Protocol Online logo
Top : New Forum Archives (2009-): : General Lab Techniques

Preparing buffers for pH testing - (Jul/09/2015 )

Hi I need help with preparing buffers. I have a pH range that i need to test my bacterium against. and for that i want to make buffers for example at each pH level.

The buffers i want to use are citric acid buffer for pH of 3.5, 4.5, 5.5, and 6.0. 

MES buffer solution for making pH of 6.5

HEPES buffer solution for making pH of 7.0, 7.5, and 8.0   and to adjust pH i will be using HCl and NaOH


So for example to make citric acid buffer at pH of 3.5 (50mM) I need 0.7119% w/v citric acid and 0.3807% w/v sodium citrate     how will i work this out in grams ?  


please help


what i do is prepare stock solutions at each pH and dilute as necessary with addition of any cofactors prior to adjusting volume.


for your citrate example, prepare equal concentration stocks of citric acid and sodium citrate then, using a table you can find with a simple search (or at, i think, where they have tables and a buffer calculator), mix them in the proportions shown in the table. if it is off then you can adjust with the necessary component without changing the concentration of the buffering component (in this case, citrate).


i don't think mes (maybe?) and hepes have similar counter agents, so you'll have to adjust with hcl and/or naoh, but be aware that you'll be increasing nacl concentration (possibly insignificantly) in your solution.


as for your question of figuring out w/v, percent is based on 100. so, 1% w/v = 1 gm/100 ml. you can figure it from there.


What if i just used Citric acid alone (concentrated citric acid) to adjust the pH of my LB broth ?    


then you won't have as much control of the concentration of the buffering compound.