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DPPH
DPPH
DPPH
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DPPH
Skeletal formula of DPPH
sample
Names
Preferred IUPAC name
2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazin-1-yl
Other names
2,2-Diphenyl-1-picrylhydrazyl
1,1-Diphenyl-2-picrylhydrazyl radical
2,2-Diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl
Diphenylpicrylhydrazyl
Identifiers
3D model (JSmol)
Abbreviations DPPH
ChemSpider
ECHA InfoCard 100.015.993 Edit this at Wikidata
UNII
  • InChI=1S/C18H12N5O6/c24-21(25)15-11-16(22(26)27)18(17(12-15)23(28)29)19-20(13-7-3-1-4-8-13)14-9-5-2-6-10-14/h1-12H checkY
    Key: HHEAADYXPMHMCT-UHFFFAOYSA-N checkY
  • InChI=1/C18H13N5O6/c24-21(25)15-11-16(22(26)27)18(17(12-15)23(28)29)19-20(13-7-3-1-4-8-13)14-9-5-2-6-10-14/h1-12,19H
    Key: WCBPJVKVIMMEQC-UHFFFAOYAG
  • InChI=1/C18H12N5O6/c24-21(25)15-11-16(22(26)27)18(17(12-15)23(28)29)19-20(13-7-3-1-4-8-13)14-9-5-2-6-10-14/h1-12H
    Key: HHEAADYXPMHMCT-UHFFFAOYAG
  • c1ccc(cc1)N(c2ccccc2)[N]c3c(cc(cc3[N+](=O)[O-])[N+](=O)[O-])[N+](=O)[O-]
Properties
C18H12N5O6
Molar mass 394.32 g/mol
Appearance Black to green powder, purple in solution
Density 1.4 g/cm3
Melting point 135 °C (275 °F; 408 K) (decomposes)
insoluble
Solubility in methanol 10 mg/mL
Hazards
NFPA 704 (fire diamond)
NFPA 704 four-colored diamondHealth 0: Exposure under fire conditions would offer no hazard beyond that of ordinary combustible material. E.g. sodium chlorideFlammability 1: Must be pre-heated before ignition can occur. Flash point over 93 °C (200 °F). E.g. canola oilInstability 0: Normally stable, even under fire exposure conditions, and is not reactive with water. E.g. liquid nitrogenSpecial hazards (white): no code
0
1
0
Safety data sheet (SDS) MSDS
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
checkY verify (what is checkY☒N ?)

DPPH is a common abbreviation for the organic chemical compound 2,2-diphenyl-1-picrylhydrazyl. It is a dark-colored crystalline powder composed of stable free radical molecules. DPPH has two major applications, both in laboratory research: one is a monitor of chemical reactions involving radicals, most notably it is a common antioxidant assay,[1] and another is a standard of the position and intensity of electron paramagnetic resonance signals.

Properties and applications

[edit]

DPPH has several crystalline forms which differ by the lattice symmetry and melting point. The commercial powder is a mixture of phases which melts at ~130 °C. DPPH-I (m.p. 106 °C) is orthorhombic, DPPH-II (m.p. 137 °C) is amorphous and DPPH-III (m.p. 128–129 °C) is triclinic.[2]

DPPH is a well-known radical and a trap ("scavenger") for other radicals. Therefore, rate reduction of a chemical reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction. Because of a strong absorption band centered at about 520 nm, the DPPH radical has a deep violet color in solution, and it becomes colorless or pale yellow when neutralized. This property allows visual monitoring of the reaction, and the number of initial radicals can be counted from the change in the optical absorption at 520 nm or in the EPR signal of the DPPH.[3]

Because DPPH is an efficient radical trap, it is also a strong inhibitor of radical-mediated polymerization.[4]

Inhibition of polymer chain, R, by DPPH.

As a stable and well-characterized solid radical source, DPPH is the traditional and perhaps the most popular standard of the position (g-marker) and intensity of electron paramagnetic resonance (EPR) signals – the number of radicals for a freshly prepared sample can be determined by weighing and the EPR splitting factor for DPPH is calibrated at g = 2.0036. DPPH signal is convenient by that it is normally concentrated in a single line, whose intensity increases linearly with the square root of microwave power in the wider power range. The dilute nature of the DPPH radicals (one unpaired spin per 41 atoms) results in a relatively small linewidth (1.5–4.7 G). The linewidth may however increase if solvent molecules remain in the crystal and if measurements are performed with a high-frequency EPR setup (~200 GHz), where the slight g-anisotropy of DPPH becomes detectable.[5][6]

Whereas DPPH is normally a paramagnetic solid, it transforms into an antiferromagnetic state upon cooling to very low temperatures of the order 0.3 K. This phenomenon was first reported by Alexander Prokhorov in 1963.[7][8][9][10]

References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

DPPH

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from Grokipedia
DPPH, or 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl, is a organic free radical compound known for its dark violet crystalline powder form and use as a standard in research. It consists of a hydrazyl group (–N–N•–) with an delocalized across the atoms, flanked by bulky 2,2-diphenyl and 2,4,6-trinitrophenyl (picryl) substituents that confer exceptional stability through steric protection and electronic conjugation. This stability prevents dimerization or reaction with molecular oxygen, allowing DPPH to persist for hours in solution or solid state, with an N–N of 1.321–1.352 indicative of partial double-bond character. Discovered in 1922 by chemists Stefan Goldschmidt and Konrad Renn, DPPH was first synthesized by oxidizing 2,2-diphenyl-1-picrylhydrazine using , marking it as one of the earliest characterized persistent radicals. Subsequent refinements in synthesis employed or other oxidants in non-polar solvents like , yielding near-quantitative results and enabling its production as a commercial . By the mid-20th century, its utility expanded beyond basic chemistry; in , Marsden S. Blois developed the DPPH radical scavenging assay to quantify activity, initially using as a model compound and measuring decolorization via loss at 517 nm. The DPPH assay operates on the principle of from an to the DPPH radical, reducing it to the pale yellow derivative (DPPH–H) and allowing evaluation of radical scavenging capacity through spectrophotometric changes. This method, later refined by Brand-Williams et al. in 1995 to incorporate EC50 values (effective concentration for 50% inhibition) and antiradical efficiency metrics, has become a cornerstone for assessing total capacity in diverse samples, including extracts, foods, and pharmaceuticals. Beyond assays, DPPH serves in electron spin resonance (ESR) spectroscopy for radical detection and in for studying oxidation processes, though its use requires caution due to sensitivities to , solvents, and that can affect reproducibility. Despite limitations, such as potential interference from sample turbidity or inability to mimic physiological radicals like hydroxyl or , DPPH remains a simple, cost-effective tool prized for its high throughput and applicability to both hydrophilic and lipophilic .

Nomenclature and structure

Chemical identity

DPPH is the widely used abbreviation for the 2,2-diphenyl-1-picrylhydrazyl, a stable free radical commonly employed in assays. The term "picryl" specifically denotes the 2,4,6-trinitrophenyl group, derived from by removal of the hydroxyl group, which imparts the compound's characteristic nitroaromatic structure. The systematic IUPAC name for DPPH is 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl, reflecting its hydrazyl core substituted with two phenyl groups and one picryl moiety. Its molecular formula is C₁₈H₁₂N₅O₆, with a of 394.32 g/mol. The compound is uniquely identified by the 1898-66-4.

Molecular geometry

The of 2,2-diphenyl-1-picrylhydrazyl (DPPH) is characterized by a central N-N bond exhibiting partial character, with lengths of 1.352(7) and 1.321(7) in the two independent molecules of ether-grown crystals, and 1.342(4) and 1.339(4) in carbon disulfide-grown crystals, corresponding to an approximate of 1.5 due to extensive . This involves multiple contributing structures that distribute electron density across the hydrazyl core, enhancing the stability of the radical. The is delocalized primarily over the two nitrogen atoms and extends into the conjugated π-systems of the adjacent phenyl and picryl (2,4,6-trinitrophenyl) rings, a feature confirmed by structural and spectroscopic analyses. Steric hindrance plays a crucial role in maintaining the monomeric radical form, as the bulky ortho-substituted phenyl groups and the electron-withdrawing picryl moiety create significant spatial barriers that prevent dimerization or disproportionation. X-ray crystallographic studies reveal that the hydrazyl moiety is largely planar, facilitating conjugation, while the phenyl rings are twisted out of this plane at dihedral angles of approximately 22° and 49° relative to the central N=N plane, and the picryl ring adopts a dihedral angle of about 33°. These twists arise from steric interactions between the substituents, further isolating the reactive nitrogen center. The overall polarity of the molecule is evident in its dipole moment of 4.88 , which is higher than that of the (3.59 ) and arises from the asymmetric charge distribution in the hybrids, with the picryl group acting as an and the diphenylhydrazyl as a donor. This push-pull electronic configuration, combined with the geometric features, underpins the exceptional persistence of the DPPH radical in both and solution states.

Synthesis

Preparation methods

The primary laboratory method for preparing the DPPH radical involves the oxidation of the precursor 2,2-diphenyl-1-picrylhydrazine (DPPH-H) using (PbO₂) as the oxidant in (CH₂Cl₂) as the solvent. This one-step process is conducted at under an inert atmosphere to minimize side reactions and ensure the stability of the resulting radical. The reaction typically proceeds with near-quantitative yields exceeding 95%, reflecting the high efficiency of PbO₂ in selectively removing two electrons and two protons from the . Alternative oxidants can be employed for similar conversions, including lead tetraacetate, (Ag₂O), or (KMnO₄), also in non-polar solvents such as or . For instance, KMnO₄ oxidation requires a like tetra-n-butylammonium bromide and proceeds by gradual addition of the oxidant, achieving high yields comparable to the PbO₂ method while offering advantages in cost and reduced toxicity. These variations maintain room-temperature conditions and inert atmospheres, with reaction completion monitored by . Following oxidation, the reaction mixture is filtered to remove insoluble byproducts such as reduced or excess PbO₂. The crude DPPH radical is then purified by recrystallization from hot or , yielding dark purple to black crystals suitable for spectroscopic and applications. The overall transformation follows the general oxidation equation: Ar2N-NH-Ar’→Ar2N-N∙-Ar’+2H++2e−\text{Ar}_2\text{N-NH-Ar'} \rightarrow \text{Ar}_2\text{N-N}^\bullet\text{-Ar'} + 2\text{H}^+ + 2\text{e}^- where Ar represents phenyl groups and Ar' denotes the picryl (2,4,6-trinitrophenyl) moiety.

Key precursors and reactions

The parent hydrazine precursor to DPPH, known as 2,2-diphenyl-1-picrylhydrazine (DPPH-H), is synthesized through the reaction of picryl chloride (2,4,6-trinitrochlorobenzene) with 1,1-diphenyl. This reaction typically proceeds in or as solvents, leveraging the electron-withdrawing nitro groups on the picryl chloride to facilitate displacement of the chloride ion by the hydrazine . The resulting DPPH-H is a solid that serves as the direct precursor for oxidation to the DPPH radical. During synthesis, side reactions can compromise yields, including over-oxidation of the intermediate to azo compounds via excessive oxidant exposure, which leads to N-N bond cleavage and formation of stable, non-radical byproducts. Additionally, occurs readily in protic solvents, where promotes instability of the nitro-substituted aromatic ring. The original preparation of DPPH traces back to , when Goldschmidt and Renn oxidized 2,2-diphenyl-1-picryl using a similar hydrazine-based method, yielding a stable violet radical solution that highlighted its persistence compared to other triarylmethyl radicals. To optimize yields, conditions are essential during the reaction and workup stages, as trace water can induce of the sensitive nitro groups on the picryl moiety, reducing the purity and quantity of the desired product.

Physical and chemical properties

Appearance and solubility

DPPH appears as a dark to black crystalline powder at . This characteristic form arises from its stable free radical nature, contributing to its utility in laboratory settings. The compound exhibits a density of approximately 1.4 g/cm³. Upon heating, DPPH decomposes above 130–140 °C without undergoing melting, as indicated by literature values around 135 °C for decomposition. DPPH is insoluble in water but readily soluble in various organic solvents, including ethanol, methanol, and chloroform, with solubility reaching up to approximately 0.1 M in these media. In ethanol, solutions of DPPH display a deep violet color due to absorption at a maximum wavelength (λ_max) of about 517 nm.

Stability and reactivity

DPPH demonstrates exceptional as a free radical, remaining intact in air and in solution for months without significant degradation. This persistence arises primarily from steric provided by the bulky phenyl and picryl substituents, which shield the nitrogen-centered radical from intermolecular interactions, and from extensive delocalization of the across the molecule, stabilizing the radical through . Unlike many hydrazyl radicals, DPPH does not undergo dimerization under ambient conditions, a feature directly attributable to these steric impediments that prevent close approach of another radical moiety. In reactive scenarios, DPPH functions as either a acceptor or a one-electron donor, enabling it to participate in radical quenching reactions typical of evaluations. Upon interaction with suitable , it is reduced to the stable, non-radical diphenylpicrylhydrazine (DPPH-H), effectively pairing the . The of the DPPH•/DPPH-H couple is approximately 0.3 V versus the (SCE) in , reflecting its moderate oxidizing ability suitable for probing capacities. Despite its overall robustness, DPPH exhibits sensitivity to certain environmental factors, decomposing gradually in the presence of or protic solvents to yield diphenylpicrylhydrazine as the primary product. This photolytic or solvolytic breakdown underscores the need for controlled storage conditions, such as darkness and aprotic media, to maintain its integrity over extended periods. The delocalization, which enhances stability, also plays a role in mitigating reactivity with atmospheric oxygen.

Spectroscopic properties

Electronic spectroscopy

The electronic spectroscopy of 2,2-diphenyl-1-picrylhydrazyl (DPPH) is dominated by its characteristic UV-Vis absorption, which arises from electronic transitions involving the delocalized unpaired electron and the conjugated π-system of the molecule. In ethanol, DPPH exhibits a strong absorption band at approximately 517 nm with a molar extinction coefficient (ε) of about 11,500 M⁻¹ cm⁻¹, responsible for its deep violet color in solution. This band is attributed to a combination of π-π* transitions and the contribution of the radical electron, making it highly sensitive to redox changes. The UV-Vis spectrum of DPPH also features additional bands in the region, which are primarily due to π-π* transitions within the aromatic picryl and phenyl moieties. These transitions reflect the extended conjugation in the hydrazyl structure, providing insight into the molecule's electronic delocalization. Upon reduction, such as during radical scavenging by antioxidants, the absorbance at 517 nm decreases markedly—a phenomenon known as bleaching—due to the pairing of the and disruption of the . This change is proportional to the extent of radical scavenging, allowing quantitative monitoring of the reaction progress. Solvent polarity influences the absorption maximum, with a bathochromic shift observed in more polar protic s; for instance, the λmax is approximately 517 nm in compared to 515 nm in . This effect arises from stabilization of the . In assays, the electronic of DPPH is routinely exploited by measuring the decrease in at 517 nm to quantify radical scavenging activity, providing a simple and rapid endpoint for evaluation.

Electron spin resonance

The electron spin resonance (ESR) spectrum of the DPPH radical in dilute solution displays a characteristic five-line hyperfine pattern with relative intensities of 1:2:3:2:1, arising from the interaction of the with the two nuclei, each having a nuclear I=1I = 1. Although the hydrazyl and picryl atoms are chemically distinct, their hyperfine constants differ by only about 1.5 G, resulting in an effectively symmetric spectrum rather than the 9 lines expected for fully inequivalent couplings. The isotropic hyperfine coupling constants have been precisely determined as aN1=9.35±0.20a_{\mathrm{N1}} = 9.35 \pm 0.20 G for the hydrazyl and aN2=7.85±0.20a_{\mathrm{N2}} = 7.85 \pm 0.20 G for the picryl , reflecting the greater spin density on the hydrazyl N atom due to its direct involvement in the radical center. These values indicate significant delocalization of the across the conjugated π\pi-system, consistent with the radical's stability. The g-factor of DPPH is 2.0036, a value typical for π\pi-delocalized organic radicals where the interacts primarily with the orbital of carbon and atoms. In solution, the ESR linewidth of DPPH is narrow, approximately 1 G, which contributes to its widespread use as a for calibrating ESR spectrometers, as the sharp lines allow accurate determination of and sensitivity. The spectrum in the solid state simplifies to a single broad line due to intermolecular interactions, but the solution-phase remains the reference for radical characterization. DPPH exhibits remarkable thermal stability in its ESR properties, with minimal linewidth broadening observed up to 100°C, confirming its reliability as a standard even under moderate heating conditions without significant radical decay or distortion. This temperature independence arises from the robust delocalization that suppresses spin-lattice relaxation mechanisms.

Applications

Antioxidant assays

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay evaluates the antioxidant capacity of compounds by measuring their ability to donate a hydrogen atom (H•) or an electron (e⁻) to the stable DPPH• free radical, resulting in the formation of the non-radical DPPH-H and a concomitant loss of the characteristic deep violet color. This decolorization is quantified spectrophotometrically at 517 nm, where the maximum absorbance of DPPH• occurs, as detailed in the electronic spectroscopy of the compound. The simplified reaction can be represented as: DPPH∙+AH→DPPH-H+A∙\text{DPPH}^\bullet + \text{AH} \to \text{DPPH-H} + \text{A}^\bullet where AH denotes the antioxidant and A• its derived radical. A standard protocol involves preparing a 0.1 mM DPPH solution in ethanol or methanol, adding varying concentrations of the sample to aliquots of this solution, incubating in the dark for 30 minutes at room temperature, and measuring the absorbance at 517 nm against a blank. The percentage inhibition of DPPH• is calculated using the formula: % inhibition=Acontrol−AsampleAcontrol×100\% \text{ inhibition} = \frac{A_{\text{control}} - A_{\text{sample}}}{A_{\text{control}}} \times 100 where AcontrolA_{\text{control}} is the absorbance of the DPPH solution without sample and AsampleA_{\text{sample}} is the absorbance with sample. This method, originally introduced by Blois and refined in subsequent studies, allows for rapid assessment of radical scavenging efficiency. Antioxidant potency is often expressed as the IC₅₀ value, defined as the sample concentration required to reduce the DPPH• by 50%, typically determined from a dose-response curve. IC₅₀ values are commonly compared to reference standards such as (a water-soluble analog) or ascorbic acid to benchmark activity, with lower IC₅₀ indicating higher scavenging capacity. The DPPH assay offers advantages including simplicity, speed (typically under 1 hour), cost-effectiveness, and independence from biological systems, making it suitable for of both hydrophilic and lipophilic . However, limitations include its reliance on non-physiological solvents like , which may not mimic biological conditions, and potential overestimation of activity for hydrophobic compounds due to biases in the reaction medium.

Radical chemistry and ESR standards

DPPH functions as an effective radical trap in free radical chemistry, reacting with short-lived species such as oxygen-, nitrogen-, sulfur-, carbon-, or phosphorus-centered radicals to form stable hydrazyl adducts detectable by electron spin resonance (ESR) spectroscopy or other analytical methods. This trapping capability arises from the delocalization of the unpaired electron across the hydrazyl and picryl moieties, allowing DPPH to undergo one-electron oxidation without rapid dimerization. Hybrid derivatives, such as DPPH-nitrones, have been developed to serve simultaneously as generators and traps for transient radicals, enhancing mechanistic insights into radical processes. In kinetic studies of radical reactions, DPPH enables monitoring of reaction rates through the decay of its characteristic UV absorption at approximately 517 nm or via ESR signal intensity, providing second-order rate constants for transfer or mechanisms in apolar solvents. These measurements are particularly valuable for elucidating the reactivity of antioxidants or substrates with radicals, avoiding complications from short-lived intermediates. For instance, the reaction of DPPH with under UV irradiation has been used to quantify free radical propagation kinetics. A dilute solution of DPPH (typically 0.1 mM in ) serves as a in ESR spectrometry due to its temperature-independent g-factor of 2.0036 and narrow linewidth, facilitating precise calibration of strength and signal intensity. The ESR of DPPH in solution exhibits a characteristic five-line hyperfine (1:2:3:2:1 intensity ) from coupling with two equivalent nitrogen nuclei (a_N1 ≈ 9.35 G, a_N2 ≈ 7.85 G), making it ideal for quantitative spin counting and instrument alignment. Beyond these roles, DPPH finds application in primarily as a potent inhibitor of free radical-mediated by trapping chain-carrying radicals, thereby allowing assessment of efficiency and transfer constants. It also acts as a in certain oxidation reactions and, in biochemical contexts, supports site-directed spin-labeling studies by serving as a paramagnetic quencher to measure parameters of nitroxide-labeled proteins in membranes. Historically, in the early 1950s, DPPH was instrumental in confirming radical mechanisms during , such as addition reactions to unsaturated systems, predating its widespread adoption in assays.

References

  1. https://pmc.ncbi.nlm.nih.gov/articles/PMC7913707/
  2. https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/cber.19220550308
  3. https://pubs.acs.org/doi/10.1021/acs.jafc.5b03839
  4. https://pmc.ncbi.nlm.nih.gov/articles/PMC3551182/
  5. https://pubchem.ncbi.nlm.nih.gov/compound/2735032
  6. https://www.merriam-webster.com/medical/picryl
  7. https://www.chemspider.com/Chemical-Structure.2016757.html
  8. https://doi.org/10.1016/j.jmmm.2009.10.012
  9. https://www.mdpi.com/1422-0067/22/4/1545
  10. https://doi.org/10.1021/ja00993a005
  11. https://doi.org/10.3390/ijms22031545
  12. https://doi.org/10.1139/v86-301
  13. https://doi.org/10.3390/ijms22041545
  14. https://www.chemicalbook.com/ChemicalProductProperty_EN_CB8753025.htm
  15. https://www.sigmaaldrich.com/US/en/sds/aldrich/d9132
  16. https://www.medchemexpress.com/dpph.html
  17. https://pmc.ncbi.nlm.nih.gov/articles/PMC8017072/
  18. https://www.mdpi.com/2227-9717/11/8/2248
  19. https://pubs.aip.org/aip/jcp/article/34/5/1693/79240/Isotropic-and-Anisotropic-Hyperfine-Interactions
  20. https://www.jeol.com/solutions/applications/assets/pdf/er230004_en.pdf
  21. https://pubs.aip.org/aip/apl/article/118/2/022407/39800/Force-detection-of-high-frequency-electron-spin
  22. https://www.semanticscholar.org/paper/DPPH-as-a-Standard-for-High-Field-EPR-Krzystek-Sienkiewicz/58504635f75258974d05d731cfad2c3f1f176422
  23. https://pmc.ncbi.nlm.nih.gov/articles/PMC3507463/
  24. https://www.researchgate.net/figure/Temperature-dependence-of-the-ESR-linewidth-and-the-g-factor-for-pressures-of-24-kbar_fig3_243435696
  25. https://doi.org/10.1007/s13197-011-0251-1
  26. https://doi.org/10.1016/S0023-6438(95)80008-5
  27. https://pubmed.ncbi.nlm.nih.gov/25511471/
  28. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913707/
  29. https://doi.org/10.1021/acs.jafc.5b03839
  30. https://core.ac.uk/download/pdf/39181581.pdf
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