j. Soc. Cosmet. Chem., 45, 257-268 (September/October 1994) FT-Raman spectroscopyApplications in hair research CHANDRA M. PANDE, Clairol Inc., 2 Blachley Road, Stamford, CT 06922. Received July 15, 1994. Synopsis FT-Raman (Fourier transform Raman) spectroscopic measurements have been performed on human hair. This technique provides a rapid and non-destructive in situ method for monitoring the disulfide bonds and their oxidation and reduction products in hair. The aromatic amino acids, by virtue of their side chains, are also clearly discernible in the spectra. It is shown that the effect of cosmetic hair treatments that modify the disulfide bonds, such as permanent waving and bleaching, can be conveniently monitored. Hair photo- damage studies reveal that besides damage to the disulfide bonds, the peptide backbone is significantly affected. These data suggest that the FT-Raman technique is ideally suited for hair research and will find practical use in the formulation and testing of milder, less damaging, cosmetic products for hair. INTRODUCTION Human hair fibers are primarily made up of keratin proteins and are roughly 50-80 in diameter. Nearly 95% of the dry hair mass is proteinaceous. The remainder is made up of lipids, pigments, and some bound ions (1). Almost 10% of the total hair protein mass derives from the disulfide-containing amino acid cystine. Under ambient condi- tions, hair fibers are hydrated and bind as much as 10% water by weight (2). The fibers have a complex morphology and show three distinct regions: the outermost cuticle layers, the inner cortical cells and, occasionally, an innermost and porous medulla. The cortical cells contain o•-helical protein arranged in supramolecular fibrillar arrange- ments. These, in turn, are embedded in an amorphous protein matrix (3). Spectroscopic methods provide a non-invasive way of analyzing molecular species in situ. These methods are non-perturbing and, therefore, do not suffer from the usual limita- tions, such as the lability of certain amino acids, that are associated with hydrolytic procedures (4). Vibrational spectroscopy, employing Raman and Fourier transform in- frared (FTIR) techniques, involves identification of molecular species based on their group vibrational frequencies. While both methods give information on the vibrational frequencies of various bonds, the mode of eliciting molecular response to the impinging photons is quite different. The IR technique is based on the absorption of photons, whereas the Raman effect is a scattering phenomenon. Thus, the two techniques com- plement each other in structure determination and identification of molecules (5). 257

258 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The success of vibrational spectroscopy in structural studies involving complex macro- molecules relies on the fact that the group vibrational frequencies show little variation from one molecule to another. Thus, comparison of the observed sample spectrum with the spectra obtained for model compounds, with known structures, leads to structural assignments (6,7). FTIR spectroscopy has been used in hair research quite extensively (8). It has found practical application in monitoring the oxidation of disulfide bonds to cysteic acid resulting from cosmetic hair treatments such as bleaching (9, 10) and oxidative dyeing (C. Pande, unpublished observations). Weathering of hair and wool has also been shown to result in the oxidation of disulfide bonds to cysteic acid and some other intermediate oxidation states of sulfur (11-13). The amount of cysteic acid formed has, therefore, been used as an index of damage to the disulfide bond. Although FTIR is a powerful technique, one of its weaknesses has been its inability to measure the disulfide bonds directly. Calculations of disulfide damage, based on measurements of cysteic acid alone, are likely to underestimate damage since other intermediate redox products may form (1, 11). Raman spectroscopy, using visible laser light, has been used to study proteins including keratins (7, 14-16). The fluorescence associated with biological tissues, particularly those containing pigments, has been one of the limitations of excitation in the visible region. Some of these problems have been resolved by the availability of FT-Raman instrumentation, which uses near-IR lasers (5). Since most biological chromophores do not absorb in this region, the Raman spectra are free of fluorescence contamination. We have obtained FT-Raman spectra of human hair. Compared to FTIR, the Raman technique provides a much more detailed analysis of hair. The measurements do not require any sample preparation and are not affected by the presence of water. Applica- tions of this technique in monitoring damage associated with cosmetic hair treatments are discussed. It is also shown that hair photodamage can be effectively studied with this method. EXPERIMENTAL Measurements were made on Piedmont hair (blonde human hair from the Piedmont region of Italy) purchased from DeMeo Brothers, New York. Hair tresses 6" long and weighing 2 g were used in the experiments. Chemical treatments were performed on one half of a tress while the other half served as the control. Dark pigmented hair was not used in these measurements due to overwhelming fluorescence of melanin detected in the initial studies. The results presented here, however, are general in nature because the presence of pigments, usually less than 2% of the fiber weight (17), is not likely to influence significantly the chemical reactivity of keratin during cosmetic treatments. Hair bleaching experiments were carried out with BW2 and 20 vol. hydrogen peroxide (4.7 g and 11.3 g, respectively, both from Clairol Inc.) for 1 h, followed by rinsing and drying. BW2 is a commercial product containing ammonium and potassium persulfates in combination with sodium metasilicate. Hair reduction was carried out with Kindness © Extra Curly Perm (Clairol Inc.) for 30