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.


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


  • 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


  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


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.



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