PHOSPHATE BUFFERS Phosphate buffer (sodium salt) is typically prepared by mixing solutions of sodium phosphate dibasic (Na2HPO4) and

and sodium phosphate monobasic (NaH2PO4). The phosphate group is a co alent unit (i.e.! the Ps and Os are co alently 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 #ill be slightly acidic (a $% solution has a pH of about 4.2) compared to the dibasic (#ith only one H" per PO4! a $% solution has a pH of about &.'). The monobasic! more acidic Solution A is mixed #ith the dibasic! more basic Solution B to get the desired pH. (f the concentration of PO4 is the same in both solutions! then the resulting solution #ill ha e the same concentration (of PO4) regardless of the proportions of each #hich are added! because) $ ml of *.2 m PO 4 " &$ ml of *.2 + PO4 , '** ml of *.2 + PO4 -

Sodium phosphate buffer, 0.1 M Solution A: 2../ g NaH2PO4 H2O per liter (*.2 +) Solution B: $0./$ g Na2HPO4 .H2O per liter (*.2 +) 1eferring to Table 2.2.0 for desired pH! mix the indicated olumes of 3olutions 2 and 4! then dilute #ith H2O to 2** ml Potassium phosphate buffer, 0.1 M Solution A: 2..2 g 5H2PO4 per liter (*.2 +) Solution B: 04.6 g 52HPO4 per liter (*.2 +) 1eferring to Table 2.2.0 for desired pH! mix the indicated olumes of 3olutions 2 and 4! then dilute #ith H2O to 2** 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. hosphate buffers sho! concentration-dependent p" changes# so chec$ concentrate p" by diluting an ali%uot to the final concentration .

Table A.2.3 7esired pH $.. $.6 $.& /.* /.' /.2 /.0 /.4 /.$ /./ /.. /.6

Preparation of *.' + 3odium and Potassium Phosphate 4uffers 3olution 2 (ml) &0.$ &2.* &*.* 6... 6$.* 6'.$ ...$ .0.$ /6.$ /2.$ $/.$ $'.* 3olution 4 (ml) /.$ 6.* '*.* '2.0 '$.* '6.$ 22.$ 2/.$ 0'.$ 0..$ 40.$ 4&.* 7esired pH /.& ..* ..' ..2 ..0 ..4 ..$ ../ ... ..6 ..& 6.* 3olution 2 (ml) 4$.* 0&.* 00.* 26.* 20.* '&.* '/.* '0.* '*.$ 6.$ ..* $.0 3olution 4 (ml) $$.* /'.* /..* .2.* ...* 6'.* 64.* 6..* &*.$ &'.$ &0.* &4..

ACETATE BUFFERS

2cetate buffers are prepared by mixing e8uimolar solutions of acidic and basic forms of acetate in order to achie e the desired pH. The acidic form is acetic acid (HO2c! or H2c)! and the basic form is the sodium or potassium salt (really the unprotonated O2c9). 2 :*.2 + acetate; buffer contains a total of *.2 mole of :acetate; per liter! #hether it is protonated or not. Potassium acetate buffer, 0.1 M Solution A: ''.$$ ml glacial acetic acid<liter (*.2 +) Solution B: '&./ g potassium acetate (5= 2H0O2<liter (*.2 +) 1eferring to Table 2.2.2 for desired pH! mix the indicated olumes of solutions 2 and 4! and then dilute #ith H2O to '** ml Sodium acetate buffer, 0.1 M Solution A: ''.$$ ml glacial acetic acid<liter (*.2 +) Solution B: 2..2 g sodium acetate (Na=2H0O2 0H2O)<liter (*.2 +) 1eferring to table 2.2.2 for desired pH! mix the indicated olumes of solutions 2 and 4! the n dilute #ith H2O to '** 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 sho! concentration-dependent p" changes# so chec$ concentrate p" by diluting an ali%uot to the final concentration To prepare buffers !ith p" intermediate bet!een the points listed in Table A.&.&# prepare closest higher p"# then titrate !ith solution A. Table A.2.2 Preparation of *.' + 3odium and Potassium 2cetate 4uffers 7esired pH 0./ 0.6 4.* 4.2 4.4 4./ 4.6 $.* $.2 $.4 $./ 3olution 2 (ml) 4/.0 44.* 4'.* 0/.6 0*.$ 2$.$ 2*.* '4.6 '*.$ 6.6 4.6 3olution 4 (ml) 0.. /.* &.* '0.2 '&.$ 24.$ 0*.* 0$.2 0&.$ 4'.2 4$.2

>ou #ill see protocols for solutions of high concentration #hich seem li?e they are prepared differently 99 but if you loo? closely you #ill see that the same principles apply) the concentration of the buffering ion (here! acetate) is #hat gi es the solution its name! and as long as the pH is ad@usted #ith either the same concentration of that buffering ion (a)! or the appropriate acid or base lac?ing that ion altogether (b)! the correct concentration #ill be maintained. (a) 3M Na Acetate, pH 5. 4*6 g Na acetate 0H2O in 6** ml of H2O 2dd H2O to ' liter 2d@ust pH to $.2 #ith 0 + acetic acid (b) 5 M ! Acetate, pH ".# 2&.$ ml glacial acetic acid 5OH pellets to 4.6 H2O to '** ml