mixed mode mechanisms in lc: curse or cure? · •the main effect of a parameter is the difference...

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Merlin K. L. Bicking, Ph.D. ACCTA, Inc. Richard A. Henry, Ph.D. Consultant Minnesota Chromatography Forum 2014 Session: 2D-LC and Novel LC Phases 1 Is A Second Retention Mechanisms in LC a Curse or Cure?

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  • Merlin K. L. Bicking, Ph.D.

    ACCTA, Inc.

    Richard A. Henry, Ph.D.

    Consultant Minnesota Chromatography Forum 2014 Session: 2D-LC and Novel LC Phases

    1

    Is A Second Retention Mechanisms in LC a

    Curse or Cure?

  • Thank You! • Thank you for your interest in our research. This work was supported by

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    2

    Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN USA 55125 Email: [email protected]

    Richard A. Henry, Ph.D. Consultant State College, PA USA 16801 Email: [email protected]

    “Analytical Solutions to Analytical Problems”

  • 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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    Curse: The Effect of Silica Purity on Selectivity

    Zorbax XDB C18 Moderate Purity Silica (fully endcapped)

    ACE C18 High Purity Silica (fully endcapped)

    Zorbax SB C18 Moderate Purity Silica (non-endcapped)

    Zorbax ODS Low Purity Silica (fully endcapped)

    9,700 pl/m

    23,100 pl/m

    63,400 pl/m

    Decreasing silica purity can change selectivity, but poor chromatography and reproducibility may result from surface interactions.

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    A second retention mechanism from the silica affected the chromatography

    Column: 250 x 4.6mm, 5m Mobile phase: 80:20 MeOH/25mM KH2PO4 (pH6.0) Flow: 1.00ml/min Components; 1: Norephedrine, 2: Nortriptyline, 3: Toluene, 4: Imipramine, 5: Amitriptyline

    1, 2, 4 and 5 not eluted

    Reproduced with kind

    permission of Mac-Mod Analytical 3

  • min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    120

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    120

    Cure: Change Some Retention Times, Not All • Decreasing the acid content causes an increase in the retention of the

    basic analytes (ionic effects). Since the acetonitrile content has not changed, the hydrophobic retention region does not change.

    1 Creatinine 2 Procainamide 3 Protriptyline

    Retention Window for Hydrophobic Components

    1 2 3

    1 2

    3 0.10% Sulfuric Acid/ 80% Acetonitrile

    0.01% Sulfuric Acid/ 80% Acetonitrile

    Column: 4.6 X 50 mm PFPP

    4

  • • When the presence of a second retention mechanism is part of the phase design, maybe we can use both mechanisms to improve the separation

    • This is possible only if,

    – We understand what the mechanisms are

    – We understand how they behave

    – We know how to control their behavior

    So, is it a curse or cure?

    5

  • Experimental Details • Equipment

    – Agilent 1100 with autosampler, column compartment, and diode array

    – OpenLab ChemStation software

    • Mobile Phase Components – Acetonitrile (HPLC Grade) – Ammonium Formate

    • 10, 20, and 30 mM

    • Operating Conditions – Flow: 1.75 mL/min – Injection: 2 uL – Temperature: 35 C – Detection: 220 nm

    Manufacturer Phase Dimensions Phase

    Supelco Ascentis Express C18 4.6X50 mm, 5 um* Hydrophobic

    Supelco Ascentis Express F5 4.6X50 mm, 5 um* Pentafluorophenylpropyl

    Supelco Ascentis Express OH5 4.6X50 mm, 5 um* OH/Diol

    Zirchrom Zirchrom-PBD 4.6X50 mm, 3 um Hydrophobic with adsorbed phosphate

    Imtakt Scherzo SM-C18 4.6X50 mm, 3 um Hydrophobic + Cation Exchange + Anion Exchange

    * Fused core design. Equivalent to 3 um particles. 6

  • • Test two mobile phase parameters that may affect retention

    – Acetonitrile Content

    – Buffer concentration

    • Test high and low levels of each parameter

    – Acetonitrile: 50% and 90%

    – Buffer Concentration: 10 mM and 30 mM

    22 Factorial Screening Design

    7

  • • Why choose this design? – Factorials evaluate two

    or more variables with a minimum number of experiments

    • Why choose these conditions? – The choice is arbitrary

    – Ideal for general LC and LC-MS applications

    22 Factorial Screening Design

    mM

    Bu

    ffer

    % Acetonitrile

    30mM

    10mM

    50% 90%

    8

    Procedure: Measure retention Calculate k Calculate Main Effects

  • • The Main Effect of a parameter is the difference between the results at high and low settings.

    • Larger values mean a larger change in retention over the range studied.

    • Small values mean that this variable does not produce a significant change in retention.

    Calculating Main Effects

    mM

    Bu

    ffer

    % Acetonitrile

    30mM

    10mM

    50% 90%

    Ace

    ton

    itri

    le H

    igh

    Ace

    ton

    itri

    le L

    ow

    Buffer Low

    Buffer High

    mM

    Bu

    ffer

    % Acetonitrile

    30mM

    10mM

    50% 90%

    High Average – Low Average

    Hig

    h A

    vera

    ge –

    Lo

    w A

    vera

    ge

    9

  • 0.407 (k=0.55)

    0.452 (k = 0.72)

    2.742 (k=9.34)

    3.076 (k=10.6)

    Example Calculations

    mM

    Bu

    ffer

    % Acetonitrile

    30mM

    10mM

    50% 90%

    Nicotinic Acid (OH5)

    Main Effect for Acetonitrile

    Main Effect for Buffer

    (9.34+10.6) 2

    (0.55+0.72) 2

    - = +9.3

    (0.55+9.34) 2

    (0.72+10.6) 2

    - = -0.7

    • There is a significant increase in retention with an increase in acetonitrile.

    • There is a small decrease in retention with an increase in buffer conc.

    10

  • What Do Main Effects Mean?

    Sign of the Main Effect

    Negative Near Zero Positive

    Acetonitrile Reversed Phase Trend

    No effect Normal Phase Trend

    Buffer Conc. Ion Exchange Trend

    No effect Salting Out Trend

    Important Note: the existence of a trend suggests, but does not prove, that the stated mechanism is responsible for the effect.

    11

  • • How do we use this information? – We want to separate the polar compounds from the less polar 1-

    ring aromatics. – Change buffer concentration to move the polar compounds, with

    little change in retention for the aromatics. – Change the acetonitrile content to move the polar compounds in

    the opposite direction from the aromatics.

    Significant Main Effects for F5 (PFP) Compound Class Main Effects Trend

    Acetonitrile Buffer

    BTEX Aromatics* Non-Polar, neutral -2.9 -0.1 RP, No ionic

    Benzyl Amine Polar, strong base +3.6 -2.6 NP, Ion Exchange

    Phenethyl Amine Polar, strong base, more hydrophobic than Benzyl Amine

    +4.2 -3.4 NP, Ion Exchange

    BTMA** Polar, cation +6.0 -5.3 NP, Ion Exchange

    12 *Benzene, Toluene, Ethyl Benzene, Xylenes **Benzyltrimethylammonium chloride

  • Improving the Separation on F5 (PFP)

    min 0 0.5 1 1.5 2

    mAU

    -10

    -5

    0

    5

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    25

    1

    2 3

    90%, 30 mM

    1 2

    3

    50%, 10 mM

    min 0 0.5 1 1.5 2

    mAU

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    0

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    1 2

    3

    50%, 30 mM

    min 0 0.5 1 1.5 2

    mAU

    -20

    -10

    0

    10

    20

    Main Effects

    Acn Buffer

    BTEX -2.9 -0.1

    Benzyl Amine (1) +3.6 -2.6

    Phenethyl Amine (2) +4.2 -3.4

    BTMA (3) +6.0 -5.3

    13

    90%, 10 mM

    1

    2

    3

    min 0 1 2 3

    mAU

    -2

    0

    2

    4

    6

    8

    10

    12

    14

    30mM

    10mM

    50% 70%

  • Improving the Separation on PBD

    1 2

    3

    50%, 10 mM

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    -15

    -10

    -5

    0

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    20

    1 2

    3

    50%, 30 mM

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    -20

    -10

    0

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    30

    70%, 10 mM

    1 2

    3

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    1 2

    3

    70%, 30 mM

    min 0.5 1 1.5 2 2.5 3 3.5

    mAU

    0

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    35

    Main Effects

    Acn Buffer

    BTEX -1.6 -0.1

    Benzyl Amine (1) +6.4 -8.0

    Phenethyl Amine (2) +6.3 -8.8

    BTMA (3) -0.1 -1.4

    14

    30mM

    10mM

    50% 70%

    Probably not a linear

    trend.

  • Improving the Separation on SM-C18 Main Effects

    Acn Buffer

    BTEX -10.0 -0.2

    Creatinine (1) +0.30 0.0

    Benzyl Amine (2) +2.1 -0.9

    Phenethyl Amine (3) +0.5 -0.5

    Nicotinic Acid (4) +2.4 -1.1

    Benzene Sulfonate (5) 0.0 -1.1

    1

    2 3

    50%, 30 mM

    min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

    mAU

    -20

    0

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    80

    100

    4

    5

    1 2 3

    50%, 10 mM

    min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

    mAU

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    min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

    mAU

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    90%, 10 mM

    1

    2 3

    4

    5

    1

    2 3

    90%, 30 mM

    min 0 0.25 0.5 0.75 1 1.25 1.5 1.75

    mAU

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    50 4

    5

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    30mM

    10mM

    50% 90%

  • Improving the Separation on OH5 Main Effects

    Acn Buffer

    BTEX 0.0 0.0

    Creatinine (1) +2.1 0.0

    Benzyl Amine (2) +2.3 -1.1

    Phenethyl Amine (3) +2.3 -0.1

    Nicotinic Acid (4) +9.3 -0.7

    Phenylalanine (5) +9.6 +0.1

    1,2,3

    50%, 30 mM

    4

    5

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    80

    100

    1,2,3

    50%, 10 mM

    4

    5

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    80

    100

    1

    2

    3

    90%, 30 mM

    4

    5

    min 0 0.5 1 1.5 2 2.5 3

    mAU

    0

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    15

    20

    90%, 10 mM

    1

    2,3

    5,4

    min 1 2 3 4

    mAU

    0

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    16

    30mM

    10mM

    50% 90%

  • Main Effects Comparisons

    17

    BTEX Main Effect: ACN

    SM-C18 -10.0

    F5 -2.9

    PBD -1.6

    OH5 0.0

    Benzyl Amine

    Main Effect: ACN

    F5 +6.4

    PBD +6.4

    OH5 +2.3

    SM-C18 +2.1

    Benzyl Amine

    Main Effect: Buffer

    PBD -8.0

    F5 -2.6

    OH5 -1.1

    SM-C18 -0.9

  • • The 22 factorial method can be used to easily identify general retention trends.

    • Each of the four columns studied demonstrated a unique combination of multiple retention mechanisms.

    • When two mechanisms are present, it is possible to separately move classes of compounds in different directions by adjusting the mobile phase composition. – On a single mode column, this would only be possible by

    changing the chemistry of the mobile phase components (e.g., change from acetonitrile to methanol).

    – On a multi-mode column, the retention time selectivity can be changed by adjusting the composition of the mobile phase, without changing the chemistry.

    Summary

    18

  • Acknowledgements

    Columns, reagents, and other resources were kindly provided by:

    For more information, contact: Merlin K. L. Bicking, Ph.D. ACCTA, Inc. St. Paul, MN 55125 Email: [email protected] Internet: www.accta.com

    19

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    20

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