User:Michael S. Bible/Notebook/CHEM-671/690 Lab Notebook/2015/09/16

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Objective

  • Remake failed samples from yesterday
  • Isolate Fibers from yesterday's successful samples
  • Calibrate pH probes
  • Create phosphate and Tris buffer solutions

Protocol

Sample Remake

10 new 1 mL fiber samples were prepared to replace the 6 unsuccessful samples created on September 15th.

Isolation of Fibers

The 1 mL fiber samples that were successfully prepared yesterday were centrifuged at 12000 RPM for 1 minute and the supernatant fluid was removed. The samples were left uncovered to allow the remaining liquid to evaporate.

Calibration of pH Meters

pH meters were calibrated using stock solutions of 4.0, 7.0 and 10.0 pH calibration standards.

Buffer Solution Preparation

  • 500 mL 10 mM Tris buffer pH 9
  1. 0.60638 g (5.006 mmols)was placed in a 500 mL volumetric flask and was filled to the line with HPLC water
  2. The pH of the resulting solution was measured and was adjusted to pH 9 using 1 M HCl
  3. The pH and conductivity of the final solution was measured.
  • 500 mL 10 mM Phosphate Buffer pH 7
  1. The following phosphate buffer calculator was used to determine the necessary quantities of dibasic and monobasic sodium phosphate to use: http://serge.engi.tripod.com/MolBio/Buffer_cal.html
  2. From the website above, the following quantities were determined:
    1. 0.1967 g Dibasic sodium phosphate
    2. 0.53413 g monobasic sodium phosphate
  3. 0.1965 g dibasic sodium phosphate and 0.53410 g monobasic sodium phosphate was placed in a volumetric flask and the flask was filled to the line with HPLC water
  4. The pH and conductivity of the final buffer solutions were measured.


Note: It was determined after the preparation of the phosphate buffer that the wrong target pH had been entered into the phosphate buffer calculator. Rather than a pH of 7.0, a pH of 7.4 was entered into the calculator. This accounts for the flawed results of the phosphate buffer preparation.

Results

  • 500 mL 10 mM Tris buffer pH 9
    • pH after adjustment: 9.014
    • conductivity after adjustment: 111.8 μS/cm


  • 500 mL 10 mM Phosphate Buffer pH 7
    • Actual pH: 6.510
    • Conductivity: 1044.4 μS/cm

Due to time restrictions and the limited availability of the pH probes, it was impossible to correct the phosphate buffer.



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