Buffer Reference Center

Section Overview

• Choosing the Right Biological Buffer
• What are Buffer Grades?
• Useful pH Ranges of Selected Biological Buffers Chart (25 °C, 0.1 M)
• Tris or Trizma^® Buffer Preparation – pH vs. Temperature
• Phosphate Buffer Preparation – 0.2 M solution
• Citric Acid – Na₂HPO₄ Buffer Preparation, pH 2.6-7.6
• Citric Acid – Sodium Citrate Buffer Preparation, pH 3.0-6.2
• Sodium Acetate – Acetic Acid Buffer Preparation, pH 3.7-5.6
• Na₂HPO₄ – NaH₂PO₄ Buffer Preparation, pH 5.8-8.0 at 25 °C
• Imidazole (glyoxaline) – HCl Buffer Preparation, pH 6.2-7.8 at 25 °C
• Sodium Carbonate – Sodium Bicarbonate Buffer Preparation, pH 9.2-10.8
• Buffer Preparation Formulas and Equations

Choosing the Right Biological Buffer

Choose a buffer based on your pH requirements as well as the pKa, a measure of acid strength that accounts for pH, concentration, and temperature. Regulatory or purity needs for your exact application should also be considered. The following tables can help you navigate preparation of many common buffer solutions by pH and pKa. 

In addition to the tables below, we've developed several buffer recipe calculators to assist your buffer preparation.

TBE and TAE for gel electrophoresis have slightly more involved recipes. We can help you pick the right one.

What are buffer grades?

Buffer grade indicates the quality and impurity levels appropriate for different uses. We provide six grades of buffers indicated for general lab use, final pharmaceutical formulation and manufacturing, and applications in between that may need trace metal testing or materials of a specified purity.

Useful pH Ranges of Selected Biological Buffers Chart (25 °C, 0.1 M)  

Buffers	Useful pH Range	pKa (at 20 °C)	pKa (at 25 °C)	pKa (at 37 °C)
MES	5.5–6.7	6.16	6.10	5.97
Bis-Tris	5.8–7.2	n/a	6.50	6.36
ADA	6.0–7.2	6.65	6.59	6.46
ACES	6.1–7.5	6.88	6.78	6.54
PIPES	6.1–7.5	6.80	6.76	6.66
MOPSO	6.2–7.6	n/a	6.90	6.75
Bis-Tris Propane	6.3–9.5	n/a	6.8, 9.0	n/a
BES	6.4–7.8	7.17	7.09	6.90
MOPS	6.5–7.9	7.28	7.20	7.02
TES	6.8–8.2	7.50	7.40	7.16
HEPES	6.8–8.2	7.55	7.48	7.31
DIPSO	7.0–8.2	n/a	7.60	7.35
MOBS	6.9–8.3	n/a	7.60	n/a
TAPSO	7.0–8.2	n/a	7.60	7.39
Tris or Trizma®	7.0–9.0	8.20	8.06	7.72
HEPPSO	7.1–8.5	n/a	7.80	6.66
POPSO	7.2–8.5	n/a	7.80	7.63
TEA	7.3–8.3	n/a	7.80	n/a
EPPS	7.3–8.7	n/a	8.00	n/a
Tricine	7.4–8.8	8.16	8.05	7.80
Gly-Gly	7.5–8.9	n/a	8.20	n/a
Bicine	7.6–9.0	8.35	8.26	8.04
HEPBS	7.6–9.0	n/a	8.30	n/a
TAPS	7.7-9.1	8.49	8.40	8.18
AMPD	7.8–9.7	n/a	8.80	n/a
TABS	8.2–9.6	n/a	8.90	n/a
AMPSO	8.3–9.7	n/a	9.00	9.10
CHES	8.6–10.0	9.55	9.49	9.36
CAPSO	8.9–10.3	n/a	9.60	9.43
AMP	9.0–10.5	n/a	9.70	n/a
CAPS	9.7–11.1	10.56	10.40	10.02
CABS	10.0–11.4	n/a	10.70	n/a

Tris or Trizma^® Buffer Preparation Table – pH vs. Temperature

pH at Temperature
5 °C	25 °C	37 °C
7.76	7.20	6.91
7.89	7.30	7.02
7.97	7.40	7.12
8.07	7.50	7.22
8.18	7.60	7.30
8.26	7.70	7.40
8.37	7.80	7.52
8.48	7.90	7.62
8.58	8.00	7.71
8.68	8.10	7.80
8.78	8.20	7.91
8.88	8.30	8.01
8.98	8.40	8.10
9.09	8.50	8.22
9.18	8.60	8.31
9.28	8.70	8.42
9.36	8.80	8.51
9.47	8.90	8.62
9.56	9.00	8.70

g/L for 0.05 M Solution
Trizma® HCL	Trizma® Base
7.02	0.67
6.85	0.80
6.61	0.97
6.35	1.18
6.06	1.39
5.72	1.66
5.32	1.97
4.88	2.30
4.44	2.65
4.02	2.97
3.54	3.34
3.07	3.70
2.64	4.03
2.21	4.36
1.83	4.65
1.50	4.90
1.23	5.13
0.96	5.32
0.76	5.47

Phosphate Buffer Preparation Table – 0.2 M solution  

Potassium  Phosphate Monobasic Anhydrous g/L	Sodium Phosphate Dibasic Heptahydrate g/L	23 °C pH
22.4	3.49	5.7
22.08	4.29	5.8
21.60	5.37	5.9
21.05	6.60	6.0
20.40	8.05	6.1
19.56	9.93	6.2
18.60	12.07	6.3
17.64	14.22	6.4
16.44	16.90	6.5
15.00	20.12	6.6
13.56	23.34	6.7
12.24	26.29	6.8

Potassium Phosphate Monobasic Anhydrous g/L	Sodium Phosphate Dibasic Heptahydrate g/L	23 °C pH
10.80	29.51	6.9
9.36	32.73	7.0
7.92	35.95	7.1
6.72	38.63	7.2
5.52	41.31	7.3
4.56	43.46	7.4
3.84	45.07	7.5
3.12	46.68	7.6
2.52	48.55	7.7
2.04	49.09	7.8
1.68	49.89	7.9
1.27	50.81	8.0

Citric Acid – Na₂HPO₄ Buffer Preparation, pH 2.6–7.6¹

Citric acid monohydrate, C₆H₈O₇ • H₂O, MW 210.14; 0.1 M contains 21.01 g/L. Na₂HPO₄, MW 141.98; 0.2 M contains 28.40 g/L, or Na₂HPO₄ • 2H₂O, MW 178.05; 0.2 M contains 35.61 g/L.

x mL 0.1 M-citric acid and y mL 0.2 M-Na₂HPO₄ mixed

pH	x mL 0.1 M-citric acid	y mL 0.2-Na2HPO4
2.6	89.10	10.90
2.8	84.15	15.85
3.0	79.45	20.55
3.2	75.30	24.70
3.4	71.50	28.50
3.6	67.80	32.20
3.8	64.50	35.50
4.0	61.45	38.55
4.2	58.60	41.40
4.4	55.90	44.10
4.6	53.25	46.75
4.8	50.70	49.30
5.0	48.50	51.50
5.2	46.40	53.60
5.4	44.25	55.75
5.6	42.00	58.00
5.8	39.55	60.45
6.0	36.85	63.15
6.2	33.90	66.10
6.4	30.75	69.25
6.6	27.25	72.75
6.8	22.75	77.25
7.0	17.65	82.35
7.2	13.05	86.95
7.4	9.15	90.85
7.6	6.35	93.65

Citric Acid – Sodium Citrate Buffer Preparation, pH 3.0-6.2¹

Citric acid monohydrate, C₆H₈O₇ • H₂O, MW 210.14; 0.1 M contains 21.01 g/L. Trisodium citrate dihydrate, C₆H₅O₇Na₃ • 2H₂O, MW 294.12; 0.1 M contains 29.41 g/L.

x mL 0.1 M-citric acid and y mL 0.1 M-trisodium citrate mixed

pH	x mL 0.1 M-citric acid	y mL 0.1 M-trisodium citrate
3.0	82.0	18.0
3.2	77.5	22.5
3.4	73.0	27.0
3.6	68.5	31.5
3.8	63.5	36.5
4.0	59.0	41.0
4.2	54.0	46.0
4.4	49.5	50.5
4.6	44.5	55.5
4.8	40.0	60.0
5.0	35.0	65.0
5.2	30.5	69.5
5.4	25.5	74.5
5.6	21.0	79.0
5.8	16.0	84.0
6.0	11.5	88.5
6.2	8.0	92.0

Sodium Acetate – Acetic Acid Buffer PREPARATION, pH 3.7–5.6¹

Sodium acetate trihydrate, CH₃COONa • 3H₂O, MW 136.09; 0.2 M contains 27.22 g/L.

x mL 0.2 M-NaOAc and y mL 0.2 M-HOAc mixed

pH, 18 °C	x mL 0.2 M-NaOAc	y mL 0.2 M-HOAc
3.7	10.0	90.0
3.8	12.0	88.0
4.0	18.0	82.0
4.2	26.5	73.5
4.4	37.0	63.0
4.6	49.0	51.0
4.8	59.0	41.0
5.0	70.0	30.0
5.2	79.0	21.0
5.4	86.0	14.0
5.6	91.0	9.0

Na₂HPO₄ – NaH₂PO₄ Buffer PREPARATION, pH 5.8-8.0 at 25 °C¹

Na₂HPO₄ • 2H₂O, MW 178.05; 0.2 M contains 35.61 g/L. Na₂HPO₄ • 12H₂O, MW 358.22; 0.2 M contains 71.64 g/L. NaH₂PO₄ • H₂O, MW 138.01; 0.2 M contains 27.6 g/L. NaH₂PO₄ • 2H₂O, MW 156.03; 0.2 M contains 31.21 g/L.

x mL 0.2 M-Na₂HPO₄, y mL 0.2 M-NaH₂PO₄; diluted to 100 mL with H₂O

pH, 25 °C	x mL 0.2 M-Na2HPO4	y mL 0.2 M-NaH2PO4
5.8	4.0	46.0
6.0	6.15	43.85
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

Imidazole (glyoxaline) – HCl Buffer PREPARATION, pH 6.2–7.8 at 25 °C¹

Imidazole, C₃H₄N₂, MW 68.08

25 mL 0.2 M-imidazole (13.62 g/L), x mL 0.2 M-HCl, diluted to 100 mL with H₂O

pH, 25 °C	x mL 0.2 M-HCl
6.2	21.45
6.4	19.9
6.6	17.75
6.8	15.2
7.0	12.15
7.2	9.3
7.4	6.8
7.6	4.65
7.8	3.0

Sodium Carbonate – Sodium Bicarbonate Buffer PREPARATION, pH 9.2-10.8¹

Na₂CO₃ • 10H₂O, MW 286.2; 0.1 M contains 28.62 g/L. NaHCO₃, MW 84.0; 0.1 M contains 8.40 g/L.

x mL 0.1 M-Na₂CO₃ and y mL 0.1 M-Na₂HCO₃ mixed

20 °C	37 °C
x mL 0.1 M-Na2CO3	y mL 0.1 M-Na2HCO3
9.2	8.8	10	90
9.4	9.1	20	80
9.5	9.4	30	70
9.8	9.5	40	60
9.9	9.7	50	50
10.1	9.9	60	40
10.3	10.1	70	30
10.5	10.3	80	20
10.8	10.6	90	10

Buffer Preparation Formulas and Equations

Percentage by weight (w/v)

(% buffer desired / 100) × final buffer volume (mL) = g of starting material needed.

Molar Solutions

desired molarity × formula weight × solution final volume (L) = grams needed

Henderson-Hasselbach Equation

Reference

1. Dawson R, Elliot D, Elliot W, Jones KM. 1986. Data for Biochemical Research. 3rd ed.. Oxford Science Publ..