DPPH

DPPH, or 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl, is a stable organic free radical compound known for its dark violet crystalline powder form and use as a standard reagent in antioxidant research.^[1] It consists of a hydrazyl group (–N–N•–) with an unpaired electron delocalized across the nitrogen atoms, flanked by bulky 2,2-diphenyl and 2,4,6-trinitrophenyl (picryl) substituents that confer exceptional stability through steric protection and electronic conjugation.^[1] This stability prevents dimerization or reaction with molecular oxygen, allowing DPPH to persist for hours in solution or solid state, with an N–N bond length of 1.321–1.352 Å indicative of partial double-bond character.^[1] Discovered in 1922 by chemists Stefan Goldschmidt and Konrad Renn,^[2] DPPH was first synthesized by oxidizing 2,2-diphenyl-1-picrylhydrazine using lead dioxide, marking it as one of the earliest characterized persistent radicals.^[3] Subsequent refinements in synthesis employed potassium permanganate or other oxidants in non-polar solvents like dichloromethane, yielding near-quantitative results and enabling its production as a commercial reagent.^[1] By the mid-20th century, its utility expanded beyond basic chemistry; in 1958, Marsden S. Blois developed the DPPH radical scavenging assay to quantify antioxidant activity, initially using cysteine as a model compound and measuring decolorization via absorbance loss at 517 nm.^[4] The DPPH assay operates on the principle of hydrogen atom transfer (HAT) from an antioxidant to the DPPH radical, reducing it to the pale yellow hydrazine derivative (DPPH–H) and allowing evaluation of radical scavenging capacity through spectrophotometric changes.^[4] 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 antioxidant capacity in diverse samples, including plant extracts, foods, and pharmaceuticals.^[4] Beyond assays, DPPH serves in electron spin resonance (ESR) spectroscopy for radical detection and in polymer chemistry for studying oxidation processes, though its use requires caution due to sensitivities to pH, solvents, and light that can affect reproducibility.^[1] Despite limitations, such as potential interference from sample turbidity or inability to mimic physiological radicals like hydroxyl or superoxide, DPPH remains a simple, cost-effective tool prized for its high throughput and applicability to both hydrophilic and lipophilic antioxidants.^[3]

Nomenclature and structure

Chemical identity

DPPH is the widely used abbreviation for the organic compound 2,2-diphenyl-1-picrylhydrazyl, a stable free radical commonly employed in antioxidant assays.^[5] The term "picryl" specifically denotes the 2,4,6-trinitrophenyl group, derived from picric acid by removal of the hydroxyl group, which imparts the compound's characteristic nitroaromatic structure.^[6] 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.^[7] Its molecular formula is C₁₈H₁₂N₅O₆, with a molar mass of 394.32 g/mol.^[5] The compound is uniquely identified by the CAS Registry Number 1898-66-4.^[5]

Molecular geometry

The molecular geometry of 2,2-diphenyl-1-picrylhydrazyl (DPPH) is characterized by a central N-N bond exhibiting partial double bond character, with lengths of 1.352(7) Å and 1.321(7) Å in the two independent molecules of ether-grown crystals, and 1.342(4) Å and 1.339(4) Å in carbon disulfide-grown crystals, corresponding to an approximate bond order of 1.5 due to extensive resonance.^[8] This resonance involves multiple contributing structures that distribute electron density across the hydrazyl core, enhancing the stability of the radical. The unpaired electron 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.^[9] 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°.^[10] 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 D, which is higher than that of the reduced form (3.59 D) and arises from the asymmetric charge distribution in the resonance hybrids, with the picryl group acting as an electron acceptor 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 solid and solution states.^[9]

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 lead dioxide (PbO₂) as the oxidant in dichloromethane (CH₂Cl₂) as the solvent.^[11] This one-step process is conducted at room temperature under an inert atmosphere to minimize side reactions and ensure the stability of the resulting radical.^[11] 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 hydrazine.^[11] Alternative oxidants can be employed for similar conversions, including lead tetraacetate, silver oxide (Ag₂O), or potassium permanganate (KMnO₄), also in non-polar solvents such as dichloromethane or benzene.^[11] For instance, KMnO₄ oxidation requires a phase-transfer catalyst 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.^[12] These variations maintain room-temperature conditions and inert atmospheres, with reaction completion monitored by thin-layer chromatography.^[12] Following oxidation, the reaction mixture is filtered to remove insoluble byproducts such as reduced manganese dioxide or excess PbO₂.^[11] The crude DPPH radical is then purified by recrystallization from hot ethanol or chloroform, yielding dark purple to black crystals suitable for spectroscopic and assay applications. The overall transformation follows the general oxidation equation:

\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.^[11]

Key precursors and reactions

The parent hydrazine precursor to DPPH, known as 2,2-diphenyl-1-picrylhydrazine (DPPH-H), is synthesized through the nucleophilic aromatic substitution reaction of picryl chloride (2,4,6-trinitrochlorobenzene) with 1,1-diphenylhydrazine.^[1] This reaction typically proceeds in ethanol or pyridine as solvents, leveraging the electron-withdrawing nitro groups on the picryl chloride to facilitate displacement of the chloride ion by the hydrazine nucleophile.^[1] The resulting DPPH-H is a yellow 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 hydrazine intermediate to azo compounds via excessive oxidant exposure, which leads to N-N bond cleavage and formation of stable, non-radical byproducts.^[1] Additionally, decomposition occurs readily in protic solvents, where protonation promotes instability of the nitro-substituted aromatic ring.^[1] The original preparation of DPPH traces back to 1922, when Goldschmidt and Renn oxidized 2,2-diphenyl-1-picrylhydrazine using a similar hydrazine-based method, yielding a stable violet radical solution that highlighted its persistence compared to other triarylmethyl radicals.^[13] To optimize yields, anhydrous conditions are essential during the reaction and workup stages, as trace water can induce hydrolysis of the sensitive nitro groups on the picryl moiety, reducing the purity and quantity of the desired hydrazine product.^[1]

Physical and chemical properties

Appearance and solubility

DPPH appears as a dark purple to black crystalline powder at room temperature.^[5]^[14] This characteristic form arises from its stable free radical nature, contributing to its utility in laboratory settings.^[15] The compound exhibits a density of approximately 1.4 g/cm³.^[14] Upon heating, DPPH decomposes above 130–140 °C without undergoing melting, as indicated by literature values around 135 °C for decomposition.^[15]^[14] 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.^[15]^[14] In ethanol, solutions of DPPH display a deep violet color due to absorption at a maximum wavelength (λ_max) of about 517 nm.^[16]^[17]

Stability and reactivity

DPPH demonstrates exceptional chemical stability as a free radical, remaining intact in air and in solution for months without significant degradation. This persistence arises primarily from steric protection provided by the bulky phenyl and picryl substituents, which shield the nitrogen-centered radical from intermolecular interactions, and from extensive delocalization of the unpaired electron across the molecule, stabilizing the radical through resonance.^[13] 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.^[13] In reactive scenarios, DPPH functions as either a hydrogen atom acceptor or a one-electron donor, enabling it to participate in radical quenching reactions typical of antioxidant evaluations. Upon interaction with suitable scavengers, it is reduced to the stable, non-radical diphenylpicrylhydrazine (DPPH-H), effectively pairing the unpaired electron.^[13] The redox potential of the DPPH•/DPPH-H couple is approximately 0.3 V versus the saturated calomel electrode (SCE) in ethanol, reflecting its moderate oxidizing ability suitable for probing antioxidant capacities. Despite its overall robustness, DPPH exhibits sensitivity to certain environmental factors, decomposing gradually in the presence of light 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 unpaired electron delocalization, which enhances stability, also plays a role in mitigating reactivity with atmospheric oxygen.^[13]

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.^[4] 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 ultraviolet region, which are primarily due to π-π* transitions within the aromatic picryl and phenyl moieties.^[18] 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 unpaired electron and disruption of the chromophore.^[4] 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 solvents; for instance, the λ_max is approximately 517 nm in methanol compared to 515 nm in ethanol.^[17] This effect arises from solvent stabilization of the excited state. In antioxidant assays, the electronic spectroscopy of DPPH is routinely exploited by measuring the decrease in absorbance at 517 nm to quantify radical scavenging activity, providing a simple and rapid endpoint for evaluation.^[18]

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 unpaired electron with the two nitrogen nuclei, each having a nuclear spin quantum number I = 1.^[19]^[1] Although the hydrazyl and picryl nitrogen atoms are chemically distinct, their hyperfine coupling constants differ by only about 1.5 G, resulting in an effectively symmetric quintet spectrum rather than the 9 lines expected for fully inequivalent couplings.^[19] The isotropic hyperfine coupling constants have been precisely determined as a_{\mathrm{N1}} = 9.35 \pm 0.20 G for the hydrazyl nitrogen and a_{\mathrm{N2}} = 7.85 \pm 0.20 G for the picryl nitrogen, reflecting the greater spin density on the hydrazyl N atom due to its direct involvement in the radical center.^[19]^[1] These values indicate significant delocalization of the unpaired electron across the conjugated \pi-system, consistent with the radical's stability. The g-factor of DPPH is 2.0036, a value typical for \pi-delocalized organic radicals where the unpaired electron interacts primarily with the orbital magnetic moment of carbon and nitrogen atoms.^[20]^[21] In solution, the ESR linewidth of DPPH is narrow, approximately 1 G, which contributes to its widespread use as a primary standard for calibrating ESR spectrometers, as the sharp lines allow accurate determination of magnetic field and sensitivity.^[22]^[23] The spectrum in the solid state simplifies to a single broad line due to intermolecular interactions, but the solution-phase quintet remains the reference for radical characterization.^[1] 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 spectral distortion.^[1] This temperature independence arises from the robust delocalization that suppresses spin-lattice relaxation mechanisms.^[24]

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.^[4] The simplified reaction can be represented as:

\text{DPPH}^\bullet + \text{AH} \to \text{DPPH-H} + \text{A}^\bullet

where AH denotes the antioxidant and A• its derived radical.^[25] 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.^[26] The percentage inhibition of DPPH• is calculated using the formula:

\% \text{ inhibition} = \frac{A_{\text{control}} - A_{\text{sample}}}{A_{\text{control}}} \times 100

where A_{\text{control}} is the absorbance of the DPPH solution without sample and A_{\text{sample}} is the absorbance with sample.^[25] 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• absorbance by 50%, typically determined from a dose-response curve.^[26] IC₅₀ values are commonly compared to reference standards such as Trolox (a water-soluble vitamin E analog) or ascorbic acid to benchmark activity, with lower IC₅₀ indicating higher scavenging capacity.^[4] The DPPH assay offers advantages including simplicity, speed (typically under 1 hour), cost-effectiveness, and independence from biological systems, making it suitable for high-throughput screening of both hydrophilic and lipophilic antioxidants.^[25] However, limitations include its reliance on non-physiological solvents like ethanol, which may not mimic biological conditions, and potential overestimation of activity for hydrophobic compounds due to solubility biases in the reaction medium.^[27]

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.^[28] 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 hydrogen atom transfer or electron transfer 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 methyl methacrylate under UV irradiation has been used to quantify free radical propagation kinetics.^[29]^[30] A dilute solution of DPPH (typically 0.1 mM in benzene) serves as a primary standard in ESR spectrometry due to its temperature-independent g-factor of 2.0036 and narrow linewidth, facilitating precise calibration of magnetic field strength and signal intensity. The ESR spectrum of DPPH in solution exhibits a characteristic five-line hyperfine pattern (1:2:3:2:1 intensity ratio) 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.^[9] Beyond these roles, DPPH finds application in polymer chemistry primarily as a potent inhibitor of free radical-mediated polymerization by trapping chain-carrying radicals, thereby allowing assessment of initiation efficiency and transfer constants. It also acts as a catalyst in certain oxidation reactions and, in biochemical contexts, supports site-directed spin-labeling studies by serving as a paramagnetic quencher to measure accessibility parameters of nitroxide-labeled proteins in membranes. Historically, in the early 1950s, DPPH was instrumental in confirming radical mechanisms during organic syntheses, such as addition reactions to unsaturated systems, predating its widespread adoption in antioxidant assays.^[29]