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

Project name

Main project page Previous entry      Next entry

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