Sensitivity issue: dissappointed from high-sensitivity cell

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UPLC_User
UPLC_User
in Instrumentation and Mass Spectrometry
<p>Hello,</p><p></p><p>we have a classical HPLC method for the determination of Tocopherol on a 250x4mm column, 5 µm.</p><p></p><p>We use an old Merck-Hitachi System with a detector cell length of 5 mm, the detection wavelength is 290 nm, we inject 20 µL</p><p></p><p>Now, we took the column and the injected solution and transferred them to an UPLC Acquity with a high-sensitivity cell in the PDA (cell length = 25 mm)</p><p></p><p>Injection of 20 µL of the transferred solution results in a chromatogram with an area that is about 5 times higher (as expected), but the S/N-ratio is worse than with the Merck-Hitachi.</p><p></p><p>See attachement for illustration.</p><p></p><p>Can somebody explain me the phenomenon?</p><p></p><p>(PS: other transfers of other analytes measured with 210 - 220 nm do not show this problem)</p><p></p><p>Thank you very much</p><p></p><p>Florian</p>

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  • lizh
    lizh
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    Hello

    Can you share the mobile phase composition and the detector settings

    many TX

    Liz

  • UPLC_User
    UPLC_User
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    Hello Liz,

    mobile phase is Acetonitrile / Water 90:10

    The detector settings are:

    General:

    Sampling rate: 40 points/sec

    Filter Time Constant: Normal

    Exposure Time: Auto

    UV blocking filter disabled

    2D Channels (Channel 1):

    Data Mode: Absorbance

    Wavelength: 290

    Resolution: 4.8 nm

    The Merck-Hitachi detector is a L-4250 (with no other settings than the wavelength)

    Thank you

    Florian

  • pattyapollo
    pattyapollo
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    An answer from a Waters RDE scientist follows:

    The peak absorbance is scaled by a factor of 5x, which is the path length ratio, and the wavelength is 290 nm where the mobile phase is not likely to be an absorber and a primary noise source.

    This looks like an isocratic  HPLC  separation with a sampling rate of 1 Hz and a 1 second time constant.  We recommend that we match the sampling rate and use a "normal" time constant.  We suspect that what we'll see is a dramatic improvement in the noise.   The HPLC data appears to be captured on a stand alone integrator which implies classical HPLC conditions.

    poated by Pat Young (Waters product management)

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