Elucidation of adsorption mechanism of bone-staining agent alizarin red S on hydroxyapatite by FT-IR microspectroscopy

To elucidate adsorption mechanism of alizarin red S (ARS), which is often used for staining bones in histology, adsorption of ARS on hydroxyapatite, Ca-10(PO4)(6)(OH)(2) (HAP), was investigated by a batch method, compared with alizarin, phenols, and benzenesulfonates. We found that ionized 1-, 2-OH groups (1-, 2-O-) of ARS can be electrostatically bound to Ca2+ on HAP, but that the 3-SO3- group of ARS hardly participates in adsorption on HAP. ARS-adsorbed HAP (ARS-HAP) in dark reddish violet was also prepared and analyzed by FT-IR microspectroscopy to gain structural information on bonding between ARS and HAP. The obtained spectrum, which was converted to difference spectra, indicated a single band of v(C=O) at 1627 cm(-1) and two types of symmetric C-O stretching bands of is (C-O) + v(C=C) at 1345 cm(-1) and vs (CO) + 8(O-C=C) at 1272 cm(-1). These bands imply the existence of a salt form in ARS-HAP via 1-, 2-OH groups of ARS. As a result of the existence of a chelate form in ARS-HAP via 1-OH and 9-C=O groups of ARS, two bands of v(C=C) + v(C=O) at 1572 cm(-1) and v(C=O) + v(C=C) at 1537 cm(-1) were also observed. In addition, ARS was almost desorbed from colored ARS-HAP at 50 degreesC by using neutral phosphate buffer to recover slightly pale pinkish HAP, or De-ARS-HAP. The desorbed ARS belongs to ARS previously adsorbed on HAP by salt formation, while the remaining color on De-ARS-HAP indicates ARS still adsorbed on HAP by chelate formation. Consequently, we elucidated two adsorption mechanisms of ARS on HAP: The major adsorption is salt formation made up with 1-, 2-O- of ARS and Ca2+ on HAP, and the minor adsorption is chelate formation made up with 1-O- and 9-C=O of ARS and Ca2+ on HAP. (C) 2003 Elsevier Science (USA). All rights reserved.