531 CHARACTERIZATION OF BLEACHED HAIR hair fiber cross-sections against the ATR crystal was used to take advantage of the 23:1 diameter-to-thickness aspect ratio, ensuring that the sections laid flat and the predominant signal originated from the cortex and medulla, with minimal contributions from the cuticle. Further, FESEM micrographs were used to estimate that the cross-sections were approximately 93.1 ± 1% (µm2/µm2) cortex, based on calculations using the average cross- sectional areas of the cortex and cuticle. Hence, the resultant spectra possessed excellent signal-to-noise ratios. Figure 6 summarizes the trends over the entire bleaching period. In the first treatment step (i.e., ≤60 min), there was an increase in cysteic acid, which was followed quickly by a short plateau after approximately 15 min of bleaching. The subsequent rate of damage (i.e., increasing cortical cysteic acid) then increased linearly through the remaining 180 min of treatment with the addition of fresh bleaching solution every 60 min. FTIR-ATR AND SPECTROFLOURESCENCE OF HAIR TRESSES To contrast FTIR-ATR analyses performed on collections of 3-µm cross-sections, the EDF ratio was also calculated from FTIR-ATR spectra measured with a single, whole hair tress. In this measurement, a portion of the uncut whole tress was pressed against the single- reflection ATR crystal. In an FTIR-ATR experiment with perfect contact, the evanescent wave from the IR source penetrates approximately 1–2 μm into the sample, meaning that only the cuticles are probed when performing FTIR-ATR on hair tresses therefore, unlike the FTIR-ATR experiment on cross-sections, this measurement provided spectra that solely represent the chemistry of the hair cuticle (see spectra in Figure 7). Further, during the bleaching process, the alkaline bleaching solution was in persistent, intimate contact with the perimeter of the fibers. Hence, we anticipated the cuticle to rapidly oxidize in the initial bleaching steps. Figure 8 summarizes the whole hair tress FTIR-ATR results and displays trends in the EDF calculated from the 1040/1548 cm−1 band ratio, showing that the rate of cysteic acid formation over the 4 h bleaching period is indeed nonlinear. Note that correlations with the 1040/1080 cm−1 band ratio obtained from hair tresses presented identical results (see EDF in Figure 8). Like hair tress ATR measurements, spectrofluorimetry of complete hair tresses is a means to study compositional changes to proteins on the fiber surface after applying cosmetic Figure 7. FTIR-ATR spectra for virgin (0 min bleached) and 240 min bleached whole-hair tresses. In previous work, the amide III (1241 cm−1) and cysteine monoxide (1080 cm−1) keratin bands had been used for cysteic acid (1040 cm−1) band normalization (16–19).
532 JOURNAL OF COSMETIC SCIENCE treatments. For example, tryptophan has been used as a fluorescence probe to monitor changes to keratin after chemical bleaching (22). In alkaline bleaching, the overall fluorescence of the fiber increases due to the degradation of melanin, which allows other chromophores in the hair to absorb light accompanied by fluorescence emission. Hence, to obtain meaningful data from fluorescence spectra, tryptophan peak intensities (I 339 ) were normalized to kynurenine (I 440 ). In our bleaching studies, the ratio of tryptophan to kynurenine fluorescence (I 339 /I 440 ) decreased, indicating that surface tryptophan had been damaged and subsequently converted into kynurenines. In contrast, the ratio of kynurenine to tryptophan fluorescence (I 440 /I 339 ) increased with increasing bleaching time. As was demonstrated with our FTIR-ATR whole fiber cystine oxidation studies, alkaline bleaching rapidly oxidizes labile surface amino acids, including keratinous tryptophan, where Figure Figure 8. Overlay of the cortical 1040/1548 cm−1 band height ratio (EDF) obtained from FTIR-ATR cross- sectional and whole hair tress FTIR-ATR analyses. The whole fiber (cuticle) cysteine-sulfonic acid ratio changed abruptly, whereas the cortical cysteic acid concentration linearly increased as a function of applied bleaching time. Figure 9. Hair tress normalized EDF (FTIR-ATR: 1040/1080 cm−1) and normalized tryptophan/kynurenine fluorescence (I339/I440) versus bleaching time. TRP: tryptophan.
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