Trends Biomater. Artif.

Organs, 25(3), 101-106 (2011)

http://www.sbaoi.org

Synthesis of Nano-crystalline Hydroxyapatite from Dead Snail Shells
for Biological Implantation
M.Dasgupta Adak1, and K. M. Purohit2
Department of Chemistry, 2Department of Life Sciences, National Institute of Technology, Rourkela, 769008
*Corresponding Author: Dr. M. Dasgupta Adak . m_dasgupta2k6@yahoo.co.in

1

Received 7 August 2010; Accepted 27 April 2011; Available online 29 May 2011
Hydroxyapatite Ca10(PO4)6(OH)2 is an important biomaterial and is the principal replacement inorganic constituent of bones and
teeth. It is also used as the replacement of heart valves, hip joints and other implants in the human body. A novel procedure to
produce porous hydroxyapatite from dead snail shells is reported. Dead snail shells which are generally discarded as a biological
waste are used as the raw material here. The thermal decomposition of the clean and dry snail shells was carried out by DTA/TG
analysis. The snail shells were thermally treated and hydroxyapatite was produced through chemical route. The powder was
characterized by X-Ray Diffraction, particle size analysis, DTA/TG analysis, scanning electron microscopy and infrared spectroscopy.
As a result, the Heap particle exhibited a micrometer-sized spherical shape where Average particle size was found to be 60 – 80 nm.
The crystalinity increased after calcinations and the size of crystallites range from 100-115 nm. Amount of hydroxyapatite obtained
from route 1 is less compare to route 2 but purity of route 1 is higher as compare to route 2 in both cases. The snail shells seems to
be a promising source of calcium for preparing nanocrystalline hydroxyapatite with excellent properties so essential for hard tissue
replacement

Introduction
Materials used for the repair and reconstruction of
diseased or damaged parts of the musco-skeletal system
is termed as ‘Biomaterials such as Hydroxyapatite;
Bioglass; Biopolymer; Ca-Phosphates etc [1]
A lot of research has been carried out to find out the
substitute to support the bone in the medical field.
Presently steel as support to bones are used but they
can react with body fluids [T = 37 OC and pH = 7.4] when
kept for many years so they need substitute. Next comes
the inert materials like bio glass etc but due to Formation
of Fibrous Tissue of Variable Thickness they can Leading
to Tumor, now ceramics specially the bio ceramics are
the better alternatives since they have high corrosion
resistance,[2] better compressive strength and relatively
low density and low weight. Porous bioactive ceramics
such as Hydroxyapatite, Ca-Phosphates are attractive
for bone regeneration and reconstruction due to their
bone bonding ability and good bone in growth property.
[3]
Among them Hydroxyapatite is taken as the best
alternative as it contains same chemical nature and
similar crystallographic structure with bone excellent Biocompatibility, [4] Bio-activity and Osteo-conductivity has
Increased its use in the clinical fields. It also allows tissue
ingrowth which helps for its replacement with bone
materials [5-6].
Formation of synthetic HAP is really critical due to the
following problems [2,7]. 1. Difficult to Sinter to Achieve

Full / Near-full Dense Body; 2. Decomposes above
12500C; 3. Difficult to Control Porosity; 4. By the virtue
Tailoring of Microstructure / Mechanical Properties.
These problems can be solved by synthesis of HAp in
nanocrystalline forms, especially by using the nano-sized
starting materials which provides [8] very high surface
energy, reduction of sintering temperature, control of
porosity tailoring of microstructure.
Commercially available HAPs are relatively expensive
due to the use of high purity of reagents. The HAP derived
from natural material has the advantages that they inherit
some properties of raw materials such as pore structures,
carbonated HAp etc. so for the present purpose natural
source of Calcium Carbonate preferably the dead snail
shells[9], a typical biological waste material is used as the
raw materials. Snail shells P. notabilis consists of 57.9
mg calcium and 31.5 mg magnesium per 100g of dry
shells.

Materials and Methods
(a) Synthesis
Dead snail shells are washed thoroughly and heated in
a box furnace at 1000oC for about 2 hours to decompose
organic matters and convert the calcium carbonate to
calcium oxide which in turn on exposure to atmosphere

The functional groups present in newly synthesized products were ascertained by FTIR.M. The first and second peaks of pH value were associated with the appearance of dicalcium phosphate anhydrate (CaHPO 4.47. The weight loss and thermal stability of the samples were also evaluated from the thermogravimetric analysis data. except a weight loss of 6% is observed at the TGA curve in the temperature range which is assumed to be resulted gradual dehydroxylation in hydroxyapatite powder. Adak. 2. The weight loss along with endothermic peak at 750 0C . CaCO 3 CaO + CO2 The TG-DTA thermograms for the hydroxyapatite powder after drying are illustrated in Fig.37. (c) 1. (d) 1.32% at temperature between 90 0C – 1200C that is due to the physically adsorbed water. 4 presents the XRD . (e) 1. Results and Discussion The TG-DTA thermogram of dead snail shell(Fig 1) shows a weight loss of 42.1then it was added to an aqueous suspension of calcium hydroxide of pH 11.The entire reaction mixture was allowed to stir constantly for 24 hours maintaining an ice cold condition. (b) 1.D.M. newly produced calcium hydroxide was weighted and was used to react with prerequisite amount of concentrated HNO 3 nitric acid to form calcium nitrate. The first endothermic region range from 90 to 295 °C with a peak at about 250°C. The surface area of the synthesized HAp powder was made by BET surface area analyzer (QUANTACHROME Model: Autosorb1). The weight loss in this region is 16%.77 seperately. So it is confirmed from the thermal analysis that Snail Shell mainly contains CaCO3 along with small amount of MgCO3 and other organic matters.57. The gelatinous product was washed repeatedly with distilled water to remove unwanted ions and dried overnight in an oven at 100 o C (b) Characterization: The particle size analysis of HAp was done by Malvern Particle Size Analyzer (Model – Micro-P. K. The gelatinous product was washed repeatedly with distilled water to remove unwanted ions and dried overnight in an oven at 100 o Fig 1: TG-DTA thermogram of snail shell According the second route. DCPA) and fibrous OCP (Ca8H2(PO4)6) respectively. UK). In order to investigate the effect of heat treatment on nano -crystallization and phase transformation in hydroxyapatite powder by precipitation method. which corresponds to the dehydration of the precipitating complex and the loss of physically adsorbed water molecules of the hydroxyapatite powder. newly produced calcium hydroxide was weighted and was used to react with preprepared acid pre-mix maintaining a pH range 2.17. This can be explained by the following reaction [10]: Ca10(PO4)6(OH)2 Ca10(PO4)6(OH)2"2xOx+xH2O The precipitates obtained from the solution at different temperatures for various lengths of time were identified by XRD and the results are shown in Fig 3. With increasing temperature from 295 to 1200 °C no peak has been observed.The specific surface area of each hydroxyapatite was determined by BET analysis which estimates of surface area by nitrogen adsorption at 77K. Over a wide range of temperature from 250 0C – 4000C the weight loss is due to the decomposition of MgCO 3 combined with the combustion of hydrocarbons.8500C indicates the decomposition of CaCo 3 following the reaction. The structural characterization of HAp powder synthesized samples were made by Scanning Electron Microscope (SEM). and the last stage corresponded to the phase transition of the precursors into HAp powder.it is allowed to stir continuously for about 24 hours at room temperature. It was then react with ammonia to form ammoniacal suspension and react with diammonium hydrogen phosphate solution corresponding to different stoichiometrioc ratio of Ca/P = ranging (a) 1.67 and (f) 1. Purohit 102 forms calcium hydroxide.01 0 interval with 1s counting time at each step. The product was finely ground in an agate pestle and mortar According the first route. 2: TG-DTA thermogram of the hydroxyapatite powder after drying at 80°C The X-Ray Powder Diffraction (XRD) analysis of the sample was done by X-ray Diffractometer using Cu-Ká radiation. Fig. The X-ray diffraction (XRD) patterns were recorded in the steps of 0. Fig.

and with different Ca/P ratio of starting materials. 3447cm -1 (better resolved) 3572cm -1 By controlling the temperature during precipitation and ripening time.e) Fig 4: XRD patterns of heat treatment hydroxyapatite powders (HAp .2 cm-1 is associated with the stretching modes of the P-O bonds of HAp14-15. (HAp. Thus. This peak is mainly due to O-H stretching vibration in HAp 13.dipyramidal phase was not transformed to the other phases up to 1200 %C. 1100 and 600cm-1..8cm -1 is due to the presence of –OH bond 10.group 16-18 . as the patterns appear very well resolved. 450. Again it rules out the formation of any new crystalline phase other than HAp.e) for different temperatures Fig 5: XRD patterns of heat treatment hydroxyapatite powders (HAp . This decomposition into tricalcium phosphate was also not observed in the present study when the HAp was heat treatment at 1200 %C. 1417. At room temperature the stable phase is hexagonaldipyramidal. The IR analysis shows a small broad peak at 1422. OH. The broad patterns around at (2 1 1) and (0 0 2) indicate that the crystallites are very tiny in nature with much atomic oscillations. XRD patterns of the as synthesized powder (HAp . up to 1200 %C (1 h holding time at each temperature). The peak at 1036. Stretching of O-H: 3447cm-1. but at 1200 %C no such transformation is observed in the present study. Again at the heat treatment temperature of 1200 %C. The patterns due to the as-prepared HAp at 100 %C bears with it the characteristic patterns of HAp but not with much resolution and intensity. the apatite . As the increase in intensity is noticed for every pattern with increase in heat treatment temperature. It contains no other crystalline phase other than HAp. (0 0 2). but the hexagonal.group in HAp is almost confirm from IR studies. 1420.Synthesis of Nano-crystalline Hydroxyapatite from Dead Snail Shells Fig 3: XRD Pattern of: (a). Weak band at 2000cm-1(Overtone). the hydroxyapatite peaks gradually increased in intensity when the sample was heated from 100.(fig 5) Infrared characterization was carried out for the sample to study the spectral characteristics indicative of the chemical bonding in the synthesized HAp-e powder. Reported transformation of HAp into oxyapatite between 1200 and 1400 %C [12].e) for different temperatures 103 spectra at various temperatures for hydroxyapatite calcined powders by precipitation method. CO 32-: 864.When the temperature was increased. powders with three different values of crystalline degree were produced. 1477cm-1. Stretching of P-O at 1040cm-1. The pH of the medium during synthesis of HAp was maintained using ammonium solution and it was removed from the suspension with repeated washing with distilled water. . The peak observed around 3431. new crystalline phases are not seen. indicating further nucleation/growth of the hexagonaldipyramidal nanocrystals inside the powder particles.e) showed the presence of an amorphous phase.4cm-1 are due to bending modes of P-O bonds in phosphate group14. This precludes even amorphous phases. Particularly noteworthy. (3 0 1). In spite of all efforts to remove ammonia from the solution.6cm-1. (2 2 2) and (2 1 3).1 cm 1 and 567. which is characteristics peak of NH4+. The double peak at 603. The sample heated at 750 oC showed broad peaks of an apatite phase. This hexagonal-phase-growth XRD data with increasing temperature is reported here for the first time. 900. (b) Calcined HAp (synthesized) 8000C/2Hr. there is a possibility of small amount of it in the HAp powder. the presence of PO4 3. 602cm-1. Bending of P-O: 567.band: 633. The XRD patterns of the heat treatment HAp at 450 and 900 %C show increase in intensity due to planes around (2 1 1). HAp (synthesized). (Fig 6) The spectrum can be divided into four regions with peaks having wave numbers around 3500.

Purohit Fig 6: FTIR Spectra of raw and calcined (HAp-e) Fig 9: SEM (Snail shell) Fig 7: XRD patterns of raw-HAp powder and that calcined at 7500C depending on the Ca/P ratio : (a) 1. (d) 1.17. (c) 1. BET plot of (HAp– e) Fig 10: SEM (hap-e) powder prepared from snail shell.D. (b) 1.M. 1st route .47. Adak. K.104 M.57 Fig 10: SEM (hap-e) powder prepared from snail shell. 1st route Fig 8.37.

however.001373 x cos (31. t = {0. 300 plane was increased. in which grains are smaller but built by larger particles.Ca/P ratio = 1. Powders are agglomerated during calcinations. The presence of hydroxyapatite was confirmed by a strong diffraction peak at 31.from fig 8.469 o and 54. The apparent size of hydroxyapatite crystallites obtained from XRD profile analysis by Scherrer method [15–17] is shown in Fig.g. (fig 7). From the figure. The calcium carbonate peak was minimized and the intensity of hydroxyapatite peaks at 211. and q is the diffraction angle. (fig 8)are 83 and 12. H 3O+. Alternatively.773 o (211) plane. The powder patterns do not indicate any peaks corresponding to CaO. In this method of broadening contribution due to the crystallites size are taken into account.because of crystal growth.96 x 1. the TCP phase was not detected at any temperature. but HAp powders have to be calcined to remove volatile impurities like ammonia.67). b stands for full width at half maximum of the peak. d = kl/b cosq.196 o and 32. 112. As expected the compaction behaviour of the powders are different. experimentally. 7. Taking mean value of b = (0. Fig 14: SEM HAp–e sintering at 1000 0C for 1 hour .0013730A). the presence of the CO 3 group also proved the formation in powder B of some amount of carbonateapatite (CO3) substituted in both positions for OH and PO 4 groups. Moreover. The crystallization of the carbonate hydroxyapatite was prepared. Also.34 m 2/gm respectively (HAp-e.902 o of equal intensities were also detected.?. The accompanying two peaks at 32. k the shape factor.399 o was present together with HAp phase. the CaO peak at 37. Where d is the average diameter in Å.029 o was not detected at any temperature. higher green density was obtained with HAp-e powder. HAp-e is characterized by larger agglomerates in turn formed by smaller primary particles in respect to HAp-a powder . A gradual increase in crystallite size was observed for hydroxyapatite-e with increasing in heat treatment temperature. (HPO4)2 – etc and they may decompose in a variety of ways leading to structural changes (Simpson1968). the secondary impurity phases such as CaO and TCP were not detected at the sintered samples of powder A. Fig 13: Crystallized HAp–e The Bragg reflections at (002) planes of HAp were considered to calculate the crystallite size. The crystallite sizes were calculated using Scherrer’s relationship.e. calcium carbonate peak at 29. On the other hand.54} / {0. It should be noted that materials prepared via low temperature wet chemical processes are known to accommodate various ionic species.Synthesis of Nano-crystalline Hydroxyapatite from Dead Snail Shells Fig 12: SEM of calcined HAp-e 105 peaks became sharper.8 nm The expansion does indicate a structural change in the lattice.00154059 nm t = 998 0A = 99.8019 / 2)} = 11200A = 112 nm Taking mean value of b =0. the O-H stretch band around 3450 cm -1 is more accentuated and shifted at lower frequency in powder C characterized by a higher solvation extent. The surface areas of the hydroxyapatite powder and calcined HAp are determined using bet plots.

Sargin. Our special thanks to Prof Sunil Sarengi. E. powders with different values of crystalline degree were produced to compare the crystallinity.Artif. Current problems in orthopedics. 4. V. Rouquet N. HAp –e (Ca/P = 1. Marquis.. Wang.R. Sevitt. ed. The snail shell seems to be a promising source of calcium for preparing nanocrystalline hydroxyapatite with excellent properties so essential for hard tissue replacement. Rodriguez-Clemente.A Suchicital. 12. Adak. T. Mater. T. Cryst.. and Hassanein. C.Torrent-Burgues . 18. Aoki H. Res. 1993.M. J. J Bone Joint Surg 25: 375-426. Das Gesstz der Transformation der Knochen. Chen.L and Alem. Mater. Kellam JF. Churchill Livingstone. P. 11. Kizilyalli.. 1941. Asher. 2008 10. with a little agglomerated structure where as calcined HAp powders are agglomerated. 14(6):423-429] 9.Victoria and F. J. 14. Bone repair and fracture healing in man. compatibility and finer particle size with respect to other newly produced HAps. S. Ceramic Processing and Sintering. Awodola. R.Gnanam. Rahaman. 7. J. and sintering behavior. S. grains are more rounded and coalscence is reduced. H. Porter S. Pizziconi.R. Biomaterials23 (2002) 1065.. A Dental Cement: X-ray Powder Diffraction and IR Studies. References 1. Fogle. Trouillet JL. 16(2002) 12-14.. Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition. at higher temperature. But In order to quantify the biodegradability of these materials. 1982. NIT. Soc. J. N. K. 4(1993) 83. Wolff. Fraile. The microstructure as reveals from SEM is in well. M. Spine 20(9): 1055-60. De Groot. Chemical and Structural Evolution of Sol-Gel-Derived Hydroxyapatite Thin Films under Rapid Thermal Processing. 36. August Hirschwald Verlag. C. Sci. Melvin. Biomaterial developments for bone tissue engineering. Azimus E. all the Staff and Faculty members of Department of Chemistry. ed. Bernache-Assollant. Med. S. Burg KJL.Trends Biomater.W. Banwart. Louis: Ishyaku EuroAmerica Inc. J. Director. 9. J. Alford.. Emmanuel. Zhou. Calcification and ossification. Boix. P.1994. Ramannan. Adubiaro and Olufemi J.M. Frayssinet P. M.. but. Pakistan Journal of Nutrition 7 (6): 741-747.B. S. J. Technol. Soc. that agrees with values higher in respect to HAp-e and the possibility to identify a preceding mechanism of particle rearrangement.A. A New Method for the Solid-State Synthesis of Tetracalcium Phosphate. K.667) samples show grains more rounded and with coalescence markedly reduced in respect to other newly produced Hap treated at the same temperature .106 M. X. E. Sci. Biomaterials. (2001) 15 19. Urist.W. After sintering at 1200 0C. 8. C. Precipitation of Stoichiometric Hydroxyapatite by a Continuous Method. Biomaterials. Raynaud. 17 (1997) 963. C. K. J. Champion. N. IV. Synthesis of Biomimetic Ca-Hydroxyapatite Powders at 37°C in Synthetic Body Fluids. A. Guler. Gomez-Morales.Organs. Biomaterials 21 (2000) 1429-1438. R.D. HAp-e samples (Ca/P ratio = 1. the densification can be improved. J. Pontoon.D. Russell. Berlin.G.B. R. 15. J Biomech 3(5): 495-511. Amount of hydroxyapatite obtained from route 1 is less compare to route 2 but purity of route 1 is higher as compare to route 2 in both cases. McKibbin. . E. New York. Ceram. Autefage A. J. Rourkela for providing laboratory and library facilities. Roberts. the ISO/FDIS 1099314:2001 degradation test should be used. Zhang. J. A Study of Hydroxyapatite Fibers Prepared via Sol–Gel Route. Synthesis and Characterisation of Biphasic Calcium Phosphate.67) shows better densification. Adeyeye. and with different Ca/P ratio of starting materials. Murray. J. Material Letters 58 (2004) 33203323 16. J.R and Johnson. 3. S. decreasing the probability of grain growth.( having lower Ca/P ratio. 17. D. Acknowledgement We are thankful to the Department of Science and Technology.S. Ceram. NIT. Conclusion Homogeneous Hydroxyapatite powders have been synthesized successfully from dead snail shells following two different routes. Purohit Scanning Electron Micrographs The morphologies of as synthesized HAp powders are shown in Fig. M. I. J. J. Edinburgh.14.T. H. Rourkela. 79 (1996) 843. Ramanan. St.21(23):2347-2359. Comparability of Chemical Composition and Functional Properties of Shell and Flesh of Penaeus notabilis. 6. Synthesized HAp powders are almost regular and round in shape. M. Thomas. Y. M. 5. R.W. Iliac crest bone graft harvest donor site morbidity. Caroline. Mater. V. Healing of fracture in man under clinical conditions. Medical applications of hydroxyapatite. Telli. 1970. 1995. 1995.. Eur. Cuneyt Tas. for financial support under the scheme WOS-A. 30(1995) 3061. A statistical evaluation. 2000.M.agreement with BET surface area analyzer results. 1981. Calcination treatment (HAp) has a delaying effect on sintering at low temperature: at temperatures in the range 950 to 10000C.) treated at the same temperature. New Delhi. Marcel Dekker. Mechanical properties on cranial bone. 13. By controlling the temperature during precipitation and ripening time. Luptak. V. Haynes. Am. Habibat O.. 2..L.L.