Apr 29, 2020
Buffers for Use in Biological Systems
- 1University of Manitoba
Protocol Citation: Neilier Junior 2020. Buffers for Use in Biological Systems. protocols.io https://dx.doi.org/10.17504/protocols.io.bfsdjna6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2020
Last Modified: April 29, 2020
Protocol Integer ID: 36389
Keywords: pH, Henderson-Hasselback, Biochemistry, Molecular Biology,
Abstract
A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.
Safety warnings
Wear personal protective equipment: gloves, lab coat and mask.
Before start
Organize your workspace.
Make sure all solutions and equipment are available. Plan the experiment!
Acetate Buffer
Acetate Buffer
pH range: 3.6 to 5.6
(a) 0.1 M Acetic acid (5.8 mL made to 1000 mL)
(b) 0.1 M Sodium acetate; 8.2 g L-1 (anhydrous; M.W. 82.0 g mol-1) or 13.6 g L-1 (trihydrate; M.W. 136.0 g mol-1)
Mix acetic acid and sodium acetate solutions in the proportions indicated
Adjust the final volume to 100 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of Acetic acid | 46.3 | 41.0 | 30.5 | 20.0 | 14.8 | 10.5 | 4.8 | |
| mL of Sodium acetate | 3.7 | 9.0 | 19.5 | 30.0 | 35.2 | 39.5 | 45.2 | |
| pH | 3.6 | 4.0 | 4.4 | 4.8 | 5.0 | 5.2 | 5.6 |
Glycine-HCl Buffer
Glycine-HCl Buffer
pH range: 2.2 to 3.6
(a) 0.1 M Glycine: 7.5 g L-1 (M.W.: 75.0 g mol-1)
(b) 0.1 M Hydrochloric acid
Mix 50 mL of glycine and indicated volume of hydrochloric acid
Mix and adjust the final volume to 100 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of HCl | 44.0 | 32.4 | 24.2 | 16.8 | 11.4 | 8.2 | 6.4 | 3.6 | |
| pH | 2.2 | 2.4 | 2.6 | 2.8 | 3.0 | 3.2 | 3.4 | 3.6 |
Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)
Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)
pH Range: 1.0 to 2.2
(a) 0.1 M Potassium chloride: 7.45 g L-1 (M.W.: 74.5 g mol-1)
(b) 0.1 M Hydrochloric acid
Mix 50 mL of potassium chloride and indicated volume of hydrochloric acid
Mix and adjust the final volume to 100 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of HCl | 97 | 64.5 | 41.5 | 26.3 | 16.6 | 10.6 | 6.7 | |
| pH | 1.0 | 1.2 | 1.4 | 1.6 | 1.8 | 2.0 | 2.2 |
Citrate-Phosphate Buffer
Citrate-Phosphate Buffer
pH range: 2.6 to 7.0
(a) 0.1 M Citric acid; 19.21 g L-1 (M.W. 192.1 g mol-1)
(b) 0.2 M Dibasic sodium phosphate; 35.6 g L-1 (dihydrate; M.W. 178.0 g mol-1) or 53.6 g L-1 (heptahydrate; M.W. 268.0 g mol-1)
Mix citric acid and sodium phosphate solutions in the proportions indicated and adjust the final volume to 100 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of Citric acid | 44.6 | 39.8 | 35.9 | 32.3 | 29.4 | 26.7 | 24.3 | 22.2 | 19.7 | 16.9 | 13.6 | 6.5 | |
| mL of Sodium phosphate | 5.4 | 10.2 | 14.1 | 17.7 | 20.6 | 23.3 | 25.7 | 27.8 | 30.3 | 33.1 | 36.4 | 43.6 | |
| pH | 2.6 | 3.0 | 3.4 | 3.8 | 4.2 | 4.6 | 5.0 | 5.4 | 5.8 | 6.2 | 6.6 | 7.0 |
Citrate Buffer
Citrate Buffer
pH range: 3.0 to 6.2
(a) 0.1 M Citric acid: 19.21 g L-1 (M.W.: 192.1 g mol-1)
(b) 0.1 M Sodium citrate dihydrate: 29.4 g/l (M.W.: 294.0 g mol-1)
Mix citric acid and sodium citrate solutions in the proportions indicated and adjust the final volume to 100 mL with deionized water
Adjust the final pH using a sensitive pH meter
The use of pentahydrate salt of sodium citrate is not recommended
| mL of Citric acid | 46.5 | 40.0 | 35.0 | 31.5 | 25.5 | 20.5 | 16.0 | 11.8 | 7.2 | |
| mL of Sodium citrate | 3.5 | 10 | 15.0 | 18.5 | 24.5 | 29.5 | 34.0 | 38.2 | 42.8 | |
| pH | 3.0 | 3.4 | 3.8 | 4.2 | 4.6 | 5.0 | 5.4 | 5.8 | 6.2 |
Phosphate Buffer
Phosphate Buffer
pH range: 5.8 to 8.0
(a) 0.1 M Sodium phosphate monobasic; 13.8 g L-1 (monohydrate, M.W. 138.0 g mol-1)
(b) 0.1 M Sodium phosphate dibasic; 26.8 g L-1 (heptahydrate, M.W. 268.0 g mol-1)
Mix sodium phosphate monobasic and dibasic solutions in the proportions indicated
Adjust the final volume to 200 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of Sodium phosphate, Monobasic | 92.0 | 81.5 | 73.5 | 62.5 | 51 .0 | 39.0 | 28.0 | 19.0 | 13.0 | 8.5 | 5.3 | |
| mL of Sodium phosphate, Dibasic | 8.0 | 18.5 | 26.5 | 37.5 | 49.0 | 61 .0 | 72.0 | 81.0 | 87.0 | 91.5 | 94.7 | |
| pH | 5.8 | 6.2 | 6.4 | 6.6 | 6.8 | 7.0 | 7.2 | 7.4 | 7.6 | 7.8 | 8.0 |
Tris-HCl Buffer
Tris-HCl Buffer
pH range: 7.2 to 9.0
(a) 0.1 M Tris(hydroxymethyl)aminomethane; 12.1 g L-1 (M.W.: 121.0 g mol-1)
(b) 0.1 M Hydrochloric acid
Mix 50 mL of Tris(hydroxymethyl)aminomethane and indicated volume of hydrochloric acid
Adjust the final volume to 200 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of HCl | 44.2 | 41 .4 | 38.4 | 32.5 | 21 .9 | 12.2 | 5.0 | |
| pH | 7.2 | 7.4 | 7.6 | 7.8 | 8.2 | 8.6 | 9.0 |
Carbonate-Bicarbonate Buffer
Carbonate-Bicarbonate Buffer
pH range: 9.2 to 10.6
(a) 0.1 M Sodium carbonate (anhydrous), 10.6 g L-1 (M.W.: 106.0 g mol-1)
(b) 0.1 M Sodium bicarbonate, 8.4 g L-1 (M.W.: 84.0 g mol-1)
Mix sodium carbonate and sodium bicarbonate solutions in the proportions indicated
Adjust the final volume to 200 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of Sodium carbonate | 4.0 | 9.5 | 16.0 | 22.0 | 27.5 | 33.0 | 38.5 | 42.5 | |
| mL of Sodium bicarbonate | 46.0 | 40.5 | 34.0 | 28.0 | 22.5 | 17.0 | 11.5 | 7.5 | |
| pH | 9.2 | 9.4 | 9.6 | 9.8 | 10.0 | 10.2 | 10.4 | 10.6 |
Glycine-Sodium Hydroxide Buffer
Glycine-Sodium Hydroxide Buffer
pH: 8.6 to 10.6
(a) 0.1 M Glycine; 7.5 g L-1 (M.W.: 75.0 g mol-1)
(b) 0.1 M Sodium hydroxide; 4.0 g L-1 (M.W.: 40.0 g mol-1)
Mix 50 mL of glycine and indicated volume of sodium hydroxide solutions
Adjust the final volume to 200 mL with deionized water
Adjust the final pH using a sensitive pH meter
| mL of Sodium hydroxide | 4.0 | 8.8 | 16.8 | 27.2 | 32.0 | 38.6 | 45.5 | |
| pH | 8.6 | 9.0 | 9.4 | 9.8 | 10.0 | 10.4 | 10.6 |
Tris Buffred Saline (TBS)
Tris Buffred Saline (TBS)
pH 7.4
10 mM Tris
150 mM NaCl
1 L of TBS can be prepared by dissolving 1.21 g of Tris base and 8.7 g of NaCl in 1 L of distilled water
Adjust the pH before use
Note: Tris has a pKa of 8.3. Hence, the buffering capacity at pH 7.4 is minimal compared to phosphate buffer (pKa = 7.21)
Phosphate Buffered Saline (PBS)
Phosphate Buffered Saline (PBS)
pH 7.4
150 mM NaCl
10 mM Potassium Phosphate buffer
1 L PBS can be prepared by dissolving 8.7 g NaCl, 1.82 g K2HPO4 • 3H2O, and 0.23 g KH2PO4 in 1 L of distilled water
Adjust the pH before use
_____________________________________
A variation of PBS can also be prepared as follows:
137 mM NaCl
2.7 mM KCl
10 mM Na2HPO4
1.76 mM KH2PO4