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

  1. Prepare 10 mL 0.5 g/L Lysozyme
    • Added 0.0050 g to 10mL volumetric flask and made up to mark with HPLC
  2. Prepare 2 mL 50 mM CaCl2 (if you read ahead, you can use your CaCl2 calibration stock)
  3. Prepare 2 mL 500 μM CaCl2 (if you read ahead, you can use a CaCl2 calibration dilution)
  4. Prepare 2 mL 50 mM NaCl (if you read ahead, you can use your NaCl calibration stock)
  5. Prepare 1 mL 0.25 mM HCl using your 2.5 mM stored stock
  6. Cut a piece of 3500 MWCO flat dialysis tubing, wet it, and insert it in the 5-well dialysis chamber
  7. Clamp the dialysis chamber shut
  8. 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
  9. 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
  10. 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
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