User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/09/30

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September 30, 2014

High Pressure Gel Permeation Chromatography

The mobile phase used was a 0.5 M HAc and 0.3 M Na2SO4 solution.

  • 21.3344 g of Na2SO4 and 14.31 mL HAc were combined in a 500 mL volumetric flask
  • This solution was filtered through a 0.2 μM membrane filter in order to remove all particulate matter and CO2

A 10 mL sample of 1 g/L lysozyme solution was made using the prepared mobile phase.

  • 0.01 g of pure lysozyme was dissolved in 10 mL of 0.5 M HAc/0.3 M Na2SO4 solution

Using the 10 mL of 1 g/L stock lysozyme solution, two additional 5 mL lysozyme samples were prepared with concentrations 0.5 and 0.1 g/L, respectively.

  • 5 mL of the stock solution was diluted with 5 mL of water in order to prepare 0.5 g/L lysozyme stock
  • 2 mL of 0.5 g/L lysozyme stock was diluted with 8 mL of water in order to prepare 0.1 g/L lysozyme stock

In order to run the lysozyme and standard (blue) samples, the HPLC machine was first conditioned with the mobile phase, after which all three samples were injected for consecutive runs with a 0.60 mL/min flow rate for 30 minutes total, each. The samples were injected via a syringe/filter combination to avoid the clotting of the machinery tubing with particulate/solid matter.

Results will be posted in the near future.

Multi-Well Dialysis

I) A piece of 3500 MWCO flat dialysis tubing was cut, wet, and inserted in between the two plates of a five well dialysis chamber. The dialysis chamber was then clamped shut.

II) Each side of each well of the chamber was filled with 1 mL of an appropriate sample (10 samples total - our experiment ended up having only 8 samples in total) using a glass Pasteur pipette and by tilting the chamber to an angle which reduces surface tension and overflow.

  • The following table demonstrates the organization/content of each side of the five well chamber:

Well 1 Well 2 Well 3 Well 4 Well 5
0.5 g/L lysozyme 0.5 g/L lysozyme 0.5 g/L lysozyme 0.5 g/L lysozyme
HPLC water 0.25 mM HCl 50 mM CaCl2 500 μM CaCl2

NOTE: Debris was found in one space of well 3 and therefore this well was not used for dialysis; eliminating the dialysis of 0.5 g/L lysozome against a 50 mM NaCl solution, as a result.

III) Top screws were inserted in the openings above each side of all five wells, preventing leakage or evaporation of sample.

The CaCl2 solutions used for (50 mM and 500 μM) dialysis were prepared last week for the determination of a CaCl2 calibration curve.

Additionally, the 0.5 g/L lysozyme samples came from the stock solution prepared for GPC earlier in the day, as mentioned above.

Conversely, a 0.25 mM HCl solution was prepared by first (accidentally) preparing 2 mL of a 2.5 mM HCl solution by diluting 10 μL of 0.5 M HCl stock solution. This solution was further diluted by diluting 100 μL (of 2.5 mM HCl) with 900 μL of water.

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