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
| 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
|
| 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
|
- 50 μM NaCl and 50 μM Cu(II)Cl2 solution gave us a -7.7 mV reading with Chloride electrode
|
Project name
