock 322
Columns for Other HPLC Techniques 323
ZORBAX HILIC Plus 324
Normal-Phase Columns 326
ZORBAX Ion-Exchange Columns – SAX and SCX 333
Hi-Plex Columns for Carbohydrate Analysis 335
appendices 343
Quick Guide to USP Designations for HPLC Columns 343
oligo Solutions 347
StratoSpheres DNA Cartridges 347
TOP, TOP-DNA and TOP-RNA Cartridges 348
Trang 4HPLC Column Selection
Tousethecolumnselectionguidediagrambelow,simplyfollowthepathforyouranalyteandmobilephase.Atthefarright,followyourfinalcolumn selectiontothepagesindicated.
Normal-Phase Mode with Bare Silica
Normal-Phase Mode with Bonded Phase
Reversed-Phase Mode with Bonded Phase
Gel Permeation (Small Molecule)
Reversed-Phase Mode with Bonded Phase
Reversed-Phase Mode with Bonded Phase Using Ionization Control
Reversed-Phase Mode with Bonded Phase Using Ion-Pair Agent
Reversed-Phase Mode with Bare Silica (HILIC)
Ion-Exchange Mode
Gel Permeation Chromatography
Gel Filtration Chromatography
Ion-Exchange Mode with Wide-Pore Material
Reversed-Phase Mode with Wide-Pore Material
Molecular Weight
< 3000
Hexane Soluble
IonicNon-Ionic
MeOH and MeOH:
H2O or ACN andACN: H2O Soluble
Page 488Page 248Pages 326-328Pages 326-328
Water Soluble
Water Soluble
Organic SolventSoluble
Trang 5Quick guide to agilent Reversed-Phase Bonded Phases
ZoRBaX RP-HPLC Columns Recommended uses and applications Page no.
Trang 6TiPS & TooLS
ZORBAXRx
AvailableinRRHD(1200bar)
andRRHT(600bar)configurations,1.8µm
ü Generalseparationofbasic,acidicandneutralcompoundsatlowpHwithdifferentselectivitythanSBcolumns
ZoRBaX RP-HPLC Columns Recommended uses and applications Page no.
Quick guide to agilent Reversed-Phase Bonded Phases
The LC Handbook: guide to LC Columns and Method development
ThishandyguidemakesiteasytochoosetherightLCcolumn,andcontainsplentyoftipsandtrickstomake
yourjobeasierandmoreproductive(publication#5990-7595EN).
Request a copy or download a mobile copy at www.agilent.com/chem/lchandbook
Trang 7Quick guide to additional agilent Reversed-Phase Columns
Pursuit Family Recommended uses and applications Page no.
ü Diphenylutilizesstrongdipole-dipolehydrogenbondingandpi-pimechanismsfordifferentselectivitywitharomaticcompounds
ü PFPprovidesexcellentseparationofpolar(halogenated)analytesandpositionalisomersunderstandardreversed-phaseconditions
Trang 8ZORBAX Reversed-Phase HPLC Column Selection Flow Chart
For small and large molecules
Mostchromatographersusereversed-phaseHPLCasoneoftheirkeyanalysistechniques.Reversed-phaseHPLCcanbeusedtoanalyzeionicandnonionic analytes.ThereforethisZORBAXColumnSelectionFlowChartwillfocusonreversed-phasecolumns.Tomoreeasilyselectareversed-phasecolumnfor methoddevelopmentofsmallandlargemolecules,followtheoutlineonthesepages.
Thisflowchartprovidesinformationonchoosinganinitialcolumnformethoddevelopmentofsmallmoleculeandproteinandpeptidesamples,andincludes decisionsonbondedphaseandcolumnconfiguration.
or very hydrophobic compounds that are difficult to elute from C18 with conventional reversed-phase solvents Start with Poroshell 120 EC-C18
or Eclipse Plus RRHT/RRHD for fast LC performance
Small Molecules Large Molecules
Starting Column Bonded Phase
Packing Pore Size
2.1 x 50 mm, 1.8 µm959757-902
4.6 x 150 mm, 5 µm883995-902
4.6 x 50 mm, 3.5 µm865973-902
2.1 x 75 mm, 5 µm660750-902
ZoRBaX 300SB-C18
Fast analysis Standard analysis
Standard analysis
Eclipse Plus C18
Poroshell 120 EC-C18
Fast analysis Poroshell 120 EC-C18
Trang 9Column and Mobile Phase Guidelines:
Trang 10Base Material
ThebasematerialforanLCcolumnismostoftenhighpuritysilicamaterialwithtotallyporousparticlessuch asthatusedinmostAgilentcolumns,includingZORBAX,Pursuit,andPolaris.However,morechoicesare available,includingpolymermaterialwithhighpHstabilityusedinPLRP-Scolumnsandsuperficiallyporous silicaparticlessuchasthoseusedinPoroshell120columns.ThehighpurityTypeBsilicas,includingthe ZORBAXRx-SilusedinZORBAXEclipsePlus,andsuperficiallyporousPoroshell120,areanexcellentfirst choiceformostmethods.TypeAsilicas,suchasZORBAXSIL,usedinOriginalZORBAXcolumns,arestill manufacturedandusedinmanymethods.
Bonded Phase
AgoodfirstchoiceforbondedphaseisC18orC8,andtherecommendedstartingcolumnchoicesare EclipsePlusC18orPoroshell120EC-C18.Thesetwochoicesprovideexcellentpeakshapeandcanbe usedoverthepHrange2-9,accommodatingmosttypicalLCandLC/MSmobilephases.Ifthesample solutesofinterestarenotadequatelyseparatedonthesecolumns,CNandPhenylcolumns–including Phenyl,Phenyl-HexylandDiphenyl–mayoffersignificantdifferencesinselectivityfromstraight-chainalkyl phasestoeffecttheseparation.
Ingeneral,largersolutes,suchasproteins,arebestseparatedonshort-chainreversed-phasecolumns (C3,CN,C8)andpeptidesandsmallmoleculesareseparatedonlonger-chaincolumns(C18).However, therearemanycaseswherethisconventionalwisdomdoesnotapply.Forexample,peptidescanalsobe effectivelyseparatedusingshort-chaincolumns,andhydrophobicpeptidescanshowbetterrecovery onlonger-chainphases.Therefore,itisbesttoinitiallyselectaphaseinthemiddleofthehydrophobic spectrum(e.g.,C8),thenchangetoamorehydrophobicphaseormorehydrophilicphasedependingon initialresultsandsolubilitypropertiesofyoursample.
Polymers
WhenacolumnisneededthatcanoperateatverylowandveryhighpH,polymericpackingsprovide analternativetosilica-basedmaterials.Polymericparticlesaregoodforsmall-scalechromatography, particularlyLC/MS,astheyarechemicallystableanddonotleachsolubleorparticulatespecies. Reversed-phasesphericalpolymericpackingsusedinAgilentPLRP-Scolumns,forexample,arebasedona styrene/divinylbenzenecopolymerwithaninherentlyhydrophobicsurface.Nobondedphaseisrequiredfor reversed-phasechromatographywithpolymericparticles.Theserigidmacroporousparticlescanbecoated andderivatizedtogivearangeoffunctionalities,includingweakandstrongcationandanionexchangers.
Silica, Polymers, and Bonded Phase
Trang 11pH and Mobile Phase
Trang 12Working with LC/MS
SincemanyLC/MSanalysesarerunatlowerflowrates(typicallyfromµL/minflowratesupto1mL/min), movingtosmallerinternaldiametercolumnsisthebestchoicefortheuser.Agilent’sSolventSaver (3.0mmid)andnarrowbore(2.1mmid)willoftenresultinlowersolventusageforthemethod,
andareexcellentoptionsforhighresolutionandhighersensitivitythanthelargeridcolumns.
Mostoften,thebestbondedphasechoiceisanendcappedC18phase.EclipsePlusC18isahigh performanceendcappedC18phaseavailableinsub-2µmRRHDandRRHTcolumnformats.Forfast high-throughputseparationswithLC/MS,Poroshell120EC-C18isanexcellentchoice.Poroshellhas alargerfrit,soit’swellsuitedfordirtierLC/MSsamples,suchasbloodplasma,whichmayoftenclog columnswithsmallerporosityfrits.
BothEclipsePlusC18andPoroshell120EC-C18phasesarestableoverawidepHrangeandare compatiblewiththevolatilebufferssuchasaceticandformicacids.
TiPS & TooLS
LC Flow Rate Calculator app
ThisFREESmartphoneappletsyouquicklyadjustyourflowratetoaccommodateothermethodchanges.
Downloadatwww.agilent.com/chem/lcapp
WhenchoosingHPLCcolumnsforLC/MS,chromatographersoftenneedtoconsiderseveralaspectsof theirmethodandseparation,typicallyincludingresolution,flowrate,andstationaryphasechoice.Often, forrelativelysimpleanalytes,shorterhighresolutioncolumnsarethebestchoice.Thesecolumnsallowfor highthroughputwhilemaintaininghighseparationefficiency.NarrowboreRapidResolutionHighDefinition (RRHD)forseparations(>600bar)andPoroshell120columns(<600bar)offerhighresolutionevenin shortercolumnsdimensions.Formoredifficultsamples,usersshouldseeklongercolumnlengths.
Trang 13Transferring your method
to a high efficiency column
optimize the data collection rate for LC and LC/MS (at least 40 Hz detector with fast
response time for uV) –Setthedetectortothefastestsetting,thentothesecondfastestsetting
Trang 14Minimize injection sample dispersion in the column –Useaninjectionsolventwithsolventstrength
thatisequivalenttoorweakerthanthemobilephase,especiallywhenusinganisocraticmethod.Thisis goodpracticeingeneralforanycolumn,andmoreimportantwithhighefficiencycolumns.
Scale your gradient profile and injection volume –Ifusinggradientelution,scalethegradient
profileandinjectionvolumetothenewsmallercolumntoquicklytransferthemethodandavoid overloading.Forisocraticandgradientelution,makesurethatyouscaletheinjectionvolumetomatch theoverallcolumnvolume.
Minimize tubing volume in the instrument –UseRed(0.12mmid)tubinginsteadofGreen
(0.17mmid)asithasonlyhalfofthevolumethatthesamplehastotravelthrough.Thiscutsdown extracolumnbandbroadening.Ensurethatyourconnectionsareasshortaspossible.Thekeylocations tocheckare:
TiPS & TooLS
Trang 1515 15
Fast analysis of cefepime
and related impurities
Column: Poroshell 120 EC-C18
2.0mL/min 300bar
1mL/min 155bar
Trang 16Start with Poroshell 120 for Fast LC performance on any HPLC – phases align with ZoRBaX family.
up to 50% less pressure than sub-2 µm; a total lab productivity enhancer
**Best Phase for Method development
1.7 µm solid core; 0.5 µm porous outer layer for a 2.7 µm particle, id's: 4.6 mm, 3.0 mm, 2.1 mm, Lengths: 30-150 mm.
New phases coming soon! Check www.agilent.com/chem/poroshell120
Compatible with HPLC and UHPLC instruments Suitable for analysis of acids, bases, and neutrals Also great for peptide mapping.
Poroshell 120 is for any lab looking for increased analytical speed and resolution with less backpressure.
Poroshell 120
ZoRBaX Family
ZoRBaX StableBond
R R R R R R
R 1
Si O OH
OH
R 1
Si O
R 1
Si O
CH3
CH3Si
CH 3
CH 3
CH3Si
O
O
O O O
CH 3
CH3Si
O
O
O O
O
SB-C18 (USP L1), SB-C8(USP L7),
SB-C3 (USP L56), SB-Phenyl (USP L11), SB-Cn (USP L10), SB-aq
RRHd:1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 20-250 mm IDs: 4.6 mm , 3.0 mm, 2.1 mm, 1.0 mm;
Prep, Capillary (C18)
High performance with acids, bases, and neutrals with superior lifetime at low pH.
Sample applications Chemical/industrial: Triton Environmental: Organic acids,
pesticides in drinking water
Food Safety: Anthocyanine,
180 m 2 /g Particle sizes:
1.8, 3.5, 5, 7 µm
pH: 1.0-8.0 (0.8-8.0 for SB-C18) Carbon Load:
C18: 10%; C8: 5.5%;
C3: 4%; Phenyl: 5.5%;
CN: 4%, Aq: Proprietary
Best for low pH mobile phases – great for method development
ZoRBaX Eclipse Plus**
C18 (USP L1), C8(USP L7),
Phenyl-Hexyl (USP L11), PaH (USP L1)
High performance and excellent peak shape with acids, bases and neutrals.
Sample applications Environmental: EPA Method 1694,
Illicit and prescribed drugs in wastewater
Food Safety: Quinolone antibiotics Pharmaceutical: Chloramphenicol,
Simvastatin, Chrysophenol (TCM), amphetamine, ranitidine
double Endcapped
(except PAH, which
is not endcapped) Temp limit: 60 °C Pore size: 95Å Surface area:
160 m 2 /g
Particle sizes:
1.8, 3.5, 5 µm pH: 2.0-9.0 for C18, C8; 2.0-8.0 for PAH, Phenyl-Hexyl Carbon load:
C18: 9%; C8: 7%;
Phenyl-Hexyl: 9% ; PAH: 14%
Best all around – exceptional peak shape, efficiency, resolution, and lifetime
ZoRBaX Eclipse XdB
C18 (USP L1), C8(USP L7),
Phenyl (USP L11),
Cn (USP L10)
Good peak shape for basic, acidic, and neutral compounds with high performance over a wide pH range (pH 2-9) eXtra Dense Bonding and double endcapping help give this column a long lifetime.
Sample applications Environmental: Herbicides/pesticides,
steroids in water
Food Safety: Food colors,
aromatic flavorings, mycotoxins, epoxyphenolic-based can coatings
Pharmaceutical: Goldenseal and related
alkaloids, antidepressants, triamcinolone
double Endcapped
Temp limit: 60 °C Pore size: 80Å Surface area:
180 m 2 /g Particle sizes:
1.8, 3.5, 5, 7 µm
pH: 2.0-9.0 (2.0-8.0 for CN) Carbon load: C18: 10%; C8: 7.6%; Phenyl: 7.2%; CN: 4.3%
High performance over a wide pH range
RRHd:1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 15-250 mm IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm; Capillary and Prep
Pursuit/
Pursuit XRs
C18 (USP L1), C8 (USP L7), diphenyl (USP L11), PFP (USP L43), PaH (USP L1), Si (USP L3)
Pursuit XRs offers higher loadability
Endcapped
(except Pursuit XRs Si) Pore Size: 200Å (Pursuit), 100Å (Pursuit XRs) Surface area: 200 m 2 /g (Pursuit); 440 m 2 /g (Pursuit XRs)
pH: 2.0-9.0 Carbon Load: Pursuit C18: 12.9%; Pursuit C8: 7.4%; Pursuit Diphenyl: 7.3%; PFP: 6.3%; XRs C18: 22%; XRs Ultra C18: 23.3%; XRs Ultra
Lengths: 30-250 mm
IDs: 2.0 mm, 3.0 mm, 4.6 mm; Prep
Poroshell 120 EC-C18** (uSP L1), EC-C8** (uSP L1), Phenyl-Hexyl (uSP L11) Carbon Load: Phenyl-Hexyl - 8%
Poroshell 120 SB-C18 (uSP L1), SB-C8
RRHd: 1.8 µm, stable to 1200 bar;
RRHT:1.8 µm, 600 bar Lengths: 30-250 mm IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm; Prep
Trang 17Poroshell 120 SB-aQ Carbon Load: Proprietary
HO N
N O
PG PG
R R R
Si Si
C18 Silica Support Si O
ZoRBaX Bonus-RP
Bonus-RP (USP-L60)
PoLaR Compounds
Polar-embedded to improve peak shapes;
for basic compounds at low and mid pH.
Sample applications Environmental: Triazine pesticides Food Safety: Hydroxymethylfurfural Pharmaceutical: Antifungal medications,
anorectics, ulcer medications
RRHd: 1.8 µm, stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 30-250 mm IDs: 4.6 mm, 3.0 mm, 2.1 mm, 1.0 mm; Prep
Triple-Endcapped
Temp limit: 60 °C Pore size: 80Å Surface area:
180 m 2 /g Particle sizes:
1.8, 3.5, 5 µm
pH: 2.0-9.0 Carbon load: 9.5%
alternative selectivity to alkyl, phenyl, cyano phases
ZoRBaX Extend-C18
C18 (USP L1)
Sample applications
Environmental: EPA 8330 (explosives)
Food Safety: Aflatoxins, mycotoxins
a good option for
See ZORBAX StableBond for specification and structure.
Polaris
C18-a (USP L1), C8-a (USP L7), C18-Ether (USP L1), C8-Ether (USP L7), amide-C18 (USP L60), nH2 (USP L8), Si-a (USP L3)
Hydrogen-bond accepting and ether group endcapping provide alternate selectivities.
Sample applications Environmental: Triazine pesticides Food Safety: Hydroxymethylfurfural Pharmaceutical: Antifungal medications,
anorectics, ulcer medications
Endcapped
Pore size: 180Å Surface Area:
200 m 2 /g Particle Sizes:
3, 5, 10 µm pH: 2.0-9.0
Carbon load: Polaris C18-A: 13.8%; Polaris C8-A: 7.4%; Polaris C18-Ether: 12.1%; Polaris C8-Ether: 7.1%
More options for Polar Compounds
Looking for a HiLiC column?
RRHd: 1.8 µm stable to 1200 bar;
RRHT: 1.8 µm, 600 bar
Lengths: 20 - 250 mm IDs: 4.6 mm, 3.0 mm, 2.1 mm; Prep
Lengths:30-250 mm, (available in
3 µm and 5 µm particles) IDs: 2.0 mm, 3.0 mm, 4.6 mm; Prep
High efficiency and long life at high pH –
up to pH 11.5 Improve retention,
resolution and peak shape of basic
compounds High sensitivity for LC/MS
separations of peptides Unique
bidendate bonding and double
endcapping provides high pH stability.
Exceptional lifetime at low pH –
Trang 18Method Development from pH 1-12
Start method development at low pH (pH 2-3)
Withsomanycolumnchoicesavailable,howdoyouknowwheretostartyourmethoddevelopment?Therecommendedstartingpointformethod developmentisusingabufferedlowpHmobilephase–aroundpH2-3.UsingalowpHmobilephasemostoftenresultsinthebestpeakshapeforbasic compoundsonsilica-basedcolumns.AtlowpH,thesilanolsonthesilicaarefullyprotonatedsopositivelychargedbasiccompoundsdonotinteractstrongly Theresultisgoodpeakshape.Manyacidiccompoundsarenon-charged,maximizingtheirretentionatlowpH.Theseobservationsarekeyadvantagesto methoddevelopmentatlowpH.
Forstandardanalyticalwork,startmethoddevelopmentwithacetonitrileasthemobilephaseorganicmodifierand20-50mMphosphatebuffer(pH2-3) astheaqueouscomponentfornon-LC/MSapplications.TheseconditionsprovidegoodpHcontrol,necessaryforthemostreproducibleanalysesofionizable compounds.ForLC/MSapplicationsformicacidorTFAaregoodmobilephaseadditivesforlowpH.
Optimize solvents and bonded phases at low pH
Theinitialmethoddevelopmentstepsmayleadveryquicklytoasatisfactoryseparation.Butifmoreoptimizationisneeded,acetonitrilecanbereplaced withmethanolortetrahydrofuranandtheseparationre-optimized.Thisstepmayleadtoasatisfactorysolution,butifstillmoreselectivityoptimization isneeded,thecolumnbondedphasecanbechanged.
AtlowpHtherearemanybondedphasechoicesavailableforoptimization.TheseincludetheEclipsePlusphasesaswellastheEclipseXDBfamilywith C18,C8,PhenylandCN.AlternatechoicesincludefivedifferentStableBondbondedphases:SB-C18,SB-C8,SB-Phenyl,SB-CN,andSB-C3.Forpolar analytes,tryBonus-RP,SB-AqorthePolarisfamily,includingC18-A,C8-A,C18-EtherandAmide-C18phases.
Trang 19Choose Agilent ZORBAX Eclipse Plus or Poroshell 120
for method development at mid pH (pH 4-9)
pHandPLRP-Scolumns,whicharemadefromapolymericmaterial.
Trang 20Poorly retained compounds
Poorly retained compounds
SAMPLE
ü ZORBAXEclipsePlusC18,C8,or Poroshell120EC-C18
Band spacing problems
Band spacing problems Band spacing problems
Band spacing problems
Band spacing problems Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems
Band spacing problems
BASICCOMPOUNDS(Ion-Pairing)
ü UseMeOHorganicmodifier,10mMhexane sulfonicacid,25-50mMpH3buffer
ü Adjust%MeOHfor0.5<k<20
STEP 1c
Increaseordecrease%oforganicmodifier by5%(v/v)
Increaseordecrease%of organicmodifierby5%(v/v)
Increaseordecrease%of organicmodifierby5%(v/v)
Increaseordecrease%oforganicmodifier by5%(v/v)
Increaseordecrease%oforganicmodifier by5%(v/v)
ü RestartatSTEP2
STEP 4
ü Changebonded-phasefunctionalitytoEclipse PlusPhenyl-Hexyl,Bonus-RP,EclipseXDB-CN, SB-CN,SB-Aq
ü Adjust%MeOHfor0.5<k<20
STEP 5b
Varytemperaturewithinrecommended rangeforbondedphase
Try different HPLC mode
Trang 22Cartridge Selection guide
icon* Type of Cartridge Features Benefits
guardcartridges
InexpensiveExtendscolumnlifetimePermitsrapidcolumnchangesCanuse2,3,4and4.6mmcartridgesCartridgeshaveauniquefilterandsieve
ateachend
HelpspreventblockageZORBAXGuardCartridge:
Standalonesystem
Highefficiency,standalone,
low-dead-volumecartridge
Sealsupto5000psi(340bar)or3000psiwithaPEEKfitting
Polymericcartridgedesigned
forleak-tightsealsagainstmetalsurfaces
NogasketsrequiredMoresolvent-resistantthanPEEK
ForallanalytetypesLowbleedSoldindividuallyorasthree-packs
ZORBAXSemi-PreparativeGuard
HPLCHardwareKit:
Standalonesystem
Easy,low-dead-volumeassembly Sealsupto2000psi(135bar,13.5MPa)Tubing(polyphenylenesulfone)designed
Completesystemsandreplacementcartridges
NotoolsrequiredModularflexibility
connectsrighttotheanalyticalcolumn
Extendscolumnlifetime
withoutimpactingperformanceAvailableinmatchingphasesforPoroshell120,
RRHDandRRHTcolumns
*Lookfortheseiconstohelpyouselecttheproperguardcartridgesandcolumns
Trang 23Cartridge/guard Cartridge Systems Compatibility guide*
icon Column Type guard Cartridge Holder
id (mm) Phases
Cartridgecolumncartridgeholder
5021-1845
Guardcartridge(internalsystem)cartridgeholder5021-1845
2.03.04.04.6
LiChrospherNucleosilPurospherSuperspherZORBAX
cartridgeholder820999-901
2.13.04.6
(Continued)
Trang 24Cartridge/guard Cartridge Systems Compatibility guide*
icon Column Type guard Cartridge Holder
id (mm) Phases
FastGuardsforUHPLC:
Singlereplacementguardcolumn
3.04.6
Poroshell120:EC-C18EC-C8SB-C18Phenyl-HexylSub-2µm:EclipsePlusC18EclipseXDB-C18SB-C18SB-C8
*StandaloneguardcartridgesfitallcartridgeandstandardfittingcolumnsavailablefromAgilent.Allcolumnswithouticonsarestandardfittingcolumns
nEw!
Trang 25Recommendations for Fast LC Columns
your Lab Situation agilent Recommends Rationale
youcan’ttransfer,theZORBAXEclipsePluscolumnwillprovideexceptionalpeakshapeandperformance
Trang 27Poroshell 120
(Maximum pressure: 600 bar)
Hardware description Size (mm)
Particle Size (µm)
EC-C18 uSP L1
EC-C8 uSP L7
Phenyl-Hexyl uSP L11
SB-C18 uSP L1
SB-C8 uSP L7 SB-aq
Bonus-RP uSP L60
Trang 281 2 3
1 2 3
4 5
8 9
Environmental phenols on Poroshell 120
Column a: Poroshell 120 EC-C18
695975-902 4.6 x 100 mm, 2.7 µm Column B: Eclipse Plus C18
959964-902 4.6 x 100 mm, 1.8 µm
Gradient: A:Water0.1%formicacid
B:Acetonitrile0.1%formicacid2mL/min
Initial:8%B10min:30%B
Detector: 275nm,2mmflowcell
Agilent1200SL40°CNopulsedamperNomixer3µLheater
uHPLC efficiency at HPLC pressures
Column a: Poroshell 120 EC-C18
695975-302 3.0 x 100 mm, 2.7 µm Column B: Eclipse Plus C18
959964-302 3.0 x 100 mm, 1.8 µm
w/50µL2mg/mLthioureainwater/acetonitrile(65:35)
Forthissampleofneutralalkylphenones,thePoroshell120columndelivered>90%oftheefficiencyattainedbythe1.8µmcolumn.Alsonotethatthepressureonthe
Poroshell120columnisabout50%ofthepressureonthe1.8µmcolumn
Trang 29HPLC separation of 12 phenols performed
in just 5 minutes – and under 400 bar –
using an agilent Poroshell 120 EC-C18 column
Column: Poroshell 120 EC-C18
0 mAU
min
12 phenols analyzed using a longer (4.6 x 100 mm)
agilent Poroshell 120 EC-C18 column
Column: Poroshell 120 EC-C18
Trang 302 4 6
0 100 200 300
0 100 200 300
for fast uHPLC separations
Column: Poroshell 120 EC-C18
695975-302 3.0 x 100 mm, 2.7 µm
MobilePhase: 65%A:0.2%Formicacid
35%B:MethanolIsocraticFlowRate: Varies
Trang 31ZoRBaX Rapid Resolution High definition
ZoRBaX RRHd Column Specifications
Bonded Phase Pore Size Surface area pH Range Endcapped Temp Limit
Trang 32min 0
mAU
0 50 100 200
A
min 0
mAU
0 50 100 200
B
min 0
mAU
0 50 100 200
C
Rs: 2.40 Rs: 1.37 Rs: 0
4 2
50 mm
Separation of licorice root on RRHd columns
Column a: ZoRBaX RRHd SB-C18
857700-902 2.1 x 50 mm, 1.8 µm Column B: 858700-902
2.1 x 100 mm, 1.8 µm Column C: 859700-902
2.1 x 150 mm, 1.8 µm
MobilePhase: 10-100%B/30min
A:0.1%Formicacid(fa)
B:Acetonitrilewith0.1%faFlowRate: F=0.4mL/min
Trang 33F=2.0mL/minP=975bar
new levels of sensitivity and resolution
Column a: ZoRBaX RRHd Eclipse Plus C18
with6410MS/MSSample: 20µL(10µLfor50mmcolumn)
Trang 34TiPS & TooLS
Selectivity comparison: C18 columns
Column a: ZoRBaX RRHd Eclipse Plus C18
959758-902 2.1 x 100 mm, 1.8 µm Column B: ZoRBaX RRHd Eclipse XdB-C18
981758-902 2.1 x 100 mm, 1.8 µm Column C: ZoRBaX RRHd Extend-C18
758700-902 2.1 x 100 mm, 1.8 µm Column d: ZoRBaX RRHd SB-C18
858700-902 2.1 x 100 mm, 1.8 µm
MobilePhase: A:0.1%HCOOHinH2O(30%)
B:0.1%HCOOHinCH3CN(70%)FlowRate: 1mL/min,isocratic
AEA
2-AG *
PEA OEA
AEA
2-AG *
×10 2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
×10 2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
×10 2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Trang 35×10 2
0 0.1 0.2 0.4 0.6 0.8 1.0
×10 2
0 0.1 0.2 0.4 0.6 0.8 1.0
Counts(%)versusAcquisitiontime(min)
TiPS & TooLS
Selectivity comparison:
Phenyl and other columns
Column a: ZoRBaX RRHd Eclipse Plus C18
Trang 36Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description Size (mm)
Particle Size (µm)
Eclipse Plus C18 uSP L1
Eclipse Plus C8 uSP L7
Eclipse Plus Phenyl-Hexyl uSP L11
Eclipse PaH uSP L1
Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description Size (mm)
Particle Size (µm)
SB-C18 uSP L1
SB-C8 uSP L7
SB-Cn uSP L10
SB-Phenyl uSP L11 SB-aq
Rapid Resolution High definition (RRHd) Columns for High Pressure use (Maximum Pressure: 1200 bar)
Hardware description Size (mm) Particle Size (µm)
Extend-C18 uSP L1
Eclipse XdB-C18 uSP L1
Bonus-RP uSP L60 HiLiC Plus
Trang 37ZoRBaX Rapid Resolution High Throughput
Trang 38doubletheefficiencyofa3.5µmcolumninthesamecolumn
length.Thishighefficiencycanbeusedforveryhigh-resolution,
highthroughputanalyses
Rapid Resolution HT (RRHT) provides double
the efficiency of Rapid Resolution columns
Column a: ZoRBaX Rapid Resolution SB-C18
835975-902 4.6 x 50 mm, 3.5 µm Column B: ZoRBaX RRHT SB-C18
827975-902 4.6 x 50 mm, 1.8 µm
0.0
1
2
3 4 1
2
3 4
Time (min)
1 0
961753-902 2.1 x 100 mm, 3.5 µm
MobilePhase: A:H2O
B:ACNPeakcapacity: A:461
B:343FlowRate: 0.5mL/min
Gradient: 0.0min50%B
20.0min100%BTemperature: 40°C
Trang 39Reduce analysis time dramatically with Rapid
6
1
4 3
1 4 3
1 4
6
1 4 2 5 7 6
Trang 40TiPS & TooLS
n (3.5 µm)*
Resolving Power