A buffer solution of HEPES can be prepared by any of several methods. The free acid can be added to water, then titrated with approximately one-half mole equivalent of sodium hydroxide or potassium hydroxide to the desired pH. A simple mixing table for preparing 0.05 M HEPES/NaOH has been published. Alternatively, equimolar concentrations of HEPES and of HEPES sodium salt can be mixed in approximately equal volumes and back-titrate with either solution to the appropriate pH. The sodium salt can be titrated with HCl to yield a half-equivalent of sodium chloride; however, the addition of the ionic strength will change the osmolality of the solution. Most solutions of HEPES hemisodium will dissolve in water forming a pH at 7.5 with little to no adjustment.
HEPES buffer commonly used in cell culture media. The addition of 10–25 mM HEPES provides extra buffering capacity when cell culture requires extended periods of manipulation outside of a CO2 incubator. HEPES is a good buffering choice for many cell culture systems because it is membrane impermeable, has limited effect on biochemical reactions, is chemically and enzymatically stable, and has very low visible and UV light absorbance. HEPES is the buffer of choice in a protein deposition technique in electron microscopy because it did not affect metal substrates. HEPES has been evaluated and shown to be suitable for use with Ampholines in generating pH gradients less than 1 pH unit wide for isolectric focusing applications. HEPES is the recommended buffer for the glutamate binding assay because it prevents binding to non-receptor materials.
HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid) is a general purpose zwitterionic organic chemical buffering agent which does not bind magnesium, calcium, manganese(II) or copper (II) ions.
Buffer strength for cell culture applications is usually in the range of 10 to 25 mM. Studies have indicated that 20 mM HEPES is the most satisfactory concentration of the buffer when both Hanks' and Earle's solutions are used.
Grade: Cell Culture Grade
Application Areas: Cell culture
Product Type: Reagents
Presentation: Clear, Colorless Solution
Formulation: HEPES: 238300.00 mg/liter
pH: 6.8 to 8.2
pKa: pKa1: ~3 pKa2 at 0°C: 7.85 pKa2 at 20°C: 7.55 pKa2 at 37°C: 7.31(Lit.)
Foreign activity: Mycoplasma free, Endotoxin ≤0.25 EU/mL
NOTES: Osmolality 1000 - 1700 mM/kg, ≤0.025 ng/mL, ΔpK/ΔT: -0.014/°C3
Solubility: Soluble at 1% solution in water at 25°C. A 1 M solution is clear and colorless. At 0°C, a saturated solution of the free acid is reportedly 2.25 M.
Sterilization of Solutions: Solutions may be autoclaved under standard conditions.
Storage & Handling: Store at Room Temperature (15-30°C). HEPES is light sensitive when added to media. Solutions should be stored in the dark to avoid possible toxic effects.