525 CHARACTERIZATION OF BLEACHED HAIR HPDSC OF HAIR FIBER SNIPPETS A TA Instruments (New Castle, DE, USA) Q2000 DSC and Perkin-Elmer high-volume pressure pans (3-part assembly, part numbers: top 0319-1526, bottom 0319-1525, and O-ring 0319-1535) were used for all HPDSC studies. The fibers were cut into 2–3 mm pieces with titanium precision shears. Approximately 10 ± 0.5 mg of fibers were loaded into high-volume steel crucibles and dried in our 60°C forced-air oven for 1 h. The fiber mass was reweighed and 50 μL of distilled water was added to the dried snippets using a micropipette. The pan was then loaded onto the center depression of the spacer insert and crimped with a Perkin-Elmer Quick Press. For each of the nine hair tresses, at least five pans were prepared (n ≥ 5). Samples were equilibrated in the sealed pans for ≥12 h prior to being individually loaded into the indium-calibrated DSC cell using an automated sample cassette and robot. Several thermal ramping rates (β) were applied in this study. As per Istrate et al. (13), the T D and ΔH D of hair in excess water were evaluated using the following protocol: 1. Equilibrate at 20°C. 2. Isothermal for 2 min. 3. Ramp β °C/min to 180°C, where β = 0.5, 1.0, 2.0, 5.0, and 10°C/min were applied in the study. The Flynn–Wall–Ozawa method with the peak in T D was applied to approximate the activation energy for denaturation of the IFKPs. The ambient humidity was 28–32% relative humidity (RH). DRY DSC OF HAIR SNIPPETS A TA Instruments Q2000 DSC was used for the dry DSC work. The temperature and cell constant were calibrated using high-purity indium, and the heat capacity (C p ) was calibrated using sapphire discs. The control and bleached fibers were cut into 1–2 mm lengths and stabilized at 35% RH. Between 4 and 6 mg of fibers were loaded into aluminum Tzero pans (TA Instruments). No lids were used to permit the rapid release of pyrolysis gases. Two experiments were performed in the dry state. In the first, the fibers were equilibrated at 150°C for 5 min (to remove free water) before ramping to 325°C at 2°C/min. The data from the first experiment were used to monitor the peak in heat capacity as a function of temperature. In the second set of experiments, the fibers were equilibrated at 150°C for 5 min before ramping to 325°C at 15°C/min. The higher ramp rate produced the doublet endotherms, where the integrated area of the second endotherm was used to estimate the enthalpy for pyrolysis of the matrix (7). MTGA OF HAIR SNIPPETS A TA Instruments Discovery Series TGA with TRIOS control and analysis software (v3.3.1.4668) was used for all testing. The experiments were performed in modulated mode with high resolution ramp constraints. In dynamic high-resolution mode, the heating ramp rate is machine-adjusted such that the faster heating rate is used in temperature regions where no mass change is occurring, and a slower ramp rate is applied with the onset of mass changes. In MGTA, a sinusoidal temperature modulation is superimposed on the

526 JOURNAL OF COSMETIC SCIENCE heating ramp, and the sample-mass change in response to the modulation is recorded. The response provides an empirical tool for studying the kinetics of sample volatilization and/or decomposition. Discrete Fourier transform of the response allows kinetic parameters such as activation energy and pre-exponential factors to be calculated on a continuous basis. Unused snippets (4–6 mg) from the HPDSC experiments were loaded onto clean platinum TGA pans and the following protocol was used to denature and pyrolyze the samples: the furnace was equilibrated at 40°C high-resolution sensitivity = 1.00 modulation temperature amplitude = 5°C period = 200 s high-resolution ramp = 5°C/min to 300°C and resolution = 6.0. The hair fibers were denatured and pyrolyzed in a dry nitrogen environment (flow rate = 25 mL/min). DVS OF 5-μm HAIR FIBER CROSS-SECTIONS Water vapor absorption curves were obtained using a DVS Advantage I sorption analyzer (Surface Measurement Systems NA, Allentown, PA, USA) and microtomed cross-sections of virgin and bleached European dark brown hair (see “Hair Cross-Section Preparation” section). All experiments were conducted at 25°C with a nitrogen gas flow of 200 mL/min. The 5-µm thick microtomed cross-sections (4–6 mg) were loaded into a stainless steel mesh sample pan, and the following sorption-desorption procedure was applied: 1. Initial drying: 60°C and 0% RH for 1 h. 2. Isothermal equilibration: 25°C and 0% RH for 15 min. 3. Absorption curve: the microtomed fibers were subjected to increasing humidity in 10% RH steps from 0% to 90% RH with dm/dt = 0.002%/min for 15 min. 4. Desorption curve: after the absorption sequence, the water vapor was progressively desorbed from the sample by lowering the humidity in 10% RH steps from 90% to 0% RH with dm/dt = 0.002%/min for 15 min. SPECTROFLUORESCENCE MEASUREMENTS OF HAIR TRESSES Tryptophan levels in chemically treated hair were determined by carrying out fluorescence measurements using a Horiba Jobin Yvon (Edison, NJ, USA) FluoroMax-4 steady-state spectrofluorometer equipped with a bifurcated fiber optic probe. Spectra were collected directly from the surface of hair, approximately 1 in from the bottom of the wax portion of the hair tress (corresponding to the area of the tress proximal to the root). The emission and excitation slits were set at 5-nm bandpasses. The measurements were performed in emission mode, where tryptophan emission spectra were obtained by irradiating hair at 290 nm and monitoring the fluorescence emission at 339 nm. Data were provided in units of counts per second (cps), and the emission at 339 nm was normalized by taking the height ratio of tryptophan to kynurenine fluorescence (I 339 /I 440 ), or the height ratio of kynurenine to tryptophan fluorescence (I 440 /I 339 ). Average values were obtained by taking three measurements in neighboring zones of one hair tress. SPECTROCOLORIMETRY OF HAIR TRESSES To quantify the degree of color changes resulting from bleaching treatment in hair, we used a HunterLab ColorQuest XE spectrocolorimeter (Hunter Associates Laboratory, Inc.,