User:Alicia Rasines Mazo/Notebook/CHEM-571 Experimental Biological Chemistry/2014/09/24

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Tasks for September 24

To run HPLC with a GPC (Gel Permeation Chromatography) column

Preparation of solutions for HPLC chromatography

To make a solution of 0.5M acetic acid and 0.3M Na2SO4:

  • (0.5mol HAc/L)(0.5L)(60.05g/mol)(1mL/1.05g)=14.3mL of acetic acid need to be added (under fume hood) to 500 mL volumetric flask
  • (0.3mol NaSO4/L)(0.5L)(142.04g/mol)=21.3060 g of NaSO4 need to be added to volumetric flask
  • Make up to 500 mL mark with HPLC water

To make dilutions of 1g/L of lsozyme:

  • 0.5g/L: (1g/L)(v1)=(0.5g/L)(10 mL)
  • v1=5 mL of 1g/L lysozyme needs to be added
  • 0.1g/L:(1g/L)(v1)=(0.1g(L)(10 mL)
  • v1=1 mL of 1g/L lysozyme needs to be added
  • Make up remainder of volume to 10 mL with distilled water

GPC

  • Prior to running HPLC, ran membrane filtration of solvent.
  • Filtered 0.5 M HAc 0.3 M NaSO4 under a vacuum to ensure all particles were <0.45 microns to degas (get rid of CO2 gas)
  • HPLC machine: Waters 1525 Binary HPLC pump, flow of A at 0.8 mL/min
  • For lysozyme and standard samples, syringe filters were used prior to injection
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