529 CHARACTERIZATION OF BLEACHED HAIR increasingly higher levels of cysteic acid. Hence, cysteic acid levels in the fibers increased with increasing bleaching time, and the intensity of the 1040/1080 cm−1 band area ratio increased accordingly. Comparing the 120 and 180 min bleaching steps, the cysteic acid spatial concentration increased from the outer cortex to the inner cortex, which corroborates that bleaching is a diffusion-controlled process (1). Interestingly, the medulla was largely free of cysteic acid, where the deficiency in -SO 3 − was likely a function of obstructed cortical diffusion, lower cystine concentrations, and lower medullar protein density, where keratin concentrations are comparably lower in the medulla (1,21). To exemplify the resultant cysteic acid gradient in the bleached cortex, Figure 4 demonstrates the spatial distribution of cysteic acid in the 120 min bleached cross-sections. Normalizing the 1040 cm−1 marker to the amide II band clearly emphasizes the compartmentalization of the fiber, where the boundaries of the cuticle, cortex, and medulla are readily observable. Additionally, the neighboring scatter plot in Figure 4 details the 1D spatial distribution of cysteic acid within the cortex of a single cross-section, which is identified by an asterisk. As judged by the color bar and 1040/1548 cm−1 scatter plot intensities, the cuticles clearly presented the highest intensities of oxidized cystine, while the centers of each cortex exhibited relatively lower levels of cysteic acid. Further, the scatter plot shows that the cysteic acid concentration was highest in the outer cortex (near the cuticle) and lowest near the center of each cross- section. Although only the 120 min bleached sample is detailed, note that all bleaching times conveyed similar 1D radial cysteic acid distributions. Figure 5A contains a partial IR imaging spectrum (1900–900 cm−1) for a virgin European dark brown tress (0 min bleached) obtained from an individual pixel from the cortex of its chemical image. The dashed vertical line is centered on the 1040 cm−1 absorption band, where the intensity changes proportionally with cysteic acid levels. Figure 5B shows the equivalent pixel spectrogram for the 240 min bleached sample in which the 1040 cm−1 band intensity has increased due to 240 min of chemical oxidation and resultant scission of keratinous disulfide. Figure 4. FTIR image and 1D EDF gradient for the 120 min bleached cross-sections. Relative to the concatenated FTIR image in Figure 3, which was normalized to 1080 cm−1, the 1040 cm−1 cysteic acid band was instead normalized to the 1548 cm−1 amide II spectral marker. A representative gradient for a single cross-section (*) is shown. The EDF is highest at the outer cortex (0 µm) and lowest at the center of the cross- section (37 µm). The results specify that the cysteic acid concentration is highest near the cuticle and lowest near the center of the cross-section.

530 JOURNAL OF COSMETIC SCIENCE FTIR-ATR OF HAIR CROSS-SECTIONS Figure 6 shows the 1040/1548 cm−1 band height ratios against bleaching time for data generated by loading layers of 3-μm thick cross-sections onto the 1.5 mm diameter ZnSe single-reflection ATR crystal. Both band height and area ratios were measured, yielding equivalent analysis results. For simplicity, the protein content was normalized without using the 1071–1080 cm−1 cystine monoxide band instead, the EDF ratio was obtained from the 1040 cm−1 absorption intensity and the α-keratin component of the amide II band at 1548 cm−1 (see Table I). Substituting the amide II band for 1080 cm−1 ensured that the calculated EDF ratios were free of interference from neighboring IR absorptions, where the 1080 cm−1 band baseline is frequently infringed upon by (1) very strong -S = O absorptions emanating from the shoulder of the asymmetric sulfonate peak at approximately 1190 cm−1 (2) unwanted contributions from nuclear remnants (PO 2 − ) and (3) -C—O- functional groups belonging to extraneous sources. Lightly compressing the cylindrical Figure 5. Example FTIR imaging spectra (1900–900 cm−1) for the unbleached control (Tress 1 in Figure 1) and 240 min bleached European dark brown hair (Tress 9 in Figure 1). The dashed line is centered on the 1040 cm−1 band, which corresponds to changes in the cysteic acid concentration. AU: arbitrary units. Figure 6. Plot of the 1040/1548 cm−1 EDF band ratio obtained from FTIR-ATR cortex studies. The 1548 cm−1 component of the amide II band was chosen to normalize protein content. The encircled plateau in first 60 min was likely due to ageing of the bleaching solution.