Buffer Selection Guide: pKa, Ranges and Choosing a Buffer

A buffer only holds pH steady close to its pKa, and the pKa itself moves with temperature and salt concentration. This guide lists pKa values, useful ranges and temperature coefficients for 14 common laboratory buffers, explains how to choose between them, and includes a buffer finder that suggests suitable buffers for any target pH and working temperature.

Find a buffer for your pH and temperature

How it works

The finder corrects each pKa from its 25 °C value using the temperature coefficient in the table below: pKa at T = pKa at 25 °C + (T − 25) × d(pKa)/dT. Tris, for example, has a pKa of 8.06 at 25 °C and a coefficient of −0.028 per °C, so its pKa is about 7.72 at 37 °C and 8.65 at 4 °C. Between about 0 and 50 °C this straight-line correction is accurate to within about 0.1 pH unit.

• Buffering capacity is greatest when the pH equals the pKa. One unit away it has fallen to about a third of the maximum, and two units away to under 5%, which is why the finder stops at ±1.
• Bicarbonate is a special case. Culture media hold pH 7.2–7.4 with bicarbonate, outside its pKa ± 1 range, because the incubator supplies a fixed level of CO₂.
• Salt concentration also shifts pKa (see the notes under the table), so always confirm the final pH with a calibrated meter at the working temperature.

pKa values and useful ranges

Buffer	pKa at 25 °C	Useful range (≈ pKa ± 1)	d(pKa)/dT (per °C)	Usually adjusted with	Metal binding and interferences	Notes
Citrate	3.13; 4.76; 6.40	2.1–7.4	−0.0024; −0.0013; +0.002	NaOH (from citric acid) or HCl (from trisodium citrate)	Chelates Ca²⁺, Mg²⁺, Fe³⁺ and other metal ions	Three overlapping pKa values; classic citrate buffers cover pH 3.0–6.2. pKa barely changes with temperature.
Acetate	4.76	3.8–5.8	+0.0002	NaOH (from acetic acid) or acetic acid (from sodium acetate)	Weak binding of some metal ions	pKa almost independent of temperature. Acetic acid is volatile, so pH can drift when solutions are dried or freeze-dried. Recipe calculator: acetate buffer.
Histidine	6.07 (side chain)	5.1–7.1	−0.017	HCl (from histidine base), or mix histidine with histidine hydrochloride	Binds Cu²⁺, Zn²⁺, Ni²⁺ and other metal ions	Common at pH 5.5–6.5 in protein formulation studies. Can degrade in light, especially with metal ions present; store protected from light.
MES	6.10	5.1–7.1	−0.011	NaOH or KOH	Negligible Cu²⁺ binding; weak binding of Ca²⁺, Mg²⁺ and Mn²⁺	Good’s buffer, most used at pH 5.5–6.7.
Bicarbonate / CO₂	6.35 (pKa1); 10.33 (pKa2)	5.4–7.4; 9.3–11.3	−0.0055; −0.009	CO₂ level near neutral pH; carbonate to bicarbonate ratio at pH 9–10.5	Calcium carbonate can precipitate if the pH rises	Needs a controlled CO₂ atmosphere: in culture medium at 37 °C the apparent pKa is about 6.1, and 5–10% CO₂ holds the pH near 7.2–7.4. Media turn alkaline once out of the incubator. Carbonate–bicarbonate buffer (for example pH 9.6) absorbs CO₂ from air, so keep it closed.
PIPES	6.76	5.8–7.8	−0.0085	NaOH or KOH (the free acid dissolves as base is added)	Negligible metal binding	The piperazine ring can form radicals; avoid in redox studies.
Phosphate	7.20 (pKa2); 2.15 (pKa1)	6.2–8.2; 1.2–3.2	−0.0028; +0.0044	Mix the mono- and dibasic salts (sodium or potassium); fine-tune with NaOH or phosphoric acid	Precipitates Ca²⁺, Mg²⁺ and other divalent metal ions; inhibits some enzymes	pKa2 is about 6.8 in 0.1–0.15 M salt, as in PBS. Sodium phosphate can fall by up to about 3 pH units on freezing as disodium phosphate crystallises; potassium phosphate shifts much less. Very low UV absorbance.
MOPS	7.20	6.2–8.2	−0.011	NaOH or KOH	Negligible metal binding	Used in RNA electrophoresis and bacterial growth media. Usually filter-sterilised; solutions can turn yellow with age or light.
HEPES	7.48	6.5–8.5	−0.014	NaOH or KOH	Negligible binding of Ca²⁺, Mg²⁺ and Mn²⁺; can form radicals	10–25 mM is added to culture media to hold the pH outside the CO₂ incubator. In light, HEPES and riboflavin (present in most media) produce hydrogen peroxide, so keep HEPES media in the dark.
Tricine	8.05	7.1–9.1	−0.021	NaOH or KOH	Binds Cu²⁺ strongly; also Ca²⁺, Mg²⁺, Zn²⁺ and other metal ions	Used in Tricine–SDS gels for small proteins. Photo-oxidised by flavins.
Tris	8.06	7.1–9.1	−0.028	HCl (from Tris base)	Binds Cu²⁺ and Ni²⁺; the primary amine reacts with aldehydes and amine-reactive reagents	pKa falls by about 0.028 per °C: pH 8.0 set at 25 °C is about 8.6 at 4 °C and 7.7 at 37 °C. Set the pH at the working temperature and use a Tris-compatible pH electrode.
Bicine	8.26	7.3–9.3	−0.018	NaOH or KOH	Binds Cu²⁺ strongly; also Ca²⁺, Mg²⁺ and other metal ions	Check compatibility before use with metal-dependent enzymes.
Glycine	2.35 (pKa1); 9.78 (pKa2)	1.4–3.4; 8.8–10.8	−0.0024; −0.026	HCl (glycine–HCl, about pH 2.2–3.6) or NaOH (glycine–NaOH, about pH 8.6–10.6)	Binds Cu²⁺ and other metal ions; primary amine	Glycine–HCl is a common low-pH elution buffer in affinity purification.
CAPS	10.40	9.4–11.4	−0.028	NaOH	Not known to bind metal ions significantly	Used for high-pH work, such as protein blotting transfer buffer at pH 11.

About these values. The pKa values are typical figures at 25 °C. For the Good’s buffers (MES, PIPES, MOPS, HEPES, Tricine, Bicine and CAPS) they are the values listed in supplier reference tables for 0.1 M solutions; for phosphate, citrate, acetate, bicarbonate, histidine, glycine and Tris they are standard values for dilute solution. Published figures differ by up to about 0.1 unit between sources.

Salt matters. Ionic strength lowers the apparent pKa of buffers whose acid form carries a negative charge. In 0.1–0.15 M salt, phosphate pKa2 falls to about 6.8 and citrate pKa3 to about 5.7–5.8, which is why PBS at pH 7.4 sits well above the effective pKa of its phosphate. For most of the other buffers here the shift is about 0.1 unit or less, and for Tris and histidine the pKa rises slightly with salt.

How to choose a buffer

1. Target pH. Pick a buffer whose pKa is within 1 unit of the pH you need, ideally within 0.5. If the experiment produces acid, a pKa slightly below the working pH gives more capacity; if it produces base, choose one slightly above.
2. Working temperature. Correct the pKa for the temperature you will actually use (the finder does this), and set the pH at that temperature. Amine buffers such as Tris change most; phosphate, citrate and acetate change least.
3. Ionic strength and concentration. 10–100 mM covers most uses. More buffer gives more capacity, but it also adds ionic strength and osmolality and changes the apparent pKa. Check the pH again after diluting a concentrated stock.
4. Assay compatibility. Primary amines (Tris, glycine) react with aldehydes and amine-reactive labelling reagents; phosphate inhibits some enzymes, including phosphatases; HEPES and PIPES can form radicals in redox work. Check the compatibility list supplied with protein assays and kits.
5. Metal ions. Citrate and histidine bind metal ions, and phosphate precipitates calcium and magnesium. Good’s buffers such as MES, PIPES, MOPS and HEPES bind metals weakly or not at all; Tricine and Bicine are the main exceptions and bind Cu²⁺ and other ions.
6. UV absorbance. Good’s buffers were selected for low absorbance above 230 nm. For measurements below that, phosphate is usually the best choice; acetate, citrate and histidine absorb more strongly at short wavelengths.
7. Sterilisation. Phosphate buffers, PBS and Tris can be autoclaved. Good’s buffers and solutions containing bicarbonate or glucose are usually filtered through a 0.22 µm PES syringe filter instead; see the syringe filter guide.

Osmolality and cell culture

Cells swell or shrink when the solution around them is not isotonic, so buffers and media for cell work are made to a set osmolality. Osmolality (mOsm/kg of water) is what an osmometer measures, usually by freezing-point depression; osmolarity (mOsm/L of solution) is what you calculate from a recipe. In dilute aqueous solutions the two are close.

• Target range. Typical culture media are roughly 260–340 mOsm/kg, and most sit between 280 and 320 mOsm/kg. Blood plasma is about 290 mOsm/kg.
• Reference point. 0.9% saline (154 mM NaCl) measures about 286–290 mOsm/kg. The simple calculation, 2 × 154 = 308 mOsm/L, overestimates it because NaCl does not behave as an ideal solute.
• What contributes. Every dissolved particle counts. Each 1 mM of NaCl adds about 1.9 mOsm/kg (two ions); each 1 mM of glucose adds about 1 mOsm/kg. Buffers count too: 25 mM HEPES brought to pH 7.4 with NaOH adds roughly 35–40 mOsm/kg, from the HEPES plus about 11–14 mM of sodium.
• Adjusting. To raise osmolality, add NaCl (about 0.3 g per litre for each 10 mOsm/kg) or, where ionic strength must not change, a non-ionic solute such as sucrose (about 1 mOsm/kg per mM). To make room for an added buffer, reduce the NaCl: for 35 mOsm/kg, use about 19 mM (1.1 g/L) less. To lower osmolality, dilute with purified water such as WFI water. Measure the final solution rather than relying on the calculation.

Buffers used in formulation research

In protein and other formulation studies, the buffer is chosen for its pH range, its behaviour during freezing and freeze-drying, its interactions with metal ions and its compatibility with the other components. Concentrations are usually low, around 10–50 mM.

• Histidine buffers around pH 5.5–6.5 and is widely used for protein solutions, including freeze-dried samples. It can degrade in light, so solutions are stored protected from light.
• Citrate covers pH 3–6.2 and chelates metal ions, which can limit metal-catalysed oxidation. Its pKa values hardly change with temperature. A ready-made 1× sodium citrate buffer, pH 6.0, is available in 500 mL bottles.
• Phosphate buffers near neutral pH and has very low UV absorbance, but sodium phosphate shifts pH strongly on freezing, and phosphate precipitates calcium and magnesium. Potassium phosphate is often chosen for samples that will be frozen. 1× PBS, pH 7.4, is available ready-made in 100 mL bottles.
• Acetate is used around pH 4–5.5. It is simple and inexpensive and its pKa barely changes with temperature, but acetic acid can evaporate during freeze-drying and shift the pH. Dilute acid such as 1% acetic acid is convenient for small pH corrections.
• Tris is used where a neutral to mildly alkaline pH (7–9) is needed. Its large temperature coefficient means the pH changes noticeably between cold storage and room temperature, and its primary amine can react with aldehydes and some reagents.

A non-ionic surfactant such as polysorbate 80 (Tween 80) is often included at a low concentration, typically below 0.1% w/v, to reduce adsorption to container surfaces.

Pros, cons and limitations

Buffer family	Strengths	Limitations
Phosphate, citrate, acetate, bicarbonate	Inexpensive and well characterised. Phosphate, citrate and acetate change little with temperature. Phosphate is transparent far into the UV.	Phosphate precipitates divalent metal ions and citrate chelates them. Sodium phosphate shifts pH on freezing. Acetate and bicarbonate can lose acetic acid or CO₂ by evaporation. Salt has a large effect on the pKa of phosphate and citrate.
Amine buffers: Tris, glycine, histidine	Inexpensive, very soluble and widely used.	Large temperature coefficients, especially Tris. Reactive primary amines (Tris, glycine). Metal binding (histidine, glycine).
Good’s buffers: MES, PIPES, MOPS, HEPES, Tricine, Bicine, CAPS	Designed for biological research: low metal binding (except Tricine and Bicine), low UV absorbance above 230 nm, and they do not readily cross cell membranes.	More expensive. Moderate temperature coefficients. HEPES and PIPES can form radicals, and HEPES forms hydrogen peroxide with riboflavin in light.

• Table pKa values and Henderson–Hasselbalch calculations are a starting point. The real pH depends on temperature, ionic strength and everything else in the solution, so finish by measuring.
• pH changes on dilution: a 10× PBS stock reads lower than its 1× dilution, so check the pH at working strength.
• Freezing concentrates solutes and can shift pH. Thaw completely and mix before use.
• Calibrate the pH meter with at least two standards that bracket the target pH, at the working temperature.
• Buffers can support microbial growth over time. Filter-sterilise, store in sterile vials and see our aliquoting guide.

Related: stock and working concentrations, with a serial dilution calculator · PBS and buffer recipes · dilution and molarity calculator · syringe filter selection guide · aliquoting reagents into sterile vials. Supplies: 1× PBS, sodium citrate buffer pH 6.0, WFI water, 0.9% saline, PES syringe filters, sterile 10 mL vials.

References

1. Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RMM. Hydrogen ion buffers for biological research. Biochemistry 1966; 5: 467–477.
2. Goldberg RN, Kishore N, Lennen RM. Thermodynamic quantities for the ionization reactions of buffers. J. Phys. Chem. Ref. Data 2002; 31: 231–370.
3. Merck (Sigma-Aldrich) Buffer Reference Center, and ITW Reagents (AppliChem) Biological Buffers: pKa and d(pKa)/dT tables at 25 °C.
4. Zigler JS, Lepe-Zuniga JL, Vistica B, Gery I. Analysis of the cytotoxic effects of light-exposed HEPES-containing culture medium. In Vitro Cell. Dev. Biol. 1985; 21: 282–287.
5. Pikal-Cleland KA, Cleland JL, Anchordoquy TJ, Carpenter JF. Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems. J. Pharm. Sci. 2002; 91: 1969–1979.

pKa values and temperature coefficients are typical published figures, and the buffer finder is a convenience tool; confirm critical values against your own reagents and validated protocols. Union Biolabs products are supplied for laboratory research use only.