BCH4160/2011:Notebook/Dr Cannon Samples/Protocol MethylRedAbs

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BCH4160/2011: Biophysical and Bioanalytical Lab at HPU

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Several parts of this protocol do not have sufficient detail and should be modified to describe exactly what you did.

Contents

Overview

Determine the pKa of methyl red by measuring the UV/Vis absorbance spectrum as a function of pH

Materials

  • Methyl Red solution provided by instructor
  • Buffer Components
    • Phosphate/Citrate buffer pH 3.5, 4.5, 5.5, 6.5
    • Phosphate buffer pH 6.5, 7.5
  • OceanOptics UV/Vis sprectrophotometer


Procedure

  1. Prepare 10 mM buffers at pH 3.5, 4.5, 5.5, 6.5, and 7.5
    • I used Phosphate/Citrate buffer for pH 3.5-6.0 and Phosphate buffer for pH 6.5-7.5
  2. Determine the optimal concentration of Methyl Red solution diluted into buffer to give a maximum absorbance between 0.7 and 1.0.
    1. Dilute 150, 30, 5, and 1μL methyl red solution with buffers at pH 3.5 and 7.5 to a final volume of 1.00 mL
    2. Measure absorbance from 320-800 nm
    3. Choose methyl red concentration for spectral measurements
  3. Prepare methyl red solutions at all pH's and measure the full absorbance spectra
    • Repeat 3 times

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

    • Jonathan G. Cannon 15:56, 13 September 2011 (EDT):OceanOptics spectrometers can vary greatly in their readings from one instrument to another, so try not to switch instruments or you may have to normalize your data.

References

Contact

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