User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/30

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Tasks for September 30 To run dyalisis To prepare calibration curves Ca2+ and Cl- Determining 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 <br.> Try measuring 50 μM and 50 mM of other salts to check for interferences (Mg2+ should, Na+ should not) CaCl2 concentration Cu(II)Cl2 concentration KCl concentration mV measurement (mV) 50mM 0 50mM 86.4 50mM 50mM 0 80.5 Note: There may be minimal interference by Cl- 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 NaSO4 Na2HPO42- mV measurement (mV) 50mM 50 mM 0 -27.0 50mM 0 50mM -30.8 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 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 pH measurements Substance pH measurement 0.25 mM HCl (diluted 10x) (2) 7.857 Lysozyme (diluted 10x) (2) 7.426