Spectroscopic determination of alkyl resorcinol concentration in hydroxyapatite composite
© Yang et al. 2016
Received: 4 February 2016
Accepted: 8 March 2016
Published: 14 March 2016
Recently, alkyl resorcinol compounds showed remarkable improvements on dental implant restoration as well as anaesthetic, antiseptic, and anthelmintic applications. In this study, we prepared biologically functional composition of 4-hexylresorcinol (4HR)-loaded hydroxyapatite (HA).
Attenuated total reflectance (ATR) Fourier-transform infrared (FT-IR) spectroscopy fully assigned vibrational absorptions of 4-hexylresorcinol as well as the HA. The absorption coefficient of 4HR aqueous solution is estimated to be 1393 ± 61 cm−1 M−1 at highly diluted concentration. The 4HR was loaded with 0.018 % (wt/wt) in the 4HR-HA composite.
We quantitatively determined the micromolar concentration of 4HR loaded in the composite based on ultraviolet-visible (UV-Vis) absorption spectroscopy.
Hydroxyapatite (HA) is the major component of bone material and a hexagonally packed crystal with hydroxyl end members (Pleshko et al. 1991). HA analogue materials have been extensively developed through a variety of chemical and physical routes (Ferraz et al. 2004; Bouyer et al. 2000; Cengiz et al. 2008). One of tremendous use of HA is bone grafting application via coating metallic dental implant surface, which stimulates bone healing and therefore improves implant integration rate and strength (Cook et al. 1987; Lange and Donath 1989). According to previous reports, however, HA-coated dental implants have often failed because of bacterial infection (Chang and Tanaka 2002; Destainville et al. 2003; Coates 2000). Hence, infection-resistant HA coating with bioinert antiseptic function is of great interest. Recently, a 4-hexylresorcinol (4HR)-treated HA-titanium dental implant showed significant improvement in both bone formation and bone-to-implant contact after implant surgery (Kim et al. 2011a).
Alkyl resorcinols, natural non-isoprenoid phenolic lipids found in plants (Tyman 1979), have attracted much attention due to a variety of biologic functions, such as being nonspecific antioxidants, antimutagens, and regulatory molecules (Kim et al. 2011b). Hexylresorcinol is an organic compound with well-known anaesthetic, antiseptic, and anthelmintic properties (Wilson and Gisvold 1954). By now, hexylresorcinol have been used in a variety of application areas, such as skincare products with anti-aging function, food additive with estrogenic activity (Amadasi et al. 2009), and anti-cancer activity by inhibiting NF-κB (Kim et al. 2011a). Hexylresorcinol inhibits the formation of graft-induced foreign body giant cells (Kweon et al. 2014). Therefore, hexylresorcinol has been used for bone substitute-related tissue engineering (Lee et al. 2015).
Several routes have been tried to deposit 4HR on the hydrophilic HA; however, precise control of 4HR loading amount is still challenging because of the amphiphilic character of 4HR (Kim et al. 2011b). Therefore, quantitative determination of the loading amount is importantly required to further optimize composite materials with a demanded function (Kweon et al. 2014; Lee et al. 2015). In this study, solution-processed 4HR-loaded HA composite were quantitatively characterized using Fourier-transform infrared (FT-IR) and ultraviolet-visible (UV-Vis) absorption spectroscopies. Infinitesimal amount of 4HR, which was loaded on HA powder, could be detected and precisely estimated based on UV-Vis absorption spectroscopy. Additionally, we entirely assigned IR absorption peaks of 4HR for the first time.
Material and methods
Chemicals and materials
Hydroxyapatite (reagent grade) and 4-hexylresorcinol (98 %) were used as received from Sigma Aldrich. 4HR-loaded HA composite (4HR-HA) was prepared in aqueous medium for FT-IR and UV-Vis absorption study. 0.5 g of hydroxyapatite was mixed with 50 ml of 0.1 M 4-hexylresorcinol aqueous solution and then stirred at 200 rpm for 1 h. The suspension was finally filtered and dried for 24 h.
FT-IR absorption spectra were obtained using a Fourier-transform spectrometer (Vertex 80, Bruker Optics) equipped with an attenuated total reflectance (ATR) accessory (MIRacle, PIKE technologies). Spectrum was recorded in the spectral range of 600 to 4000 cm−1 at a resolution of 2 cm−1 with a mercury cadmium telluride detectors (MCT detector), and 128 repeated scans were averaged for each spectrum. The UV-Vis absorption spectra were obtained using a spectrophotometer (S-3100, Scinco).
Results and discussion
Characteristic IR absorptions of hydroxyapatite
(Meejoo et al. 2006)
PO4 3− (ν3)
PO4 3− (ν1)
Labile PO4 3−
Characteristic IR absorptions of 4-hexylresorcinol
Hydrogen bonded OH stretching
(Kim et al. 2012)
Aromatic CH stretching
(Kim et al. 2012)
Aliphatic CH3 asymmetric stretching
Aliphatic CH2 asymmetric stretching
Aliphatic CH3 symmetric stretching
Aliphatic CH2 symmetric stretching
Aromatic ring C–C stretching
Aliphatic CH2 bending
Aliphatic CH3 asymmetric bending
Phenolic OH bending
Aliphatic CH3 symmetric bending
Aliphatic CH2 wagging (disordered phase)
(Krimm et al. 1956)
1234, 1217, 1192, 1155
OH deformation stretching
(Evanoff and Chumanov 2004)
CH2 wagging or twisting
(Kozubek and Tyman 1999)
(Krimm et al. 1956)
864, 839, 800, 791, 779
Aromatic C–H out-of-plane bending
Out-of-plane ring C=C bending
(Silverstein et al. 1991)
OH out-of-plane bending
(Silverstein et al. 1991)
FT-IR spectrum of the 4HR-HA composite showed the majority absorptions for the HA without any distinct change (Fig. 1). Additionally, very weak C–H stretching absorption corresponding to the 4HR is observed in the region of 2800–3000 cm−1 (asterisk indicated). After 4-hexylresorcinol treatment, the 4HR-HA composite showed enhanced absorption at 3260 cm−1 (arrow indicated). This can be attributed to hydrogen bonding between 4HR and HA. However, it is still ambiguous to precisely estimate 4HR concentration in this composite.
The additional absorption was also shown at ~430 nm, which is attributed to the scattering of HA powder. A colloidal powder suspension typically scatters light in the visible region. The scattering absorption can vary as a function of colloidal particle size (Evanoff and Chumanov 2004; Chae et al. 2009). In this study, the bulk HA powder showed a broad scattering with a maximum at 535 nm. As the HA was treated with 4HR, the scattering peak was observed at 430 nm. This blue-shifted scattering seems to be attributed to the fragmentation of the HA powder after 4HR treatment.
It is noteworthy that the absorption spectrum of the 4HR-loaded HA powder clearly reveals the presence of 4HA. The measured absorbance of 0.0065 is corresponding to 4.7 μM concentration and 3.5 μg 4HR in a standard cuvette. Regarding the total 4HR-HA composite powder mass of 19 mg for the UV-Vis measurement, the 4HR was loaded with 0.018 % (wt/wt) in the 4HR-HA composite. This small loading amount reasonably explains why IR spectroscopy need to take extra special care on sampling to determine the 4HR concentration even in ATR mode with a typical detection limit of ~0.1 wt % or 10−4 M.
We estimated an amount of 4-hexylresorcinol compound from its composite form with hydroxyapatite using FT-IR and UV-Vis absorption spectroscopies. Because of the majority IR absorptions of HA, the precise estimation of 4HR concentration in the 4HR-HA composite is a little ambiguous using FT-IR spectroscopy. On the other hand, the 4HR-HA composite showed clear absorption for 4HR in the ultraviolet region. Based on UV-Vis absorption spectroscopy, we effectively estimated the micromolar quantity of the loaded 4HR in the 4HR-HA composite.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01058935).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Amadasi A, Mozzarelli A, Meda C, Maggi A, Cozzini P. Identification of xenoestrogens in food additives by an integrated in silico and in vitro approach. Chem Res Toxicol. 2009;22:52–63.View ArticleGoogle Scholar
- Bouyer E, Gitzhofer F, Boulos MI. Morphological study of hydroxyapatite nanocrystal suspension. J Mater Sci: Mater Med. 2000;11:523–31.Google Scholar
- Cengiz B, Gokce Y, Yildiz N, Aktas Z, Calimli A. Synthesis and characterization of hydroxyapatite nanoparticle. Colloids Surf A: Physicochem Eng Aspects. 2008;322:29–33.View ArticleGoogle Scholar
- Chae WS, Kershner RJ, Braun PV. Fabrication of 50 to 1000 nm monodisperse ZnS colloids. Bull Korean Chem Soc. 2009;30:129–32.View ArticleGoogle Scholar
- Chang MC, Tanaka JZ. FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde. Biomater. 2002;23:4811–8.View ArticleGoogle Scholar
- Choo ESG, Tang XS, Sheng Y, Shuter B, Xue JM. Controlled loading of superparamagnetic nanoparticles in fluorescent nanogels as effective T2-weighted MRI contrast agents. J Mater Chem. 2011;21:2310–9.View ArticleGoogle Scholar
- Coates J. In Encyclopedia of Analytical Chemistry, Interpretation of Infrared Spectra, A Practical Approach. Chichester: John Wiley & Sons; 2000.Google Scholar
- Cook SD, Kay JF, Thomas KA. Interface mechanics and histology of titanium and HA-coated titanium for dental implant applications. Int J Oral Maxillofac Implants. 1987;2:15–22.Google Scholar
- Cui CZ, Park DH, Kim JY, Joo JS, Ahn DJ. Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes. Chem Commun. 2013;49:5360–2.View ArticleGoogle Scholar
- Destainville A, Champion E, Bernache-Assollant D, Laborde E. Synthesis, characterization and thermal behavior of apatitic tricalcium phosphate. Mater Chem Phys. 2003;80:269–77.View ArticleGoogle Scholar
- Evanoff DD, Chumanov G. Size-controlled synthesis of nanoparticles. 2. Measurement of extinction, scattering, and absorption cross sections. J Phys Chem B. 2004;108:13957–62.View ArticleGoogle Scholar
- Ferraz MP, Monteiro FJ, Manuel CM. Hydroxyapatite nanoparticles: a review of preparation methodologies. J Appl Biomater Biomech. 2004;2:74–80.Google Scholar
- Frost RL, Wain DL, Martens WN, Reddy BJ. Vibrational spectroscopy of selected minerals of the rosasite group. Spectrochim Acta Part A: Mol Biomol Spectrosc. 2007;66:1068–74.View ArticleGoogle Scholar
- Gómez-Sánchez E, Simon S, Koch LC, Wiedmann A, Weber T, Mengel M. ATR-FTIR spectroscopy for the characterization of magnetic tape materials. e-Preserv Sci. 2011;8:2–9.Google Scholar
- Hallos RS. A “chain fold band” in the infrared spectra of nylon 6. J Appl Polym Sci. 1984;29:3907–14.View ArticleGoogle Scholar
- Han JK, Song HY, Saito FM, Lee BT. Synthesis of high purity nano-sized hydroxyapatite powder by microwave-hydrothermal method. Mater Chem Phys. 2006;99:235–9.View ArticleGoogle Scholar
- Kim SG, Hahn BD, Park DS, Lee YC, Choi EJ, Chae WS, et al. Aerosol deposition of hydroxyapatite and 4-hexylresorcinol coatings on titanium alloys for dental implants. J Oral Maxillofac Surg. 2011a;69:354–63.View ArticleGoogle Scholar
- Kim SG, Lee SW, Park YW, Jeong JH, Choi JY. 4-hexylresorcinol inhibits NF-κB phosphorylation and has a synergistic effect with cisplatin in KB cells. Oncol Rep. 2011b;26:1527–32.Google Scholar
- Kim MK, Park YT, Kim SG, Park YW, Lee SK, Choi WS. The effect of a hydroxyapatite and 4-hexylresorcinol combination graft on bone regeneration in the rabbit calvarial defect model. Korean Assoc Maxillofac Plast Reconstr Surg. 2012;34:377–83.Google Scholar
- Kozubek A, Tyman JHP. Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem Rev. 1999;99:1–25.View ArticleGoogle Scholar
- Krimm SCYL, Liang CY, Sutherland GBBM. Infrared spectra of high polymers. II. Polyethylene. J Chem Phys. 1956;25:549–62.View ArticleGoogle Scholar
- Kweon H, Kim SG, Choi JY. Inhibition of foreign body giant cell formation by 4-hexylresorcinol through suppression of diacylglycerol kinase delta gene expression. Biomater. 2014;35:8576–84.View ArticleGoogle Scholar
- Lange GL, Donath K. Interface between bone tissue and implants of solid hydroxyapatite or hydroxyapatite-coated titanium implants. Biomater. 1989;10:121–5.View ArticleGoogle Scholar
- Lee SW, Um IC, Kim SG, Cha MS. Evaluation of bone formation and membrane degradation in guided bone regeneration using a 4-hexylresorcinol-incorporated silk fabric membrane. Maxillofac Plast Reconstr Surg. 2015;37:32–6.View ArticleGoogle Scholar
- Meejoo S, Maneeprakorn W, Winotai P. Phase and thermal stability of nanocrystalline hydroxyapatite prepared via microwave heating. Thermochim Acta. 2006;447:115–20.View ArticleGoogle Scholar
- Pleshko N, Boskey A, Mendelsohn R. Novel infrared spectroscopic method for the determination of crystallinity of hydroxyapatite minerals. Biophys J. 1991;60:786–93.View ArticleGoogle Scholar
- Raynaud S, Champion E, Bernache-Assollant D, Thomas P. Calcium phosphate apatites with variable Ca/P atomic ratio I. Synthesis, characterisation and thermal stability of powders. Biomater. 2002;23:1065–72.View ArticleGoogle Scholar
- Silverstein RM, Bassler GC, Morrill TC. In Spectrometric identification of organic compounds, Infrared Spectrometry. New York: John Wiley & Sons; 1991Google Scholar
- Tyman JHP. Non-isoprenoid long chain phenols. Chem Soc Rev. 1979;8:499–537.View ArticleGoogle Scholar
- Wilson CO, Gisvold O. Textbook of organic medicinal and pharmaceutical chemistry. Philadelphia: Lippincott; 1954. 237-262Google Scholar