Tasks for September 30
- To run dyalisis
- To prepare calibration curves Ca2+ and Cl- calibration curves
Making up calibration curves for Ca2+ and Cl-
Procedure followed as described by Dr. Fox
- Ca2+ calibration curve
- Prepare 25 mL 50 mM CaCl2 using HPLC water
- Added 0.13876g CaCl2 to a 25 mL volumetric flask and made up to the mark with HPLC
- Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM CaCl2
- Measure Ca2+ using ISE
| CaCl2 concentration
|
mV measurement (mV)
|
| 5μM |
-44.6
|
| 50μM |
8.2
|
| 500μM |
39.5
|
| 5mM |
63.5
|
| 50mM |
86.6
|
- Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg2+ should, Na+ should not)
- Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
| Substance
|
mV measurement (mV)
|
| Tap water |
51.3
|
| HPLC water |
-23.0
|
| DI water |
-51.5
|
| Lysozyme 0.5g/L |
-19.1
|
- Cl- calibration curve
- Prepare 25 mL 50 mM NaCl using HPLC water
- Added 0.07091 g NaCl to a 25 mL volumetric flask and made up to the mark with HPLC
- Using serial dilutions, prepare 25 mL each of 5 mM, 500 μM, 50 μM, & 5 μM NaCl
- Measure Cl- using ISE
| NaCl concentration
|
mV measurement (mV)
|
| 5μM |
146.5
|
| 50μM |
129.6
|
| 500μM |
81.3
|
| 5mM |
27.5
|
| 50mM |
-23.9
|
- Try measuring 50 μM and 50 mM of other salts to check for interferences & ion-pairing
| NaCl concentration
|
Cu(II)Cl2 concentration
|
mV measurement (mV)
|
| 50μM |
50 μM |
-7.7
|
- Be sure to measure HPLC water, DI water, tap water, and your lysozyme stock solution.
| Substance
|
mV measurement (mV)
|
| Tap water |
65.5
|
| HPLC water |
163.5
|
| DI water |
158.8
|
| Lysozyme 0.5g/L |
89.4
|
Dyalisis
- Dialysis
- Prepare 10 mL 0.5 g/L Lysozyme
- Added 0.0050 g to 10mL volumetric flask and made up to mark with HPLC
- Prepare 2 mL 50 mM CaCl2 (if you read ahead, you can use your CaCl2 calibration stock)
- Prepare 2 mL 500 μM CaCl2 (if you read ahead, you can use a CaCl2 calibration dilution)
- Prepare 2 mL 50 mM NaCl (if you read ahead, you can use your NaCl calibration stock)
- Prepare 1 mL 0.25 mM HCl using your 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
- 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
- Carefully 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
|
Project name
