BCH4160/2011:Notebook/Tiffany Byerly Biochemistry Lab/Methyl Red Lab

Overview

Determine the pK_a 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 4.0, 4.23, 5.16, 6.0, 6.7, 7.16, and 7.40
• OceanOptics UV/Vis sprectrophotometer

Procedure

1. Prepare 7 mM buffers at pH 4.0, 4.23, 5.16, 6.0, 6.7, 7.16, and 7.40
   • I used Phosphate/Citrate buffer
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 150μL methyl red solution with buffers at pH 4.0, 4.23, 5.16, 6.0, 6.7, 7.16, and 7.40 to a final volume of 1.00 mL
   2. Measure the full absorbance spectra
   3. Choose methyl red concentration for spectral measurements
      • Repeat 3 times