Acetate and 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 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 PO4 + 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 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

pH (ml) (ml) 3.6 46.3 3.7 3.8 44.0 6.0 4.0 41.0 9.0 4.2 36.8 13.2 4.4 30.5 19.5 4.6 25.5 24.5 4.8 20.0 30.0 5.0 14.8 35.2 5.2 10.5 39.5 5.4 8.8 41.2

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 (b) 5 M K Acetate, pH 4.8 408 g Na acetate 3H2O in 800 ml of H2O 29.5 ml glacial acetic acid Add H2O to 1 liter KOH pellets to 4.8 Adjust pH to 5.2 with 3 M acetic acid H2O to 100 ml