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MOPS

MOPS (3-(N-morpholino)propanesulfonic acid) is a zwitterionic buffering agent used in molecular biology and biochemistry. It is one of the twenty Good's buffers developed in the 1960s to provide alternatives to traditional buffers like phosphate and Tris for biological research. With a chemical formula of C7H15NO4S and a pKa of 7.20 at 20 °C, MOPS is effective for maintaining pH in the range of 6.5–7.9, making it suitable for applications such as cell culture, electrophoresis, and enzyme assays at near-neutral pH. It is a structural analog of MES buffer, featuring a morpholine ring.) MOPS is not recommended for use above 20 mM concentration in mammalian cell culture due to potential toxicity.

Chemical Properties

Molecular Structure and Formula

MOPS, formally known as 3-(N-morpholino)propanesulfonic acid, is one of the zwitterionic buffers introduced in the series developed by Norman E. Good and colleagues in the to address limitations in biological buffering agents. Its systematic IUPAC name is 3-morpholin-4-ylpropane-1-sulfonic acid. The molecular formula is \ce{C7H15NO4S}, corresponding to a molecular weight of 209.26 g/mol. The core structure features a ring—a saturated six-membered heterocycle with a nitrogen atom opposite an oxygen atom—covalently linked through the nitrogen to a chain (-\ce{CH2-CH2-CH2}-). This chain ends with a functional group (-\ce{SO3H}), characterized by a atom bonded to three oxygen atoms, including a hydroxyl and two with double bonds. The nitrogen in the enables (\ce{N -> NH+}), which, in conjunction with the strongly acidic sulfonic group that readily deprotonates to \ce{SO3-}, imparts the zwitterionic nature essential for its buffering properties. Compared to the related buffer MES (2-(N-morpholino)ethanesulfonic acid, \ce{C6H13NO4S}), MOPS differs by incorporating an additional \ce{CH2} unit in the alkyl chain, extending it from two to three carbons while retaining the morpholine and sulfonic acid moieties. This structural extension influences the molecule's conformational flexibility and solubility characteristics.

Physical Characteristics

MOPS is typically observed as a to off-white crystalline powder, which is and free-flowing in its pure form. This appearance facilitates its handling in settings, where it is commonly supplied at high purity levels exceeding 99%. The compound exhibits high in , approximately 600 g/L at 20°C, allowing for the preparation of concentrated solutions without precipitation under standard conditions. In contrast, it shows limited solubility in organic solvents, being sparingly soluble in and acetone, which limits its use in non-aqueous environments. Its melting point is approximately 277–282°C. MOPS demonstrates chemical stability under normal laboratory conditions, remaining unaffected by typical ambient temperatures and pressures, and it does not form complexes with common metal ions such as , , or . It is non-hygroscopic, with low (typically ≤0.1 at 250 nm and ≤0.2 at 260–280 nm for 1 M aqueous solutions), making it suitable for spectroscopic applications without interference. For optimal preservation, storage is recommended in a , environment at to minimize any potential degradation over time.

Acid-Base Behavior

MOPS acts as a zwitterionic , with its group fully dissociated at physiological due to its strong acidity (pKa ≈ -2), while the buffering action arises from the of the nitrogen, which behaves as a monoprotic acid with = 7.20 at 20°C. This allows MOPS to maintain stability near neutral values, governed by the Henderson-Hasselbalch : \text{pH} = \text{p}K_\text{a} + \log_{10}\left(\frac{[\text{A}^-]}{[\text{HA}]}\right), where [\text{A}^-] represents the deprotonated (zwitterionic) form and [\text{HA}] the protonated form. The effective buffering range of MOPS spans 6.5–7.9, with optimal performance around the physiological range of 7.0–7.4, where the concentrations of protonated and deprotonated forms are roughly equal at = . This range makes MOPS particularly suitable for applications requiring precise control near neutral without significant interference from the buffer itself. The of MOPS exhibits a temperature dependence of Δ ≈ -0.011 per °C, resulting in a modest decrease in buffering capacity at elevated temperatures typical of biological systems. Additionally, the remains stable across moderate ionic strengths (up to 0.5 M), as the zwitterionic structure minimizes electrostatic interactions that could otherwise shift the . MOPS demonstrates minimal chelation of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, with binding constants below detectable levels at physiological concentrations, ensuring it does not disrupt metal-dependent processes.

Preparation and Synthesis

Laboratory Synthesis

MOPS, or 3-(N-morpholino)propanesulfonic acid, is synthesized in the laboratory primarily through the nucleophilic ring-opening reaction of morpholine with 1,3-propanesultone, a method that forms the zwitterionic sulfonic acid structure efficiently. This approach was developed in the 1960s as part of the Good's buffer series by Norman E. Good and colleagues, who sought buffering agents with minimal interference in biological systems. In a standard procedure, and 1,3-propanesultone are combined in a ratio of approximately 1.02–1.2:1 in an alcoholic solvent such as , with the mixture stirred at 20–30°C for 2–4 hours to complete the exothermic . The reaction is typically conducted under inert atmosphere to prevent side reactions, followed by cooling to 0–5°C for 10 hours to promote of the crude product, which is isolated by or . Yields range from 80–85% based on the sultone reactant. Purification involves recrystallization from an -water mixture to remove impurities and achieve high purity. The crude solid is dissolved in deionized water at 70°C, treated with (about 3% by weight) and a like sodium hydrosulfite for decolorization over 1 hour, then filtered while hot. Upon cooling to 35–40°C and further to 0–5°C, the product crystallizes, is filtered, washed with cold absolute , and dried under vacuum at less than 80°C. This yields white crystals with purity exceeding 99%, verified by alkaline for acid content and NMR spectroscopy for structural integrity; UV of a 0.5 M is typically below 0.03 at 260 nm and 280 nm to ensure suitability for biochemical use.

Preparation of Buffer Solutions

MOPS buffer solutions are typically formulated at concentrations between 10 and 100 mM to suit various biochemical applications, providing effective stabilization within the range of 6.5 to 7.9. For media, a concentration of 20 mM MOPS is commonly employed to maintain physiological without to mammalian cells. To prepare a using the free acid form, dissolve the required quantity of MOPS (molecular weight 209.26 g/) in deionized or to reach the desired concentration, such as 20 mM by adding approximately 4.19 g per liter. Adjust the to the target value, for example 7.2 (near its of 7.2 at 25°C), by slowly adding 1 M NaOH or HCl while monitoring with a ; the solution's is temperature-sensitive, decreasing by about 0.015 units per °C. Sterilize the buffer by through a 0.2 μm to remove and microbes, as autoclaving is not recommended due to potential , especially in the presence of glucose. The sodium salt variant, MOPS-Na (molecular weight 231.25 g/), offers an alternative for preparing buffers closer to neutral without extensive initial adjustment, dissolving readily in at up to 231 mg/mL to yield a clear solution. For formulations requiring conditions, a common recipe includes 50 mM MOPS supplemented with 150 mM NaCl, adjusted to 7.2, which mimics physiological for applications like protein extraction. Prepared buffers should be stored at 2–8°C in sterile containers, where they remain stable for up to six months if protected from light and contamination.

Applications in Biochemistry

Use in Cell Culture and Media

MOPS serves as an essential buffering agent in bacterial and culture media, particularly in defined minimal formulations that require stable control without reliance on CO₂ gassing. In the Neidhardt MOPS minimal medium, originally developed for and other enterobacteria, MOPS is included at a concentration of 40 mM and adjusted to 7.2, enabling reproducible growth rates comparable to those in richer media while avoiding pH instability associated with buffers in ambient atmospheres. This formulation supports metabolic and genetic studies by providing a chemically defined environment that minimizes variability from undefined components. In mammalian , MOPS is incorporated into serum-free media at concentrations of 10–20 mM to stabilize during prolonged incubation, particularly in systems where CO₂ levels may fluctuate. It is often combined with in hybrid buffering setups to extend the effective range and enhance stability under atmospheric conditions, as demonstrated in expansion without compromising growth rates over multiple passages. Usage above 20 mM is generally avoided to prevent potential . The advantages of MOPS in cell culture stem from its low toxicity and lack of interference with cellular signaling pathways, attributes inherent to Good's zwitterionic buffers designed for biological compatibility. For instance, it maintains high cell viability in Chinese hamster ovary (CHO) cells during hypoxic conditions by ensuring consistent pH without introducing metabolic stressors. Historically, MOPS gained adoption in the post-1960s era as a superior alternative to phosphate buffers in defined media, reducing issues like metal ion precipitation and improving overall culture reproducibility.

Role in Electrophoresis and Purification

MOPS buffer plays a crucial role in denaturing for analysis, particularly in formaldehyde-agarose gels designed to separate intact molecules by size under denaturing conditions. The standard running buffer consists of 20 mM MOPS, 8 mM , and 1 mM EDTA at 7.0, which maintains a stable near physiological levels while minimizing degradation and ensuring sharp band resolution for transcripts ranging from small RNAs to large mRNAs. This formulation is widely adopted because MOPS exhibits low UV absorbance, allowing clear visualization of bands without interference during staining or post-electrophoretic analysis. In protein purification, MOPS serves as an effective equilibration and binding buffer for ion-exchange chromatography, typically at concentrations around 50 and 7.0, to facilitate selective binding of target proteins based on charge differences while preserving their native structure. Its utility extends to immobilized metal affinity chromatography () for His-tagged proteins, where MOPS avoids of divalent metal ions like , preventing disruption of the resin-protein interaction and enabling efficient capture without column stripping. By stabilizing in the physiological range (6.5–7.9), MOPS enhances rates in affinity columns compared to buffers prone to pH shifts. For , variants incorporating MOPS, such as the Tris-MOPS-SDS running buffer (50 mM MOPS, 50 mM Tris, 0.1% , 7.7), are employed with Bis-Tris gels to achieve superior resolution of low-molecular-weight proteins (below 30 kDa) by optimizing ion mobility and reducing band distortion. This buffer system supports faster run times and sharper separation in the low mass range, making it preferable for analyzing small peptides or fragments in denaturing conditions.

Other Biochemical Techniques

MOPS buffer is widely employed in UV/VIS spectrophotometry for the quantification of proteins and DNA due to its low UV absorptivity, which minimizes interference in the relevant wavelength ranges. Typically, concentrations of 50–100 mM are used, as MOPS exhibits negligible absorbance above 230 nm, allowing accurate measurements at 260 nm for nucleic acids and 280 nm for proteins without significant background noise. In enzyme assays, particularly for pH-sensitive enzymes such as , MOPS serves as an effective buffering agent at concentrations around 20–25 mM and 7.2, often supplemented with Mg²⁺ to support activity. For instance, in assays of C-mediated , 20 mM MOPS ( 7.2) with 15 mM MgCl₂ enables precise measurement of without observed inhibition by the buffer itself. Similarly, kinase activity evaluations for RPS6KB1 use 20 mM MOPS ( 7.2) with 5 mM MgCl₂, demonstrating MOPS's compatibility and stability in these systems. For protein extraction from mammalian tissues, MOPS-based lysis buffers are commonly formulated at 20 mM with 150 mM NaCl and protease inhibitors to maintain physiological ionic strength and prevent degradation. This composition effectively solubilizes proteins from cell lysates while preserving native structures, as seen in protocols for transfecting mammalian cells where 20 mM MOPS (pH 7.4), 120 mM KCl (comparable to NaCl in osmolarity), and inhibitors yield high-quality extracts for downstream analysis. Emerging applications include the use of MOPS in nanoparticle synthesis for biomedicine, where it provides precise pH control during bioconjugation processes. For example, 0.1 M MOPS (pH 7.4) is utilized to resuspend iron oxide nanoparticles post-modification, facilitating stable attachment of biomolecules like antibodies without altering surface chemistry.

Within Good's Buffers

Good's buffers refer to a set of buffers introduced by Norman Good and colleagues in their 1966 paper, later expanded to over 20 zwitterionic compounds developed through 1980 specifically for biological research applications within physiological pH ranges. These buffers were designed to meet stringent criteria, including a between 6 and 8 to align with typical cellular environments, high to facilitate use in dilute solutions, minimal in the range to avoid interference with spectrophotometric assays, and low effects to prevent perturbations in enzymatic or macromolecular interactions. MOPS, or 3-(N-morpholino)propanesulfonic acid, is one of the original buffers in this set, with a of 7.2 that strategically fills the buffering gap between (pKa 6.8) and (pKa 7.5). It was introduced as a superior alternative to traditional buffers like cacodylate and phosphate, which suffer from toxicity concerns, metal chelation, or precipitation issues in biological systems. The development of Good's buffers, including MOPS, had a profound historical impact by providing researchers with tools for precise pH control in early molecular biology experiments, such as enzyme kinetics and chloroplast studies, thereby advancing reproducibility and accuracy in vitro assays. This foundational work is detailed in the seminal publication "Hydrogen Ion Buffers for Biological Research" by Good et al. (1966).

Analogs and Alternatives

One key analog of MOPS (3-(N-morpholino)propanesulfonic acid) is (2-(N-morpholino)ethanesulfonic acid), which differs structurally by having a shorter ethanesulfonic acid chain instead of the propanesulfonic acid moiety in MOPS. This structural variation results in a lower of 6.10 for MES at 25°C, compared to 7.20 for MOPS, making MES more suitable for buffering in acidic ranges ( 5.5–6.7) while MOPS is preferred for neutral applications ( 6.5–7.9). Another close analog is MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid), which shares the propanesulfonic acid backbone with MOPS but includes an additional hydroxyl group at the 2-position of the propane chain, enhancing its solubility. MOPSO has a of 6.90 at 25°C, providing a slightly broader buffering range ( 6.2–7.6) that overlaps with MOPS, allowing the two to be used interchangeably in many biochemical protocols. Among broader alternatives, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) offers a of 7.48 at 25°C and a buffering range of 6.8–8.2, making it suitable for physiological similar to MOPS, but its group leads to higher UV absorption (notably around 230 ), which can interfere in spectrophotometric assays where MOPS's minimal UV is advantageous. Phosphate buffers, with a relevant around 7.2 for the second dissociation, provide effective buffering in the 5.8–8.0 range but are prone to chelating divalent metal ions, potentially disrupting activities or metal-dependent reactions, whereas MOPS shows negligible metal coordination and is thus selected for low-metal environments. In selection guides for biochemical applications, MOPS is often recommended over these alternatives when stability and non-interference with metals or UV readings are priorities. Regarding performance, MOPS offers better stability than Tris (tris(hydroxymethyl)aminomethane, 8.06 at 25°C) in some biochemical assays, particularly due to Tris's higher temperature sensitivity and hygroscopic nature.

References

  1. [1]
    About - The MomCo
    then, simply known as MOPS (Mothers of Preschoolers)—was founded in 1973 when seven women in Colorado invited moms to meet regularly to grow together as ...
  2. [2]
    MOPS International Inc. - GuideStar Profile
    MOPS International encourages and equips moms of young children to realize their potential as mothers, women and leaders.
  3. [3]
    Hydrogen Ion Buffers for Biological Research* | Biochemistry
    Article February 1, 1966. Hydrogen Ion Buffers for Biological Research*. Click to copy article linkArticle link copied! Norman E. Good · G. Douglas Winget ...
  4. [4]
  5. [5]
  6. [6]
    4-Morpholineethanesulfonic acid | C6H13NO4S | CID 78165
    4-Morpholineethanesulfonic acid ... 2-(N-morpholino)ethanesulfonic acid is a Good's buffer substance, pKa = 6.15 at 20 ℃. It is an organosulfonic acid and a MES.
  7. [7]
    MOPS | 1132-61-2 - ChemicalBook
    Sep 24, 2025 · MOPS is capable of buffering within a pH range of 6.5-7.9. MOPS is widely used in biological and biochemical studies due to its inert ...
  8. [8]
    M4610 MOPS Free Acid CAS: 1132-61-2 - United States Biological
    Solubility (1M). Colorless, clear, complete. pH (1%). 3.6-4.4. pKa. 7.1-7.2. Water (KF). ≤1%. Melting Point. 277-281°C. Absorbance (A260, 1M H2O). As Reported.
  9. [9]
    Cas 1132-61-2,3-Morpholinopropanesulfonic acid | lookchem
    1132-61-2 ; Storage Temp.: Store at RT. ; Solubility: H2O: 1 M at 20 °C, clear ; PKA: 7.2(at 25℃) ; Water Solubility: 1000 g/L (20 ºC) ; Stability: Stable.<|control11|><|separator|>
  10. [10]
    [PDF] SAFETY DATA SHEET - Fisher Scientific
    Mar 1, 2010 · Melting Point/Range. 277 - 282 °C / 530.6 - 539.6 °F. Boiling Point ... Will likely be mobile in the environment due to its water solubility.
  11. [11]
  12. [12]
    Cas 1132-61-2 MOPS Good Buffer Solutions 3-Morpholine Propane ...
    Rating 4.8 · Review by anonymous6. No complex or precipitation with metal ions;. 7. The buffer is chemically stable;. 8. Absorption in the ultraviolet region of 0.1 mol/L aqueous solution ...
  13. [13]
    Calculator for pH buffers
    The tool designs thermodynamically corrected buffers, making temperature and ionic strength corrections. Users input buffer species, volume, pH, concentration, ...
  14. [14]
    MOPS synthesis - ChemicalBook
    Synthesis of MOPS from Morpholine and 1,3-Propanesultone.Chemicalbook can provide 2 synthetic routes.
  15. [15]
  16. [16]
    [PDF] MOPS Free acid - HiMedia Laboratories
    MOPS buffer is used in biochemistry, cell culture and molecular biology ... culture medium should not exceed 20mM as it can be toxic to cells. MOPS ...
  17. [17]
    None
    ### Summary of MOPS Buffer Preparation
  18. [18]
  19. [19]
    None
    **Summary of MOPS Sodium Salt (Product Number M5789):**
  20. [20]
  21. [21]
    Culture Medium for Enterobacteria - ASM Journals
    Interestingly, a mutant of E. coli B/r has been isolated in which the micronutrient components of MOPS medium are growth stimulatory (results not shown).
  22. [22]
    Culture medium for enterobacteria - PubMed
    A new minimal medium for enterobacteria has been developed. It supports growth of Escherichia coli and Salmonella typhimurium at rates comparable to those ...
  23. [23]
    MOPS Minimal Medium Recipe - E. coli Genome Project
    MOPS Minimal Medium includes 10X MOPS mixture, K2HPO4, H2O, and optional thiamine. Add carbon source before use. Adjust pH to 7.2.
  24. [24]
    Expansion of Mesenchymal Stem Cells under Atmospheric Carbon ...
    The MSC cultured in the MOPS-HEPES buffer under atmospheric CO2 were able to maintain high/optimal growth rates for five passages, which would allow for large- ...Real Time Qrt-Pcr · Figure 2. Adipogenic... · Figure 3. Osteogenic...
  25. [25]
  26. [26]
    Cell line development for continuous high cell density ... - NIH
    Jul 29, 2021 · Here we present an effort to create novel engineered Chinese hamster ovary (CHO) cell lines by exploiting encountered hypoxic bioprocess conditions.
  27. [27]
    MOPS - an overview | ScienceDirect Topics
    The most commonly used buffer system in cell culture media is sodium bicarbonate (NaHCO3) and CO2 gas; however the low pKa of NaHCO3 results in suboptimal ...
  28. [28]
    [PDF] Denaturing Formaldehyde Gels in MOPS Buffer
    Prepare 10X MOPS buffer, 1% agarose gel with formaldehyde, heat RNA samples, then load onto the gel in 1X MOPS buffer.
  29. [29]
    Separation of long RNA by agarose-formaldehyde gel electrophoresis
    The commonly used procedures for agarose-formaldehyde gel electrophoresis of RNA traditionally use the combination of 3-(N-morpholino)propanesulfonic acid (MOPS) ...
  30. [30]
    6 Biological buffers recommended for protein purification - Blog
    Nov 6, 2019 · 1) BIS-TRIS. CAS No.: 6976-37-0. Useful pH range: 5.8 - 7.2 · 2) MOPS. CAS No.: 1132-61-2. Useful pH range: 6.5 - 7.9 · 3) PIPES. CAS No.: 5625-37 ...
  31. [31]
  32. [32]
    NuPAGE Bis-Tris and Bolt Bis-Tris Plus Gels - Thermo Fisher Scientific
    The gels can be run using MES SDS running buffer to better resolve smaller proteins (1–200 kDa) and MOPS SDS running buffer to resolve medium- to large-size ...
  33. [33]
    MOPS, Free Acid - BioSpectra
    MOPS can be used as a “Good's” buffer because it has low UV absorptivity, minimal reactivity, stable pH and is soluble in water. These are general ...Missing: concentration | Show results with:concentration<|separator|>
  34. [34]
    Protein Kinase C-mediated Phosphorylation and Activation of ... - NIH
    In Vitro Phosphorylation—PDE3A immunoprecipitates were washed with kinase buffer (20 mm MOPS pH 7.2, 20 mm sodium β-glycerophosphate, 15 mm MgCl2, 1 mm sodium ...
  35. [35]
    Co-expression of the RPS6KB1 and PDPK1 genes for production of ...
    Jan 17, 2025 · The Assay Buffer consisted of 20 mM MOPS (pH 7.2), 2.5 mM β-glycerophosphate, 5 mM MgCl2, 0.05% BSA, 0.05% Brij-35, and 5 mM β-mercaptoethanol.
  36. [36]
    An RNA-Binding Protein Secreted by a Bacterial Pathogen ...
    Nov 21, 2019 · 24 h after transfection, the cells were washed twice with PBS and lysed using 1 mL lysis buffer per dish (20 mM MOPS-KOH pH 7.4, 120 mM KCl, 0.2 ...
  37. [37]
    Cross-reactivities in conjugation reactions involving iron oxide ...
    The products were pelleted by centrifugation, and the recovered nanoparticles were washed twice with methanol, and then resuspended in 1.0 mL of 0.1 M MOPS (pH ...
  38. [38]
  39. [39]
    MES vs. MOPS: Understanding the Differences in Biological Buffers
    Oct 17, 2025 · The core difference between MES and MOPS lies in their ethanesulfonic and propanesulfonic acid moieties, respectively. MES has an ethanesulfonic ...
  40. [40]
    The Difference between MOPS and MOPSO as Buffer
    Aug 30, 2019 · As mentioned above, MOPS and MOPSO have different useful pH range. And they have respective advantages in the biochemical studies. So, buffer ...Missing: His- | Show results with:His-
  41. [41]
    Blog - Why use MOPS? - Hopax Fine Chemicals
    Mar 12, 2020 · It is a white powder solid at normal temperature, with high hydrophilicity and excellent water solubility (1000 g/L at 20℃).
  42. [42]
    Biological buffers and their interactions with metal ions - Blog
    Oct 5, 2018 · Some biological buffers have very low metal-binding constants, making them excellent choices to investigate metal-dependent enzymes.Missing: issues | Show results with:issues
  43. [43]
    [PDF] Comparing pH and Buffer Solutions for Stabilising a Monoclonal ...
    The high quality, detailed denaturation data produced with the SUPR-CM highlights the overall improvement in stability of the mAb when using a MOPS buffer at pH ...Missing: chelation issues