User:Beatriz Gimenez De C./Notebook/571/2014/09/30

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Tasks

  • Make a Dialysis of lysozyme in HPLC
    • Prepare 10 mL 0.5 g/L Lysozyme (added 0.0050 g to 10 mL volumetric flask filling the rest with HPLC)
    • Prepare 2 mL 50 mM CaCl2
    • Prepare 2 mL 500 μM CaCl2
    • Prepare 2 mL 50 mM NaCl
    • Prepared 50 mL 0.25 mM HCl with HPLC
    • Cut a piece of 3500 MWCO flat dialysis tubing, wet it, and insert it in the 5-well dialysis chamber
    • Clamp the dialysis chamber shut
    • Fill one side of each well with 1 mL of your 0.5 g/L Lysozyme solution
      • Transfer 1 mL to a plastic test tube using a pippetter, then
      • Fill each well with with a glass Pasteur pipette
      • You may need to tip the chamber on an angle to reduce water surface tension
    • Fill the other side of the well with one of the following solutions
      • HPLC water, 0.25 mM HCl, 50 mM CaCl2, 500 μM CaCl2, or 50 mM NaCl
      • Each well has a different solution, which will be tested tomorrow
    • Insert top screws to prevent leaking/evaporation.
  • Make Ca2+ calibration curve
    • Prepare 25 mL 50 mM CaCl2 using HPLC water (0.13876 g CaCl2 in 10 mL volumetric flask with HPLC)
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
    • Measure Ca2+ using ISE
    • Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg2+ should, Na+ should not)
    • Measure HPLC water, DI water, tap water, and your lysozyme stock solution.
  • Make Cl- calibration curve
    • Prepare 25 mL 50 mM NaCl using HPLC water (0.0730 g in 10 mL volumetric flask with HPLC)
    • Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
    • Measure Cl- using ISE
    • Measure 50 μM and 50 mM of other salts to check for interferences & ion-pairing
    • Measure HPLC water, DI water, tap water, and your lysozyme stock solution.

Results

  • Ca2+ calibration
CaCl2 Concentration (μM) mV measurement (mV)
5 -44.6
50 8.2
500 39.5
5000 63.5
50000 86.6
Substance mV measurement (mV)
TAP WATER 51.3
HPLC water -23.0
DI water -51.5
Lysozyme 0.5g/L -19.1
  • Cl- calibration
NaCl concentration (μM) mV measurement (mV)
5 146.5
50 129.6
500 81.3
5000 27.5
50000 -23.9
Substance mV measurement (mV)
Tap water 65.5
HPLC water 163.5
DI water 158.8
Lysozyme 0.5g/L 89.4
  • readings with Chloride electrode
    • 50 mM CaCl2 and 50 mM Cu(II)Cl2 solution gave us a 80.5mV
    • 50 mM CaCl2 and 50 mM KCl solution gave 86.4 mV reading
  • Readings with Sodium electrode
    • 50mM NaCl and 50 mM NaSO4 solution gave -27.0 mV
    • 50mM NaCl and 50 mM Na2HPSO42- solution gave -30.8 mV