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  • Agilent 1290 Infinity LC

    Next Generation Technology for highest Performance and Flexibility.

    HPLC 2009HPLC 2009Dresden, GermanyStefan SchuetteAgilent TechnologiesAgilent Technologies

  • Short History of UHPLC

    2003 Agilent 1200 Binary 1.8um RRHT Columns2004 Waters Acquity 1.7um BEH Columns2006 Agilent 1200 RRLC2006 Agilent 1200 RRLC

    Thermo Accela 2007 Shimadzu UFLCxr

    Jasco Xtreme-LCHitachi LaChrom UltraScient. Syst. Ultra HPy

    2008 Dionex RSLCKnauer Platin Blue

    2009 PE Fl2009 PE Flexar

    10 New UHPLC Systems in 5 Years All different.

  • UHPLC Situation 2009Power Rangesg

    1200

    bar Power = Pressure x Flow

    Pressure Resolution

    1000WatersAcquity

    Pressure ResolutionFlow Speed

    600

    800Acquity

    Dionex RSLC

    400

    Agilent RRLCThermoAccela

    Shimadzu UFLCXR

    0

    200

    Standard LC

    3

    0 1 2 3 4 5 ml/min

  • UHPLC Situation 200910 Systems in 5 Years All Different!y

    Problems

    Method Transfer

    Differences

    System Volumes

    Debate

    Methods: transferable? Not possible Requires re-devel.

    and -validationP f Li it ti

    y

    Pressures

    Flow Rates

    T t

    ID: 4.6, 3 or 2.1 mm?

    Particle: TP or SP? Performance Limitations

    Speed Resolution Sensitivity

    Temperatures

    Data Rates

    Sensitivity

    Size: STM or larger?

    Flow: low or high?

    T: low or high? Sensitivity Precision Linearity

    Injection principle

    ...

    T: low or high?

    P: 600 or 1000 bar?

    4

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • Agilent 1290 Infinity LC- Development Objectives- Development Objectives

    System Requirements The Debate is Over

    1. Power Range:encloses any (U)HPLC

    Methods: transferable !

    ID: 4 6 3 and 2 1 mm !2. Delay Volume:

    lowest, adjustable

    3. Sensitivity, Precision:

    ID: 4.6, 3 and 2.1 mm !

    Size: STM and larger !

    Particle: TP and SP !better than any (U)HPLC

    4. Injection Principle:variable and fixed loop

    a c e a d S

    Flow: low and high !

    T: low and high !5. Gradient Behaviour:

    emulate any (U)HPLC P: 600 and 1000 bar !

    5

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • Agilent 1290 Infinity LCPower Range

    All analytical columns

    Method Transfer

    1200

    bar

    Higher Resolution

    Faster Analysis

    Higher (MS) Sensitivity

    1000WatersAcquity

    Agilent 1290 Infinity LC More viscous solvents

    Lower temperatures

    600

    800Acquity

    Dionex RSLC

    400

    Agilent RRLCThermoAccela

    Shimadzu UFLCXR

    0

    200

    Standard LC

    6

    0 1 2 3 4 5 ml/min

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • Agilent 1290 Infinity LC Power Range and 4.6mm, 1.8um TP Columns 50 x 4.6 mmg ,

    1200

    bar

    100 x 4.6 mm150 x 4.6 mm250 x 4.6 mm

    1000H2O/ACN, 40 C

    600

    800

    400

    0

    200

    7

    00 1 2 3 4 5 ml/min

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • Agilent 1290 Infinity LC Power Range and 2.1mm, 1.8um TP Columns

    50 x 2.1 mm100 x 2.1 mm

    1200

    bar

    150 x 2.1 mm250 x 2.1 mm

    0.45 mL/min

    H O/ACN 40 C

    1000

    H2O/ACN, 40 C

    600

    800

    400

    600

    ZORBAX RRHT SB-C182.1 x 50mmH2O/ACN2 4 l/ i

    0

    2002.4ml/minT=95 C560bar

    8

    00 1 2 3 4 5 ml/min

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • How to achieve highest Peak Capacity at given Gradient Time and System Pressure?

    t_g < 5min: 50 mm Column Length

    By applying maximum Flow Rate (and Temperature)!

    5min < t_g < 30min: 100 mm Column Length

    t_g > 30min: 150mm Column Length

    P Petersson et al (AZ) J Sep Sci D Guillarme et al Journal of Chromatography

    June 06, 2005Page 9

    P. Petersson et al (AZ), J.Sep.Sci, 31, 2346-2357, 2008

    D. Guillarme et al, Journal of Chromatography A, 1216 (2009) 32323243, 2009

  • Critical Comparison of TP 1.7um and SP 2.7um Particle Columns under Optimized Ultra-high Pressure Conditions. Xiaoli Wang et al, AstraZeneca, High-Speed LC Symposium, Pittcon 2009

    Theory Experiment

    Wed, LH5, 11:30am

  • Agilent 1290 Infinity LC Power Range and 2.7um SP Columns

    50 x 4.6 mm100 x 4.6 mm150 x 4 6 mm

    1200

    g

    bar

    150 x 4.6 mm250 x 4.6 mm150 x 3 mm

    1000

    250 x 3 mm150 x 2.1

    H2O/ACN, 40 C

    600

    800 2.1x50, T=40C

    400

    6002.1x50, T=80C

    0

    200

    00 1 2 3 4 5

    11

    ml/min

    STM = Sub-2um SP = Superficially PorousTP = Totally Porous

  • Agilent 1290 Infinity LC- Resolution per Time: Superficially Porous Columnsp p y

    Peak Capacity* ~ 170 in 24 sec.World Record *Pc = based on 2nd peak W(1/2)World Record.

    0.095sec PW

    0.140secPW

    Tranquilizer Sample

    Column: Superficially Porous

    50 x 2.1 mm, 2.7 m,

    F = 3.5 ml/min, P = 700 bar

    Gradient 2-92 % ACN in 0.4 min

    T= 80 CT= 80 C

    12

  • 1290 Infinity LC and 6460 QQQ Jet Stream- Resolution per Time: Totally Porous Columns

    5x10

    4

    6

    8

    + MRM (455.29999 -> 165.09961) std-t0-1500ulmin-column3-60C-r003.d

    1

    Metabolic Stability Assay Column: ZORBAX RRHD SB-C18

    VerapamilT = 0.54 min

    Avg W1/2 = 0.44 sec 11 points across WRel. Area RSD [%] = 4.0

    p y

    2

    5x10

    2

    3

    + MRM (386.00000 -> 122.09961) std-t0-1500ulmin-column3-60C-r003.d

    1

    Column: ZORBAX RRHD SB-C18 Dimension: 2.1x50, 1.8um

    T = 60CBuspironeAvg W1/2 = 0.70 sec 20 points across W1

    2

    4x10

    2

    + MRM (272.20001 -> 215.19922) std-t0-1500ulmin-column3-60C-r003.d

    1

    F = 1.5 mL/min P = 1070 bar

    PW = 0 37 0 98 sec

    pT = 0.22 min

    pRel. Area RSD [%] = 4.2

    Avg W = 0 98 sec

    0.5

    1

    1.5

    2

    3x10- MRM (294.00000 -> 250.00000) std-t0-1500ulmin-column3-60C-r003.d

    1

    PW = 0.37 0.98 sec114 msec CT, 6 Transitions, 30ms polarity switching

    9 28 Data Points across Peak Area RSD = 2.2 7.8%

    DextromethorphanT = 0.30 min

    Avg W1/2 = 0.98 sec 28 points across WRel. Area RSD [%] = 2.2

    1

    2

    3

    41

    0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 1 1 1 1 2

    Area RSD 2.2 7.8%

    Analysis time < 0.8 min Run time = 1.5 min

    DiclofenacT = 0.73 min

    Avg W1/2 = 0.37 sec 9 points across WRel. Area RSD [%] = 7.8

    Counts vs. Acquisition Time (min)0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2

  • Pharma Impurity Analysis: HPLC to UHPLC- Highest Speed with Highest Sensitivity & Precisiong p g y

    150 x 3.0 mm, 3.5 mMethod Successfully Validated Metoclopramide PN 5990-3981EN

    14 min

    Metoclopramide, PN 5990-3981EN, . G. Vanhoenacker, F. David, P. Sandra

    LOD: 0.001 - 0.005 % w/w relative to main compound (0 5 2 5ng on column)

    100 x 2 1 mm 1 7 m

    main compound (0.5-2.5ng on-column) 10 - 50x more sensitive than required

    RSD 0.05% Impurities: 0.5 - 2.7%100 x 2.1 mm, 1.7 mPmax = 880 bar

    RSD 0.005% Impurities: 1.4 - 4.5%3x / 2x more precise than required

    3 min

    14

  • 1290 Infinity LC with 1200bar ZORBAX RRHD Resolution: Totally Porous Columns

    Rs3,2: 4.92N6 : 29600P 560 b

    20:80 H2O:ACN, F = 0.5 mL/min, T = ambient

    150 mmmAU

    200

    250

    300

    P: 560 bar3,2: 1.15Pw2: 0.015 minPw6: 0.023 min

    50

    100

    150

    200

    Rs (3,2): 6.75N6 49200

    150 +100 mm = 250mm1. Uracil2 A t h

    min0 0.5 1 1.5 2 2.5 3 3.5

    0

    mAU

    250

    300

    N6 : 49200P: 1000 bar3,2: 1.15Pw2: 0.017 min

    2. Acetophenone3. Propiophenone4. Butyrophenone5. Valerophenone6. Hexanophenone

    2

    6

    543

    100

    150

    200

    250

    Pw6: 0.029 min11

    min0 0.5 1 1.5 2 2.5 3 3.5

    0

    50

    N = 50 000 in less than 3 minutes

    Group/Presentation TitleAgilent Restricted

    Month ##, 200XPage 15

    N = 50,000 in less than 3 minutes

  • 1290 Infinity LC + 6460 QQQ Jet Stream- Analysis of 300 Pesticides in 15 min.

    600 Dynamic MRM Transitions9 replicates analyzed at 200 pg level

    Column: 2.1 x 150mm, 1.7umMobile phase: A = H2O w/5 mM ammonium formate + 0.01% formic

    B = MeOH w/5 mM ammonium formate + 0.01% formicLC Gradient: 8%B (T=0) to 92%B (T=15 min), hold 3 min

    40 Pesticides in 1 minPW ~2 sec FWHM

    p y pgMean area %RSD = 3.2 Mean height %RSD= 3.6

    ( ) ( ),Column Temperature: 70CLC flow rate: 0.5 ml/min

    Agilent MassHunter Workstation

    February-2009Page 16

  • Agilent 1290 Infinity LCNext Generation Technology

    for highest Flexibility and Performance

    New! 1290 Infinity Binary Pump

    New! 1290 Infinity Autosampler

    New! 1290 Infinity FlexCubeNew! 1290 Infinity FlexCube

    New! 1290 Infinity Column Compartment

    N ! 1290 I fi it Di d A D t kt New! 1290 Infinity Diode-Array Detektor

    New! ZORBAX RRHD Columns

    17

  • 1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump1290 Infinity LC Binary Pump

    Page 18

  • 1290 Infinity LC Binary Pump-The Next Generation in LC Pump Technology

    Highest Speed and Resolution1200 bar @ 2.0 mL/min

    1000bar @ 3.5ml/min

    800 bar @ 5.0 mL/min

    Lowest Delay Volume: 10 l / 45 ly

    Highest Precision and SensitivityLowest Mixin

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