521 CHARACTERIZATION OF BLEACHED HAIR highly bleached fibers, where the compromised fibers were additionally found to have a 48% reduction in native cystine (1). More recently, thermal analysis and vibrational spectroscopy have been used to further the thermochemical understanding of oxidative bleaching damage. Thermal analysis techniques, specifically DSC, differential thermal analysis, and TGA, have been extensively employed to probe melting and pyrolysis mechanisms in keratinous materials (6–14). In dry DSC experiments, snippets of dry hair fibers are heated to 250°–300°C and the intermediate filaments are consequently denatured and pyrolyzed in a coupled thermal event. Comparatively, in HPDSC experiments, water is added to the volume of hair fibers in hermetically sealed crucibles, wherein heating the cortex in excess water plasticizes the matrix and denatures the intermediate filaments at lower temperatures (e.g., 130°–155°C) than pyrolysis events (e.g., 210°C). Because the wet-matrix viscosity and associated cystine cross-links are directly related to denaturation temperatures measured in wet DSC, HPDSC provides a means to quantify the covalent cross-link density of the matrix (6,13). Vibrational spectroscopy has also been routinely used for studying oxidative hair damage and is typically less invasive and labor intensive than tensile testing protocols. Techniques including FTIR spectroscopy, FTIR imaging, and Raman spectroscopy have been applied to evaluate increases in cysteic acid, whereas confocal Raman spectroscopic imaging has been used to chemically image complementary decreases in disulfides bonds (16–18). In the present work, results from FTIR chemical imaging, FTIR-ATR, and Raman spectroscopic measurements were compared with results from HPDSC, dry DSC, DVS, MTGA, colorimetry, and spectrofluorescence analyses to discern fundamental chemical structure/physical property relationships in chemically bleached hair fibers. Spectroscopic results were then leveraged to calculate the equivalent damage factor (EDF), which was applied to comparatively index bleaching damage in the cuticular and cortical compartments of the hair fiber. MATERIALS AND METHODS European dark brown and natural white hair tresses were bleached for 15, 30, 45, 60, 90, 120, and 240 min. Cysteic acid formation in peroxide-bleached whole fibers and cryotomed cross- sections was monitored using Raman and two FTIR spectroscopic techniques. In Raman analyses, the EDF was observed by following trends in the symmetric sulfonate (S = O) to phenylalanine ratio (1040/1003 cm−1) and the attenuation in cystine (505–510 cm−1) via the 509/1003 cm−1 band area ratio. Similar approaches were taken in FTIR-ATR spectroscopy and FTIR imaging, where the ratios of symmetric sulfonate (1040 cm−1) to the cystine monoxide stretching band at 1080 cm−1 or amide II band (1548 cm−1) were used to monitor cystine oxidation. In this work, both raw EDF and normalized EDF indices are reported. Raw EDF indices, which hereafter are referred to as EDF, were taken directly from protein- normalized cysteic acid spectral intensities, whereas normalized EDF results were evaluated to enable comparison of bleaching damage between spectroscopic techniques and band area/ intensity normalizations. Normalized EDF calculations define EDF = 1.00 for nonbleached fibers, and accordingly the EDF indices for bleached fibers were always ≥1. These data were compared to results obtained from HPDSC, where endothermic transitions such as the denaturation temperature (T D ) and denaturation enthalpy (ΔH D ) were monitored. The T­ D parameter correlates with changes in matrix viscosity and cross-link density, whereas ΔH D

522 JOURNAL OF COSMETIC SCIENCE measures the energy required to break the interface between the matrix and intermediate filament keratin proteins (IFKPs) and the energy needed to denature IFKPs. Additionally, dry DSC, DVS, and MTGA were performed to further the understanding of bleaching kinetics and to provide correlations between keratin oxidation, cortical swellability, and cortical pyrolysis. Spectrofluorescence and colorimetry measurements were carried out on hair tresses to correlate changes in cuticular EDF with tryptophan degradation and ΔE. MATERIALS Studies were carried out on medium density European dark brown and natural white hair tresses that were purchased from International Hair Importers & Products Inc. (Glendale, NY, USA). The natural white fibers were used solely for the Raman scattering studies. The hair was supplied as large tresses constructed with rectangular pieces of wax, which secured the root ends of the hair fiber. From this large tress, ¾-in wide tresses were sampled as a function of bleaching time. Bleaching was carried out by mixing 120 g of Clairol Professional BW2 powder lightener (The Wella Corporation, Woodland Hills, CA, USA) with 147 mL of Salon Care Professional 20 Volume Clear developer (Arcadia Beauty Labs LLC, Reno, NV, USA). The resulting mixture was applied to damp hair. Bleaching was carried out for time periods of 15, 30, 45, 60, 90, 120, and 240 min. The 15, 30, 45, and 60 min bleaching experiments were accomplished using the same bleach mix and removing hair samples at selected intervals. The tresses bleached for 120, 180, and 240 min were placed in a freshly prepared bleach mix at 60, 120, and 180 min, respectively, and the 90 min sample was taken midway between the 60−120 min bleaching treatment. After each hour, the remaining tresses were thoroughly rinsed in warm tap water to remove water- soluble material prior to reimmersing the tress in fresh bleaching solution and restarting the bleaching clock. After the last bleaching step, each tress was thoroughly rinsed with 40°C tap water and then soaked in distilled water for 3 h to remove soluble leachate. The leachate removal step was repeated a total of five times for each tress. Finally, the tresses were air-dried overnight prior to subsequent analyses. HAIR CROSS-SECTION PREPARATION Quarter-inch wide excised samples of the tress were subjected to sectioning using a Leica CM3050 S (Leica Microsystems GmbH, Wetzlar, Germany) cryostat equipped with a high- profile sectioning head and Leica 818 high-profile cutting blades. Note that the cryostat blade was changed after each sample. For each sample, a 25-mm aluminum specimen disc was pre-equilibrated in dry ice (−78.5°C). A small section (1/8–1/4 in wide x 1−2 in length) of the ¾-in wide tress was removed from the center of the tress length. The tip end of the damp fiber section was then held straight and perpendicular against the platform of the disc. At the interface between the disc and tip end of the fiber bundle, a drop of distilled water was then added. The drop instantly crystallized, hence causing the tip end of the fiber bundle to adhere to the aluminum specimen disc. By pulling gently upward on the root end of the fibers and slowly adding water to the fiber bundle, a straight rod of ice-embedded hair was produced. Separately, each embedded sample was then mounted onto the specimen head (−30°C) of the cryostat, where the chamber temperature was equilibrated at −25°C.