Preparation of Buffer Solutions

Preparation of Buffer Solutions - RAJARSHI PATEL

Standard
Buffer Solutions

Standard
Buffer Solutions are solutions of standard pH. They are used for reference
purposes in pH measurements and for carrying out many pharmacopoeial
tests which require adjustments to or maintenance of a specified pH. They may
be prepared by the methods described below. The preparation of special buffer
solutions is described in the sections in which their use is specified as in
the microbiological assay of antibiotics or in the individual
monographs where the use of such solutions is indicated.

The
reagents required for the preparation of standard buffer solutions are
described in Appendix 4.2. All the crystalline reagents except boric acid
should be dried at 110° to 120° for 1 hour before use. Carbon dioxide-free
water should be used for preparing buffer solutions and wherever water is
mentioned for preparation of such solutions the use of carbon dioxide-free
water is implied. The prepared solutions should be stored in chemically
resistant, glass-stoppered bottles of alkakli-free glass and used within 3 months of preparation.
Any solution which has become cloudy or shows any other evidence of
deterioration should be discarded.

Standard
buffer solutions for various ranges of pH values 1.2 to 10.0 may be prepared by
appropriate combinations of 0.2 M hydrochloric acid or 0.2 M sodium
hydroxide and of solutions described below, used in the proportions shown
in the accompanying tables. The standard pH values given in the tables and
elsewhere in the Appendix are considered to be reproducible within ±
0.02
Unit at 25°.

1.
Boric Acid and Potassium Chloride, 0.2 M: Dissolve 12.366 g of boric
acid and 14.911 g of potassium chloride in

water
and
dilute with water to 1000 ml.

2.
Disodium Hydrogen Phosphate, 0.2 M: Dissolve 71.630
g of disodium hydrogen phosphate in water
and dilute with water to 1000 ml.

3.
Hydrochloric Acid, 0.2 M: Hydrochloric acid diluted with water to
contain 7.292 g of HCl in 1000 ml.

4.
Potassium Chloride, 0.2 M: Dissolve 14.911 g of potassium chloride in water and
dilute with water to 1000 ml.

5.
Potassium Dihydrogen Phosphate, 0.2 M: Dissolve 27.218
g of potassium dihydrogen phosphate in water
and dilute with water to 1000 ml.

6.
Potassium Hydrogen Phthalate, 0.2 M: Dissolve 40.846 g of potassium
hydrogen phthalate in water and dilute with water to 1000 ml.

7.
Sodium Hydroxide, 0.2 M: Dissolve sodium hydroxide in water to produce
a 40 to 60 per cent w/v solution and allow to stand. Taking precautions to
avoid absorption of carbon dioxide, siphon off the clear supernatant liquid and
dilute with carbon dioxide-free water, a suitable volume of the liquid
to contain 8.0 g of NaOH in 1000 ml.

NOTE - 0.2 M Sodium hydroxide must
not be used later than one month after preparation.

 

Composition
of Standard Buffer Solutions

Hydrochloric
Acid Buffer: Place 50.0 mI of the 0.2Mpotassium
chloride in a 200 ml volumetric flask, add the specified volume of 0.2
M hydrochloric acid (see Table I) and then add water to volume.

Acid
Phthalate Buffer: Place 50.0 ml of 0.2 M potassium hydrogen phthalate in
a 200 ml volumetric flask, add the specified volume of 0.2 M
hydrochloric acid (see Table 2) and then add water to volume.

Neutralised Phthalate
Buffer; Phthalate Buffer: Place 50.0 ml of 0.2 M potassium hydrogen phthalate in
a 200 ml volumetric flask, add the specified volume of 0.2 M sodium hydroxide
(see Table 3) and then add water to volume.

Phosphate
Buffer:
Place 50.0 ml of 0.2 M potassium dihydrogen
phosphate in a 200 ml volumetric flask, add the specified volume of 0.2
M sodium hydroxide (see Table 4) and then add water to
volume.

Alkaline Borate Buffer: Place 50.0 ml
of 0.2 M boric acid and potassium chloride in a 200 ml volumetric
flask, add the specified volume of 0.2 M sodium hydroxide (see
Table 5) and then add water to volume.

Other
Buffer solutions

Acetate
Buffer pH 2.8: Dissolve 4 g of anhydrous sodium acetate in about
840 ml of water, add sufficient glacial acetic acid to adjust the
pH to 2.8 (about 155 ml) and dilute with water to 1000 ml.

Acetate
Buffer pH 3.4: Mix 50 ml of 0.1 M sodium acetate with 950 ml of 0.1
M acetic acid.

Acetate
Buffer pH 3.5: Dissolve 25 g of ammonium acetate in 25 ml of water
and add 38 ml of 7 M hydrochloric acid. Adjust the pH to 3.5 with
either 2 M hydrochloric acid or 6 M ammonia and dilute with water
to 100 ml.

Acetate
Buffer pH 3.7: Dissolve 10 g of anhydrous sodium acetate in 300 ml
of water, adjust the pH to 3.7 with glacial acetic acid and
dilute with water to 1000 ml. Before use adjust to pH 3.7, if
necessary, with glacial acetic acid or anhydrous sodium acetate, as
required.

Acetate
Buffer pH 4.0: Place 2.86 ml of glacial acetic acid and 1.0 ml of a
50 per cent w/v solution of sodium hydroxide in a 1000 ml volumetric
flask, add water to volume and mix. Adjust the pH, if necessary.

Acetate
Buffer pH 4.4: Dissolve 136 g of sodium acetate and 77 g of ammonium
acetate in water and dilute with water to 1000 ml. Add 250 ml
of glacial acetic acid and mix.

Acetate
Buffer pH 4.6: Dissolve 5.4 g of sodium acetate in 50 ml of water,
add 2.4 ml of glacial acetic acid and dilute with water to
100 ml. Adjust the pH, if necessary.

Acetate
Buffer pH 4.7: Dissolve 8.4 g of sodium acetate and 3.35 ml of glacial
acetic acid in sufficient water to produce 1000 ml. Adjust the pH,
if necessary.

Acetate
Buffer pH 5.0: Dissolve 13.6 g of sodium acetate and6 ml of glacial
acetic acid in sufficient water to produce 1000 ml. Adjust the pH,
if necessary.

Acetate
Buffer pH 5.5: Dissolve 272 g of sodium acetate in 500 ml of water
by heating to 35°, cool and add slowly 50 ml of glacial acetic acid and
sufficient water to produce 1000 ml. Adjust the pH, if necessary.

Acetate
Buffer pH 6.0: Dissol ve
100 g of ammonium acetate in 300 ml of water, add 4.1 ml of glacial
acetic acid, adjust the pH, if necessary, using 10M ammonia or
5 M acetic acid and dilute with water to 500 ml.

Acetate
Buffer Solution: Dissolve 14 g of potassium acetate and 20.5 ml of glacial
acetic acid in sufficient water to produce 1000 ml.

Acetate-Edetate Buffer pH 5.5: Dissolve 250 g of ammonium
acetate and 15 g of disodium edetate in 400 ml of water and add 125 ml
of glacial acetic acid in sufficient water to produce 1000ml.

Acetic
Acid-Ammonium Acetate Buffer: Dissolve 77.1 g of ammonium acetate
in water, add 57 ml of glacial acetic acid and dilute with water
to 1000 ml.

Acetic
Ammonia Buffer pH 3.7, Ethanolic: To 15 ml of 5
M acetic acid add 60 ml of ethanol (95 per cent) and 24 ml of
water. Adjust the pH to 3.7 with 10 M ammonia and dilute with water
to 100 ml.

Acetone
Solution, Buffered: Dissolve 8. 15 g of sodium acetate and 42 g of sodium
chloride in water, add 68 ml of 0.1 M hydrochloric acid and
150 ml of acetone and dilute with water to 500 ml.

Albumin
Phosphate Buffer pH 7.2; Phosphate-albumin

Buffered
Saline pH 7.2: Dissolve 10.75 g of disodium
hydrogen phosphate, 7.6 g of sodium chloride and 10 g of bovine
albumin in sufficient water to produce 1000 ml. Before use
adjust to pH 7.2 with 2 M sodium hydroxide or a 10 per cent w/v
solution of phosphoric acid as required.

Ammonia-Ammonium
Chloride Buffer: Dissolve 67.5 g of ammonium chloride in about 200 ml
of water, add 570 ml of strong ammonia solution and dilute with water
to 1000 ml.

Ammonia
Buffer pH 9.5: Dissolve 33.5 g of ammonium chloride in ISO ml of water,
and 42 ml of 10M ammonia and dilute with water to 250 ml.
Store in polyethylene containers.

Ammonia
Buffer pH 10.0: Dissolve 5.4 g of ammonium chloride in 20 ml of water,
add 35 ml of 10 M ammonia and dilute with water to 100 ml.

Ammonia
Buffer pH 10.9: Dissolve 67.5 g of ammonium chloride in sufficient 10
M ammonia to produce 1000 ml.

Barbitone Buffer pH 7.4: Mix 50 ml of
solution containing 1.944 per cent w/v of sodium acetate and 2.946 per
cent w/v of barbitone sodium with 50.5
ml of 0.1 M hydrochloric acid, add 20 ml of an 8.5 per cent w/v solution
of sodium chloride and dilute with water to 250 ml.

Barbitone Buffer pH 8.6,
Mixed;
Barbitone Buffer pH 8.6: Dissolve 1.38
g of barbitone, 8.76 g of barbitone sodium and 0.38 g of calcium
lactate in sufficient water to produce 1000 ml.

Boric
Buffer pH 9.0; Borate Buffer pH 9.0: Dissolve 6.20 g of boric acid in
500 ml of water, adjustto pH 9.0 with] M
sodium hydroxide (about 41.5 ml) and dilute with water to
1000 ml.

Buffer
Solution pH 2.5: To 25.0 ml of 0.2 M potassium hydrogen phthalate add
37.0 ml of 0.1 M hydrochloric acid and dilute with sufficient water to
produce 100.0 ml.

Buffer
(HEPES) solution pH 7.5: Dissolve 2.38 g of 2[4-( hydroxyethyl)piperazin-1-yljethanesulphonic
acid in about 90 ml of water. Adjust the pH to 7.5 with sodium
hydroxide solution. Dilute to 100 ml with water.

Carbonate
Buffer pH 9.7: Dissolve 8.4 g of sodium bicarbonate and 10.6 g of sodium
carbonate in sufficient water to produce 500 ml.

Chloride
Buffer pH 2.0: Dissolve 6.57 g of potassium chloride in water, add
119.0 ml of 0.1 M hydrochloric acid and dilute with water to
1000 ml.

Citrate
Buffer:
Dissolve 0.5 g of citric acid monohydrate and 0.4 g of dibasic sodium
phosphate in sufficient water to produce 1000 ml.

Citro-phosphate
Buffer pH 5.0: Mix 48.5 ml of 0.1 M citric acid with sufficient 0.2
M disodium hydrogen phosphate to produce
100 ml.

Citro-phosphate
Buffer pH 6.0: Mix 36.8 ml of a 2.1 per cent w/v solution of citric
acid with 63.2 ml of a 7.15 per cent w/v solution of disodium
hydrogen phosphate.

Citro-phosphate
Buffer pH 7.0: Mix 17.6 ml of a 2.1 percent w/v solution of citric acid
with 82.4 ml of a 7.15 per cent w/v solution of disodium
hydrogen phosphate.

Citro-phosphate
Buffer pH 7.2: Mix 13.0 ml of a 2.1 percent w/v solution of citric acid
with 87.0 ml of a 7.15 per cent w/v solution of disodium
hydrogen phosphate.

Citro-phosphate
Buffer pH 7.6: Dissolve 1.33 g of citric acid and 67.1 g of disodium hydrogen phosphate in sufficient water
to produce 1000 ml.

Cupric
Sulphate Solution pH 2.0, Buffered: Mix 5.3 ml of 0.2
M hydrochloric acid and 25 ml of 0.2 M potassium chloride, add
4 ml of a 0.393 per cent w/v solution of cupric sulphate
and dilute to 100 ml of water.

Cupric
Sulphate Solution pH 4.0, Buffered: Dissolve 0.25 g
cupric sulphate and 4.5 g of ammonium
acetate in sufficient water to produce 100 ml.

Cupric
Sulphate Solution pH 5.2, Buffered: Dissolve 1.522
g of anhydrous disodium hydrogen phosphate in
sufficient water to produce 53.6 ml and add a 2.1 per cent solution of citric
acid until the pH of the solution is between 5.15 and 5.25 (about 46 ml).
Mix 98.5 ml of the resulting solution with 1.5 ml of a 0.393 per cent solution
of cupric sulphate.

Diethanolamine Buffer pH 10.0: Dissolve 96.4 g
of diethanolamine in sufficient water
to produce 400 ml. Add 0.5 ml of an 18.6 per cent w/v solution of magnesium
chloride, adjust the pH to 10.0 with 1 M hydrochloric acid and
dilute with water to 500 ml.

Glycine Buffer pH 11.3: Mix a solution
containing 0.75 per cent w/v of glycine and
0.58 per cent w/v of sodium chloride with an equal volume of 0.1 M
sodium hydroxide. Adjust the pH if necessary.

Glycine Buffer Solution: Mix 42 g of sodium
bicarbonate and 50 g of potassium bicarbonate with 180 ml of water
and add a solution containing 37.5 g of glycine
and IS ml of strong ammonia in 180 ml of water. Dilute with water
to 500 ml and stir until solution is complete.

Imidazole Buffer pH 6.5: Dissolve 6.81 g
of imidazole and 1.23 g of magnesium
sulphate in 752 ml of 0.] M hydrochloric acid,
adjust the pH if necessary and dilute with water to produce 1000 ml.

Imidazole Buffer pH 7.4: Dissolve 3.40 g
of imidazole and 5.84 g of sodium
chloride in water, and 18.6 ml of] M hydrochloric acid and
dilute with water to produce 1000 ml.

Palladium
Chloride Solution, Buffered: To 0.5 g of palladium chloride add 5 ml of hydrochloric
acid and warm on a waterbath. Add 200 ml
of hot water in small portions with continued heating until
solution is complete. Cool and dilute with sufficient water to
produce 250.0 ml. To 50.0 ml of the resulting solution add 10.0 ml of] M
sodium acetate, 9.6 ml of] M hydrochloric acid and sufficient water
to produce 100.0 ml.

Phosphate-albumin
buffered saline pH 7.2: Dissolve 10.75 g of disodium
hydrogen phosphate, 7.6 g of sodium chloride and 10 g of bovine
albumin in water and dilute to 1000.0 ml with the same solvent.
Immediately before use adjust the pH using dilute sodium hydrogen solution or
dilute phosphoric acid.

Phosphate
Buffer pH 2.0: Dissolve 0.136 g of potassium dihydrogen
phosphate in 800 ml of water, adjust the pH to 2.0 with hydrochloric
acid and add sufficient water to produce l000ml.

Phosphate
Buffer pH 2.5:
Dissolve 100 g of potassium dihydrogen phosphate in
800 ml of water, adjust the pH to 2.5 with hydrochloric acid and
add sufficient water to produce l000ml.

Phosphate
Buffer pH 3.0: Dissolve 1.36 g of potassium dihydrogen
orthophosphate and 2 ml of triethylamine
in 800 ml of water, adjust the pH to 3.0 with orthophosphoric acid and add sufficient water
to produce 1000 ml.

Phosphate
Buffer pH 3.6: Dissolve 0.900 g of anhydrous disodium
hydrogen phosphate and 1.298 g of citric acid monohydrate in
sufficient water to produce 1000 ml.

Phosphate
Buffer pH 4.0, Mixed: Dissolve 5.04 g disodium
hydrogen phosphate and 3.01 g of potassium dihydrogen
phosphate in sufficient water to produce 1000 ml. Adjust the pH
with glacial acetic acid.

Phosphate
Buffer pH 4.9: Dissolve 40 g of sodium dihydrogen
phosphate and 1.2 g of sodium hydroxide in sufficient water to
produce 100 ml. If necessary, adjust the pH with 1 M sulphuric
acid or 1 M sodium hydroxide as required.

Phosphate
Buffer pH 5.0: Dissolve 6.8 g of potassium dihydrogen
phosphate in 1000 ml of water and adjust the pH to 5.0 with
]0 M potassium hydroxide.

Phosphate
Buffer pH 5.5, Mixed:

SOLUTION
I -
Dissolve
13.61 g of potassium dihydrogen phosphate in
sufficient water to produce 1000 ml.

SOLUTION
II -
Dissolve
35.81 g of disodium hydrogen phosphate in
sufficient water to produce 1000 ml.

Mix
96.4 ml of solution I with 3.6 ml of solution II.

Phosphate
Buffer pH 6.5: Dissolve 60.5 g of disodium

hydrogen
phosphate and
46 g of potassium dihydrogen phosphate in water,
add 100 ml of 0.02 M disodium edentate and
20 mg of mercuric chloride and dilute with water to produce I000
ml.

Phosphate
Buffer pH 6.8, Mixed: Dissolve 28.?0 g of disodium
hydrogen phosphate and 11.45 g of potassium dihydrogen
phosphate in sufficient water to produce 1000 ml.

Phosphate
Buffer pH 6.8, 0.2 M Mixed: Dissolve 13.872 g of potassium dihydrogen
phosphate and 35.084 g of disodium hydrogen
phosphate in sufficient water to produce 1000 ml. Store in a cold
place.

Phosphate
Buffer pH 7.0, Mixed: Dissolve 0.50 g of anhydrous disodium
hydrogen phosphate 0.301 g of potassium

dihydrogen phosphate in sufficient water
to produce

Phosphate
Buffer pH 7.0 with Azide, Mixed: To 1000 ml of a
solution containing 1.8 per cent w/v of disodium
hydrogen phosphate and 2.3 per cent w/v of sodium chloride, add
sufficient of a solution containing 0.78 per cent w/v of sodium dihydrogen phosphate and 2.3 per cent w/v of sodium
chloride (about 280 ml) to produce a pH of 7.0. Dissolve sufficient
sodium azide in the resulting solution to give a
0.02 per cent w/v solution.

Phosphate
Buffer pH 7.0, 0.067 M Mixed: Dissolve 3.532 g of potassium dihydrogen phosphate and 14.542 g of disodium hydrogen phosphate in sufficient water
to produce 1000 ml.

Phosphate
Buffer pH 7.0, 0.1 M Mixed; Phosphate Buffer pH 7.0, 0.1 M: Dissolve 1.361
g of potassium dihydrogen orthophosphate
in sufficient water to produce 100 ml and adjust the pH using 3.5
per cent w/v solution of disodium hydrogen
orthophosphate.

Phosphate
Buffer pH 7.5: Dissolve 6.8 g of potassium dihydrogen
orthophosphate and 1.56 g of sodium hydroxide in 900 ml of water adjust
the pH 7.5 with sodium hydroxide solution and dilute with water to
produce 1000 ml.

Phosphate
Buffer pH 7.5, 0.2 M: Dissolve 27.2 g of potassium dihydrogen
phosphate with 930 ml of water adjust the pH 7.5 with 0.3 per
cent w/v solution of potassium hydroxide and add sufficient water
to produce 1000 ml.

Phosphate
Buffer pH 7.5, 0.33 M Mixed:

SOLUTION
I -
Dissolve
119.3 I g of disodium hydrogen phosphate in
sufficient water to produce 1000 ml.

SOLUTION
II -
Dissolve
45.36 g of potassium dihydrogen phosphate in
sufficient water to produce 1000 ml.

Mix
85 ml of solution I and 15 ml of solution II and adjust the pH if necessary.

Phosphate
Buffer pH 8.0, 0.02 M: Mix 50 ml of 0.2 M potassium dihydrogen phosphate with 46.8 ml of 0.2 M sodium
hydroxide
and
add sufficient water to produce 500 ml.

Phosphate
Buffer, 0.025 M Standard: Dissolve 3.40 g of potassium dihydrogen
phosphate and 3.55 g of anhydrous disodium
hydrogen phosphate, both previously dried at 1100 to 1300 for 2 hours, in
sufficient water to produce 1000 ml.

Phosphate
Buffer, 0.05 M: Dissolve 6.8 g of potassium dihydrogen
orthophosphate in sufficient water to produce l000ml.

Saline,
Phosphate-buffered: Dissolve 2.5 g of sodium dihydrogen
phosphate, 2.523 g of disodium hydrogen
phosphate and 8.2 g of sodium chloride in sufficient water to
produce 1000 ml.

Saline
pH 6.4, Phosphate-buffered: Dissolve 1.79g of disodium
hydrogen phosphate, 1.36 g of potassium dihydrogen
phosphate and 7.02 g of sodium chloride in sufficient water to
produce 1000 ml.

Saline
pH 7.4, Phosphate-buffered: Dissolve 2.38 g of disodium
hydrogen phosphate, 0.19 g of potassium dihydrogen
phosphate and 8.0 g of sodium chloride in sufficient water to
produce 1000 ml. Adjust the pH, if necessary.

Tris-Acetate Buffer
pH 8.5:
Dissolve 0.294 g of calcium chloride and 12.11 g of tris(hydroxymethyl)aminomethane in
water. Adjust the pH with 5 M acetic acid and dilute to 1000.0
ml with water.

Tris-Chloride Buffer
pH 7.4:
Dissolve 7.27 g of tris(hydroxymethyl)methylamine
and 5.27 g of sodium chloride adjust the pH, if necessary and
dilute with water to produce 1000 ml.

Tris(hydroxymethyl)aminomethane Buffer
pH 7.4:
Dissolve 30.3 g of tris(hydroxymethyl)aminimethane in approximately 200 ml of water. Add
183 ml of I M hydrochloric acid. Dilute to 500.0 ml with water.

NOTE - The pH is 7.
7-7.8 at
room temperature and 7.4 at 37°. This solution is stable
for several months at 4°.

Tris(hydroxymethyl)aminomethane Buffer
pH 8.1:
Dissolve 2.9 g of calcium chloride with 400 ml of tris(
hydroxymethyl)aminomethane
solution adjust the pH with] M hydrochloric acid and dilute with water
to produce l000 ml.