519 Address all correspondence to Timothy Gillece, tgillece@yahoo.com Characterization of Bleached Hair: Vibrational Spectroscopy, Thermal Analysis, and Determination of Equivalent Damage Factor TIMOTHY GILLECE, LARRY SENAK AND ROGER L. MCMULLEN Ashland LLC, Bridgewater, New Jersey, USA (T.G., L.S., R.L.M) Accepted for publication May 27, 2021. Synopsis In this study, we sought to determine a practical correlation between disulfide bond oxidation and the thermal response of chemically bleached hair fibers. Bleaching processes, and the alkaline environment under which they are applied, cause scission of native covalent cystine cross-links in virgin hair fibers to form cysteine-sulfenic, cysteine-sulfinic, and cysteine-sulfonic (cysteic) acids in the cuticle, cortex, and, to a lesser extent, in the medulla. To further our understanding of hair bleaching kinetics, results from Fourier transform infrared (FTIR) chemical imaging, FTIR-attenuated total reflectance (FTIR-ATR), and Raman spectroscopic measurements were correlated with results from high pressure differential scanning calorimetry (HPDSC), dry differential scanning calorimetry (DSC), dynamic vapor sorption (DVS), and modulated thermogravimetric analysis (MTGA). Spectroscopic results were used to calculate an equivalent damage factor (EDF), which was used to index bleaching damage to the cuticular and cortical compartments of the hair fiber. Spectrofluorescence and colorimetry measurements were performed on bleached whole fiber hair tresses. Fluorescence measurements provided a means to monitor changes in the tryptophan and kynurenine levels, and colorimetry measurements were conducted to quantify the overall color change (ΔE) of hair at various bleaching intervals. FTIR imaging showed that cysteic acid levels in the fibers increased with increasing bleaching time and that the spatial distribution of cysteic acid builds from the outer cortex to the inner cortex, which further validates that bleaching is a diffusion-controlled process. FTIR- ATR studies with whole fiber hair tresses and 3-µm cross-sections showed that the cuticular cysteic acid concentration changes abruptly, whereas conversion of cortical cystine to cysteic acid is diffusion limited. Raman spectroscopy perfectly complemented FTIR-ATR and FTIR imaging, in which case Raman was used to directly follow changes in cystine (509 cm−1) as a function of bleaching time, whereas FTIR spectroscopy monitored increases in cysteic acid concentration (1040 cm−1). The cortical EDF values for Raman and FTIR spectroscopic techniques correlated linearly (R2= 0.93–0.99), whereas the association between whole tress and cortical EDF results was poor (R2= 0.61–0.73). For the series of bleached fibers, changes in the denaturation temperature (TD) from HPDSC analyses obeyed Fick’s laws of diffusion (R2= 0.99), where the diffusion constant was estimated to be 1.1 × 10−8 cm2min−1. Using the peak in TD, the model-free Ozawa method was applied to approximate changes in the activation energy of intermediate filament denaturation as a function of increasing bleaching time. After 90 min of bleaching, the HPDSC activation energies plateaued at 180 ± 8 kJ/mol against increasing cysteic acid concentration. Dry DSC results showed that conversion of cystine to cysteic acid increased the cortical mobility temperature, advocating that ionic and hydrogen-bonded networks stabilized components of the dry cortex during excessive heating. The MTGA pyrolysis onset temperatures ranged from 237°C to 248°C for virgin and 240 min bleached hair tresses, J. Cosmet. Sci., 72, 519–546 (September/October 2021)

520 JOURNAL OF COSMETIC SCIENCE respectively, where the onsets positively and linearly correlated with increases in cysteic acid concentration (R2= 0.95) however, the activation energy for pyrolysis of dry fibers showed a curvilinear correlation with Raman EDF, with a peak activation energy (554 ± 9 kJ/mol) corresponding to 60–90 min bleaching times. To establish connections between water management properties and cystine oxidation, linear trends in denaturation temperature against the normalized Raman cystine band at 509 cm−1 demonstrated that decreased cross-link density is directly connected to greater steady-state moisture regains (R2= 0.94). For hair tresses, low EDF correlated with high tryptophan levels however, with increased bleaching, tryptophan and cystine levels rapidly decreased. As expected, longer bleaching times produced increased differences in color, as indexed by ΔE. INTRODUCTION Hair bleaching is one of the most common chemical treatments of hair. This cosmetic procedure is carried out to lighten hair that is naturally dark. Therefore, the objective of such treatments is to destroy and remove melanin granules from the hair fiber. This is an oxidative process that is typically achieved with hydrogen peroxide (H 2 O 2 ) and ammonia. Since melanin granules are in the cortex of hair, hair bleaching formulations must penetrate the fiber integument. Unfortunately, bleaching of hair results in collateral damage to lipids, proteins, and other structural components of the fiber. Regarding oxidative susceptibility, some of the most labile amino acids in keratin are methionine, tyrosine, threonine, and tryptophan. Even more susceptible to bleaching damage are cystine residues, which predominantly undergo oxidative fission of sulfur-sulfur (-S–S-) cross-links, resulting in the formation of salts of sulfur acids, including sulfenic acid (-SOH), sulfinic acid (-SO 2 H), and sulfonic acid (-SO 3 H). Most of the species are intermediates while sulfonic acid, which is frequently referred to as cysteic acid, is the predominate moiety remaining after normal bleaching cycles (1). The oxidation of disulfide bonds in hair has a significant influence on the various components of the fiber. In fact, the highest concentration of disulfide bonds in the hair fiber is in distinct lamellar layers of cuticle cells including the A-layer and exocuticle however, in comparison to the cortex, the cuticle does not significantly contribute to the mechanical properties of hair since it is only a fraction of the total cross- sectional area. The cortex of hair is comprised of elongated cortical cells that are separated by a cell membrane complex. The cells are filled with macrofibrils containing low-sulfur intermediate filaments (crystalline phase) embedded in an amorphous matrix of cystine- rich proteins. Therefore, major targets of bleaching damage in the cortex are the disulfide bonds of the amorphous phase proteins. Measurements including tensile strength, fatigue testing, vibrational spectroscopy, liquid retention, DSC, thermogravimetric analysis (TGA), scanning electron microscopy, and amino acid analysis, have been extensively used by researchers to survey chemical and structural damage to hair fibers as a function of alkaline bleaching treatments (2–19). In one of the earliest studies, Edman and Marti provided empirical evidence that hydrogen peroxide degrades disulfide bonds and diminishes the work required to stretch fibers (5). The investigators performed their tensile studies in distilled water to exclusively evaluate the influence of disulfide cross-links on the fiber modulus and to eliminate strength and resilience contributions from hydrogen and ionic bonding. Specifically, tensile testing provides modulus and breaking strength values for the hair fiber, where the overall hair fiber modulus involves contributions from hydrogen bonds, ionic bonds, and disulfide bonds (6). With this understanding, Robbins evaluated the wet tensile properties of bleached fibers from a single source and found that the wet tensile strength decreased by nearly 60% for