PHOSPHATE BUFFERS

Phosphate buffer (sodium salt) is typically prepared by mixing solutions of sodium phosphate dibasic
(Na2HPO4) and sodium phosphate monobasic (NaH2PO4).

The phosphate group is a covalent unit (i.e., the Ps and Os are covalently bound to each other). The Na and H
atoms dissociate, and the H contributes the pH of the solution.

The monobasic form has more H+ to contribute (per PO4 unit) than the dibasic, and a solution of monobasic
will be slightly acidic (a 5% solution has a pH of about 4.2) compared to the dibasic (with only one H+ per
PO4, a 5% solution has a pH of about 9.1).

The monobasic, more acidic Solution A is mixed with the dibasic, more basic Solution B to get the desired
pH.

If the concentration of PO4 is the same in both solutions, then the resulting solution will have the same
concentration (of PO4) regardless of the proportions of each which are added, because: 5 ml of 0.2 m PO 4 + 95
ml of 0.2 M PO4 = 100 ml of 0.2 M PO4 !
Sodium phosphate buffer, 0.1 M
Solution A: 27.6 g NaH2PO4 H2O per liter (0.2 M)
Solution B: 53.65 g Na2HPO4 7H2O per liter (0.2 M)
Referring to Table A.2.3 for desired pH, mix the indicated volumes of Solutions A and B, then
dilute with H2O to 200 ml
Potassium phosphate buffer, 0.1 M
Solution A: 27.2 g KH2PO4 per liter (0.2 M)
Solution B: 34.8 g K2HPO4 per liter (0.2 M)
Referring to Table A.2.3 for desired pH, mix the indicated volumes of Solutions A and B, then
dilute with H2O to 200 ml
This may be made as a 5 or 10-fold concentrate by scaling up the amount of potassium or sodium
phosphate in the same volume. Phosphate buffers show concentration-dependent pH changes, so check
concentrate pH by diluting an aliquot to the final concentration.
Table A.2.3
Desired
pH
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
Preparation of 0.1 M Sodium and Potassium Phosphate Buffers
Solution A
(ml)
93.5
92.0
90.0
87.7
85.0
81.5
77.5
73.5
68.5
62.5
56.5
51.0
Solution B
(ml)
6.5
8.0
10.0
12.3
15.0
18.5
22.5
26.5
31.5
37.5
43.5
49.0
Desired
pH
6.9
7.0
7.1
7.2
7.3
7.4
7.5
7.6
7.7
7.8
7.9
8.0
Solution A
(ml)
45.0
39.0
33.0
28.0
23.0
19.0
16.0
13.0
10.5
8.5
7.0
5.3
Solution B
(ml)
55.0
61.0
67.0
72.0
77.0
81.0
84.0
87.0
90.5
91.5
93.0
94.7
ACETATE BUFFERS
Acetate buffers are prepared by mixing equimolar solutions of acidic and basic forms of acetate in order to achieve
the desired pH. The acidic form is acetic acid (HOAc, or HAc), and the basic form is the sodium or potassium salt
(really the unprotonated OAc-). A “0.2 M acetate” buffer contains a total of 0.2 mole of “acetate” per liter, whether
it is protonated or not.
Potassium acetate buffer, 0.1 M
Solution A: 11.55 ml glacial acetic acid/liter (0.2 M)
Solution B: 19.6 g potassium acetate (KC2H3O2/liter (0.2 M)
Referring to Table A.2.2 for desired pH, mix the indicated volumes of solutions A and B, and then
dilute with H2O to 100 ml
Sodium acetate buffer, 0.1 M
Solution A: 11.55 ml glacial acetic acid/liter (0.2 M)
Solution B: 27.2 g sodium acetate (NaC2H3O2 3H2O)/liter (0.2 M)
Referring to table A.2.2 for desired pH, mix the indicated volumes of solutions A and B, the n
dilute with H2O to 100 ml
This may be made as a 5-or 10-fold concentrate by scaling up the amount of potassium or sodium acetate in the
same volume. Acetate buffers show concentration-dependent pH changes, so check concentrate pH by diluting an
aliquot to the final concentration
To prepare buffers with pH intermediate between the points listed in Table A.2.2, prepare closest higher pH, then
titrate with solution A.
Table A.2.2 Preparation of 0.1 M Sodium and
Potassium Acetate Buffers
Desired
pH
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
Solution A
(ml)
46.3
44.0
41.0
36.8
30.5
25.5
20.0
14.8
10.5
8.8
4.8
Solution B
(ml)
3.7
6.0
9.0
13.2
19.5
24.5
30.0
35.2
39.5
41.2
45.2
You will see protocols for solutions of high concentration which seem like they are prepared differently -- but if you
look closely you will see that the same principles apply: the concentration of the buffering ion (here, acetate) is what
gives the solution its name, and as long as the pH is adjusted with either the same concentration of that buffering ion
(a), or the appropriate acid or base lacking that ion altogether (b), the correct concentration will be maintained.
(a) 3M Na Acetate, pH 5.2
408 g Na acetate 3H2O in 800 ml of H2O
Add H2O to 1 liter
Adjust pH to 5.2 with 3 M acetic acid
(b) 5 M K Acetate, pH 4.8
29.5 ml glacial acetic acid
KOH pellets to 4.8
H2O to 100 ml