User:Khyra A. Neal/Notebook/Chem 571/2014/09/24

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September 25,2014

High Pressure Liquid Chromatography (HPLC)

Running HPLC with Gel Permeation Column (GPC)

Tasklist

1. Prepare 0.5 g/L and 0.1 g/L Lysozyme from 1 g/L Lysozyme Stock

  • (1 g/L)v1= (0.5 g/L)(10 mL)
    • Volume of 1 g/L Lysozyme needed: 5 mL need
    • Dilute to 10 mL in volumetric flask
  • (1 g/L)v1= (0.1 g/L)(10 mL)
    • Volume of 1 g/L Lysozyme needed: 1 mL
    • Dilute to 10 mL in volumetric flask

2. Prepare 0.5 M acetic acid and 0.3 M Na2SO4 HPLC Solvent

  • 0.5mol HAc/L)(0.5 L)(60.05 g/mol)(1mL/1.05g)=14.3mL of acetic acid needed
  • (0.3mol NaSO4/L)(0.5L)(142.04g/mol)=21.3060 g of NaSO4 need to be added to 500 mL volumetric flask under fume hood
    • Add 14.3 mL of acetic acid to 500 mL volumetric flask (ADD SMALL AMOUNT OF HPLC WATER FIRST)
    • Dilute to 500 mL with HPLC water.

3. Filtered 0.5 M acetic acid and 0.3 M Na2SO4 by membrane filtration to removed particles and remove CO2

4. We ran our yellow standard and Lysozyme samples at 0.8 mL/min for 12 minutes initially. We noticed that all of the sample didn't leave the column with the time frame so we extended the remaining samples up to 16 minutes. We also ran our [30]:[1] Lysozyme colloid sample at 0.8 mL/min for 16 minutes. All of the samples were injected into the HPLC using a syringe and syringe filter.



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