Three component buffer - (Jul/30/2015 )
I need to prepare such a buffer buffer. Its composition is:
20 mM citrate/ 20 mM phosphate/20 mM Tris pH 3.5 and the same with pH 7.
Should I prepare this my doing the following steps:
1. Add 20 mmol/L of Sodium citrate dihydrate
2. Add 20 mmol/L of sodium phosphate dibasic (heptahydrate)
3. Add 20mmol/L of Trizma base
4. And then adjust pH to 3.5 and pH 7 for the second buffer with HCL and NaOH respectively and add water to 1L?
Or maybe I oversimplified and to make 20mM phosphate I have to mix sodium phosphate monobasic and dibasic in the correct ratio the same way as when making phosphate buffer, then prepare citrate buffer by mixing sodium citrate dihydrate with citric acid and then prepare Tris, and mix them but then which base or acid to use to adjust pH?
Any ideas will be appreaciated.
The presence of HPO4-2 and H2PO4- in the solution depends on the pH, the same with other protonated and unprotonated species. You could even use phosphoric acid and neutralise it with NaOH. It will be the same.
The use of mono- and dibasic salts is to make things easier and safer, given that we know that the mixing of equimolar concentrations in known ratios produces solutions with a fixed pH, or at least very close. Also, manipulating concentrated acids is a pain respect to the safety if you compare to the granulated salts.
how you prepare the mixture depends on what you can have present in the final mixture (ie salts).
you can prepare the citrate to pH 3.5 with acid/base components of citrate but not tris or phosphate (unless, maybe, you use equimolar phosphoric acid and monobasic but will be outside the buffering range of phosphate's pK1).
you can prepare phosphate to pH 7 with mono and dibasic components and, maybe, tris with tris base and tris-hcl (but this is below the buffering range of tris) but not with citrate.
if salt is of no concern the you should prepare the buffer as you first propose (i, however, prefer preparing a stock at a higher concentration so that other components may be added).