535 CHARACTERIZATION OF BLEACHED HAIR areas against the area of the phenylalanine band at 1003 cm−1 allowed for a quantitative probe of hair bleaching effects. Quantitatively, Figure 11 traces the content of the two essential indicators of the hair cortex structural integrity, namely the states of disulfide and cysteic acid plotted as a function of bleaching time. These indicators are visualized through the intensity (area) of vibrational bands for disulfide linkages (-S—S-) at 509–511 cm−1 and the cysteic acid band (-S—O- stretch) at approximately 1040 cm−1. The plotted intensity response of these bands is elegantly complimentary, displaying the disulfide intensity loss due to cystine-bond scission, accompanied by the increase in intensity of the cysteic acid band at 1040 cm−1, which together corroborate oxidative bleaching damage. To verify the integrity of the band normalization, the ratio of the 509 cm−1 and 1040 cm−1 bands to the 1450 cm−1 band were also calculated, yielding equivalent trends (17). The progression in intensity for the 509 cm−1 and 1040 cm−1 indicator bands conveys a distinct sigmoidal response to applied bleaching time. This change is particularly pronounced at shorter bleaching times, with an acceleration and apparent plateauing noted in the first hour. As mentioned in the infrared results, the nonlinear response segment between 15 and 60 min of bleaching may relate to the effects of bleach solution viscosity, sampling artifacts, or to the delayed swelling behavior of the fiber (i.e., due to increased alkalinity) in the early moments of the treatment. Agreement between Raman and FTIR bleaching kinetics is demonstrated by comparing the FTIR 1040 cm−1 cysteic acid band intensities, which are measurable with FTIR and Raman, to the 509 cm−1 -S—S- band intensity obtained exclusively from Raman scattering. Comparing the spectral markers in the virgin and 180-min bleached dark brown and natural white hair cross-sections, Figure 12A indicates that melanin granules and/or solubilized melanin remnants in dark brown hair negligibly influenced the height and area of the FTIR-ATR 1040 and 1080 cm−1 spectral EDF bands. In addition, Figure 12B compares the 1040/1080 cm−1 integrated band intensity ratio from FTIR imaging (using dark brown hair cross-sections) against the Raman 509/1003 cm−1 integrated shift ratio (using natural white hair cross-sections). The results for two completely different sets of experiments yielded a reasonably linear result (R2= 0.94), thus validating the ability to interweave EDF data from the Raman and FTIR techniques. Summarizing, equivalent damage factors were calculated from cortical and cuticular cysteic acid versus keratin band area normalizations. To contrast spectroscopic techniques and Figure 12. (A) Overlay of FTIR spectra for virgin European dark brown and natural white hair cross-sections bleached for 0 and 180 min. For each hair type, the spectra of virgin and bleached cross-sections are virtually identical and (B) comparison of EDF and EDF–1 evaluations from FTIR imaging and Raman scattering, respectively. EDB: European dark brown White: Natural white.

536 JOURNAL OF COSMETIC SCIENCE band intensity evaluations, normalized EDF values for each bleaching time are displayed in Tables II and III, where normalized EDF values were calculated using Equation 2. Table II includes normalized cortical EDF values, and Table III contains normalized whole hair tress EDF indices. Normalized Band normalized cm-1 intensity for bleach EDF = 1040 fibers Band normalized cm-1 intensityfor unbleached 1040 fibers (2) where normalized EDF ≥1 for bleached hair, and band normalized 1040 cm−1 refers to indexing the 1040 cm−1 intensity to the 1003, 1080, 1548, and 1650 cm−1 protein bands, as described in the “Raman Spectroscopy of Cross-Sectioned Hair” and “FTIR-ATR of Hair Cross-Sections” sections. Overall, EDF values increased with increasing bleaching time. The cortical EDF values for Raman against FTIR techniques correlate linearly (R2=0.95–0.99), independent of the band chosen for normalization. Similarly, if the 0 and 15 min bleaching Table II Normalized Cortical EDF Ratios Versus Bleaching Time as Indexed from Various Spectroscopic Methods Bleaching time (min) Raman cortexa 1040/1003 cm−1 ATR cortexa 1040/1548 cm−1 Imaging cortexa 1040/1548 cm−1 Imaging cortexb 1040/1650 cm−1 Imaging cortexb 1040/1080 cm−1 0 1.00 1.00 1.00 1.00 1.00 15 2.25 1.34 1.36 1.10 1.68 30 3.04 1.49 1.44 1.14 2.04 45 3.66 1.61 1.69 1.21 2.20 60 4.00 1.83 1.97 1.26 2.23 90 5.03 2.44 2.42 1.32 3.04 120 5.99 3.29 3.72 1.39 3.29 180 6.68 4.27 4.44 1.43 4.04 240 9.19 5.34 5.21 1.50 5.09 a Normalized by band area. b Normalized by band height. Table III Normalized Whole Hair Tress EDF Ratios Versus Bleaching Time as Indexed from the Various Spectroscopic Methods Bleaching time (min) Raman whole fibera 1040/1003 cm−1 ATR whole fibera 1040/1080 cm−1 ATR whole fibera 1040/1548 cm−1 Raman whole fibera 1040/1650 cm−1 Spectrofluorimetry (TRP)b 440/339 nm intensity 0 1.00 1.00 1.00 1.00 1.00 15 1.65 1.23 1.08 1.71 1.26 30 4.49 1.53 1.23 4.19 1.52 45 4.97 2.25 1.53 5.93 1.65 60 6.84 2.89 2.57 8.46 1.93 90 7.93 3.70 3.52 9.79 1.99 120 8.31 4.00 4.06 10.55 2.17 180 8.19 4.35 4.03 10.89 2.18 240 8.34 4.40 4.02 11.74 2.52 a Normalized by band area. b Normalized by band height.