Created a create a Ca2+ calibration curve and began a new dialysis
- Prepared 10 mL 0.5 g/L Lysozyme
- Prepared 2 mL 50 mM CaCl2 (used our CaCl2 calibration stock)
- Prepared 2 mL 500 μM CaCl2 (used a CaCl2 calibration dilution)
- Prepared 2 mL 50 mM NaCl (used our NaCl calibration stock)
- Prepared 1 mL 0.25 mM HCl using our 2.5 mM stored stock
- 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
- Filled one side of each well with 1 mL of our 0.5 g/L Lysozyme solution
- Transfered 1 mL to a plastic test tube using a pippetter, then
- Carefully filled each well with with a glass Pasteur pipette
- Filled 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
- Inserted top screws to prevent leaking/evaporation
- Tomorrow, UV-vis analysis will be done on all of the solutions from each well.
- GPC will be conducted later
- Ca2+ calibration curve
- Prepared 25 mL 50 mM CaCl2 using HPLC water
- Using serial dilutions, prepared 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
- Measured Ca2+ using ISE
- Measured 50 mM of other salts (MgCl2, CuCl2, KCl, and NaCl) added to 50 mM CaCl2 to check for interferences. Also added some HCl solution.
- Measured HPLC water, DI water, tap water, and our lysozyme stock solution.
- Cl- calibration curve
- Prepared 25 mL 50 mM NaCl using HPLC water
- Using serial dilutions, prepared 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
- Measured Cl- using ISE
- Measured 50 mM of other salts to check for interferences & ion-pairing
- Also measured HPLC water, DI water, tap water, and your lysozyme stock solution.
The protocol above was adapted from Dr. Fox's Notebook.
Ca2 calibration curve
- For the 0.5 g/L Lysozyme, 0.00570g Lysozyme used.
- For NaCl serial dilution 0.0713g NaCl was placed in 25 mL of water
- For the 0.25 mM HCl, a new solution was made containing HPLC water rather than DI water
The figure below shows the calibration curve for CaCl2. A logarithmic scale was used because the concentration values of the salt vary by an order of magnitude between each value.
In the figure below, the values of the potential were taken after the addition of ions. If the ion added first did not affect the value of the potential, the next ion was added to that same solution. As can be seen below, there is essentially no change in the potential value measured after the addition of any of the ions. This means that the presence of other ions has no effect on the potential reading of CaCl2.
Cl-1 Calibration Curve