527 CHARACTERIZATION OF BLEACHED HAIR Reston, VA, USA). The use of the spectrocolorimeter enabled us to obtain the tristimulus (L, a, b) values, which were utilized to calculate discoloration parameters for describing color changes in the bleached hair. The data are reported in terms of the total color difference: ∆ = ∆ + ∆ + ∆b)2 E L)2 a ( ( )2 ( (1) Like the spectrofluorescence studies, three measurements obtained from one hair tress (approximately 1 in below the wax tab) represent the average reported values. RESULTS AND DISCUSSION In the current work, chemical bleaching was carried out on large (10 × 8 in) European dark brown hair tresses for a total of 4 h. At designated time intervals, including 15, 30, 45, 60, 90, 120, 180, and 240 min of continuous oxidative bleaching, samples were taken from the larger tress to assess the relationship between bleaching application times and changes to the chemical components of the hair fiber. For each sample, various spectroscopic and thermal analyses were conducted on whole fibers, short fiber snippets, and cryotomed fiber cross-sections. As much of this study utilized vibrational spectroscopy for insights into the molecular and kinetic aspects of hair bleaching, Table I (16–19) provides some convenient and relevant mid-IR and Raman shift band assignments. We report an EDF parameter that indexes the degree of damage to cystine residues (-S—S-) in hair (19). The EDF parameter is based on the 1040 cm−1 symmetric -S = O sulfonate (-SO 3 − ) stretching vibration band. However, as the intensity of individual FTIR-ATR spectra may vary, the 1040 cm−1 band intensity must be normalized to another invariable band in the mid-IR spectrum of the protein to ensure meaningful sample-to-sample comparisons. One approach is to ratio the 1040 cm−1 band to the 1080 cm−1 band (cystine monoxide stretch) in the mid-IR region. A potential difficulty in choosing the 1080 cm−1 band for normalization is that confounding Table I Mid-IR and Raman Band Assignments Pertinent to Hair Bleaching Band assignment Mid-IR (cm−1)(16,19) Raman shift (cm−1)(16–18) Amide I, α-helix 1650 1652 (16), 1671 (cuticle) and 1666 (cortex) Sulfonate, S–O symmetrical stretch 1040, 1042 (19) 1040 Cystine monoxide (R–SO–S–R) 1075–1080 — Sulfonate, S–O asymmetrical stretch 1175 — Cystine dioxide (R–SO2–S–R) 1229 — Amide III (N–H stretch) 1247 1245 (cuticle) and 1243 (cortex) CH2 scissoring 1448–1454 1440 (16), 1451 and 1465 (cuticle) and 1448 (cortex) (18) Amide II (C–N stretch, α-helix) 1547–1548 — Amide I (C = O stretch, α-helix) 1650–1655 1652 (α-helix) (16), 1671 (cuticle) and 1659 (cortex) N–H (primary amine) 3315 — -S—S- — 505 (cuticle) (17) and 507–509 (cortex) -C—S- — 664 (cuticle and cortex) Phenylalanine of keratin — 1004 (16), 1001–1003 (cuticle and cortex)

528 JOURNAL OF COSMETIC SCIENCE contributions from hair, including various sources of C—O stretching and phosphates from nuclear remnants, may interfere with accuracy additionally, using the 1080 cm−1 absorption band may be inappropriate when formulated additives are included in the studies (20). For this reason, the ratio between 1040 cm−1 and the amide II band at 1548 cm−1 was used as an alternate method for normalizing the 1040 cm−1 spectral marker. Note that the applied measurement technique, specific analyses, and local conformations slightly shifted the peaks representing the protein vibrational bands. FTIR IMAGING OF HAIR CROSS-SECTIONS Samples from the bleached tresses, which are displayed in order of increasing bleaching time in Figure 2, were sectioned as described in the “Hair Cross-Section Preparation” experimental section and arranged on CaF 2 crystals for optimal staging in the FTIR microscope. FTIR spatial maps were then collected, and the series of images were concatenated to produce a single standardized chemical image. The color bar spectrum in Figure 3 represents the sulfonate concentration range in the concatenated image, where dark blue specifies the lowest concentration, and green, yellow, and red specify Figure 2. Photograph of (1) European dark brown hair, and European dark brown hair bleached for (2) 15 min, (3) 30 min, (4) 45 min, (5) 60 min, (6) 90 min, (7) 120 min, (8) 180 min, and (9) 240 min. For the series of tresses, a plot of ΔE as a function of the kynurenine fluorescence intensity (I440/I339) resulted in a linear relationship of increasing slope with very good correlation (R2=0.91). Figure 3. Concatenated images of hair fiber cross-sections treated for the indicated times with a bleaching formula. The color bar denotes the mid-IR 1040/1080 cm−1 normalized band area intensity, where the dark- red color specifies the highest level of cysteic acid in the 2D image of a cross-section.