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  • Transferring Yesterdays Methodsto Tomorrows Technology:

    The Evolution from HPLC to UPLCTM

    Eric S. Grumbach

    Thomas E. Wheat

    Chuck Phoebe

    Joe Arsenault

    Jeffrey R. Mazzeo

    Diane M. Diehl

  • 2005 Waters Corporation

    UPLC TECHNOLOGY

    A new class of separation science Based on chromatography columns with very small particles Based on instruments designed to take advantage of the small

    particles

    Provides improved Resolution, Speed, and Sensitivity without compromise

    Suitable for chromatographic applications in general Appropriate for developing new methods Appropriate for improving existing methods

  • 2005 Waters Corporation

    Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00

    HPLC vs. UPLCTMSpeed, Sensitivity and Resolution

    2.1 x 150 mm, 5 mRs (2,3) = 4.29

    12 3

    HPLC

    20.00

    0.26

    Abs

    orba

    nce

    at 2

    70 n

    m

    0.00

    Minutes0.40 0.80 1.20 1.60 2.00 2.50

    2.1 x 50 mm, 1.7 mRs (2,3) = 4.281

    23

    8X Speed3.4X SensitivitySame Resolution

    0.26

    Abs

    orba

    nce

    at 2

    70 n

    m

    0.00

    UPLCTM

    Faster, More Sensitive Methods

    Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

    3

    2.1 x 100 mm, 1.7 mRs (2,3) = 6.38

    1

    2

    4.5X Speed2X Sensitivity1.5X Resolution

    4.50

    0.26

    Abs

    orba

    nce

    at 2

    70 n

    m

    0.00

    UPLCTM

    Faster, More Sensitive, Higher Resolution Methods

  • 2005 Waters Corporation

    What Does Ultra Performance LC Bring to the Chromatographic Laboratory?

    Choices available: Increased speed and sensitivity

    with the same resolution

    Increased sensitivity and speed with resolution

    Increased resolution and sensitivity at the same speed

    Speed Resolution

    Sensitivity

  • 2005 Waters Corporation

    HPLC to Optimized UPLC

    Abso

    rban

    ce 2

    54 n

    m

    Minutes0 10 20 30

    1

    23

    Abso

    rban

    ce 2

    54 n

    m

    Minutes0 2.6 5.2

    1

    23

    HPLC

    Optimized UPLC

    Magnification ofOptimized UPLC

    Abso

    rban

    ce 2

    54 n

    m

    Minutes0 10 20 30

    1

    23

    An Example of aSuccessful Method Transfer

  • 2005 Waters Corporation

    Methods Transfer Considerations

    Classes of methods transfer Replace a column brand with another Replace an instrument with another Replace both the instrument and the column

    The benefits of UPLC can only be realized with a new column on a new instrument

    As with any project to transfer a Liquid Chromatographic method, proper consideration of all the factors which control the separation process is essential for success

  • 2005 Waters Corporation

    UPLCTM Column SelectionInternal diameter and length

    Internal diameter2.1 mm or 1.0 mm

    Length If primary goal is speed, choose 50 mm length If primary goal is resolution, choose 100 mm length

  • 2005 Waters Corporation

    Ratio of Column Length to Particle Size

    If you keep the L/dp ratio the SAME for 2 columns, you will obtain the SAME Resolution. With smaller particle sizes in shorter columns, you will achieve the same separation, but in LESS TIME!

    100mm1.7m

    300mm10 m = 30,000

    30,000150mm =5m100mm =3m 33,300

    50mm1.7m

    = 29,500

    = 58,820

    1970s ~ 30+ min.

    1990s ~ 5-10 min.

    2004 ~1- 2 min.

    1980s ~10-15 min.

    L/dp RATIO Typical Run Times

    2x Maximum Resolution Capability

  • 2005 Waters Corporation

    UPLCTM Column SelectionLigand Selection

    Available Ligands: ACQUITY UPLCTM BEH C18

    Straight chain alkyl C18 ACQUITY UPLCTM BEH Shield RP18

    Embedded polar group (carbamate)

    ACQUITY UPLCTM BEH C8 Straight chain alkyl C8

    ACQUITY UPLCTM BEH Phenyl

    Why Multiple Ligands?: Changes in hydrophobicity

    Changes in silanol activity

    Changes in hydrolytic stability

    Changes in ligand density

    Changes in selectivity

  • 2005 Waters Corporation

    Reversed-Phase Column Selectivity Chart

    Retentivity(ln [k] acenaphthene)

    012005

    SunFire C18

    YMC-Pack PolymerC18

    Hypersil CPS Cyano

    YMC-Pack CN

    Waters Spherisorb S5 P

    Hypersil BDS PhenylNova-Pak Phenyl

    YMC-Pack Phenyl

    Hypersil PhenylInertsil Ph-3

    YMC-Pack Pro C4

    YMCbasic

    Symmetry C8YMC-Pack Pro C8

    Nova-Pak C8

    XTerra MS C18 Symmetry C18

    YMC-Pack Pro C18

    Inertsil ODS-3

    YMC-Pack ODS-A

    Nova-PakC18

    YMC J'sphereODSL80 Nucleosil C18

    Waters Spherisorb ODS2

    Waters Spherisorb ODS1Resolve C18

    Bondapak C18

    YMC-Pack ODSAQ

    YMC J'sphere ODSH80YMC J'sphere ODSM80

    Inertsil CN-3

    Waters Spherisorb S5CN

    Nova-Pak CN HP

    SymmetryShield RP8

    SymmetryShield RP18

    XTerra RP8

    XTerra RP18

    -0.6

    -0.3

    0

    0.3

    0.6

    0.9

    1.2

    1.5

    1.8

    2.1

    2.4

    2.7

    3

    3.3

    3.6

    -1.5 -0.5 0.5 1.5 2.5 3.5

    Sele

    ctiv

    ity(ln

    [] a

    mitr

    ipty

    line/

    acen

    apht

    hene

    )

    XTerra MS C8

    Luna C18 (2)

    ACQUITY UPLC BEH C18

    XTerra Phenyl Luna

    Phenyl Hexyl

    ChromolithTMRP-18

    Atlantis dC18

    Zorbax XDB C18ACT Ace C18

    Zorbax SB C18

    SunFire C8

    LunaC8 (2)

    ACQUITY UPLC Shield RP18

    ACQUITY UPLC BEH C8

    ACQUITY UPLC BEH Phenyl

  • 2005 Waters Corporation

    Method Transfer Consideration

    Solvent delivery Scale flow rate appropriate to column dimensions Scale linear velocity appropriate to particle size Adjust all segments of method, including equilibration

    Sample injection Scale injection volume

    Detection Ensure proper data sampling rate Ensure proper time constant

  • 2005 Waters Corporation

    Original Gradient Profile

    1

    1

    6

    *

    Curve

    9551.5152

    5951.5304

    9551.5203

    5951.50Initial

    %B%AFlow RateTime Since

    InjectionGradient

    Step

    4.6 x 150 mm, 5 m HPLC Column

  • 2005 Waters Corporation

    Target Conditions Gradient Profile

    Express gradient duration in % change per column volume (cv) units

    Calculate each segment as a number of column volumes

    Calculate time required to deliver the same number of column volumes to the UPLCTM column at the chosen flow rate.

  • 2005 Waters Corporation

    Express Gradient Segments (Steps) in Units of Column Volumes

    For 15 min at 1.5 mL/min on a 4.6 x 150 mm column

    Gradient Volume = Flow Rate x Time = 1.5 mL/min x 15 min = 22.5 mL

    Column Volume = x r2 x L = 3.14 x 2.32 x 150 = 2.49 mL

    Gradient Duration (cv) = Gradient VolumeColumn Volume

    Gradient Duration = 22.5 mL2.49 mL

    = 9.03 cv

  • 2005 Waters Corporation

    Original Gradient Profile for Scaling

    1

    1

    6

    *

    Curve

    10

    5

    15

    0

    Segment Duration

    (min)

    9.039551.5152

    6.025951.5304

    3.019551.5203

    05951.50Initial

    Segment Duration (Col.Vol.)

    %B%A

    Flow Rate

    Time Since Injection

    GradientStep

    4.6 x 150 mm, 5 m HPLC Column

  • 2005 Waters Corporation

    Estimate Optimum UPLC Flow Rate

    Consider 1.7 m target particle (2.1 mm i.d. column)Assume temperature and viscosity transferredAdjust flow rate based on van Deemter curve and

    approximate molecular weight

    ~0.6 mL/min for average 500 dalton (molecular weight) molecules

    ~0.1 mL/min for larger molecules because diffusion is slower, e.g., ~2,000 dalton peptides

  • 2005 Waters Corporation

    Scaling Gradient Step Time for UPLC Flow Rate - Maintain Duration (cv)

    Original Step 2: 15 min @ 1.5 mL/min with Duration of 9.03cv

    Calculate Target Step 2: (keeping duration @ 9.03cv)

    Target Column Volume (2.1 x 50) = 0.17 mL

    Gradient Step Volume = Duration (cv) x Target Column Volume

    = 9.03cv x 0.17 mL = 1.54 mL

    Gradient Step Time = Gradient Step Volume / UPLC Flow Rate

    = 1.54 mL / 0.60 mL/min. =

    2.6 min.

  • 2005 Waters Corporation

    Optimized Gradient Profile

    0015950.60InitialHold

    1

    1

    6

    *

    Curve

    1.74

    0.87

    2.61

    0

    Segment Duration

    (min)

    9.039550.62.612

    6.025950.65.224

    3.019550.63.483

    05950.60Initial

    Segment Duration (Col.Vol.)

    %B

    %A

    Flow Rate

    Time Since

    Injection

    Gradient

    Step

    2.1 x 50 mm, 1.7 m UPLCTM Column

  • 2005 Waters Corporation

    Comparing Results:Does the new method meet resolution criteria?

    4.6 x 150 mmResolution 1/2 = 12Resolution 2/3 = 28

    Abs

    orba

    nce

    254

    nm

    Minutes

    12

    3

    0 10 20 30

    Abs

    orba

    nce

    254

    nm

    Minutes0 10 20 30

    1

    23 UPLC optimized 2.1 x 50 mm

    Resolution 1/2 = 11Resolution 2/3 = 26

  • 2005 Waters Corporation

    Comparing Results:Does the new method meet resolution criteria?

    4.6 x 150 mm, 5mResolution 1/2 = 12Resolution 2/3 = 28

    Abs

    orba

    nce

    254

    nm

    Minutes

    12

    3

    0 10 20 30UPLC optimized 2.1 x 50 mm

    Resolution 1/2 = 11Resolution 2/3 = 26

    Abs

    orba

    nce

    254

    nm

    Minutes0 2.6 5.2

    1

    23

  • 2005 Waters Corporation

    Original HPLC Method:Caffeic Acid Derivatives in Echinacea Purpurea

    Chromatographic Conditions :Columns: XTerra MS C18 4.6 x 150 mm, 5.0 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve

    (min) %A %B0.0 92 8 62.0 92 8 732.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6

    Injection Volume: 10.0 LWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance 2695 Separations Module with

    2996 PDA detector

    Analyte1. Caftaric acid2. Chlorogenic acid3. Cynarin4. Echinacoside5. Cichoric acid

    AU

    0.00

    0.10

    0.20

    0.30

    0.40

    Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

    1 24

    5

    3

    AU

    -0.001

    0.000

    0.001

    0.002

    0.003

    0.004

    0.005

    Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00

    Enhanced BaselineE.G.

    35 min.

  • 2005 Waters Corporation

    HPLC-to-UPLCTM Method Transfer:Chromatographic Conditions

    Chromatographic Conditions :Columns: XTerra MS C18 4.6 x 150 mm, 5.0 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 1.0 mL/min Gradient: Time Profile Curve

    (min) %A %B0.0 92 8 62.0 92 8 7

    32.0 50 50 635.0 10 90 636.0 92 8 641.0 92 8 6

    Injection Volume: 10.0 LSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 10 pts/secTime Constant: 0.1Instrument: Alliance 2695 Separations Module with

    2996 PDA detector

    Chromatographic Conditions :Columns: ACQUITY UPLCTM BEH C18 2.1 x 50 mm, 1.7 mMobile Phase A: 0.1% CF3COOH in H2OMobile Phase B: 0.08% CF3COOH in ACNFlow Rate: 0.5 mL/min Gradient: Time Profile Curve

    (min) %A %B0.0 92 8 60.1 92 8 7

    4.45 50 50 64.86 10 90 65.0 92 8 66.0 92 8 6

    Injection Volume: 1.0 LWeak Needle Wash: 0.1% CF3COOH in 8% ACNSample: Caffeic Acid Derivatives in Echinacea PurpureaSample Diluent: 50:50 H2O: MeOH with 0.05% CF3COOH Sample Concentration: 100 g/mLTemperature: 40 oCDetection: UV @ 330 nmSampling rate: 40 pts/secTime Constant: 0.1Instrument: Waters ACQUITY UPLCTM, with TUV detector

    Original HPLC Method Final UPLCTM Method

  • 2005 Waters Corporation

    AU

    Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

    HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea

    AU

    Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

    Final UPLCTM Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m

    Original HPLC Method = 35 minXTerra MS C184.6 x 150 mm, 5 m

    1

    1

    2

    2

    3

    3

    4

    4

    5

    5

    35.00

    5.00

  • 2005 Waters Corporation

    HPLC-to-UPLCTM Method Transfer:Caffeic Acid Derivatives in Echinacea Purpurea

    AU

    -0.001

    0.000

    0.001

    0.002

    0.003

    0.004

    0.005

    Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

    au

    -0.001

    0.000

    0.001

    0.002

    0.003

    0.004

    0.005

    Minutes

    0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

    Original HPLC Method = 35 minXTerra MS C184.6 x 150 mm, 5 m

    Final UPLCTM Method = 5 minACQUITY UPLCTM BEH C182.1 x 50 mm, 1.7 m

    35.00

    5.00

  • 2005 Waters Corporation

    Conclusions

    Methods can be moved directly from HPLC to ACQUITY UPLC Improved resolution Improved speed Improved detectability

    Many parameters can and must be transferred to preserve results

    Attention to detail leads to success

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