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Mobile Phase pH Analyte pK a Shift. Lecture 4. Yuri Kazakevich Seton Hall University. Measuring pH of Hydro-organic Mixtures. There are three different pH scales that are employed in pH measurement of reversed phase HPLC mobile phases. - PowerPoint PPT Presentation

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  • Mobile Phase pH Analyte pKa ShiftLecture 4Yuri KazakevichSeton Hall University

  • Measuring pH of Hydro-organic MixturesThere are three different pH scales that are employed in pH measurement of reversed phase HPLC mobile phases.

    wwpH scale - the electrode system is calibrated with aqueous buffers. The pH of the aqueous portion of the mobile phase is measured prior to the addition of the organic modifier.

    swpH scale - the electrode system is calibrated with aqueous buffers. The pH of the hydro-organics mobile phase is measured after the addition of the organic modifier.

    sspH scale - the electrode system is calibrated with buffer-organic mixtures of the same composition as the mobile phase. The pH of the hydro-organic mobile phase is measured after the addition of the organic modifier.

  • pH Shift of Hydro-Organic Eluents

    Chart1

    2.093.1145.026.117.018.9

    2.23.264.185.26.297.229.04

    2.423.494.445.466.557.499.26

    2.563.654.595.616.77.659.39

    2.783.894.825.846.937.879.73

    3.064.145.096.117.28.150

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    v/v% MeCN

    sspH

    Sheet1

    Note

    d =Ej-log(swgH0)=swpH-sspH10 mM Na2HPO4 adjusted with phosphoric acid.Alex make a table of chromatographic pKa determined from k' vs. aqeous pH and k' vs. sspKap.

    delta= liquid junction potential + primary medium effectWe need to determine sspKap for 50% pH 2,4,7, and 30% pH 2,4,7

    (calibrate pH meter with 2,4,7 standards in 50% and analyze hydroorganic solutions. and calibrate pH meter with 2,4,7 standards in 10% and then analyze hydro-organic solutions

    w spKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9Roses, anal.chem. 2002, 74, 3809-3818.

    d02.093.1145.026.117.018.92.217.23

    100.01-0.01102.213.274.195.216.37.239.052.387.39

    200.020.06202.363.434.385.46.497.439.22.597.57

    300.060.05302.513.64.545.566.657.69.342.767.78

    400.130.12402.663.774.75.726.817.759.612.977.94

    500.260.18502.883.964.915.937.027.923.28.16

    slope0.01557142860.01691428570.01782857140.01782857140.01782857140.01794285710.01710.01968571430.0186

    R20.99167552350.99906117560.9984170670.9984170670.9984170670.99506484330.9805835010.99767981930.9979341227

    AnilinepH2pH74.455.466.47

    104.514.480.03-0.014.665.666.66

    204.354.280.070.064.765.836.83

    304.154.110.040.054.955.986.98

    404.053.870.180.125.156.127.11

    503.993.670.320.18

    2.092.212.362.512.662.88

    05.175.220.053.113.273.433.63.773.96

    105.075.06-0.0144.194.384.544.74.91

    204.934.91-0.025.025.215.45.565.725.93

    304.744.69-0.056.116.36.496.656.817.02

    404.634.46-0.177.017.237.437.67.757.92

    504.554.22-0.338.99.059.29.349.610

    sspKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9

    02.093.1145.026.117.018.9

    102.23.264.185.456.467.229.040.21

    202.423.494.725.726.727.499.260.27

    302.563.654.815.886.887.659.390.16

    402.783.895.076.17.17.879.730.22

    503.064.145.336.37.298.10.23

    0.0222142857slope0.01922857140.02057142860.02688571430.02431428570.02280.02160.0201

    R20.98322111470.99352217410.96906795660.97092849430.98348108110.99724778110.9718320023

    These in yellow should be used as the correction factors for pH shift

    sspKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9

    02.093.1145.026.117.018.9

    102.23.264.185.26.297.229.040.21

    202.423.494.445.466.557.499.260.27

    302.563.654.595.616.77.659.390.16

    402.783.894.825.846.937.879.730.22

    503.064.145.096.117.28.10.23

    2.092.22.422.562.783.060

    3.113.263.493.653.894.140

    44.184.444.594.825.090

    5.025.25.465.615.846.110

    6.116.296.556.76.937.20

    7.017.227.497.657.878.10

    8.99.049.269.399.7300

    Sheet1

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    % MeCN (v/v)

    w spKap.

    10mM Na2HPO4 adj. w/H3PO4

    Sheet2

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    %MeCN (v/v)

    sspKap.

    10mM Na2HPO4 adj. w/H3PO4

    Sheet3

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    % MeCN

    sspH

    Sheet1

    Note

    d =Ej-log(swgH0)=swpH-sspH10 mM Na2HPO4 adjusted with phosphoric acid.Alex make a table of chromatographic pKa determined from k' vs. aqeous pH and k' vs. sspKap.

    delta= liquid junction potential + primary medium effectWe need to determine sspKap for 50% pH 2,4,7, and 30% pH 2,4,7

    (calibrate pH meter with 2,4,7 standards in 50% and analyze hydroorganic solutions. and calibrate pH meter with 2,4,7 standards in 10% and then analyze hydro-organic solutions

    w spKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9Roses, anal.chem. 2002, 74, 3809-3818.

    d02.093.1145.026.117.018.92.217.23

    100.01-0.01102.213.274.195.216.37.239.052.387.39

    200.020.06202.363.434.385.46.497.439.22.597.57

    300.060.05302.513.64.545.566.657.69.342.767.78

    400.130.12402.663.774.75.726.817.759.612.977.94

    500.260.18502.883.964.915.937.027.929.453.28.16

    slope0.01557142860.01691428570.01782857140.01782857140.01782857140.01794285710.01305714290.01968571430.0186

    R20.99167552350.99906117560.9984170670.9984170670.9984170670.99506484330.87166299150.99767981930.9979341227

    AnilinepH2pH74.455.466.47

    104.514.480.03-0.014.665.666.66

    204.354.280.070.064.765.836.83

    304.154.110.040.054.955.986.98

    404.053.870.180.125.156.127.11

    503.993.670.320.18

    2.092.212.362.512.662.88

    05.175.220.053.113.273.433.63.773.96

    105.075.06-0.0144.194.384.544.74.91

    204.934.91-0.025.025.215.45.565.725.93

    304.744.69-0.056.116.36.496.656.817.02

    404.634.46-0.177.017.237.437.67.757.92

    504.554.22-0.338.99.059.29.349.619.45

    sspKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9

    02.093.1145.026.117.018.9

    102.23.264.185.456.467.229.040.21

    202.423.494.725.726.727.499.260.27

    302.563.654.815.886.887.659.390.16

    402.783.895.076.17.17.879.730.22

    503.064.145.336.37.298.10.23

    0.0222142857slope0.01922857140.02057142860.02688571430.02431428570.02280.02160.0201

    R20.98322111470.99352217410.96906795660.97092849430.98348108110.99724778110.9718320023

    These in yellow should be used as the correction factors for pH shift

    sspKap.

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9

    02.093.1145.026.117.018.9

    102.23.264.185.26.297.229.040.21

    202.423.494.445.466.557.499.260.27

    302.563.654.595.616.77.659.390.16

    402.783.894.825.846.937.879.730.22

    503.064.145.096.117.28.19.90.23

    slope0.01922857140.02057142860.02148571430.02148571430.02148571430.02160.0205714286

    R20.98322111470.99352217410.99466000630.99466000630.99466000630.99724778110.9837558828

    2.092.22.422.562.783.060

    3.113.263.493.653.894.140

    44.184.444.594.825.090

    5.025.25.465.615.846.110

    6.116.296.556.76.937.20

    7.017.227.497.657.878.10

    8.99.049.269.399.739.90

    pH 2pH 3pH 4pH 5pH 6pH 7pH 9

    slope0.0190.0210.0210.0210.0210.0220.020

    R20.9830.9940.9950.9950.9950.9970.972

    Sheet1

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    % MeCN (v/v)

    w spKap.

    10mM Na2HPO4 adj. w/H3PO4

    Sheet2

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    %MeCN (v/v)

    sspKap.

    10mM Na2HPO4 adj. w/H3PO4

    Sheet3

    0000000

    0000000

    0000000

    0000000

    0000000

    0000000

    pH 2

    pH 3

    pH 4

    pH 5

    pH 6

    pH 7

    pH 9

    % MeCN

    sspH

  • Determination of the Chromatographic pKaHPLC can be used as a powerful technique for the determination of dissociation constants.

    Only requires small quantity of compoundsCompounds do not need to be pureSolubility is not a serious concern

    To properly describe the effect of pH on the retention of ionizable analytes the actual pH of the hydro-organic mobile phase must be known.

  • Determination of the Chromatographic pKakok1Potentiometric pKa = 4.6Eluent50% Buffer50% MeCN

    ko= Retention factor of analyte in its neutral formk1 = Retention factor of analyte in its ionized form

  • Chromatographic Conditions Column: 15 cm x 0.46 cm Luna C18(2)Eluent: Aqueous/ 10, 20, 30, 40, 50% MeCNAqueous: 15 mM K2HPO47H2O adj. to pH 1 - 9 with H3PO4Flow rate: 1 ml/minTemp: 25oCWavelength: 220 nmEffect of pH on Aniline Retention

  • Effect of Organic Content on Analyte pKa ShiftA decrease of ~0.2 pKa units per 10% v/v MeCN for aniline was determined.The slope could be used to estimate sspKa of pharmaceutical compounds containing aromatic amine functionalities at a certain organic composition after adjusting for the mobile phase pH shift.

  • Example: 2-4 dimethylpyridine (base) has a pKa of 6.7 and initial eluent conditions are: 50% MeCN and 50% Buffer.

    What should the pH of the buffer be in order to obtain the basic analyte in its fully ionized form?pH Shift and pKa ShiftThe downward pKa shift for basic analytes must be accounted for.The working pH should be at least 2 pH units below the basic analyte pKa to be fully ionized. The upward pH shift of the aqueous acidic buffer upon addition of the organic must be accounted for.

    Downward analyte pKa shift.pH at which basic analyte would be protonated

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