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| Product Name | HEPES Buffer, 1 M, pH 7.4, Sterile-Filtered, Cell Culture Grade |
| Catalog No. | ATR-B-YJL-0201 |
| Description | Ready-to-use 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution at pH 7.4, prepared with ultrapure water and 0.1 um sterile-filtered for cell culture and biochemical applications. HEPES is a zwitterionic organic buffer with a pKa of 7.55 at 20 C (7.31 at 37 C), providing excellent buffering capacity in the physiological pH range (6.8-8.2) without the cytotoxicity and metabolic effects associated with bicarbonate/CO2 and phosphate buffer systems. HEPES is widely used as a supplemental buffer in cell culture media (10-25 mM final) to maintain pH stability during handling outside the CO2 incubator, and as the primary buffer in biochemical and biophysical assays requiring precise pH control. This 1 M stock solution allows convenient addition to media, buffers, and reaction mixtures at the desired working concentration. |
| Intended Use | pH buffering agent for: supplementation of cell culture media (10-25 mM final) to maintain physiological pH during bench-top handling, microscopy, flow cytometry, and cell sorting; primary buffer in enzyme assays, protein purification, and biochemical reactions at pH 6.8-8.2; buffering component for fluorescence microscopy live-cell imaging in CO2-independent medium; electrophysiology recording solutions; NMR and X-ray crystallography samples (HEPES has minimal metal-binding capacity compared to phosphate and Tris); organ and tissue preservation solutions. |
| Principle / Technology | HEPES is a zwitterionic N-substituted amino acid buffer (Good's buffer) designed by Norman Good for biological research. Its pKa of 7.55 at 20 C (7.31 at 37 C) places its maximum buffering capacity in the physiological range. The zwitterionic structure renders it membrane-impermeable, preventing intracellular accumulation and metabolic interference. HEPES does not chelate divalent cations significantly (log K for Mg2+ approximately 0, Ca2+ approximately 0), making it compatible with metal-dependent enzymes and processes. Unlike bicarbonate/CO2 buffers, HEPES maintains pH independently of atmospheric CO2, providing stable pH during open-air handling of cell cultures. |
| Detection Method | Ready-to-use 1 M stock solution; add directly to culture medium or assay buffer at the desired final concentration: typical final concentration 10-25 mM for cell culture supplementation (e.g., add 10-25 mL 1 M HEPES per 1 L medium for 10-25 mM final); typical use 10-100 mM for biochemical assays; mix thoroughly after addition; 1 M stock is approximately 238 mg/mL. |
| Sample Type | 1 M HEPES stock solution; dilute to desired working concentration in cell culture medium, assay buffer, or reaction mixture. |
| Performance Range / Specifications | Concentration: 1.0 M (+/-0.01 M); pH: 7.40 +/-0.05 at 25 C (adjusted with NaOH); pKa: 7.55 at 20 C, 7.48 at 25 C, 7.31 at 37 C (delta pKa/C approximately -0.014); osmolarity contribution at 25 mM: approximately 50 mOsm/L; sterility: 0.1 um filtered; endotoxin: <0.05 EU/mL; heavy metals: Fe <5 ppm, Cu <2 ppm, Pb <2 ppm. |
| Sensitivity / LOD | Not applicable — buffer solution; buffer pH accuracy verified with calibrated pH meter (NIST-traceable standards). |
| Specificity | HEPES specifically buffers in the pH 6.8-8.2 range with maximum capacity at pH 7.55 (pKa). Unlike Tris (pKa 8.07), HEPES does not have significant temperature-dependent pH drift. Unlike phosphate, HEPES does not participate in precipitation reactions with divalent cations. Unlike bicarbonate/CO2, HEPES does not equilibrate with atmospheric CO2. |
| Reaction Conditions / Protocol | Buffering action via protonation/deprotonation of the piperazine nitrogen and sulfonate groups; HEPES is non-reactive and stable under standard biological conditions; HEPES may generate hydrogen peroxide when exposed to light in the presence of riboflavin and other photosensitizers — protect from light for long-term storage. |
| Components / Formulation | 1 M HEPES Buffer, pH 7.40: 238.3 g/L HEPES (free acid, C8H18N2O4S, MW 238.30), adjusted to pH 7.40 +/-0.05 with NaOH, in ultrapure water (cell culture grade, 18.2 MOhm-cm), 0.1 um sterile-filtered. No preservatives or antimicrobials added. |
| Storage Conditions | Store at 2-8 C or RT (15-25 C); protect from light (amber bottle or dark storage); sterile-filtered: use aseptic technique after opening. |
| Shelf Life | 24 months from date of manufacture at RT or 2-8 C protected from light; after opening: 6 months at 2-8 C with aseptic technique. |
| Package Specifications | 100 mL, 500 mL, 1 L. |
| Product Form | Clear, colorless sterile liquid; 1 M aqueous solution. |
| Quality Control | Each lot: pH 7.38-7.42 (25 C, calibrated meter); concentration 0.99-1.01 M (titration); A260 <0.05, A280 <0.05; sterility: bacterial and fungal <0.1 CFU/mL; endotoxin: <0.05 EU/mL; mycoplasma: negative (PCR); cell toxicity: >90% viability of HEK293T after 48 h in DMEM + 10% FBS + 25 mM HEPES; DNase/RNase: not detected; heavy metals: within specification. |
| Key Features | 1 M ready-to-use sterile stock; pH 7.40 for physiological applications; Good's buffer — minimal metal chelation; membrane impermeable; cell culture grade; endotoxin <0.05 EU/mL; maintains pH independent of CO2 atmosphere; 0.1 um sterile filtered. |
| Purity | HEPES: >=99.5% purity (anhydrous assay); water: ultrapure (18.2 MOhm-cm); no preservatives; heavy metals within specification. |
| Concentration | 1.0 M (+/-0.01 M); typical working concentration: 10-25 mM cell culture, 10-100 mM biochemical assays. |
| Activity / Unit Definition | Not applicable — buffer solution. |
| Molecular Weight | HEPES free acid: 238.30 g/mol; HEPES sodium salt: 260.29 g/mol; 1 M solution contains 238.3 g/L free acid (pH adjusted with NaOH, introducing Na+ counterions). |
| Source / Origin | HEPES: synthetic (chemical synthesis from piperazine and ethylene oxide-derived reagents); NaOH: synthetic (chloralkali process); water: ultrapure deionized; no animal-derived components. |
| pH Range / Optimal pH | pKa 7.55 at 20 C, 7.48 at 25 C, 7.31 at 37 C; effective buffering range pH 6.8-8.2 (pKa +/-0.7); working pH 7.4. |
| Shipping Conditions | Ambient temperature. |
| Expiration Date / Stability | 24 months at RT protected from light; 24 months at 2-8 C protected from light; HEPES is chemically stable but may develop microbial growth if contaminated — use aseptic technique; if discoloration occurs (yellowing), discard (indicates oxidative degradation). |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. Cell culture grade but not GMP-certified for clinical manufacturing. |
| Compatibility | Compatible with all common cell culture media (DMEM, RPMI 1640, MEM, Ham's F-12, IMDM, Neurobasal). Compatible with serum, growth factors, cytokines, and antibiotics at standard concentrations. Compatible with enzymatic assays: low metal-binding capacity makes it suitable for kinase, phosphatase, and protease assays with Mg2+, Mn2+, Ca2+ requirements. Compatible with fluorescence-based assays: HEPES has negligible absorbance >240 nm and negligible fluorescence, making it suitable for UV and fluorescence detection. Compatible with BCA and Bradford protein assays. Incompatible with: oxidative reactions involving Fenton chemistry (HEPES is a weak radical scavenger — avoid in hydroxyl radical studies); photodynamic therapy studies (HEPES can generate H2O2 under light in the presence of riboflavin). |
| Recommended Buffer System | 1 M HEPES, pH 7.40 (NaOH-adjusted); pKa 7.55 at 20 C; buffering range pH 6.8-8.2. |
| Application Notes / Precautions | When supplementing cell culture media with HEPES: the typical final concentration is 10-25 mM. For a 500 mL bottle of complete medium, add 5-12.5 mL 1 M HEPES. HEPES adds approximately 2 mOsm/L per mM, so 25 mM HEPES contributes approximately 50 mOsm/L — account for this when calculating final medium osmolarity. HEPES-buffered medium maintains pH for 2-4 hours of bench-top handling in ambient air, making it essential for flow cytometry, cell sorting, time-lapse microscopy, and microinjection. HEPES can generate hydrogen peroxide when exposed to ambient light for extended periods; for long-term incubations (>24 h at 37 C), supplement with 50 ug/mL catalase or wrap tubes in foil. HEPES may interfere with Lowry protein assays (copper-HEPES complex formation); use BCA or Bradford assay instead. For electrophysiology, HEPES solutions should be prepared fresh weekly and stored at 2-8 C. HEPES is not metabolized by mammalian cells, unlike some alternative buffers. |
| Batch-to-Batch Consistency | pH 7.38-7.42 for every lot; concentration 0.99-1.01 M; cell toxicity: >90% viability confirmed; endotoxin <0.05 EU/mL. |
For research use only, not for clinical use.
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