Posted by: ericmjl | August 15, 2009

3M Sodium Acetate

As part of our lab maintenance duties, I spent today testing the new miniprep reagents made yesterday. Since the yield (141.8 ng/µL) and purity (1.69) were not up to scratch, I decided to make a fresh batch of reagents.

Halfway through, I realized I needed 3M sodium acetate, pH 5.5 So at first, I thought, why not make the 3M solution, top up to 40 mL, then pH using HCl? Not a bad idea at first glance, and I tried it – and my yield and purity didn’t improve much (241.7 ng/µL; 1.77).

So I sat down and thought about this problem. It wasn’t right to introduce the counter ion Cl- into the solution, and that may have been the cause of much chagrin. Thus, I decided to try making the solution the other way – using acetic acid and then raising the pH to 5.5 using NaOH. However, no matter how I tried to calculate it, I couldn’t recall my Analytical Chem (CHEM211) concepts for calculating a titration. The volumes I had attempted to calculate were ridiculously large too – 17 mL of 1M NaOH?! That sounded like too much. How about using 6M rather than glacial acetic acid instead? That cut the volume, but would be too troublesome.

3 scrap sheets of paper and 2 hours later, I gave up and decided to look online. And bingo, the revelation came – 12.31g sodium acetate + water to 35 or 40 mL, then lower pH using glacial acetic acid, and then top up to 50 mL. I had been using the right reagents the wrong way. However, after 3 hours of exposure to glacial acetic acid, with an irritated nose, throat and personality, the yield went up to 392.03 ng/µL with a purity of 1.85. Purer, but not much more encouraging since I usually get yields of 2000 ng/µL, rather than 400 ng/µL, from 1.5 mL of culture.

Perhaps I am a biologist, not a chemist.

Update: 5 minutes after writing this post, I decided to just type up the protocol anyways. And that’s where I caught where I may have gone wrong – I didn’t adjust the pH of the Tris-Cl buffer to 8.0. Grr, I really need a protocol to start with.

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