Research of Calcium Phosphates Using Fourier Transform Infrared Spectroscopy

In the biomaterial research field, nowadays a great attention is driven onto calcium phosphates synthesis and obtaining of ceramics that can be used in orthopedics and dentistry, in the form of coatings, granules, porous or solid blocks, as well as in the form of various composite materials. The most frequently studied, clinically tested and used synthetic materials based on calcium phosphate (CaP) are hydroxyapatite [HAp Ca10(PO4)6(OH)2], ┚-tricalcium phosphate [┚-TCP Ca3(PO4)2] and biphasic HAp/┚-TCP mixture. CaP ceramic demonstrates high biocompatibility and bioactivity while it contacts bone cells, builds a direct chemical connection between bone tissues and ceramic implant. As practice shows, purchased materials, most often commercial CaP materials, not always have properties and qualities defined by the manufacturer. Frequently, the manufacturer’s information about the offered product is not complete or precise, by it troubling usage of raw CaP material for development of implants. The most common imperfections of CaP materials are – unpredictable properties after the high temperature treatment (composition and clarity of crystal phases, chemical composition, thermal stability, etc.) which become clear only after the high temperature treatment of the ready implant material has occurred. For several years, Riga Biomaterial Innovation and Development Centre (RBIDC) of Riga Technical University perform a wide range of property studies of various commercial CaP raw materials.


Introduction
In the biomaterial research field, nowadays a great attention is driven onto calcium phosphates synthesis and obtaining of ceramics that can be used in orthopedics and dentistry, in the form of coatings, granules, porous or solid blocks, as well as in the form of various composite materials.The most frequently studied, clinically tested and used synthetic materials based on calcium phosphate (CaP) are hydroxyapatite [HAp -Ca 10 (PO 4 ) 6 (OH) 2 ], -tricalcium phosphate [ -TCP -Ca 3 (PO 4 ) 2 ] and biphasic HAp/ -TCP mixture.CaP ceramic demonstrates high biocompatibility and bioactivity while it contacts bone cells, builds a direct chemical connection between bone tissues and ceramic implant.As practice shows, purchased materials, most often commercial CaP materials, not always have properties and qualities defined by the manufacturer.Frequently, the manufacturer's information about the offered product is not complete or precise, by it troubling usage of raw CaP material for development of implants.The most common imperfections of CaP materials are -unpredictable properties after the high temperature treatment (composition and clarity of crystal phases, chemical composition, thermal stability, etc.) which become clear only after the high temperature treatment of the ready implant material has occurred.For several years, Riga Biomaterial Innovation and Development Centre (RBIDC) of Riga Technical University perform a wide range of property studies of various commercial CaP raw materials.
Properties of bioceramic implants obtained from various commercial and laboratory synthesized calcium phosphate precursors are different, since behavior of those precursors is different within the thermal treatment processes, which are a significant stage of obtaining ceramics.
CaP synthesis methods and their technological parameters can significantly impact stoichiometry of the synthesis product, its grade of crystallization, particle size, bioceramic phase composition, thermal stability, microstructure and mechanical properties.The important technologic parameters that impact properties of calcium phosphate synthesis product and then also of bioceramic, are temperature of synthesis, pH of synthesis environment, reagent type and concentration, as well as selection of raw materials, their purity and quality.All of the above mentioned also brings a significant impact on the tissue response of these bioceramic implants.
Fourier transform infrared spectroscopy (FTIR) is one of the methods which, systematically monitoring variations of structural characteristic groups and vibrations bonds, can provide an indirect evaluation of the synthesized Ca/P implant materials from TCP up to HAp and bioceramics, obtained from these materials.
FTIR spectroscopy has numerous advantages when used for chemical analysis of CaP products.First of all, an obtained spectrogram provides useful information about location of peaks, their intensity, width and shape in the required wave number range.Secondly, FTIR is also a very sensitive technique for determining phase composition.In the third place, FTIR is a comparatively quick and easy everyday approach.
During recent years, many authors' attention is turned onto synthesis of CaP and research of structure of the synthesized products depending on their technological parameters, with various methods, including an X-ray diffraction (XRD) and FTIR methods.However, the data of the literary sources is often incomplete or sometimes even contradictory.Studying various literary sources and analyzing the taken spectra of laboratory synthesized and commercial CaP products was aimed onto creating summary IR spectrum tables for the characteristic calcium phosphate chemical groups absorption bands.HAp stoichiometry is very important if the material has undergone a high temperature treatment.
A minor misbalance of synthesis product in a stoichiometric ratio (standard molar ratio of Ca/P is 1.67) during high temperature treatment can lead to composition of -, -TCP, or other phases.Thermally treating the stoichiometric calcium phosphates, it is possible to obtain stable phases at temperatures up to 1300 o C. One of the main non-stoichiometry reasons is inclusion of impurities, often substitutions of Ca 2+ or interpenetration of other ions in the crystal lattice.In total, biological calcium phosphates are defined as calcium hydroxyapatites with deficient of calcium, Ca 10-x (PO 4 ) 6-x (HPO 4 ) x (OH) 2-x (0<x<2), including the substituting atoms or groups, as, for example, Mg 2+ , Na + , K + , Sr 2+ , or Ba 2+ substitute Ca 2+ , CO 3 2-, H 2 PO 4 -, HPO 4 2-; SO 4 2-substitute PO 4 3-; F -, Cl -, CO 3 2-, PO 4 3-substitute OH -.
The main target of our work was to perform a FTIR spectroscopy analysis of the CaP products synthesized in RBIDC laboratory and make summarizing conclusions about chemical groups of calcium phosphates, their variations under impact of synthesis parameters and further thermal treatment, as well as creating summary tables for:

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CaP powders synthesized in the laboratory with a chemical solution precipitation method with different synthesis parameters (temperature, final suspension pH, maturation time) as-synthesized and then thermally treated at various temperatures from 200 o C up to 1400 o C;  Commercial CaP products;  CaO containing materials of various origins (marble, eggshells, land snail shells) and FTIR spectra of obtained products;

Calcium phosphates
Calcium phosphates as chemical compounds arise interest of the numerous fields of science, like geology, chemistry, biology and medicine.Many forms of calcium phosphates are determined by their Ca/P molar ratio.From the point of view of chemistry, they are formed by three main elements: calcium, phosphorus and oxygen.Many calcium phosphates also contain hydrogen in an acidic phosphate anion (for example, HPO 4 2-), hydroxyl groups (for example, Ca 10 (PO 4 ) 6 (OH) 2 ) or in a form of bonded water (for example, CaHPO 4 •2H 2 O).Majority of compounds of this class are poorly soluble in the water and non-soluble in alkaline solutions, but all of them easily dissolve in acids.Chemically pure calcium orthophosphates are white crystals with an average hardness, while natural materials are always of some other color which depends on the type and amount of impurities.Biologic calcium phosphates are main mineral components in the calcified tissues of the vertebrates (Dorozhkin, 2009a).
Main components of the natural bone tissues are calcium phosphates which, along with the other elements (Na, K, F, and Cl) form ~ 70% of the bone tissue mass.Also, bone tissues contain water (10% of mass) and collagen along with the other organic materials in small amounts.In living system CaP are found in the form of crystalline hydroxyapatite (HAp) and in the amorphous calcium phosphate (ACP) form.
As bone substitution materials, calcium orthophosphates are researched for more than 80 years.The most significant characteristics of calcium phosphates are their bioresorbtion and bioactivity.They are non-toxic and biocompatible.Bioactivity shows as an ability to create a physical chemical bond between an implant and a bone.This process is called ostheointegration (Dorozhkin, 2009b).
Depending on the calcium/phosphorus (Ca/P) molar ratio and solubility of the compound, it is possible to obtain numerous calcium phosphates of different composition.Molar Ca/P ratio and solubility are connected with the pH of the solution.Majority of materials of this class are resorbable and dissolve when inserted in a physical environment.Calcium phosphates that are most frequently used in the biomaterial field are demonstrated in Table 1 (Dorozhkin, 2009c;El Kady, 2009;Shi, 2006).
For biomedical application, the following calcium phosphates are most frequently used: HAp (Ca/P = 1.67) and -TCP (Ca/P=1.5),as well as biphasic calcium phosphate which mainly consists of HAp and -TCP mixture in various ratios.

FTIR absorption bands of the synthesized HAp
Hydroxyapatite Ca 10 (PO 4 ) 6 (OH) 2 is dominating and the most significant mineral phase in the solid tissues of the vertebrates.It consists of the same ions that form mineral part of teeth and bones.
A biological HAp usually has a calcium deficient; it is always substituted with a carbonate.Two types of carbonate substitution are possible: (1) direct substitution of OH -with CO 3 2- (A-type substitution (CO 3 ) 2-↔ 2OH -) and (2) necessity after charge compensation, PO 4 3- substituting a tetrahedral group with CO 3 2-(B-type substitution).Substitution groups may provoke characteristic changes in the lattice parameters, crystallinity, crystal symmetry, thermal stability, morphology, and solubility, physical, chemical and biological characteristics (Shi, 2006).
The most characteristic chemical groups in the FTIR spectrum of synthesized HAp are PO 4 3-, OH -, CO 3 2-, as well as HPO 4 2-that characterize non-stoichiometric HAp.A typical FTIR spectrum of hydroxyapatite (Ratner, 2004) PO 4 3-group forms intensive IR absorption bands at 560 and 600 cm -1 and at 1000 -1100 cm -1 .Adsorbed water band is relatively wide, from 3600 to 2600 cm -1 , with an explicit peak at 3570 cm -1 , a weaker peak is formed at 630 cm -1 .CO 3 2-group forms weak peaks between 870 and 880 cm -1 and more intensive peaks between 1460 and 1530 cm -1 .Absorption bands of chemical bonds of the synthesized HAp spectrum are summarized in Table 2.

FTIR absorption bands of thermally treated calcium phosphates
As a biomaterial, HAp is mainly used in its ceramic form that was obtained by sintering the powder at 1000 -1350 °C or as a coating on the implant surface.During the process of thermal decomposition of HAp, sintering of ceramic or obtaining the coating, physical, chemical, mechanical and, most important, biomedical properties may be negatively affected.Thus, HAp and other CaP materials should be thoroughly studied while thermally treated.

Chemical groups and phases
Absorption bands, (cm -1 ) Description
During the thermal treatment, behavior of CaP is affected by various factors, like, atmosphere of sintering, ratio of Ca/P, method and conditions of powder synthesis, type and amount of impurities, sample size, particle size, etc.
During thermal treating HAp undergoes the following processes:  dehydration (separation of adsorbed water);  dehydroxylation (separation of structured water), forming oxy-hydroxyapatite (OHAp) and oxyapatite (OAp);  HAp decomposition with formation of other phases.
In Tables 3-5, the data obtained from literary sources about the FTIR absorption bands of thermally treated CaP chemical groups, is summarized.

Chemical groups and phases
Absorption bands, (cm -1 ) Description
Losing the adsorbed water do not impact lattice parameters.The water adsorbed on the surface discharges under temperature of less than 250°C, when the moisture is discharged from pores up to 500°C.With temperature rising, wide water bands at 3540 cm -1 become narrower and gradually disappear, but the sharp narrow peaks at 630 and 3570 cm -1 refer to variations of structural OH -groups, which is characteristic to structure of HAp.Depending on the synthesis condition, a carbonate containing apatite is often obtained.Then, it should be considered that CO 2 is discharged from the sample between 450-950°C.
Thermal stability is characterized by the decomposition temperature of HAp sample.The decomposition occurs when a critical dehydration point is achieved.In the temperatures less than the critical point, crystal structure of HAp remains unchanged in spite of the stage of dehydration.Achieving the critical point, a complete and irreversible dehydroxillation occurs, which results damage of HAp structure, decomposing onto tricalcium phosphate ( -TCP under 1200 °C and -TCP in higher temperatures) and tetracalcium phosphate (TTCP).
At 900 o C, -TCP shoulders begin to show up at 947, 974 and 1120 cm -1 , but during heating at higher temperature as, for example, 1200 o C, -TCP phase becomes more visible and PO 4 3- peaks shift from 603 and 565 cm -1 to 601 and 571 cm -1 , also from 1094 and 1032 to 1090 and 1046 cm -1 .
Various studies show that in the result of HAp (OAp) decomposition, apart from TCP and TTCP, also other calcium compounds may form, like calcium pyrophosphate (CPP, -Ca 2 P 2 O 7 ) and calcium oxide (CaO).
During the synthesis of TCP, the most important controllable parameters are temperature and pH.According to the literary sources, pH is almost neutral or slightly acidic, and is synthesized at lower temperatures.
A pure stoichiometric -TCP with a molar ratio Ca/P=1.500, is formed in the result of temperature treatment.If Ca/P > 1.500, HAp is formed as the second phase.When Ca/P ratio is changed for 1%, HAp is formed for 10 wt%.If Ca/P < 1.500, then DCPA is formed, this is proven by presence of calcium pyrophosphate Ca 2 P 2 O 7 .
In order to control speed of biodegradation, a biphasic calcium phosphate (BCP) bioceramic is developed, containing both HAp and TCP.By variation HAp/TCP ratio, it is possible to control bioactivity and biodegradation of implant.Since -TCP is more soluble and HAp allows a biological precipitation of apatites, solubility of BCP depends on the ratio of HAp/TCP.Osteoconductivity among BCP, HAp and TCP does not significantly differ.
BCP is formed, if 1.500 < Ca/P < 1.667, which refers to a hydroxyapatite with calcium deficient, its chemical formula is Ca 10-x (PO 4 ) 6-x (HPO 4 ) x (OH) 2-x (0< x <2).Ca/P ratio of synthesis sedimentary is not directly connected with the initial Ca/P ratio.At the constant pH, molar ratio of calcium phosphates may be varied by changing the temperature during the synthesis process.

Synthesis of calcium phosphates
In order to achieve higher assay of the obtained material, its thermal stability and predictability of other properties, the calcium phosphates were synthesized in a laboratory using a wet chemical precipitation method from CaO (or calcium hydroxide Ca(OH) 2 ) of various origins (commercial, marble, eggshells, land snails shells) as precursors and orthophosphoric acid H 3 PO 4 .
Main advantages of this method are a simple synthesis process, a relatively quick obtaining of end product, possibility to obtain large quantities of end product, relatively cheap raw materials and the only by-product it gives is water.It is also important that calcium phosphates with nanometric crystal size can be obtained at a low process temperature (from room to water boiling temperature).
During synthesis, technological parameters like final pH of calcium phosphate suspension and synthesis temperature (T,°C) were changed.Final suspension pH was stabilized in the range of 5-11, using solution of acid.Synthesis temperature was changed in the range from room temperature (21°C) up to 70°C, the following parameters were controlled: acid solution adding speed (ml/min), stirring speed (rpm), synthesis temperature (T,°C), final suspension pH, stabilization time (h), maturity time (τ, h), drying temperature and time (T,°C; h), calcifying temperature and time (T,°C; h).
For further obtaining HAp or biphasic bioceramic synthesized under impact of various parameters (final pH and synthesis temperature), powder is thermally treated.Samples are heated in different environments (air, vacuum and water vapor), variating thermal treatment temperature in range 200-1400°C and processing time.

Analysis methods and sample preparation
In order to determine raw materials, structure of synthesized and heated powder, phase composition and functional groups, two important methods are used, complementing each other: Fourier transform infrared spectroscopy (FTIR) and X-ray difractometry (XRD).XRD method is widely used for apatite characterization, for it provides data about the crystal structure of material and its phase composition, however, it is not convenient to determine www.intechopen.comamount of [OH] or [CO 3 ] groups in hydroxyapatite.FTIR method, in many cases is more sensitive than XRD when determining presence of new phases.Using FTIR, CaP can be characterized, considering three spectrum parameters:  Location of absorption maximum indicates material composition, even slight variations of the composition influence energy of material bondings and, as follows, frequency of variations;  Peak width shows degree of the atoms' order in the apatite elementary cell.

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Considering the absorption maximum of [OH] vibrations, presence of HAp and its thermal stability can be determined, as well as hydroxyl group concentration in the sample.
During the research process, X " Pert PRO X-ray diffractometer has been used (PANalitical, the Netherlands).Samples were measured in a spinning mode, in the 2θ angle range from 20-90º, with a scanning step 0,0334 o , with a CuK radiation.Ratio of HAp/TCP is determined using a XRD semi-quantitative method after calibration line.
Calcium phosphate spectra are measured and analyzed with a FTIR spectrometer "Varian 800" of Scimitar Series, with a wave length range from 400 -4000 cm -1 , with precision of 4 cm -1 and RESOLUTION software.
Samples are prepared by mixing powder with KBr and pressing the pellet.This method of sample preparation has some complications and requires a certain experience, in order to obtain a good quality spectrum in everyday work routine.Special factors should be considered in order to perform invariable sample analysis by using a KBr method, and these include pellet thickness, particle dispersion, ensuring vacuum state during the pressing, pressure influence, ion exchange, etc.
In a prepared KBr pellet, there should be material concentration of 2-10% from the total weight.For preparing a 300g KBr pellet, from 1 to 5mg of the sample is required, and the pellet size will be 13 mm.
Powder grain size should be ~150 m.The analyzed sample is crashed in a powder and thoroughly mixed with the KBr powder.A powder mixer "Pulverisette 23" was used for crashing and mixing of the powder.Prepared powder was located in a specific SPECAC (d = 13 mm) mould, and the required pressure was achieved by applying a uniaxial press (required pressure is ~5. 10 3 kg/cm 2 , pressing time 1 min).
KBr attracts water molecules from the environment and they create wide water bands in the spectrum, so they are hard to or even impossible to analyze.KBr powder should be of the highest assay, Riedel-de-Haen KBr (Lot 51520) brand was used with an assay in the range of 99.5-100.5%.Usually, absorption bands of the main impurities in the KBr are: OH -groups and H 2 O molecules (3500 cm -1 and 1630 cm -1 ), NO 2 (1390 cm -1 ), SO 4 2-(1160-1140 cm -1 ).Spectrum of the KBr used in our research is demonstrated on the Fig. 2. Considering that the powder is hygroscopic, KBr powder is dried at 105 o C and kept in special hermetic containers.Prepared pellets with the analyzed material are dried once again for 24 h.The obtained pellets should be transparent and equally colored.Weak bands connected with water can also be compensated by using a KBr pellet of the same thickness, but not containing the analyzed material, for background spectrum measuring.

Commercial HAp products description
While preparing bioceramic samples from various commercial materials available on the market, we have come across hardly predictable properties of the end product, like crystallinity degree, phase composition and, following, bioactivity and mechanical characteristics.One of the disadvantages while purchasing commercial calcium phosphate powders or commercial calcium phosphate ceramic materials is insufficient information about synthesis conditions of these calcium phosphates, raw materials and in which proportions these materials are taken.
It is significant to know if the purchased powder is thermally treated, and in which temperature range this thermal treatment was performed.Exactly temperature, at which the sample was obtained and processed, is one of the conditions that influences outcome of ceramic and phase composition.
For example, in spectra of several overviewed commercial hydroxyapatites, OH -and PO 4 3- groups are observed, but band shape, width and intensity are different.Differences in spectra are also observed at CO 3 2-and HPO 4 2-groups location and intensity.On Fig. 3, there are three spectra from different commercial HAp compared: "Fluka"(F), "Riedel-de Haën ® " Fig. 3. FTIR spectra of commercial HAp products (1 -S-A; 2 -R-dH; 3 -Fluka) www.intechopen.com(R-dH) and "Sigma Aldrich" (S-A).IR spectrum of commercial S-A product has a lower CO 3 2-and HPO 4 2-intensity that could mean a higher assay degree than the other materials have.These spectra also demonstrate that the products have a low crystallinity degree and they were not thermally treated.
Characteristic chemical groups of the commercial synthesized products are summarized in table 6.In the FTIR spectra of commercial -TCP products, PO 4 3-groups are observed, which is characteristic to -TCP (Fig. 4).In HAp IR spectrum, OH -group peaks are observed (at 630 and 3570 cm -1 ), but there are no such in the IR spectrum of commercial -TCP, which means that there is no HAp phase in this -TCP product.In addition, at 725 cm -1 , presence of P 2 O 7 4- group can be observed, which is characteristic to calcium pyrophosphate phase.

Phase composition control during synthesis reaction
As above mentioned, CaP material synthesis was performed in our laboratory, variating synthesis and further sample heating parameters, searching for their optimal combination depending on the required properties of the obtained material.
In order to control reaction process and possible appearance of by-products, control samples were taken every 5 minutes and analyzed by measuring their spectra.After taking a sample from reactor with a plastic dropper, it was inserted in a glass bottle, hermetically sealed and frozen by putting it in the mixture of dry ice and acetone.After full freezing, the temperature is supported by storing the bottle in the dry ice.Before inserting in the cryogenic drying device, bottles are covered with a perforated plastic film.From the spectra of samples prepared this way (Fig. 5), synthesis with a final pH=9,3 and synthesis temperature 45 o C, it can be seen that the synthesized material is formed with an apatitic HAp structure with a slight Ca deficient, which is proven by presence of the CO 3 2-group, amount of which is constantly reducing along with reaction approaching its end.No by-products were detected, during the reaction, CaO has reacted fully which is proven by an OH -peak disappearing at 3642 cm -1 and forming OH -peaks at 3571 cm -1 and 631 cm -1 which is characteristic for HAp phase.
Such control is very important for scaling the synthesis, relatively increasing amount of synthesis and amount of obtained CaP.

Selection of CaO containing materials of various origins (marble, eggshells, snail shells) and its influence on the CaP product properties
Scientific literature contains very few information about research of how raw materials' (for example, CaO, Ca(OH) 2 ) quality (chemical and physical properties) impacts properties of the obtained bioceramics.
Before starting synthesis of CaP products, selection of CaO containing raw material and complex research were performed.For "Ca" precursors, two commercial available synthetic www.intechopen.com CaO powders from "Riedel-de Haën ® " (CaO R ) and "Fluka" (CaO F ) and two materials of biogenic origin, widespread in the nature -egg shells and land snail (Arianta arbustorum) shells, were chosen.These materials were selected as raw materials for obtaining CaO and further usage in the synthesis process of CaP.
In composition of commercial CaO, presence of Ca(OH) 2 phase, small amount of MgO phase, as well as small amount of polymorphous CaCO 3 modification -calcite phase (Fig. 6), is detected.Presence of CaCO 3 is undesirable in CaO, for during the process of suspension obtaining, Ca(OH) 2 creates an error in preparation of precise amount of the reagent and, as follows, product reproduction, also preventing obtaining a homogenous Ca(OH) 2 suspension which is required for further synthesis of CaP.After heating biogenic and commercial CaO at 1000ºC, X-ray diagrams of all the synthesis materials demonstrated a CaO crystal phase with a small amount of MgO phase, along with Ca(OH) 2 that formed in the result of CaO contact with air moisture; considering that CaO is hygroscopic (Siva Rama Krishna, et al., 2007).
Fig. 6.Commercial CaO X-ray diffractograms before calcifying FTIR spectra were also taken for the thermally treated CaO samples, and their compositions were similar (Fig. 7.).In all the IR spectra, an explicit absorption peak is visible at 3642 cm -1 , that indicates stretching variations of Ca(OH) 2 (Ji, et al., 2009) and [OH] groups (Siva Rama Krishna, et al., 2007).Presence of CaO is also proven by a wide intensive absorption band of [Ca-O] group, which is centered at ~ 400 cm -1 (Ji, et al., 2009).Absorption peaks at 874 cm -1 , 1080 cm -1 [113], as well as at 1420 cm -1 prove presence of the [CO 3 ] groups in the samples which, therefore, shows a slight carboxilation of Ca(OH) 2 from CO 2 of the atmosphere.Absorption band from 3430-3550 cm -1 proves presence of adsorbed water molecules in the samples.
FTIR spectra of the synthesized powders demonstrate absorption bands of the chemical functional groups, characteristic to HAp phase (Fig. 8.).Number of [CO 3 ] groups in those is different, but after thermal treatment at 1100ºC for 1 h, the spectra become very similar (Fig. 9).Using CaO containing materials of various origins, including the commercial CaO available on the market that seemingly correspond with the assay specification, it is still impossible to obtain a completely reproductable HAp bioceramic materials.XRD and FTIR of the HAp products synthesized and heated at 1100ºC, produce similar pictures, and post-synthesis morphology, phase composition and molecular structure of these bioceramics are identical.However, analyzing it with FE-SEM micrographs, it can be observed that obtained HAp products demonstrate different microstructures.
A homogenous, fine-grained microstructure with a small grain size -about 150-200 nm, is observed at the HAp bioceramic from commercial reagents, and non-homogenic grainy structure with irregular grain size in the range from 200 nm up to 1 mcm is observed at the ceramic which was synthesized using Ca of natural origin.
Therefore, the samples from commercial reagents demonstrated color changing, from white in the synthesized and just dried powder to average aquamarine, at ceramic samples heated at 1000 °C, up to light blue color at 1300 °C.It can be explained by oxidizing of the manganese impurities from Mn 2+ up to Mn 5+ and substitution of hydroxyapatite (PO 4 3-) group with (MnO 4 3-) (Ślósarczyk, et al., 2010).Color change can be also explained by other microelements or defects in the crystal lattice, but, in this case, FTIR and XRD analysis cannot give a precise answer to this.

Thermal behavior of calcium phosphates depending on synthesis parameters
Sample structure, phase composition and thermal stability after heating depend on synthesis parameters, especially from final pH and synthesis temperature.Also, connection between pH value and temperature (with temperature increasing, pH decreases); so, by combining and analyzing those parameters, both pure HAp and TCP materials with a good thermal stability are obtained and biphasic materials with various Ca/P ratios and various percentages of phase composition.
Thermal behavior data of laboratory synthesed calcium phosphates with various synthesis parameters were summarized.Final reaction pH was variated in the range from 5.0 up to 10.7 and synthesis temperature was chosen as room (22 o C), 45 o C and 70 o C (Table 7).7. Variable parameters of some calcium phosphate synthesis and calcium phosphate products phase composition after thermal treatment at 1100ºC for 1 h

Nr. of synthesis
It can be ascertained that, in spite of variable parameters of synthesis, all the spectra of samples and XRD diffraction diagrams, are similar, all the functional groups correspond with non-stoichiometric apatitic HAp structure with a low crystallinity degree (Fig. 10).The only slight differences that can be observed between spectra, are intensities of CO 3 2-group bands, however, no significant differences in the number of OH -groups, depending on the synthesis parameters at the non-calcified samples, are detected (Table 8).; absorption band at 963 cm -1 corresponds to a symmetric stretching mode; intensive absorption band in the range of 1040-1090 cm -1 corresponds to a band characteristic to [PO 4 ] 3 groups ( 3 P-O) at 1040 cm -1 and 1090 cm -1 asymmetrical stretching mode, which, as explicit maximums, can be observed after thermal treatment of the samples;  An absorption band of weak intensity within the range between 1950-2100 cm -1 is connected with combinations of [PO 4 ] 3 , 1 modes.
Weak absorption bands at 2365 cm -1 and 2344 cm -1 appeared due to attraction of CO 2 from the atmosphere.
Presence of [CO 3 ] bands can be identified with explicitly visible absorption bands within the range between 1600-1400 cm -1 and at 875 cm -1 , which are observed in the spectra of synthesized samples.Absorption bands of weak intensity, centered at 1418 cm -1 and 1458 cm -1 correspond to symmetrical and asymmetrical stretching modes of the [CO 3 ] 3 groups (C-O).Absorption band at ~875 cm -1 can prove presence of [CO 3 ] 2 stretching mode (at ~872 cm -1 ), intensity of which is approximately 1/5 share of [CO 3 ] 3, or presence of [HPO 4 ] group absorption maximum (Siva Rama Krishna, et al., 2007;Landi, et al., 2000).Considering that the [HPO 4 ] group band partially covers [CO 3 ] 2 , it is complicated to detect, of which group is this band.Presence of this absorption band ascertains solution of atmosphere CO 2 in the suspension, if synthesis occurs in the alkaline environment.
Absorption maximum of [CO ] group at 875 cm -1 can also prove that "AB-type" ([PO 4 ] and [OH] groups) substitution in the structure of HAp, as well as a weak absorption band at 3571 cm -1 in the synthesized HAp samples can mean the "AB-type" substitution (Barinov, et al., 2006).
A wide absorption band within the range from ~3600 cm -1 up to 3100 cm -1 points on 3 and 1 with H 2 O molecules bonded with hydrogen for stretching modes and an absorption band at 1629 cm -1 is referable onto deformation mode 2 of H 2 O molecules (Siva Rama Krishna, et al., 2007), that proves presence of physically adsorbed water in the synthesized samples.
Processing the samples in the temperature range between 200 ºC to 1400 ºC in the air atmosphere, a similar sample behavior, even up to characteristic bioceramic sintering temperatures, is observed in all the syntheses.Dehydratation of the samples occurs up to 500 o C, and at 600 o C, the spectra demonstrate that adsorbed water band disappears from the spectrum, adsorbed and capillary water is eliminated from CaP.Within the temperature range between 500 o C and 800 o C, amount of CO 3 2-groups in the samples also reduces, and bands of CO 3 2-group fully disappear at 900 o C. FTIR spectra (Fig. 12) and XRD diffractograms (Fig. 13) of the thermally treated at 1100ºC samples considerably differ, comparing with the samples that just have been synthesized.A restructurization of functional [PO 4 ] groups have occurred, and sample phase composition is considerably different from the combination of the initial synthesis parameters.It can be concluded that phases with a high crystallinity degree were formed.
A pure, stoichiometric and stable HAp in a wide temperature range is obtained using the following synthesis parameters: final pH=9,3 and synthesis temperature Ts=45 o C (synthesis 6).Sample thermal treatment is performed in the air atmosphere, in the temperature range 200 o C -1400 o C for 1 h.HAp spectra at various heating temperatures are demonstrated on the Fig. 14 and 15.As can be seen after FTIR analysis which is often more sensitive than XRD, HAp begins to decompose when a sample is heated for 1 h at more than 1200°C, forming TCP shoulders at 948 cm -1 , 975 cm -1 and poorly intensive band at 432 cm -1 , which proves dehydroxilation of HAp and forming of -TCP or OHAp phases.Therefore, XRD analysis shows that the decomposition occurs only at 1400°C, and is resulted with mixture of -tricalcium phosphate ( -TCP, Ca 3 (PO 4 ) 2 ), tetracalcium phosphate (TTCP, Ca 4 (PO 4 ) 2 O) and HAp.By heating and then cooling a HAp sample in the air atmosphere, a complete HAp decomposition cannot be achieved, for during cooling [OH] groups get back in the structure from the air and therefore a partial reversible -TCP and TTCPtransform into HAp.A pure -tricalcium phosphate phase Ca 3 (PO 4 ) 2, obtained using the following synthesis parameters: final pH=5,8 and T= 22 o C (synthesis 4), it is synthesized at low temperatures and pH is acidic.
OH -group bands completely disappear at 700 o C with a wave length 3569 cm -1 and 631 cm -1, which is characteristical for HAp.It can also be observed that at 900 o C, bands that correspond to a CO 3 2-functional group, disappear.At 700 o C, a -TCP phase begins to form, which is shown by characteristic shoulders which become more sharply explicit with increasing of temperature.Until 1100 o C, also poorly intensive pyrophosphate (CPP) group bands at 727 and 1212 cm -1 , that disappear at higher temperatures.
Since the sample does not contain CPP and HAp any longer, a pure TCP is obtained in the result, and it is stable up to 1400 o C (Fig. 16 and 17).A -TCP phase can be identified in the spectrum by appearance of characteristic bands at 947 cm -1 and 975 cm -1 .In the synthesis with final pH=5,00 and T=22 o C (synthesis 1), a complicated phase composition is formed from -TCP, CaHPO 4 and TTCP phases, and it remains stable up to 1200 o C. CaHPO 4 phase can be recognized after a HPO 4 2-group band at 897 cm -1 , which is visible in the spectra already at 200 o C.
In the synthesis with a final pH=10,74 and T=45 o C (synthesis 7), a HAp structure is formed but at 1000 o C, and additional OH -peak appears at 3644 cm -1 , which means that there is a unreacted CaO left.The reaction did not occur completely and this synthesis, as well as the above mentioned synthesis 1, cannot be used in practice.] absorption band at 601 cm -1 , when [OH] bonding at 3572 cm -1 is more stable in the higher temperatures (Fig. 18).Stability of hydroxyapatite phase depends on the partial pressure of the water in the atmosphere, so, in an environment with no presence of water, by addition of sufficient amount of energy, HAp will turn into more stable calcium phosphates in the waterless environment.
After thermally treating the sample in vacuum, there is an absorption band detected at 948 cm -1 in the FTIR spectrum already at 600 °C.It appears in the result of [PO 4 ] group fluctuations and usually points onto TCP phase, however the literary sources mention that the absorption band at this wave length could also be characteristic to oxyapatite phase.In the result of thermal treatment, in vacuum at 1300 °C, HAp phase has completely decomposed and absorption bands, characteristic to TCP and TTCP phases, are visible in the FTIR spectrum (overlapping of numerous [PO 4 ] groups vibrations occurs, so it is not possible to distinguish, which absorption bands belong to TCP, and which to TTCP phase).According to XRD data, supplying water vapor during heating of hydroxyapatite does not change phase composition, however slight changes in the sample structure are detected with FTIR analysis.After thermal treatment in the water vapor, absorption intensity of [OH] group increases in ratio to [PO 4 ], comparing with a sample heated in the air, which could improve HAp properties and make this phase more stable.

Conclusions
This summarizing work can help to evaluate both synthesed CaP and structure, phase composition and properties of the CaP bioceramic products.FTIR spectrometry along with XRD is one of the most important, quickest and most available methods for studying CaP materials and in many cases more sensitive than XRD in order to detect forming of new phases.Using these methods, it was possible to detect synthesis parameters in order to obtain a pure and thermally stable HAp and -TCP, as well as biphasic mixtures with controlable ratio.From the results of above mentioned studies it can be concluded that a thorough selection of environment is required for processing HAp powder and it is also required to monitor behavior of the material during the heating, in order to obtain the desirable product.By variating conditions of thermal treatment, it is possible to improve structure of the synthesized HAp, for example, eliminate carbonate groups included in the structure during synthesis, increase number of [OH] groups, as well as slow (by thermal treatment with water vapor presence) or quicken (by thermal treatment in vacuum) HAp decomposition.

Fig. 5 .
Fig. 5. FTIR spectra of the samples from the synthesis series with final pH=9,3 at temperature 45 o C, depending on the reaction occurrence time (from 1 to18).

Fig. 9 .
Fig. 9. FTIR spectra of HAp bioceramics obtained from various CaO after thermal treatment at 1100ºC for 1 h.

Fig. 14 .
Fig. 14.FTIR spectra of HAp (synthesis 6) depending of thermal treatment temperature in range 200 -1000 o C for 1 h

Table 1 .
Calcium phosphates used in the biomaterial field.

Table 6 .
Characteristic chemical groups of commercial HAp FTIR absorption bands to temperature changes and at higher heating temperatures (over 1300°C) is only shown as shoulder of[PO 4 sensitive

Table 11 .
Phase composition after thermal treatment of HAp in the vacuum oven.