User:Brigette D. Black/Notebook/Brigettes Notebook/2009/06/22/Making PEM/BRB80/KochLab Buffer
We began today by making a strong acid (HCl) and base (NaOH) so that we could adjust the pH of the buffer when that time came.
HCl
I made 40 mL of 1M HCl. I added about 20mL of DI water to a tube, then pipetted in 3.306 mL of a stock solution at 12.1M. I then added about 36 mL of water (until the volume reached the 40 mL mark). This was then vortexed and set aside for further use.
NaOH
This mixture was done by Linh and myself. For 40mL at 1M we need 1.6 g of NaOH pellets(molar mass = 40). We measured out 1.63 g and added this to about 20 mL of water in the tube, then added the rest of the water needed (about 40mL total). Andy told us that NaOH pellets were hygroscopic and thus the container could only be opened for a very short time, and after it had been opened, we needed to blow nitrogen into the container so to remove any air that had seeped in.
BRB80 Recipe
While the name is debatable, the ingredients remain the same.
- 1mM MgCl_2
- 1mM EGTA
- 80mM PIPES
We wanted about 200mL of buffer total, so we added...
- 200uL MgCl_2 (at 1M)
- 0.0761g EGTA
- 4.8379g PIPES
The EGTA and PIPES were weighted out on the scale and added to a small bit of water in a glass jar. We then added the MgCl_2 and filled the jar up to about 190 mL.
pH-ing the Buffer
We calibrated the pH meter (which had been set up by Andy) by first sticking it in a solution known to be at 7, and hten one known to be at 10.
We want our final solution to be at at pH of 6.9, we initially measured the pH of our buffer solution to be at 2.78. We then began added the NaOH, initially by pipetting in unit of 1mL (1000uL) until the solution was clear, which was about 20mL of NaOH. The pH of the solution at this point was about 6.32. We than began adding in incriments of 200 uL, however the change in pH between the additions was almost nonexisent. We then added in incriments of 500 uL. We had added a total of 25.9 mL of NaOH when the pH meter read 6.89 (oscillating up to 6.9) and we called it a day.
Final Recipe
- 1mM MgCl_2
- 1mM EGTA
- 80mM PIPES
- 125mM NaOH
