Abstract

Nile red, systematically named 9-(diethylamino)-5H-benzo[a]phenoxazin-5-one with molecular formula C₂₀H₁₈N₂O₂ and molecular mass 318.376 g/mol, represents a highly solvatochromic phenoxazinone dye of significant analytical utility. The compound exhibits a melting point range of 203–205 °C and demonstrates exceptional photophysical properties characterized by environment-dependent fluorescence emission. Nile red manifests intense fluorescence exclusively in nonpolar environments with emission maxima shifting from approximately 585 nm in neutral lipids to 638 nm in polar phospholipids. Its pronounced solvatochromism enables applications as a selective stain for lipid detection and as a molecular probe for microenvironment polarity assessment. The compound's synthesis typically proceeds through acid-catalyzed condensation reactions or hydrolysis of Nile blue precursors.

Introduction

Nile red belongs to the class of organic heterotricyclic compounds specifically classified as benzo[a]phenoxazin-5-ones. This lipophilic dye possesses substantial scientific importance due to its extraordinary solvatochromic properties and selective fluorescence in hydrophobic environments. First developed as a derivative of Nile blue, the compound has found extensive application as a fluorescent probe in materials science and analytical chemistry. The structural foundation consists of a planar phenoxazinone core system substituted with a diethylamino electron-donating group, creating a pronounced push-pull electronic system responsible for its unique photophysical behavior. The compound's capacity to serve as a microenvironment polarity sensor stems from its sensitivity to solvent relaxation processes and dielectric constant variations.

Molecular Structure and Bonding

Molecular Geometry and Electronic Structure

The molecular architecture of Nile red features a rigid, planar benzo[a]phenoxazinone tricyclic system with approximate C_s point group symmetry. The diethylamino substituent at position 9 adopts a conformation where the ethyl groups rotate out of the molecular plane to minimize steric interactions with the aromatic system. X-ray crystallographic analysis reveals bond lengths typical for aromatic systems: C-C bonds in the range of 1.38–1.42 Å, C-N bonds of approximately 1.35 Å, and C-O bonds of 1.36 Å. The carbonyl group at position 5 exhibits a bond length of 1.22 Å, characteristic of polarized carbonyl functionality. Molecular orbital calculations indicate highest occupied molecular orbital (HOMO) localization on the diethylamino-substituted aromatic ring and lowest unoccupied molecular orbital (LUMO) predominance on the carbonyl-containing ring system, creating a substantial intramolecular charge transfer character.

Chemical Bonding and Intermolecular Forces

Covalent bonding in Nile red follows typical aromatic patterns with sp² hybridization throughout the tricyclic system. The molecule exhibits a calculated dipole moment of approximately 5.2 D in the gas phase, oriented from the electron-donating diethylamino group toward the electron-withdrawing carbonyl functionality. Intermolecular forces include van der Waals interactions, dipole-dipole forces, and limited hydrogen bonding capacity through the carbonyl oxygen atom. The compound demonstrates moderate π-π stacking capability in solid state with interplanar distances of approximately 3.5 Å. Crystallographic analysis reveals herringbone packing motifs with molecules arranged in slipped parallel stacks. The diethylamino group provides limited hydrogen bond acceptance capacity through the nitrogen lone pair, with calculated hydrogen bond acceptance ability of approximately 8.5 kJ/mol.

Physical Properties

Phase Behavior and Thermodynamic Properties

Nile red presents as a dark red to purple crystalline solid at room temperature with a characteristic metallic green sheen in reflected light. The compound melts sharply between 203 °C and 205 °C with decomposition observed above 250 °C. Differential scanning calorimetry reveals a heat of fusion of 28.5 kJ/mol ± 0.5 kJ/mol. The density of crystalline Nile red measures 1.32 g/cm³ at 20 °C. Solubility parameters vary considerably with solvent polarity: solubility in water is negligible (less than 0.01 mg/mL), while solubility in nonpolar solvents such as hexane reaches 15 mg/mL at 25 °C. The refractive index of crystalline material measures 1.78 at 589 nm. Molar volume calculations yield 241 cm³/mol with a molecular parachor of 812. The compound exhibits no known polymorphic forms under standard conditions.

Spectroscopic Characteristics

Nile red exhibits pronounced solvatochromic behavior in ultraviolet-visible spectroscopy with absorption maxima ranging from 450 nm in polar solvents to 552 nm in nonpolar environments. Molar extinction coefficients reach 45,000 M⁻¹cm⁻¹ in hexane at 552 nm. Fluorescence emission demonstrates exceptional solvent dependence with quantum yields varying from near zero in water to 0.68 in nonpolar solvents. Emission maxima shift from 585 nm in triglycerides to 638 nm in phospholipids. Infrared spectroscopy reveals characteristic vibrations: carbonyl stretch at 1650 cm⁻¹, aromatic C=C stretches between 1580–1600 cm⁻¹, and C-N stretch at 1340 cm⁻¹. Nuclear magnetic resonance spectroscopy shows proton signals at δ 8.5–8.7 ppm for H-4 and H-6, δ 7.2–7.8 ppm for aromatic protons, and δ 3.4 ppm for methylene protons of the diethylamino group, with carbon-13 signals including δ 180.5 ppm for the carbonyl carbon and δ 44.2 ppm for the methylene carbons.

Chemical Properties and Reactivity

Reaction Mechanisms and Kinetics

Nile red demonstrates moderate chemical stability under ambient conditions but undergoes photodegradation under prolonged ultraviolet irradiation with a quantum yield of photodecomposition of 0.012 in aerated solutions. The compound exhibits resistance to hydrolysis across pH 3–9 with degradation observed under strongly acidic (pH < 2) or basic (pH > 10) conditions. Acid-catalyzed hydrolysis proceeds through protonation of the carbonyl oxygen followed by nucleophilic attack at the adjacent carbon, resulting in ring opening with a rate constant of 3.2 × 10⁻⁴ s⁻¹ in 1 M HCl at 25 °C. The diethylamino group undergoes gradual N-dealkylation under oxidative conditions with hydrogen peroxide, yielding the monoethyl and subsequently the primary amine derivatives. Reduction with sodium borohydride produces the corresponding hydroxyl derivative at position 5 with complete loss of fluorescence properties.

Acid-Base and Redox Properties

The compound exhibits basic character through the diethylamino group with a pK_a of 3.1 in water, protonating to form a cationic species with altered absorption characteristics. The carbonyl group demonstrates extremely weak acidity with pK_a estimated above 15. Electrochemical analysis reveals a reversible one-electron reduction wave at -0.85 V versus standard calomel electrode corresponding to formation of the radical anion, and an irreversible oxidation wave at +1.12 V associated with formation of the radical cation. The compound demonstrates stability toward common oxidants including molecular oxygen but undergoes rapid degradation in the presence of strong oxidizing agents such as potassium permanganate or ceric ammonium nitrate. Cyclic voltammetry shows a HOMO-LUMO gap of 2.07 eV in acetonitrile solution.

Synthesis and Preparation Methods

Laboratory Synthesis Routes

The most common laboratory synthesis involves acid-catalyzed condensation of 5-(diethylamino)-2-nitrosophenol with 2-naphthol. The reaction typically employs concentrated sulfuric acid as both catalyst and solvent at temperatures between 80–100 °C for 2–4 hours. This method yields Nile red in moderate quantities (typically 45–55%) with the major byproduct being unreacted starting materials and oxidation products. Purification proceeds through column chromatography on silica gel using dichloromethane as eluent, followed by recrystallization from ethanol. An alternative synthetic pathway involves acid hydrolysis of Nile blue sulfate with concentrated sulfuric acid at reflux temperatures for 1–2 hours. This method produces Nile red in higher purity but lower overall yield (35–40%) due to competing decomposition pathways. The reaction mechanism proceeds through initial protonation of the iminium nitrogen followed by nucleophilic water attack and elimination of aniline derivative.

Analytical Methods and Characterization

Identification and Quantification

Nile red identification typically employs reversed-phase high-performance liquid chromatography with C18 stationary phase and acetonitrile-water mobile phase gradients. Retention time under standard conditions (70% acetonitrile, 30% water) is 6.8 minutes with detection at 550 nm. Mass spectrometric analysis shows molecular ion peak at m/z 318.1367 with characteristic fragmentation patterns including loss of ethyl groups (m/z 290, 262) and cleavage of the phenoxazinone system. Quantitative analysis utilizes fluorescence detection with excitation at 530 nm and emission at 590–640 nm depending on solvent environment. The limit of detection by fluorescence spectroscopy reaches 0.1 nM in nonpolar solvents. Thin-layer chromatography on silica gel plates with ethyl acetate:hexane (1:1) mobile phase yields an R_f value of 0.45 with visible red spot under daylight and intense yellow fluorescence under 365 nm illumination.

Purity Assessment and Quality Control

Commercial Nile red typically contains impurities including unreacted precursors, decomposition products, and isomeric compounds. High-purity material exhibits absorbance ratios A₅₅₂/A₅₀₀ > 4.5 in hexane solution. HPLC purity assessment requires >98% area percentage under specified chromatographic conditions. Common impurities include monoethylamino derivative (retention time 5.2 minutes) and deoxygenated byproducts (retention time 7.5 minutes). The compound demonstrates stability for at least two years when stored protected from light at -20 °C in inert atmosphere. Accelerated stability testing shows less than 5% decomposition after 30 days at 40 °C. Elemental analysis for pure compound requires carbon 75.45%, hydrogen 5.70%, nitrogen 8.80%, and oxygen 10.05% within ±0.4% of theoretical values.

Applications and Uses

Industrial and Commercial Applications

Nile red serves primarily as a fluorescent stain for detection and quantification of neutral lipids in various industrial contexts. The dye finds application in biodiesel production monitoring as a rapid indicator of triglyceride content in feedstocks with detection limits of 0.01% (w/w). Petroleum industry applications include use as a tracer in enhanced oil recovery processes due to its partition coefficient favoring oil phases. The compound functions as a solvatochromic probe in polymer science for characterizing microenvironments in polymeric materials, particularly for assessing hydrophobicity of polymer surfaces and interfaces. Manufacturing applications include quality control in lubricant production where Nile red detects hydrocarbon contamination in synthetic lubricants at concentrations as low as 10 ppm. The global market for Nile red remains specialized with annual production estimated at 100–200 kg worldwide.

Research Applications and Emerging Uses

Research applications predominantly exploit Nile red's solvatochromic properties for microenvironment characterization. The compound serves as a molecular probe for studying micelle formation, critical micelle concentration determination, and microemulsion characterization. Materials science applications include incorporation into sensor systems for detection of organic vapors through fluorescence changes, with particular sensitivity to polar volatile organic compounds. Emerging applications involve use in organic electronics as a dopant in light-emitting devices, leveraging its efficient fluorescence in solid state. Recent research explores Nile red derivatives with modified substituents for tuned photophysical properties, particularly for application in fluorescence lifetime imaging microscopy. The compound's capacity for intersystem crossing with reasonable triplet yield (Φ_T = 0.15) suggests potential applications in photodynamic therapy research, though this area remains exploratory.

Historical Development and Discovery

Nile red originated from early twentieth-century dye chemistry research focused on oxazine and phenoxazine derivatives. The compound was first reported in the chemical literature in the 1960s as a hydrolysis product of Nile blue, a histological stain known since the late nineteenth century. Systematic investigation of its photophysical properties began in the 1980s when researchers recognized its exceptional solvatochromic behavior and environment-sensitive fluorescence. The application as a specific lipid stain was developed in 1985 through pioneering work demonstrating selective fluorescence in intracellular lipid droplets. Throughout the 1990s, research expanded to include applications in polymer science and materials characterization. The twenty-first century has seen renewed interest in Nile red derivatives with modified spectral properties and applications in advanced analytical techniques including single-molecule spectroscopy and super-resolution microscopy.

Conclusion

Nile red represents a chemically distinctive phenoxazinone dye with exceptional solvatochromic properties that enable diverse applications in analytical chemistry and materials science. The compound's rigid planar structure and push-pull electronic system create pronounced environment-dependent photophysical behavior characterized by fluorescence emission shifts exceeding 50 nm between polar and nonpolar environments. Its selective fluorescence in hydrophobic media establishes utility as a sensitive probe for lipid detection and microenvironment polarity assessment. Synthetic accessibility through straightforward condensation or hydrolysis reactions permits widespread availability despite moderate yields. Current research continues to explore modified derivatives with enhanced photostability and tuned spectral properties, suggesting ongoing relevance in developing analytical methodologies and sensor technologies. The fundamental photophysical principles demonstrated by Nile red continue to inform design strategies for environment-responsive molecular probes across chemical and biological sciences.