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Buffer preparation and selection

Buffer Calculator — pH, Concentration, Volume, and Preparation Recipe

Generate theoretical preparation starting estimates for the eight systems and modes approved in the signed scientific decision, or review selection-only authority records for the remaining systems.

Calculations run locally in your browser. Values are only placed in a URL when you choose “Copy Shareable Link.”

Buffer parameters

Choose a buffer to see reagent forms, useful range, and temperature-adjusted pKa context.

Worked example: 50 mM Tris at pH 8.0

For 100 mL of 50 mM Tris at 25 °C, the signed thermodynamic reference pK is 8.072. The base/acid ratio is 10(8.000 − 8.072) ≈ 0.847. The 5 mmol total therefore partitions to about 2.71 mmol Tris hydrochloride and 2.29 mmol Tris base.

Using 0.1 M Tris hydrochloride and 0.1 M Tris base stocks, the theoretical estimate is about 27.1 mL acid-form stock and 22.9 mL base-form stock. Prepare near final volume, measure pH with a calibrated meter at 25 °C, adjust experimentally, then bring to 100 mL.

Load this Tris example

How to use the calculator

  1. Choose a buffer system. Selection/reference-only records show authority data without recipe controls.
  2. For a preparation-enabled record, enter a target within pK(T) ± 1, concentration, final volume, and one of the six approved temperatures.
  3. Select only a preparation mode shown for that system. Tris solids use the exact reviewed anhydrous Tris hydrochloride and Tris base forms.
  4. Create the theoretical starting estimate, check feasibility and caveats, then verify reagent identity and final pH before use.

The result is a planning record, not a validated laboratory SOP.

Signed scientific authority catalog

Buffer systems

Eight systems are preparation-enabled only for their explicitly approved modes. Twelve additional canonical records are selection/reference-only and expose no recipe controls. Every card is rendered at build time from the same signed authority data used by the calculator.

Sodium phosphate (NaH₂PO₄/Na₂HPO₄)

Also called: Phosphate buffer, Sodium phosphate

Preparation-enabled

Chemical identity
Phosphate; H3PO4; CAS 7664-38-2; 98 g/mol
Theoretical pK(25 °C) ± 1 ratio window
pH 6.198–8.198
Thermodynamic reference pK
pKa2 / reaction 2: 7.27863 at 4 °C, 7.22333 at 16 °C, 7.198 at 25 °C, 7.18925 at 30 °C, 7.18283 at 37 °C, 7.18713 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 6.198–8.198. These are reference windows, not preparation ranges.
Ionization reaction
H2PO4− = H+ + HPO4²−, reaction 2, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks

Scientific and preparation caveat: Check pH after dilution; ionic strength shifts pKa slightly. Avoid with Ca²⁺/Mg²⁺ to prevent precipitation.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: Use pK 7.198 at 25 °C and NIST Equation (12); approve only mixing user-declared molar NaH2PO4 and Na2HPO4 stocks. No solid recipe.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Standard Reference Materials: Standardization of pH Measurements, Buffer Reference Center

Load a signed 50 mM exampleRead the Sodium phosphate (NaH₂PO₄/Na₂HPO₄) guide

Citrate (Citric acid / Trisodium citrate)

Also called: Citrate, Citric acid–citrate, Sodium citrate

Selection/reference only

Chemical identity
Citrate; C6H8O7; CAS 77-92-9; 192.13 g/mol
Historical selection range; not preparation authority
pH 3–6.2
Thermodynamic reference pK
pKa / reaction 2: 4.81602 at 4 °C, 4.77757 at 16 °C, 4.761 at 25 °C, 4.75584 at 30 °C, 4.75303 at 37 °C, 4.76012 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 3.761–5.761. These are reference windows, not preparation ranges.
Ionization reaction
H2L− = H+ + HL²−, reaction 2, evaluation grade AAB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Chelates metals; may inhibit metalloproteins. Monitor ionic strength when combining with multivalent cations.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Do not use citric acid plus trisodium citrate in a single-step Henderson–Hasselbalch recipe.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Standard Reference Materials: Standardization of pH Measurements, Buffer Reference Center

View scientific referenceRead the Citrate (Citric acid / Trisodium citrate) guide

Acetate (Acetic acid / Sodium acetate)

Also called: Acetic acid/acetate, Sodium acetate buffer, Acetate buffer

Preparation-enabled

Chemical identity
Acetate; C2H4O2; CAS 64-19-7; 60.052 g/mol
Theoretical pK(25 °C) ± 1 ratio window
pH 3.756–5.756
Thermodynamic reference pK
pKa / reaction 1: 4.77083 at 4 °C, 4.75728 at 16 °C, 4.756 at 25 °C, 4.7582 at 30 °C, 4.76448 at 37 °C, 4.78497 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 3.756–5.756. These are reference windows, not preparation ranges.
Ionization reaction
CH3COOH = H+ + CH3COO−, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks

Scientific and preparation caveat: Volatile—prepare fresh and keep tightly capped. Compatible with many enzyme assays near pH 4–5.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Buffer Reference Center

Load a signed 50 mM exampleRead the Acetate (Acetic acid / Sodium acetate) guide

Carbonate–Bicarbonate (Na₂CO₃ / NaHCO₃)

Also called: Carbonate–bicarbonate, Sodium carbonate/bicarbonate

Selection/reference only

Chemical identity
Carbonate; CO2; CAS 124-38-9 (CO2); 463-79-6 (carbonic acid); 44.01 g/mol
Historical selection range; not preparation authority
pH 9.2–10.8
Thermodynamic reference pK
pKa / reaction 2: 10.5597 at 4 °C, 10.4153 at 16 °C, 10.329 at 25 °C, 10.2883 at 30 °C, 10.2393 at 37 °C, 10.1708 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 9.329–11.329. These are reference windows, not preparation ranges.
Ionization reaction
HCO3− = H+ + CO3²−, reaction 2, evaluation grade BAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: CO₂ sensitive—store capped; verify pH at working temperature. Useful for ELISA blocking; avoid long exposure to air.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: No open-vessel recipe until a dissolved-CO2/gas-equilibrium policy and conditions-matched model are approved.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Standard Reference Materials: Standardization of pH Measurements, Buffer Reference Center

View scientific reference

Glycine

Also called: Glycine buffer, Glycine-NaOH, Glycine-HCl

Selection/reference only

Chemical identity
Glycine; C2H5NO2; CAS 56-40-6; 75.067 g/mol
Historical selection range; not preparation authority
pH 2.3–10.6
Thermodynamic reference pK
pKa1 / reaction 1: 2.42394 at 4 °C, 2.37626 at 16 °C, 2.351 at 25 °C, 2.34044 at 30 °C, 2.32947 at 37 °C, 2.3197 at 50 °C | pKa2 / reaction 2: 10.3749 at 4 °C, 10.0224 at 16 °C, 9.78 at 25 °C, 9.65269 at 30 °C, 9.48269 at 37 °C, 9.19034 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 1.351–3.351; reaction 2: pH 8.78–10.78. These are reference windows, not preparation ranges.
Ionization reaction
H2L+ = H+ + HL±, reaction 1, evaluation grade AAA; HL± = H+ + L−, reaction 2, evaluation grade AAB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Zwitterionic—buffers near both pKa values; common in SDS-PAGE transfer buffers. Can weakly chelate metals; supplement where required.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Preserve historical glycine ID, display low- and high-pH reactions separately, and expose no preparation modes until distinct reagent models are implemented and reviewed.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Henderson–Hasselbalch equation

View scientific reference

Imidazole / HCl

Also called: Imidazole, Imidazole buffer

Selection/reference only

Chemical identity
Imidazole; C3H4N2; CAS 288-32-4; 68.077 g/mol
Historical selection range; not preparation authority
pH 6.2–7.8
Thermodynamic reference pK
pKa / reaction 1: 7.48066 at 4 °C, 7.19302 at 16 °C, 6.993 at 25 °C, 6.88719 at 30 °C, 6.745 at 37 °C, 6.49789 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 5.993–7.993. These are reference windows, not preparation ranges.
Ionization reaction
HL+ = H+ + L, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Common in His-tag workflows; absorbs strongly at 230–280 nm—monitor when measuring proteins.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: The current catalog and evaluated thermodynamic pK differ by more than 0.03; operational conditions are not declared.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Buffer Reference Center

View scientific referenceRead the Imidazole / HCl guide

Tris / Tris·HCl

Also called: Trizma, Tris base, Tris(hydroxymethyl)aminomethane

Preparation-enabled

Chemical identity
Tris; C4H11NO3; CAS 77-86-1; 121.14 g/mol
Manufacturer-recommended useful range
pH 7–9
Thermodynamic reference pK
pKa / reaction 1: 8.7103 at 4 °C, 8.33221 at 16 °C, 8.072 at 25 °C, 7.93532 at 30 °C, 7.75274 at 37 °C, 7.43862 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 7.072–9.072. These are reference windows, not preparation ranges.
Ionization reaction
HL+ = H+ + L, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks, Weigh solids, Titrate

Scientific and preparation caveat: Strong temperature dependence (≈ −0.028 pH/°C); verify working temperature. Primary amine interferes with NHS-esters and aldehyde labeling.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Standard Reference Materials: Standardization of pH Measurements, Biological Buffers Selection Guide

Load a signed 50 mM exampleRead the Tris / Tris·HCl guide

HEPES

Also called: N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), HEPES-Na, HEPES buffer

Preparation-enabled

Chemical identity
HEPES; C8H18N2O4S; CAS 7365-45-9; 238.31 g/mol
Manufacturer-recommended useful range
pH 6.8–8.2
Thermodynamic reference pK
pKa / reaction 2: 7.82809 at 4 °C, 7.67408 at 16 °C, 7.564 at 25 °C, 7.50472 at 30 °C, 7.42384 at 37 °C, 7.27976 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 6.564–8.564. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 2, evaluation grade ABB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks, Titrate

Scientific and preparation caveat: Minimal metal binding; excellent buffering for cell culture media. dpH/dT ≈ -0.014 pH/°C—adjust for incubator temperature.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Hydrogen Ion Buffers for Biological Research, Biological Buffers Selection Guide

Load a signed 50 mM exampleRead the HEPES guide

MES

Also called: 2-(N-Morpholino)ethanesulfonic acid, MES buffer

Selection/reference only

Chemical identity
MES; C6H13NO4S; CAS 4432-31-9; 195.24 g/mol
Manufacturer-recommended useful range
pH 5.5–6.7
Thermodynamic reference pK
pKa / reaction 1: 6.46575 at 4 °C, 6.35058 at 16 °C, 6.27 at 25 °C, 6.2272 at 30 °C, 6.16948 at 37 °C, 6.06859 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 5.27–7.27. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Low UV absorbance; ideal for spectrophotometric assays. Limited buffering above 37 °C due to lower pKa.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Keep 6.10, 6.15, and 6.270 as separately conditioned evidence; do not average or select an undeclared operational model.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Hydrogen Ion Buffers for Biological Research, Biological Buffers Selection Guide

View scientific referenceRead the MES guide

MOPS

Also called: 3-(N-Morpholino)propanesulfonic acid, MOPS buffer

Preparation-enabled

Chemical identity
MOPS; C7H15NO4S; CAS 1132-61-2; 209.26 g/mol
Manufacturer-recommended useful range
pH 6.5–7.9
Thermodynamic reference pK
pKa / reaction 1: 7.46052 at 4 °C, 7.29844 at 16 °C, 7.184 at 25 °C, 7.12285 at 30 °C, 7.03997 at 37 °C, 6.8939 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 6.184–8.184. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks, Titrate

Scientific and preparation caveat: Zwitterionic; compatible with most enzyme assays. Low metal binding and low temperature coefficient.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Hydrogen Ion Buffers for Biological Research, Biological Buffers Selection Guide

Load a signed 50 mM example

PIPES

Also called: Piperazine-N,N′-bis(2-ethanesulfonic acid), PIPES buffer

Selection/reference only

Chemical identity
PIPES; C8H18N2O6S2; CAS 5625-37-6; 302.37 g/mol
Manufacturer-recommended useful range
pH 6.1–7.5
Thermodynamic reference pK
pKa / reaction 1: 7.28653 at 4 °C, 7.20153 at 16 °C, 7.141 at 25 °C, 7.10848 at 30 °C, 7.0642 at 37 °C, 6.98557 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 6.141–8.141. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade ABA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Good near physiological pH with minimal metal binding. Avoid high concentrations with divalent cations due to precipitation risk.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Keep NIST I=0 pK 7.141 separate from finite-ionic-strength observations near 6.7–6.8; no preparation output.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Hydrogen Ion Buffers for Biological Research, Biological Buffers Selection Guide

View scientific reference

MOPSO

Also called: 3-(N-Morpholino)-2-hydroxypropanesulfonic acid, MOPSO buffer

Preparation-enabled

Chemical identity
MOPSO; C7H15NO5S; CAS 68399-77-9; 225.26 g/mol
Manufacturer-recommended useful range
pH 6.2–7.6
Thermodynamic reference pK
pKa / reaction 2: 7.22644 at 4 °C, 7.03538 at 16 °C, 6.9 at 25 °C, 6.82749 at 30 °C, 6.72902 at 37 °C, 6.5549 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 5.9–7.9. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 2, evaluation grade BCB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Mix stocks, Titrate

Scientific and preparation caveat: Good choice for enzyme assays near neutral pH. Low absorbance above 260 nm.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

Load a signed 50 mM example

TES

Also called: N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, TES buffer

Selection/reference only

Chemical identity
TES; C6H15NO6S; CAS 7365-44-8; 229.25 g/mol
Manufacturer-recommended useful range
pH 6.8–8.2
Thermodynamic reference pK
pKa / reaction 1: 7.55 at 25 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 6.55–8.55. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Maintains osmolarity; popular for mammalian cell culture. Temperature coefficient about −0.02 pH/°C.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Do not select between delta_rCp 0 and -15 J K^-1 mol^-1 until the source inconsistency is resolved.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Hydrogen Ion Buffers for Biological Research, Biological Buffers Selection Guide

View scientific reference

TAPS

Also called: N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, TAPS buffer

Selection/reference only

Chemical identity
TAPS; C7H17NO6S; CAS 29915-38-6; 243.28 g/mol
Manufacturer-recommended useful range
pH 7.7–9.1
Thermodynamic reference pK
pKa / reaction 1: 8.97415 at 4 °C, 8.65993 at 16 °C, 8.44 at 25 °C, 8.32316 at 30 °C, 8.16555 at 37 °C, 7.88997 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 7.44–9.44. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade BBB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Low metal binding; alternative to Tris above neutral pH. Slight inhibition of some enzymes—verify compatibility.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: The current catalog and evaluated thermodynamic pK differ by more than 0.03; operational conditions are not declared.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

View scientific reference

EPPS (HEPPS)

Also called: EPPS, HEPPS, N-[2-hydroxyethyl]piperazine-N′-[3-propanesulfonic acid]

Selection/reference only

Chemical identity
HEPPS; C9H20N2O4S; CAS 16052-06-5; 252.33 g/mol
Manufacturer-recommended useful range
pH 7.6–8.6
Thermodynamic reference pK
pKa / reaction 1: 8.23289 at 4 °C, 8.07196 at 16 °C, 7.957 at 25 °C, 7.89511 at 30 °C, 7.8107 at 37 °C, 7.6604 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 6.957–8.957. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Good buffering around pH 8; low UV absorbance. Compatible with many amine-reactive chemistries.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: Preserve both historical IDs and prove identical restoration. Do not copy the NIST C6 formula typo; use the independently supported C9 identity.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

View scientific reference

HEPPSO

Also called: N-(2-Hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid), HEPPSO buffer

Selection/reference only

Chemical identity
HEPPSO; C9H20N2O5S; CAS 68399-78-0; 268.33 g/mol
Historical selection range; not preparation authority
pH 7.4–8.6
Thermodynamic reference pK
pKa / reaction 1: 8.34989 at 4 °C, 8.17007 at 16 °C, 8.042 at 25 °C, 7.97318 at 30 °C, 7.87947 at 37 °C, 7.71304 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 7.042–9.042. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Improved solubility variant of HEPPS; low metal binding. Slightly higher buffering capacity above pH 8.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: The current catalog and evaluated thermodynamic pK differ by more than 0.03; operational conditions are not declared.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

View scientific reference

Tricine

Also called: N-(Tri(hydroxymethyl)methyl)glycine, Tricine buffer

Preparation-enabled

Chemical identity
Tricine; C6H13NO5; CAS 5704-04-1; 179.17 g/mol
Manufacturer-recommended useful range
pH 7.4–8.8
Thermodynamic reference pK
pKa / reaction 2: 8.55899 at 4 °C, 8.30737 at 16 °C, 8.135 at 25 °C, 8.04474 at 30 °C, 7.92449 at 37 °C, 7.71857 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 7.135–9.135. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 2, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Titrate

Scientific and preparation caveat: Chelates divalent metals; supplement with Mg²⁺ when needed. Popular for SDS-PAGE running buffers.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

Load a signed 50 mM example

Bicine

Also called: N,N-Bis(2-hydroxyethyl)glycine, Bicine buffer

Preparation-enabled

Chemical identity
Bicine; C6H13NO4; CAS 150-25-4; 163.17 g/mol
Manufacturer-recommended useful range
pH 7.6–9
Thermodynamic reference pK
pKa / reaction 2: 8.68365 at 4 °C, 8.47763 at 16 °C, 8.334 at 25 °C, 8.25789 at 30 °C, 8.15546 at 37 °C, 7.977 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 2: pH 7.334–9.334. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 2, evaluation grade AAA
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
Titrate

Scientific and preparation caveat: Useful for chromogenic assays; moderate metal complexation. Maintain temperature control due to dpH/dT ≈ −0.015.

Preparation-enabled: Signed Pack 6C decision authorizes only the listed modes using the explicit NIST thermodynamic reference model.

Decision rationale: The evaluated thermodynamic model is supportable as a reference, but practical preparation requires explicit standard-state scope and final-pH verification.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

Load a signed 50 mM example

CHES

Also called: 2-(Cyclohexylamino)ethanesulfonic acid, CHES buffer

Selection/reference only

Chemical identity
CHES; C8H17NO3S; CAS 103-47-9; 207.29 g/mol
Manufacturer-recommended useful range
pH 8.6–10
Thermodynamic reference pK
pKa / reaction 1: 9.91772 at 4 °C, 9.60944 at 16 °C, 9.394 at 25 °C, 9.27965 at 30 °C, 9.12556 at 37 °C, 8.85648 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 8.394–10.394. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAB
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Hydrophobic buffer useful for systems near pH 9–10. Avoid CO₂ uptake; store tightly sealed.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: The current catalog and evaluated thermodynamic pK differ by more than 0.03; operational conditions are not declared.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

View scientific reference

CAPS

Also called: 3-(Cyclohexylamino)-1-propanesulfonic acid, CAPS buffer

Selection/reference only

Chemical identity
CAPS; C9H19NO3S; CAS 1135-40-6; 221.32 g/mol
Manufacturer-recommended useful range
pH 9.7–11.1
Thermodynamic reference pK
pKa / reaction 1: 11.1294 at 4 °C, 10.7599 at 16 °C, 10.499 at 25 °C, 10.3596 at 30 °C, 10.1707 at 37 °C, 9.83768 at 50 °C
Separate theoretical pK ± 1 windows at 25 °C
reaction 1: pH 9.499–11.499. These are reference windows, not preparation ranges.
Ionization reaction
HL± = H+ + L−, reaction 1, evaluation grade AAC
Reference conditions
hypothetical ideal aqueous solution of unit molality, I = 0, 0.1 MPa; Goldberg et al. Equation (12)
Approved preparation modes
None — scientific reference and selection content only

Scientific and preparation caveat: Stable at alkaline pH; common for blotting buffers. Can absorb CO₂; verify pH just before use.

Selection/reference only: Scientific reference and selection content only; the signed Pack 6C decision authorizes no preparation mode.

Decision rationale: The current catalog and evaluated thermodynamic pK differ by more than 0.03; operational conditions are not declared.

Sources: Thermodynamic Quantities for the Ionization Reactions of Buffers, Biological Buffers Selection Guide

View scientific reference

System-specific context

Choosing and preparing common buffers

These guides are limited to systems with enough source support for substantive, system-specific guidance. They explain when a system may fit, what to watch, why it is useful, and what the calculator does not model.

Sodium phosphate buffer

Sodium phosphate uses the H₂PO₄⁻/HPO₄²⁻ conjugate pair for near-neutral buffering.

When to consider it

Consider it when a phosphate system is compatible with the assay and the target pH falls within the catalog range.

What to watch

Phosphate can precipitate with calcium or magnesium, and ionic strength can shift measured pH.

Why it is useful

It is a familiar, water-soluble system with a useful range around neutral pH.

Model limitation

This calculator models sodium phosphate only. It does not calculate phosphate-buffered saline (PBS), which is a multicomponent formulation that also includes salts.

Back to Sodium phosphate (NaH₂PO₄/Na₂HPO₄) data

Citrate buffer

Citrate is a polyprotic system; this calculator uses the catalog-selected pKa₂ pair as a local Henderson–Hasselbalch approximation.

When to consider it

Consider it for acidic targets within the catalog range when citrate is compatible with the sample and downstream chemistry.

What to watch

A single-pKa partition does not model every citrate protonation state, and citrate can bind metal ions.

Why it is useful

Its broad acidic range makes it a common option for formulation and biochemical work.

Model limitation

The output is a pairwise estimate, not a full polyprotic equilibrium or activity-corrected model.

Back to Citrate (Citric acid / Trisodium citrate) data

Acetate buffer

Acetate uses acetic acid and acetate as a monoprotic conjugate pair for acidic pH targets.

When to consider it

Consider it when the target is near the acetic-acid pKa and acetate is compatible with the experiment.

What to watch

Volatility, odor, biological effects, and ionic-strength shifts may matter for the intended workflow.

Why it is useful

The conjugate pair is simple and its useful range covers many acidic preparations.

Model limitation

The calculation assumes concentration ratios approximate activity ratios and does not model matrix effects.

Back to Acetate (Acetic acid / Sodium acetate) data

Imidazole buffer

Imidazole can be protonated with hydrochloric acid to create a buffer pair in the mildly acidic-to-neutral range.

When to consider it

Consider it when imidazole is compatible with the biochemical system and the target falls inside the catalog range.

What to watch

Imidazole can interact with metal-affinity workflows and may not be inert in every protein assay.

Why it is useful

It provides buffering near neutral pH and can be prepared by mixing stocks or controlled titration.

Model limitation

The calculator estimates stoichiometry; it does not prescribe an affinity-purification recipe or vendor protocol.

Back to Imidazole / HCl data

Tris buffer

Tris is a widely used amine buffer whose pKa—and therefore prepared pH—changes appreciably with temperature.

When to consider it

Consider it for targets in its useful range when temperature can be controlled and Tris is compatible with the assay.

What to watch

Prepare and verify pH at the intended working temperature; temperature changes can move the measured pH.

Why it is useful

Tris is familiar, soluble, and available as base and hydrochloride forms.

Model limitation

Goldberg Equation (12) supplies thermodynamic reference pK values at six signed temperatures; the model does not correct a real preparation for ionic strength or activity.

Back to Tris / Tris·HCl data

HEPES buffer

HEPES is a Good's buffer designed for biological work near neutral pH.

When to consider it

Consider it for near-neutral targets when HEPES is compatible with the cells, proteins, detection chemistry, and downstream steps.

What to watch

Light exposure, reactive species, metal interactions, temperature, and assay-specific compatibility may matter.

Why it is useful

Its useful range covers many biological workflows and its temperature coefficient is smaller than Tris.

Model limitation

The calculator does not model photochemistry, metal binding, ionic strength, or activity coefficients.

Back to HEPES data

MES buffer

MES is a Good's buffer with a useful range in the mildly acidic region.

When to consider it

Consider it when the target is around pH 6 and MES is compatible with the biological or analytical workflow.

What to watch

Confirm compatibility with metal-dependent systems and with the intended detection chemistry.

Why it is useful

It offers biological buffering below the neutral range with a documented temperature coefficient.

Model limitation

The output is an idealized conjugate-pair estimate and the current temperature series remains under provenance review.

Back to MES data

Method and equations

The calculator uses the Henderson–Hasselbalch equation for the signed conjugate acid/base pair:

pH = pK(T) + log10([base] / [acid])

ratio = 10^(pH − pK(T))

acid moles = total moles / (1 + ratio)

base moles = total moles × ratio / (1 + ratio)

The thermodynamic pK(T) is evaluated with Goldberg et al. Equation (12) from the signed pK at 298.15 K, reaction enthalpy, and heat-capacity change. Only the signed oracle temperatures 4, 16, 25, 30, 37, and 50 °C are accepted.

Assumptions

  • The NIST thermodynamic reference is a hypothetical ideal aqueous solution of unit molality at zero ionic strength and 0.1 MPa.
  • Concentration ratios are used as practical approximations for activity ratios; calculated values are theoretical starting estimates, not operational pK values.
  • The preparation guardrail is pK(T) ± 1. Manufacturer-recommended useful ranges are displayed separately as selection guidance.
  • Only one explicitly reviewed ionization reaction and one explicitly reviewed preparation model are used per preparation-enabled record.
  • Stock concentrations and exact solid identities must match the user’s actual reagents.

Validation and limitations

  • Selection/reference-only records expose no preparation controls or results.
  • Stock and titration plans are blocked when required liquid volume exceeds final capacity.
  • Tris solid mode is restricted to anhydrous Tris hydrochloride (157.60 g/mol) and anhydrous Tris base (121.14 g/mol); users must verify the bottle label.
  • Ionic strength, activities, matrix composition, counterions, purity, and nonideal equilibria can shift final pH. MOPSO uses an explicitly labeled heat-capacity approximation.
  • The engine does not perform full charge balance, buffer-capacity optimization, mixed-buffer modeling, precipitation prediction, or protocol validation.

Safety and final-pH verification

Confirm the chemical name, salt, hydration state, formula, molecular weight, purity, hazards, and compatibility against the reagent label and your laboratory controls. Add titrants gradually with appropriate PPE and local procedures.

Prepare near final volume, measure pH with a calibrated meter at the working temperature, adjust experimentally, then bring to final volume. This calculation aid does not replace a validated SOP, risk assessment, or instrument calibration.

Coverage decisions and scientific gaps

Not every familiar buffer name is safe to add to a single-pair calculator. The following systems were investigated but intentionally not added in this pack.

Why sodium phosphate is not labeled PBS

The supported phosphate model calculates the NaH₂PO₄/Na₂HPO₄ conjugate pair. Phosphate-buffered saline is a multicomponent formulation that also includes salts and may use different target compositions. The site therefore does not use “PBS” as a synonym and does not present a phosphate pair calculation as a complete PBS recipe.

Scientific references and provenance

References are attached to the fields they support. Preparation-enabled records follow the exact mode matrix in the signed decision. Selection/reference-only records retain thermodynamic and operational evidence for buffer choice but intentionally expose no preparation result.

  1. Henderson–Hasselbalch equation. International Union of Pure and Applied Chemistry (IUPAC), IUPAC Gold Book, 2025. DOI: 10.1351/goldbook.H02781. Used for: equation definition. Accessed 2026-09-01.
  2. Hydrogen Ion Buffers for Biological Research. Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, R. M., Biochemistry, 1966. DOI: 10.1021/bi00866a011. Used for: Good's buffer design context; HEPES and MES scientific background.
  3. Biological Buffers Selection Guide. Merck KGaA / MilliporeSigma, MilliporeSigma, 2026. Used for: useful pH ranges; selected pKa values by temperature; temperature coefficients; buffer identities. Accessed 2026-09-01.
  4. Buffer Reference Center. Merck KGaA / MilliporeSigma, MilliporeSigma, 2026. Used for: selected useful pH ranges; preparation-pair identities; selected stock preparation tables. Accessed 2026-09-01.
  5. Standard Reference Materials: Standardization of pH Measurements. National Bureau of Standards (now National Institute of Standards and Technology), U.S. National Bureau of Standards, Special Publication 260-53, 1977. Used for: standard phosphate compositions; Tris standard composition; temperature and activity context; meter standardization context.
  6. Thermodynamic Quantities for the Ionization Reactions of Buffers. Goldberg, R. N.; Kishore, N.; Lennen, R. M., National Institute of Standards and Technology / Journal of Physical and Chemical Reference Data, 2002. DOI: 10.1063/1.1416902. Used for: chemical identity; ionization reactions; thermodynamic pK; delta rH; delta rCp; evaluation grade; temperature equation; ionic-strength treatment. Accessed 2026-09-01.

Calculation record

Methodology and scientific authority

Generate a theoretical starting estimate for an approved buffer mode or review a selection-only authority record.

Engine
buffer-plan 2.0.0
Content updated

Methodology

Uses the signed NIST thermodynamic reference model, Goldberg Equation (12), and Henderson–Hasselbalch to create theoretical starting estimates only for explicitly approved preparation modes.

Inputs

  • buffer system
  • target pH
  • concentration
  • final volume
  • temperature
  • preparation mode

Outputs

  • acid/base ratio
  • component amounts
  • stock volumes or solid masses
  • theoretical preparation starting estimate with final-pH verification warning

Validation examples

These scientific fixtures are reproducible tests of the calculation engine, not build-only checks.

  • pH equal to pKa produces a 1:1 acid/base ratio. Test: equal-ph-pka
  • A requested final concentration above available stock capacity is rejected. Test: stock-feasibility

Assumptions and limitations

Assumptions

  • Thermodynamic pK values describe a hypothetical ideal aqueous standard state; manufacturer useful ranges remain separate selection guidance.
  • The final pH must be verified under the actual working conditions.

Limitations

  • Activity, ionic strength, matrix composition, and volume non-additivity may make real solutions depart from the ideal model.
  • The plan does not replace a validated buffer recipe or SOP.

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