Henderson-Hasselbalch above gives you the acid:base ratio at one pH. This titrates the whole buffer. Strong acid or base goes in drop by drop, the pH curve draws itself, and the beaker changes colour like universal indicator. The flat stretch is where the buffer is actually working. Drag the dashed ADD line across the curve, or focus it and use the arrow keys, to read any point of the titration. Every control updates the numbers live. Run titration just replays it from zero. Volumes are per 1 litre of buffer.
Select a buffer, enter volume and concentration, get the exact mass to weigh — Sigma-Aldrich (Merck)
Thermodynamically corrected buffer recipes (temperature, ionic strength) — University of Liverpool
Curated buffer recipes by application, with preparation notes — AAT Bioquest
Biopharma buffer formulation is the design and preparation of buffer solutions used across biopharmaceutical manufacturing: upstream cell culture media, downstream purification (chromatography equilibration, wash, elution, regeneration) and final drug product formulation. Typical components are: a buffering species matched to the target pH within ±1 unit of its pKa (phosphate for pH 6–8, acetate for pH 4–5, Tris for pH 7–9, citrate for pH 3–6, HEPES for pH 7–8), a salt for ionic strength (often NaCl 50–500 mM), optional stabilisers (sucrose, trehalose, arginine, polysorbate), and adjustments to reach the exact target pH and conductivity.
Typical biopharma buffer formulations for mAb downstream processing: Protein A equilibration 25 mM Tris, 150 mM NaCl, pH 7.2; Protein A wash 25 mM Tris, 500 mM NaCl, pH 7.2 (optionally with 10–20% ethylene glycol for HCP removal); Protein A elution 100 mM glycine pH 3.5 or 100 mM acetate pH 3.5; low-pH inactivation 100 mM acetate pH 3.5–3.7 (30–60 min hold); CEX equilibration 25 mM acetate pH 5.0; CEX elution linear gradient 50–500 mM NaCl; AEX flow-through 50 mM Tris pH 7.8, 100 mM NaCl; UF/DF formulation buffer 20 mM histidine, 240 mM sucrose, 0.02% polysorbate 20, pH 6.0. Each formulation is validated for the specific molecule and scale.
Open the mode that matches your task. (1) Stock Solution: enter MW, target molarity and volume → returns mass to weigh and water to add. (2) Dilution (C1V1 = C2V2): enter any three of stock conc, stock volume, final conc, final volume → returns the fourth. (3) Henderson-Hasselbalch: choose a buffer species (acetate pKa 4.76, MES 6.15, phosphate 7.20, HEPES 7.55, Tris 8.06, glycine 9.78, CHES 9.5, CAPS 10.4), enter the target pH within ±1 unit of the pKa → returns the [acid]:[base] ratio + mass of each form. (4) Serial Dilution: enter starting conc, dilution factor, number of steps, well volume → returns the full table. (5) Recipe Library: pick from PBS, TBS, TAE, TBE, Tris-HCl, sodium acetate, sodium citrate, HEPES, phosphate buffer, Tris-glycine, RIPA, lysis buffer, ammonium acetate, ammonium bicarbonate, MOPS, Tricine, citrate-phosphate (McIlvaine), or protein-A elution glycine, scaled to any final volume.
(1) Calculate the mass of each solute: mass (g) = Molarity (M) × Volume (L) × MW (g/mol). (2) Dissolve each solute in about 80% of the final volume of Milli-Q water, stirring until fully dissolved. (3) Add adjustment acids/bases (HCl, NaOH) to reach target pH using a calibrated pH meter. (4) Adjust conductivity if critical (measure with conductivity meter, adjust NaCl as needed). (5) Bring to final volume with Milli-Q water. (6) Filter through a 0.2 µm membrane into a sterile container. (7) Record lot numbers, pH, conductivity, osmolality and operator initials in the batch record. Prepare fresh for pH-sensitive applications — many buffers have limited shelf life once filter-sterilised.
Protein A elution buffers exploit acid-induced dissociation. Two formulations dominate biopharma: (1) 100 mM glycine pH 3.0–3.5 (most common, sharp elution peak, well-tolerated viral inactivation hold), or (2) 100 mM sodium acetate pH 3.5 (gentler on labile mAbs, slightly broader peak). Add 1–2 M arginine when aggregation is a concern at low pH. Elute into a tube pre-loaded with 1 M Tris-HCl pH 8.5 (about 10% of the elution volume) to neutralise rapidly to pH 5.0–5.5 within 60 min of low-pH hold. The Henderson-Hasselbalch mode of this calculator can compute the exact glycine free-acid/sodium ratio for any target elution pH between 2.5 and 4.5.
Use the formula: mass (g) = Molarity (M) × Volume (L) × Molecular Weight (g/mol). For example, to make 500 mL of 1 M NaCl (MW = 58.44 g/mol): mass = 1 × 0.5 × 58.44 = 29.22 g. Dissolve 29.22 g NaCl in water and bring to 500 mL final volume.
The Henderson-Hasselbalch equation relates pH to the pKa of a buffer and the ratio of conjugate base to acid: pH = pKa + log([A-]/[HA]). It is used to calculate how much acid and base forms of a buffer to mix to achieve a target pH. The equation works best within ±1 pH unit of the buffer's pKa.
The dilution equation C1×V1 = C2×V2 states that the moles of solute before and after dilution are equal. C1 is the initial concentration, V1 is the volume of stock to use, C2 is the desired final concentration, and V2 is the desired final volume. Solve for any one unknown given the other three.
For biological applications at pH 7.4, phosphate buffer (pKa 7.20) and HEPES (pKa 7.55) are excellent choices. PBS (phosphate-buffered saline) is the most common buffer for cell culture and protein work at physiological pH. HEPES is preferred when phosphate interference is a concern.
For 1 L of 1× PBS (pH 7.4): dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 in ~800 mL distilled water. Adjust pH to 7.4 with HCl if needed, then bring to 1 L final volume. Autoclave or filter-sterilize before use.
A serial dilution is a stepwise dilution of a substance, where each step uses the same dilution factor. It is widely used in bioprocessing for preparing standard curves (e.g., ELISA, Bradford assay), determining MIC values for antibiotics, cell counting, and titer determination. Common dilution factors are 1:2, 1:5, and 1:10.
HEPES (pKa ~7.55) is a zwitterionic Good's buffer with strong buffering across ~pH 6.8–8.2, minimal binding of divalent metals, and small pH shifts on dilution or temperature change, so it suits cell culture and metal-sensitive work. Phosphate (pKa2 ~7.2) is cheap and physiological but precipitates with Ca2+/Mg2+ and shows a larger pH shift with temperature and dilution. Choose HEPES for metal-sensitive or temperature-variable systems, and phosphate where cost and physiological relevance dominate.
Yes for some buffers. Diluting changes ionic strength, which shifts activity coefficients and the effective pKa, so a concentrated stock does not simply match the working-strength pH. Phosphate and citrate are especially prone to this. Always titrate to the target pH at the final working concentration and temperature rather than assuming a concentrate scales linearly.
Because pKa is itself temperature dependent, and for amine buffers the dependence is large. Tris has a dpKa/dT of about −0.028 per °C, so a Tris buffer titrated to pH 7.40 on the bench at 25 °C reads about pH 7.99 in a 4 °C cold room (nearly 0.6 units higher) and about pH 7.06 at 37 °C.
Coefficients per °C for the common biological buffers: HEPES −0.014, MES −0.011, MOPS −0.011, glycine (amine) −0.025, histidine (imidazole) −0.020. Phosphate is nearly flat at −0.0028 for pKa2, and acetate and citrate are effectively temperature independent. The dissociation constant of water shifts too (pKw is 14.94 at 0 °C, 14.00 at 25 °C, 13.53 at 40 °C), so even neutral pH is not 7.00 at every temperature.
Two rules follow. Titrate at the temperature you will actually run at, not at whatever the bench happens to be. And if a step has to move between bench and cold room, phosphate, acetate and citrate survive the transfer far better than Tris. The live titration explorer above computes the 4 °C and 37 °C readings for your own buffer, concentration and target pH.
Buffer capacity β is the amount of strong acid or base needed to move the pH by one unit: β = dCb/dpH, in mol L−1 per pH unit. For a monoprotic buffer β = 2.303 · Ct · α(1 − α), where α is the fraction in the base form. It peaks exactly at the pKa, where α = 0.5, giving βmax = 0.576 · Ct. A 50 mM buffer at its pKa therefore holds about 28.8 mM per pH unit; a 100 mM buffer holds 57.6.
Capacity falls off fast away from the pKa: at pKa ± 1 you keep only about 33% of the peak, which is precisely why the effective buffering range is quoted as pKa ± 1. Capacity also scales linearly with concentration, so doubling the buffer doubles the resistance to drift, and diluting a 10× stock to 1× leaves one tenth the capacity even when the pH is right.
Worked example: spiking 10 mmol/L of NaOH into 50 mM phosphate at pH 7.4 moves the pH by only about 0.44 units. The same spike into unbuffered water takes it to pH 12. That gap is the entire point of the buffer, and the explorer above computes it for whatever you are actually making.