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Acetate buffer solutions pH 3 - 6 ³

Make up the following solutions

(1) 0.1M acetic acid

(2) 0.1M sodium acetate (tri-hydrate) (13.6g / l)

Mix in the following proportions to get the required pH

pH Vol. of 0.1M acetic acid Vol. of 0.1M sodium acetate

3 982.3 mls 17.7 mls

4 847.0 mls 153.0 mls

5 357.0 mls 643.0 mls

6 52.2 mls 947.8 mls

Acetate buffer solutions pH 3.6 - 5.6

Make up the following solutions

(1) 0.1M acetic acid

(2) 0.1M sodium acetate (tri-hydrate) (13.6g / l)

Mix in the following proportions to get the required pH [Pearse 1980]

pH Vol. of 0.1M acetic acid Vol. of 0.1M sodium acetate

3.6 185 mls 15 mls

3.8 176 mls 24 mls

4.0 164 mls 36 mls

4.2 147 mls 53 mls

4.4 126 mls 74 mls

4.6 102 mls 98 mls

4.8 80 mls 120 mls

5.0 59 mls 141 mls

5.2 42 mls 158 mls

5.4 29 mls 171 mls

5.6 19 mls 181 mls

Common Buffers

I. Phosphate Buffer (Sorenson's buffer) pH 5.8-8

Advantages:

1. Most physiological of common buffers. Mimics certain components of extracellular fluids.

2. Non-toxic to cells.

3. pH changes little with temperature.

4. Stable for several weeks at 4 C.

Disadvantages:

1. Precipitates more likely to occur during fixation. Tends to form precipitates in presence of
calcium ions. Precipitates uranyl acetate and tends to react with lead salts.

2. Becomes slowly contaminated with micro-organisms

Preparation of Buffer

Stock solutions:

0.2M dibasic sodium phosphate 1 liter

Na2HPO4*2H20 (MW = 178.05) 35.61 gm

or

Na2HPO4*7H20 (MW = 268.07) 53.65 gm

or

Na2HPO4*12H20 (MW = 358.14) 71.64 gm

+ ddH20 to make 1 liter

0.2M monobasic sodium phosphate 1 litter

NaH2PO4*H20 (MW = 138.01) 27.6 gm

or

NaH2PO4*2H20 (MW = 156.03) 31.21 gm

+ ddH20 to make 1 liter

Working buffer: 0.1M 100 ml

Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100
ml with ddH20 or dilute 1:1 with fixative.
pH (25 C) X ml Y ml

5.8 4.0 46.0

6.0 6.15 43.75

6.2 9.25 40.75

6.4 13.25 36.75

6.6 18.75 31.25

6.8 24.5 25.5

7.0 30.5 19.5

7.2 36.0 14.0

7.4 40.5 9.5

7.6 43.5 6.5

7.8 45.75 4.25

8.0 47.35 2.65

Osmolarity is adjusted by varying the molarity of phosphates or by the addition of sucrose, glucose
or sodium chloride.

At pH 7.2:

0.10M = 226 mOs (milliosmoles)

0.05M = 118 mOs

0.075 = 180 mOs

0.15M = 350 mOs

https://www.shimadzu.com/an/hplc/support/lib/lctalk/38/38lab.html

100 mM phosphoric acid (sodium) buffer solution (pH=6.8)

Sodium dihydrogen phosphate dihydrate (M.W.=156.01)..50 mmol (7.8 g)

Sodium dihydrogen phosphate 12-hydrate (M.W.=358.14)..50 mmol (17.9 g)

Add water to make up to 1 L.

10 mM phosphoric acid (sodium) buffer solution (pH=6.9)

Sodium dihydrogen phosphate dihydrate (M.W.=156.01)..5 mmol (0.78 g)

Sodium dihydrogen phosphate 12-hydrate (M.W.=358.14)..5 mmol (1.79 g)

Add water to make up to 1 L.

(Alternatively, dilute 100 mM phosphoric acid (sodium) buffer solution (pH=6.8) ten times.)

100 mM acetic acid (sodium) buffer solution (pH=4.7)

Acetic acid (glacial acetic acid) (99.5 %, 17.4 mol/L)..................50 mmol (2.87 mL)

Sodium acetate trihydrate (M.W.=136.08)........50 mmol (6.80 g)

Add water to make up to