2-[4-(2-Hydroxyethyl)-1-Piperazinyl]ethanesulfonic acid, best known to scientists as HEPES, first made its way into lab work during research efforts in the 1960s. Chemists, always on the hunt for improved buffer systems that keep pH steady in experiments, found earlier buffers lacking, either due to instability or toxicity. HEPES was a welcome breakthrough, thanks in part to the work of Norman Good and his team. Back then, life science research shifted gears and demanded tools that wouldn’t interfere with cell growth or protein purification. HEPES answered this call. As research demands picked up through the decades, HEPES stuck around, proving its value every time biochemists reached for a buffer that worked in the physiological pH range.
Walk through the aisles of any scientific supply company, and you’ll see HEPES—white or off-white powder, available in several package sizes. Its main draw among lab workers is how reliably it keeps solutions at the right pH, holding tight between pH 6.8 and 8.2, even if you have to add other chemicals or if temperature shifts during work. Its taste for neutrality ensures research doesn’t get hijacked by pH changes. HEPES packs a punch thanks to its low toxicity and non-interference with biological reactions, which makes it a favorite for sensitive applications like cell culture work.
HEPES comes as a solid powder, dissolving easily in water to make clear solutions. Its molecular weight clocks in at 238.3 g/mol. Chemists enjoy its low UV absorbance, so it doesn’t mess with spectrophotometric measurements. At room temperature, it stays stable, resisting breakdown. Unlike many similar compounds, it holds steady as temperatures rise to body heat or fall near freezing. Its pKa hovers around 7.5 at 25°C. That makes it slot perfectly into protocols where simulated physiological conditions matter, like when growing animal cells or running enzyme tests. Thanks to the sulfonic acid group, HEPES behaves as a zwitterion in solution, not carrying a net charge, and steering clear of cell membranes, unlike other buffers that might build up inside cells.
Lab workers expect full traceability and clear information. Companies selling HEPES provide thorough details, listing chemical purity, water content, and heavy metal limits right on the packaging. Certificates of analysis come standard, showing batch-specific results. Researchers look for powder that offers at least 99% purity; lower grades risk introducing unwanted reactions. The chemical’s shelf life, batch number, and supplier contact all find a spot on the label. Storage guidelines steer clear of moisture or extreme temperatures, so the powder performs predictably, even after months on the shelf.
Manufacturing HEPES starts with piperazine, a cyclic amine, undergoing alkylation to tack on an ethanol group. Follow that up with a sulfonation reaction that introduces the ethanesulfonic acid side chain. Filtration and multiple rounds of crystallization help dial up purity. Each step in production needs watching—residual solvents, incomplete conversion, and byproducts get flushed out with rigorous quality control. Big industrial plants run sophisticated purification, but even small-batch synthesis in university labs follows this roadmap, only scaled down.
HEPES holds up to most reaction conditions in biochemistry labs. That resilience means scientists can tweak the buffer’s properties by swapping in isotopes for experimental tracing or by linking fluorescent groups for cell imaging studies. For protein labeling, biochemists sometimes attach tags via amide bond formation at the amine moiety. Under strong acid or base, the piperazine ring can open, but most standard reaction conditions don’t faze this compound. HEPES doesn’t chelate metals as strongly as other buffers, so it won’t yank ions away from enzyme active sites, protecting data from strange interference.
Buyers might stumble across HEPES under more formal names: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), or just N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid). Commercial listings keep things clearer with the plain “HEPES.” The consistency helps researchers stick with the right product, minimizing mix-ups in shared lab spaces.
Solid HEPES doesn’t come with major toxicity, but good lab practice keeps people safe. Gloves and goggles do the trick during weighing and mixing. Though the powder doesn’t throw off harmful fumes, scientists still work with it in ventilated spaces. If the compound lands on the skin, washing with soap and water removes it. HEPES solutions dispose of down standard drains provided local wastewater rules don’t forbid it, since the buffer doesn’t persist in the environment or show acute hazard to aquatic life at typical concentrations. Long-term exposure studies on lab animals haven’t linked the compound to cancer or reproductive harm. Safety data sheets spell out handling, cleanup, and emergency response, making it easy to bring new technicians up to speed.
Anywhere pH matters, HEPES comes into play. In my own work with mammalian cell lines, cell health fell apart any time we swapped out HEPES for a less sturdy buffer—acid built up, cells struggled, and costly experiments had to start over. HEPES stands guard in enzyme assays, letting proteins churn out products without interference from buffer breakdown products. Electrophysiology, fluorescence microscopy, and tissue engineering protocols lean on its stable character. Vaccine researchers lean on it, too, since its presence in culture media makes sure results aren’t skewed by CO2 build-up. In diagnostics and molecular biology, HEPES buffers show up in PCR master mixes, helping enzymes amplify DNA without missing a beat.
Through the years, research on HEPES keeps turning up new uses. Molecular diagnostics, high-throughput screening, and bioprinting each draw on its properties. Analytical chemists probe HEPES-modified surfaces for biosensors. Some groups investigate custom derivatives to tailor buffering ranges or attach bioconjugates with site specificity. As automation and miniaturization push the demands for chemical stability higher, more industries look to tweak HEPES or combine it with other stabilizers. Modern labs devote significant effort to validate their buffers, and many publish side-by-side studies scouting for hidden effects on data reproducibility.
Researchers run standard toxicity panels before using any reagent, and HEPES passes most with flying colors. Animal studies, both acute and chronic, offer little evidence that HEPES disrupts organ function. It doesn’t build up inside tissues, and urine eliminates most of it. Some cell lines show sensitivity if concentrations climb above typical levels, but the vast majority tolerate HEPES just fine. In my daily work, I’ve watched undisturbed growth even as cell cultures spent weeks in HEPES-based buffers. Even so, researchers always recheck toxicity in specific applications, since cell type and protocol tweak the buffer’s behavior.
Demand for HEPES isn’t slowing down, as labs worldwide ramp up work on stem cells, advanced therapies, and biosensors. As cell therapies and personalized medicine get more sophisticated, regulatory guidelines tighten. Manufacturers invest in cleaner synthesis and better documentation to keep pace. Automation calls for even stricter reproducibility—and buffers like HEPES make that possible. Conversations in trade journals hint at opportunities for even better derivatives: compounds that work at more extreme pH, or that avoid any residual impact on metabolic pathways. Sustainability also pushes innovators to look at bio-based synthesis routes or green chemistry approaches for large-scale manufacture. As more scientific fields converge, the place of HEPES expands—anchoring research now, with eyes on technical evolution.
A lot of experiments look simple from the outside. You pipette a little here, add a bit over there, and expect an answer at the end. But real work faces problems hiding out of sight. pH is one of them. Small changes can wreck months of progress, especially in biology. Cells don’t appreciate surprises. Bacteria freak out if the pH drifts by even half a notch—suddenly nothing grows the way you need. That’s why so many labs turn to HEPES. I’ve watched scientists rely on it time and again. HEPES works as a buffer, keeping pH stable, and that job is tougher than it sounds.
Sure, there are a bunch of buffers out there—Tris, phosphate, bicarbonate. But HEPES became famous for a reason. Research keeps chipping away at the surface and finds HEPES can do what others can’t. Take cell culture, for example. Carbon dioxide shifts how most buffers perform. But HEPES holds the line. Media prepped with HEPES won’t start sliding in pH just because you pull the flask out for a few minutes. So, if you forget to close the incubator door or spend a little too long observing under the microscope, the conditions will still stay the same.
I’ve seen HEPES shine in biochemistry labs and cell biology spaces. Trying to run enzyme assays and needing the results to mean something? HEPES won’t react with most enzymes and won’t pull tricks on you by introducing artifacts. It’s colorless, doesn’t soak up light in UV, and won’t mess with optical readings. That makes it a favorite in places running protein purification, studying enzyme behaviors, or running fluorescence-based measurements. DNA, RNA, proteins—all play nicer in HEPES than in many alternatives. Even electrophysiology thrives on it. In patch-clamp studies, the tiny currents measured get thrown off by unstable pH, but HEPES helps shield those experiments from swings that would otherwise send folks back to square one.
Not every chemical that works well in the lab is easy on people or on the ground it spills onto. Researchers trust HEPES partly because it’s less toxic than many other buffers. No heavy metal worries, no complicated hazardous waste streams if you’re careful. HEPES isn’t edible, but accidental contact won’t lock down a whole workspace for decontamination. Everyone can focus on what matters—the science.
A stable pH means clearer results and repeated success. Breakthroughs build on hundreds of regular days when nothing goes wrong. HEPES quietly powers those days. From genetics research to cancer studies and even industrial biomanufacturing, the same lesson holds: predictable pH is a building block for discovery. Labs looking to save money or reduce complexity still keep a stash of HEPES for the projects that demand consistency above all.
Science needs reliability, and HEPES delivers just that. Some people debate newer buffer systems or seek cheaper alternatives, but time and again, they circles back for the peace of mind HEPES brings. Sure, not every experiment has to use the gold standard. But for those that do, choosing HEPES often means fewer headaches, more robust data, and smoother progress.
Anyone who’s spent time in a lab knows the shelf above the bench fills up fast. Glass bottles compete with boxes of pipette tips, each with its job. In all this organized chaos, buffer solutions like HEPES tend to blend in. But behind that unassuming bottle sits a backbone for countless assays and experiments. HEPES—short for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid—gains fans in biology labs because it keeps pH steady over a wide range. It’s the kind of dependability every researcher hopes for in a buffer.
The label suggests storing HEPES at room temperature, dry and out of direct sunlight. That simple. Strict refrigeration isn’t required. What’s more important is keeping the buffer away from moisture. I made the mistake of storing a half-used container near the sink once. After a few weeks, the powder began to clump. Whenever you open a bottle, never leave it exposed to air for too long. Recap quickly, and store it on a stable surface—preferably not that wobbly metal cart every lab seems to have.
Stability makes HEPES valuable. It doesn’t break down or oxidize fast at room temperature, but direct sun can cause degradation. UV light triggers slow decomposition. So, keep the bottle in a cabinet or drawer. Refrigerators may sound good in theory, especially for folks used to babying certain enzymes. Yet, HEPES doesn’t require those cold temperatures. Cold storage won’t hurt it, but in practice, opening a chilly bottle introduces condensation and moisture each time. That triggers clumping or in worst cases, microbial contamination.
I always use high-purity water—ultrapure or distilled—when making HEPES solutions. Impurities present in tap water can cause cloudiness, especially when mixing at higher concentrations. Once in solution, HEPES stands strong for weeks at 4°C in a fridge, sealed tightly. It fights off oxidation reasonably well. Over time, sometimes the slightest yellow tint can form. That points to slow breakdown, likely from light or contaminant exposure. A tinted solution signals it’s time to whip up a fresh batch.
If you plan to sterilize HEPES by autoclaving, it holds up at 121°C for short durations. Still, filter sterilization (using a 0.22 µm filter) has always given me better peace of mind. It also avoids the odd caramel smell that autoclaving can bring. After preparation, aliquoting the solution prevents recurrent opening and contamination. Label each aliquot with date and concentration. It sounds basic, but those details save headaches weeks later.
A buffer can pick up contamination if left open or handled carelessly. Fungi, bacteria, or even small bugs aren’t strangers in shared spaces. Mold growth on buffer solutions ruined one of my controls a few years ago, forcing me to backtrack on two weeks’ work. Single-use aliquots prevent this drama. If you suspect any contamination, dump it—never risk cross-contaminating precious samples.
Peer-reviewed studies and manufacturer guidelines echo the basics: dry, room temperature, with a tight lid and no direct light. Organic chemistry texts and product data sheets all mirror these steps. I’ve checked with Sigma and Thermo reference materials. Nobody demands cold storage, and nobody recommends sunlight. Out in the real world, that translates to a powerfully simple message: keep it dry, stop the light, seal the bottle, use clean water, and prepare smaller batches.
Lab folks seem to swear by HEPES. Carrying out cell culture or protein work gets much easier with a buffer that keeps pH steady, even if carbon dioxide wavers or samples spend time outside the incubator. Many protocols even recommend it over classic choices like phosphate buffer. I understand why people reach for it so often—the pH stability really does seem unbeatable for sensitive biological samples.
It's normal to wonder if that level of convenience brings downsides. HEPES shows up in all sorts of cell culture recipes. People rarely mention toxicity issues within the standard range—concentrations between 10 and 25 millimolars seem fine for most mammalian cells in practice. Some experiments even use more. But not all cell types react the same. Stem cells, primary neurons, and other delicate cultures can show subtle changes in function if the buffer concentration climbs higher. I've seen neurons lose certain branching patterns in HEPES media, though growth rate looked normal at first glance.
Researchers have flagged another risk. HEPES can become toxic under bright, strong light—the sort used in fluorescence imaging or certain types of microscopy. Blue or UV exposure can break HEPES down and lead to hydrogen peroxide formation. If the cells rely on antioxidant defenses already, this extra oxidative stress disrupts cellular health. Some studies show DNA damage, fixable by catalase or by swapping HEPES for a gentler buffer like bicarbonate. For light-driven protocols, using HEPES without considering phototoxicity risks leads to skewed data or dying cultures.
Toxicity questions don't stop with cell lines. Animal experiments using HEPES for buffer injections or perfusions also face concerns. Injected in moderate amounts, HEPES usually clears out of a rodent's body within a few hours. But sudden or excessive exposure to the buffer's ion load can drive kidney stress, urinary acid-base shifts, or even acute organ injury. My colleagues who do in vivo work now avoid high doses or long-term infusions, instead choosing more physiological buffers.
The environment also feels effects, even if the impacts show up less dramatically. Laboratory discharge with HEPES residue washes down drains into municipal treatment systems. Unlike simple saline, complex chemicals resist quick breakdown, adding to the challenge of environmental management for research facilities.
Careful concentration control remains the best approach. Dialing in the minimum effective HEPES level for a given cell type, and checking for subtle toxicity in initial trials, helps avoid hidden pitfalls. Limiting buffer exposure during strong light microscopy protects sensitive cultures from oxidative bursts. Some researchers switch to alternatives like BES or MOPS where possible. Adding antioxidants such as catalase in imaging experiments limits hydrogen peroxide accumulation.
On the animal side, using the lowest effective dose and checking osmolarity or acid-base changes reduces acute organ strain. Water treatment systems for labs can add filtration steps to catch complex chemical residues before release.
HEPES simplifies many experiments—but recognizing its risks keeps cells, animals, and the surrounding world a lot safer.
HEPES shows up all over biology labs. Its job? Keeping the pH steady so cells and molecules don’t flip out. Some buffers handle acids well but fall apart with bases, or the other way around. HEPES hangs tight near neutral pH; it doesn’t mess with most biological reactions. Scientists worry about tiny shifts in pH changing results. That’s where HEPES comes in with its solid performance between pH 6.8 and 8.2. It has a pKa of 7.5 at room temperature, which puts it near the sweet spot for most mammalian work.
I spent grad school prepping more liters of HEPES than I can count. The steps barely changed. Start by adding the right amount of HEPES powder to clean water. For 1 liter of 1M HEPES, that’s 238.3 grams. Use a glass beaker, not a plastic one, since HEPES can leave residues. Stir it well to dissolve. Don’t rely on swirling — use a magnetic stirrer for full mixing. Lots of us forget HEPES powder sometimes clumps and takes time to fully go into solution if you’re impatient.
Once dissolved, the real work begins: adjusting the pH. Here’s the thing — HEPES itself is acidic. To hit the usual pH 7.2-7.4, add sodium hydroxide. Drop in NaOH slowly, using a freshly calibrated pH meter. Overshooting the target wastes time and can make you start over. Some folks rush and dump in base too fast, which heats the mix and gives off weird readings. Patience pays off here. Once the pH is right, top up to the volume you want with more distilled water. For cell work, don’t skip filtering through a 0.22-micron filter. Dust, bacteria, or fungi can sneak in at any step and ruin batches or experiments later.
People pick HEPES because it doesn’t interact with most metal ions. Tris buffer, which some labs still use, can mess with enzyme activity and give unexpected results. HEPES holds up under heavy laboratory use. Heat or light don’t break it down easily. In one immunology project, we saw that HEPES carried cells through an afternoon in the incubator where other buffers would have drifted off target. The focus stays on your experiment, not fussy chemistry.
HEPES costs more than cheap buffers—something labs on a budget notice. Still, weighing low cost against the risk of ruined cell culture means most groups bite the bullet and buy it. There are reports of HEPES forming toxic breakdown products under strong ultraviolet light. Overexposed cultures can show weird toxicity, which reminds us to keep solutions clear of direct UV and to label bottles with the prep date. Routine practices—like aliquoting and not leaving buffer open for days—help keep everything safe.
Automation in buffer prep helps where high-quality batches matter. pH meters connected to dosing pumps speed things along, cut down on mistakes, and let researchers focus on experiments, not babysitting solutions for an hour. For smaller labs, just practicing careful weighing and pH monitoring will avoid problems.
Most research can’t shrug off pH problems. Reliable HEPES buffer is a foundation for reproducible science. Taking shortcuts shows up in bad data sooner or later. As experiments get more complex, small technical steps like buffer prep decide whether results stand up or just vanish on repeat. Respecting the basics goes a long way, and HEPES lets scientists focus on the biology instead of firefighting chemistry mishaps.
HEPES finds a natural place in many science labs where cell cultures line incubators. Its main draw comes from its capacity to stabilize pH better than the chalky old sodium bicarbonate buffer, especially outside of the incubator environment. In a personal stint working with sensitive neuronal lines, short periods outside the CO2 incubator used to raise all kinds of problems: pH drops, cells stressing out, and experiments unraveling. With HEPES in the media, those issues all but disappeared. The difference can be obvious to any scientist juggling time between the microscope and the tissue culture hood.
Cells won’t tolerate too much fluctuation in pH. Small changes lead to stress, strange morphology, and unreliable results. HEPES holds the line within a tight pH window (about 7.2–7.6), even if the lid isn’t shut all day. That quality isn’t a luxury for cell culture work; it's a baseline necessity for success. Cultures that see frequent handling outside of CO2 control often rely on HEPES for this reason. Some researchers favor HEPES for imaging work, as it doesn’t fizz off CO2 or shift color while sitting on a microscope stage.
Not every buffer pairs smoothly with others, and mixing HEPES with sodium bicarbonate has trade-offs. Together, both can be used, but there's a sweet spot. Adding too much HEPES leads to an unnecessary chemical load for cells. Combining both often means lowering the amount of each, not doubling up. High HEPES concentrations might even become toxic. Careful balancing and routine pH checks protect the culture from disaster.
In media that already include sodium bicarbonate, HEPES can still serve for short-term experiments outside of CO2 ovens. That said, several textbooks and journals recommend not pushing much past 10–25 mM HEPES. Reason: metabolic by-products can build up faster than in regular buffered medium. Excess HEPES also raises concerns during autoclaving, where it breaks down and can form cytotoxic compounds.
Peer-reviewed work supports the use of HEPES for cell lines like HEK293, CHO, and several primary cells. Many labs post detailed protocols mentioning HEPES concentrations and why they use it over other buffers. A study in PLoS ONE (2017) highlighted how HEPES kept pH stable for fibroblasts during extended imaging without CO2. The data show improved morphology and survival. That kind of evidence brings confidence for those considering HEPES for tricky culture work.
Sticking to guided concentrations and keeping track of what’s in the media matters a lot. Use a reliable source and double-check HEPES pH after dissolving. Avoid routine autoclaving and filter-sterilize instead. Watch for unwanted side effects if mixing with other buffers or adding extra supplements. Cells speak quickly through their appearance and growth, so checking cultures after changes in buffer type is just smart science.
As cell culture work moves toward higher standards, more peer sharing, and reproducibility, buffer choices like this get more attention. With the right vigilance, HEPES stands out as a buffer that offers more control and reliability without a mystery factor—so long as one respects its limits.
![2-[4-(2-Hydroxyethyl)-1-Piperazinyl]Ethanesulfonic Acid](/static/81c4c90b-3bfe-4770-bbec-fcbf5e0a3809/images/2d/2-4-2-hydroxyethyl-1-piperazinyl-ethanesulfonic-acid.png)
![2-[4-(2-Hydroxyethyl)-1-Piperazinyl]Ethanesulfonic Acid](/static/81c4c90b-3bfe-4770-bbec-fcbf5e0a3809/images/3d/2-4-2-hydroxyethyl-1-piperazinyl-ethanesulfonic-acid.gif)
| Names | |
| Preferred IUPAC name | 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid |
| Other names |
HEPES N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) Hepes acid 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid |
| Pronunciation | /tuː fɔːr tuː haɪˌdrɒksiˈɛθɪl wʌn pɪpəˈreɪzɪnɪl ˈɛθeɪnˌsʌlfɒnɪk ˈæsɪd/ |
| Identifiers | |
| CAS Number | 7365-45-9 |
| Beilstein Reference | 1721399 |
| ChEBI | CHEBI:39028 |
| ChEMBL | CHEMBL1236 |
| ChemSpider | 21234 |
| DrugBank | DB03754 |
| ECHA InfoCard | 17e4e832-cb50-46fa-a175-8d46b2d74b7b |
| EC Number | EC 252-529-6 |
| Gmelin Reference | 82287 |
| KEGG | C00059 |
| MeSH | D010615 |
| PubChem CID | 85881 |
| RTECS number | RH0410000 |
| UNII | YDJ94O6H6U |
| UN number | UN2811 |
| CompTox Dashboard (EPA) | DTXSID2023568 |
| Properties | |
| Chemical formula | C8H18N2O4S |
| Molar mass | 260.33 g/mol |
| Appearance | White crystalline powder |
| Odor | Odorless |
| Density | 1.05 g/cm³ |
| Solubility in water | freely soluble |
| log P | -1.85 |
| Vapor pressure | <0.01 hPa (20 °C) |
| Acidity (pKa) | 7.5 |
| Basicity (pKb) | 7.55 |
| Magnetic susceptibility (χ) | -7.24e-6 |
| Refractive index (nD) | 1.512 |
| Dipole moment | 3.16 D |
| Thermochemistry | |
| Std molar entropy (S⦵298) | 324 J·mol⁻¹·K⁻¹ |
| Std enthalpy of formation (ΔfH⦵298) | -1196.5 kJ/mol |
| Std enthalpy of combustion (ΔcH⦵298) | -1907 kJ·mol⁻¹ |
| Hazards | |
| Main hazards | Causes serious eye irritation. |
| GHS labelling | GHS07 |
| Pictograms | GHS07 |
| Signal word | Warning |
| Hazard statements | H319: Causes serious eye irritation. |
| Precautionary statements | P264, P270, P301+P312, P330, P501 |
| NFPA 704 (fire diamond) | 1-1-0 |
| Flash point | > 230 °C |
| LD50 (median dose) | LD50 (median dose): >10,000 mg/kg (oral, rat) |
| NIOSH | MGV19910 |
| PEL (Permissible) | PEL (Permissible Exposure Limit) for 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid: Not established |
| REL (Recommended) | 50 mg/m³ |
| IDLH (Immediate danger) | Unknown |
| Related compounds | |
| Related compounds |
ACES BES CHES HEPES MES MOPS PIPES TES Tricine |
