Journal of Molecular Structure 1202 (2020) 127225

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Journal of Molecular Structure

journal homepage: http://www.elsevier.com/locate/molstruc

Adsorption and structural properties of hydroxy- and new lacunar

apatites
Said Louihi a, Hassan Noukrati b, c, Youssef Tamraoui d, Hamid Ait Said b,
Hicham Ben youcef d, Bouchaib Manoun a, d, *, Allal Barroug b, c
a
University Hassan 1st, FST, Rayonnement-Mati
ere et Instrumentation, S3M, Settat, Morocco
b
Cadi Ayyad University, Faculty of Sciences Semlalia, LPCME, Marrakech, Morocco
c
Mohammed VI Polytechnic University, CIAM, Benguerir, Morocco
d
Materials Science and Nanoengineering Department, Mohamed VI Polytechnic University, Benguerir, Morocco

a r t i c l e i n f o a b s t r a c t

Article history: Design of novel phosphorus apatites, K2Pb6Zn2(PO4)6 and K2Pb4Ca4(PO4)6, zinc hydroxyapatite
Received 16 August 2019 Ca8Zn2(PO4)6(OH)2, as well as hydroxyapatite Ca10(PO4)6(OH)2, and its corresponding calcined powder
Received in revised form were conducted by means of two different synthesis protocols, i.e. solid state and wet chemical methods.
9 October 2019
The prepared materials adopt P63/m (No 176) as a space group. Structural refinements for K2Pb6Zn2(PO4)6
Accepted 11 October 2019
and K2Pb4Ca4(PO4)6 lacunar apatites, and that for the calcined hydroxyapatite, were performed using
Available online 14 October 2019
Rietveld method. For K2Pb6Zn2(PO4)6, the refinement results showed that the majority of Pb2þ cations
occupy (6h) sites.
Keywords:
adsorption
This study examines the impact of the synthesis conditions on the physico-chemical properties and on
Crystal structure the surface reactivity of the prepared materials. The analysis of the interactions of the samples with
Lacunar zinc-apatite respect to a protein model (Bovin Serum Albumin), evaluated under the same experimental conditions,
Rietveld refinement revealed a fast-kinetic process for the precipitated samples, compared to the crystals obtained by solid
Zinc-hydroxyapatite chemical reaction. Thus, the equilibrium conditions were reached in less than 20 min for the former
while 6 h of contact were requested for the latter. However, all the obtained isotherms show a Lang-
muirian shape. The difference in the adsorption parameters of the materials, i.e. constant affinity and
amount adsorbed at saturation, is discussed in terms of surface characteristics, chemical composition,
crystals size and specific surface area. It is concluded that the interaction with the Bovin Serum Albumin
(BSA) molecules is highly sensitive to the microstructure of the crystals.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction general formula is Me10(XO4)6Y2, Me being a divalent cation (Ca2þ,

Materials such as perovskites (ABO3), garnets (A3B2(SiO4)3),
spinels (AB2O4) and apatites (Me5(XO4)3Y) are the archetypal
structures for a large number of materials having different chemical
compositions [1e4]. These types of oxides can accommodate a wide
variety of metal cations while retaining their unique structure and
the wide range of properties shown makes them very versatile
materials. Properties can often be tuned or tailored by elemental
substitution, so materials with the same structure can be applied
for different purposes.
Apatites constitute a large family of solid compounds whose
* Corresponding author. University Hassan 1st, FST, RMI, S3M, Settat, Morocco.
E-mail address: manounb@gmail.com (B. Manoun).
3
Sr2þ, Pb2þ, …), XO3 3 3
4 a trivalent anion (PO4 , VO4 ; AsO4 ; ….) and
Y a monovalent anion (F¡, OH, Cl, …..) [5e8]. Their structure
consists of two non-equivalent sites 6h and 4f with cations labelled
Me(1) and Me(2), respectively. This particular architecture allows
them to form solid solutions and accept many substitutions.
The lead system is the only system where the apatite structure
can be synthesized without anion Y [9]. The apatites having va-
cancies in the Y anion sites, with the general formula APb4(XO4)3
(A: Na, Ag, K), have been studied by several authors [10e13]. The
Pb2þ electron pairs 6s2 play a key role to maintain the ideal apatitic
network, since they can offset the imbalanced Coulombic forces
created by anion vacancies in the tunnels of the apatite [6,13]. A key
feature of this type of structure is the ability to form solid solutions
of a large number of elements.

https://doi.org/10.1016/j.molstruc.2019.127225
0022-2860/© 2019 Elsevier B.V. All rights reserved.
2 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225
Apatites materials are very abundant in nature and in the living
organisms, that is why researchers have reported on their design,
structure, chemical composition doping and surface properties
[8,14,15]. The intrinsic characteristics of apatites compounds allow
them to have a wide range of applications in various fields and
these can be controlled by adapting the synthesis conditions. That
fact is the main motivation of the present work, which deals with
the structural characterization of lacunar lead phosphate apatites of
potassium without anions (K2Pb4Ca4(PO4)6 and K2Pb6Zn2(PO4)6)
and a calcium zinc hydroxyapatite (Ca8Zn2(PO4)6(OH)2), together
with the reference hydroxyapatite (Ca10(PO4)6(OH)2). The speci-
mens were prepared by solid state reaction and by aqueous solu-
tion precipitation to examine the effect of the synthesis routes on
the final structural and microstructural properties of phosphorus
apatites as well as their surface reactivity with respect to a bio-
logical medium.
2. Materials and methods
2.1. Synthesis of the apatite materials
The targeted apatite specimens were synthesized under fixed
conditions in order to evaluate the role of the physicochemical
factors on their structural and surface properties.
2.1.1. Dry method
The synthesis of the lacunar apatites K2Pb4Ca4(PO4)6 and
K2Pb6Zn2(PO4)6, named respectively Ap-Ca and Ap-Zn, was carried
out using the following commercial grade reactants: lead oxide
(PbO), potassium carbonate (K2CO3), zinc oxide (ZnO), calcium
hydroxide (Ca(OH)2) and diammonium hydrogenophosphate
((NH4)2HPO4). These reagents were mixed in the appropriate
amounts to react accordingly to:
K2 CO3 þ 4 CaðOHÞ2 þ 4 PbO þ 6 ðNH4 Þ2 HPO4 ƒƒƒƒƒ!D
700 C K2 Pb4 Ca4 PO4
and
D

K2 CO3 þ 2 ZnO þ 6 PbO þ 6 ðNH4 Þ2 HPO4 ƒƒƒƒƒ! K2 Pb6 Zn2 PO4 6 þ CO2 þ 12 NH3 þ 9 H2 O
 700 C
The obtained mixtures were ground in an agate mortar, placed
in alumina crucibles and heated progressively up to 500  C in order
to improve their homogeneity and increase the kinetics of the solid
state reaction. The resulting powders were reground and sintered
at a temperature between 700  C and 800  C for 48 h. After each
thermal treatment, the products were air quenched to room
temperature.
2.1.2. Wet method
The hydroxyapatite (HA) was synthesized by double decompo-
sition at pH around 10 as reported earlier [16]. The calcium nitrate
solution Ca(NO3)2.4H2O (1 M, 100 mL deionized water) was added
drop by drop during 3h using a peristaltic pump to a phosphate
ammonium aqueous solution (NH4)2HPO4 (0.06 M, 1000 mL
deionized water) at a temperature close to 80  C under continuous
stirring. After filtration, the precipitate was washed with deionized
water and lyophilized for 68 h.
For the preparation of the calcined apatite, named HA900, a
portion of the as-obtained HA powder was heated at 900  C for
3 h at a rate of 5  C/min. The reaction leading to the preparation of a
stoichiometric hydroxyapatite is as follows:
10 Ca(NO3)2 þ 6 (NH4)2HPO4 þ 8 NH4OH / Ca10(PO4)6(OH)2 þ 20
NH4NO3 þ 6H2O
The zinc-substituted hydroxyapatite (Zn-HA) was obtained us-
ing the same wet chemical route as for HA, by mixing 100 mL of a
cationic solution containing calcium nitrate (Ca(NO3)2$4H2O;
0.758 M) and zinc nitrate hexahydrate (Zn(NO3)2$6H2O; 0.189 M)
with 1000 mL of a solution of ammonia and phosphate
((NH4)2HPO4; 1000 mL, 0.058 M). The reaction corresponding to
the formation of Zn-HA is given by:
8 Ca(NO3)2 þ 2 Zn(NO3)2 þ 6 (NH4)2HPO4 þ 8 NH4OH /
Ca8Zn2(PO4)6(OH)2 þ 20 NH4NO3 þ 6H2O
2.2. Physico-chemical characterization
The structural characterization of the prepared specimens was
carried out by X-ray powder diffraction, infrared spectroscopy and
chemical analyses, while the surface area of the crystals was
determined by the BET method.
The structural refinements of the final products (Ap-Ca, Ap-Zn,
HA, HA900 and Zn-HA) were undertaken using the powder data.
Diffraction data were collected at room temperature on a D2
PHASER diffractometer, having the Bragg-Brentano geometry, using
CuKa radiation (l ¼ 1.5406 Å) at 30 kV and 10 mA. The XRD patterns
were scanned through 2q range 10e100 with step size of 0.01 and
a fixed-time counting of 10 s. Rietveld’s profile analysis method was
employed for refinements using FULLPROF program [11].


6
þ CO2 þ 12 NH3 þ 13 H2 O
The infrared spectra (1 mg sample/99 mg KBr) were recorded
over the 400-4000 cm1 range using a PerkinElmer frontier
Fourier-transform infrared (FTIR) spectrometer. The surface specific
area (SSA) of the crystals was determined by the BET method, using
N2 adsorption (Micromeritics Gemini VII 1014 Instrument).
The concentration of calcium-zinc ions and phosphate species in
the hydroxyapatites obtained by the wet method (HA, HA900 and
Zn-HA) were determined, respectively, by complexometry with
EDTA and spectrophotometry using the phosphovanadomolybdic
complex at l ¼ 460 nm [17]. The content of the elements in the
samples obtained by the dry method (Ap-Ca and Ap-Zn) was
determined using the refinement of the occupation parameters.
Bovine Serum Albumin (BSA) was purchased from Sigma (frac-
tion V powder). This protein exhibits a molecular weight of 67000,
a size of 11.6  2.7  2.7 nm, and has an isoelectric point around 4.7
[18].
 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225 3

2.3. Adsorption experiments Table 1

Main characteristics of the prepared apatites: structural parameters, chemical
composition, crystals size and specific surface area.
The interaction of the protein with the apatite samples was
carried out at room temperature (25 ± 1  C) by dispersing the Sample Unit cell Volume (A3) Me/P (±0.02) Crystals SSA
adsorbent (25 mg) into 5 mL of the adsorption solution in a poly- parameters Dimension (m2/g)
(nm) ±3%
ethylene tube. The adsorbate was dissolved in 1 mM potassium
nitrate solution with a concentration varying from 0 to 3.5 mg/mL. a (Å) c (Å) Length Width

The pH of the prepared solutions was adjusted to a value close to 7.4
by adding potassium hydroxide and hydrochloric acid. After soni-
cation for 1e2 min, the suspensions were incubated for a period of
2 he8 h without stirring and centrifuged for 10 min at 4000 rpm.
This equilibration time was previously determined from kinetic
experiments using BSA concentrations of 2.5 mg/mL. Blanks
without apatite solid, containing only the protein, were incubated
similarly as the adsorption tests and used as control.
The resulting supernatants were filtered through Millipore fil-
ters (pore size 0.2 mm) and the BSA concentration in the filtrates
was determined using UV spectrophotometric absorption at
l ¼ 280 nm (BECKMAN type 24 with dual beam), that corresponds
to the maximum absorbance of the BSA molecules. The calibration
curve ranged from 0 to 2 mg/mL. The molar absorption coefficient
deduced from all calibration curves was 0.66 ± 0.02 L g1 cm1 at
pH 7.4. This value is close to that reported (0.65 ± 0.04.L g1 cm1
at pH 7) in the literature [18].
The quantity of BSA adsorbed Q (mg/g) was then determined
following the relationship:
Q ¼ (Ci - Ce)*V / W
where Ci and Ce (mg/L) are BSA solution concentration at initial and
equilibrium time, V (L) is the volume of the solution in contact with
W(g) of apatite. The reproducibility of the adsorbed BSA, as average
of 3 measurements, is 7%.
3. results and discussion
3.1. X-ray diffraction study
Fig. 1 shows the X-ray powder diffraction patterns of the syn-
thesized apatites, that are comparable to those reported earlier
[9,15]. Indexing of the X-ray powder patterns was performed by
means of the software DICVOL [12]. The first 20 peak positions,
Fig. 1. X-ray powder diffraction patterns of the synthesized apatites Ap-Ca, Ap-Zn, HA,
HA900 and Zn-HA. The inset shows a comparison of the crystallinity in the hydroxy-
apatites prepared under different conditions.
HA900 9.435 6.890 531.12 1.67 54.2 42.2 8.00
HA 9.419 6.875 528.22 1.65 36.9 9.9 79.00
Zn-HA 9.431 6.853 527.89 1.66 27.6 7.6 120.00
Ap-Ca 9.671 7.159 579.89 1.67 117.0 126.4 0.69
Ap-Zn 9.853 7.319 615.40 1.67 62.6 e 0.62
Me: Ca or (Ca þ Zn).
with a maximal absolute error of 0.03 (2q), were used as input
data. The data were calibrated externally using the zero shift of the
Rietveld refinement. All the found compositions were assigned to a
hexagonal apatite structure.
The diffraction peaks of the precipitated calcium phosphate
powders are typical of hydroxyapatites. As it can be seen in Fig. 1,
the peaks of the calcined sample (HA900) are sharper and more
defined which reflect a well crystallized apatitic phase. The pattern
indicates the presence of a small quantity of beta-tricalcium
phosphate (less than 5%), due to the slight calcium deficiency of
the reference HA. The calcination of the powder HA results in a
significant increase in the intensity of peaks and decrease in the
width compared to the phase HA. However, an opposite tendency
occurred upon substitution of calcium ions by zinc species in the
lattice of the mother sample HA.
The unit cell parameters obtained for the prepared composi-
tions (Ap-Ca, Ap-Zn, HA, HA900 and Zn-HA) are given in Table 1 and
they are close to those reported in the literature [7,19]. The unit cell
parameters of the lacunar apatites (Ap-Ca and Ap-Zn) are much
bigger than those of the precipitated samples (HA, HA900 and Zn-
HA); this fact can be simply explained by the size of lead and po-
tassium atoms which have bigger radii compared to that of calcium
species.
The mean crystallite size derived from Scherrer’s formula
applied to the apatitic diffraction lines (002) and (310) was calcu-
lated, after deconvolution of the peaks related to lka1 and lka2
(Table 1). The values of the crystals length (L002) and width (L310)
obtained for the specimen HA are respectively 36.9 nm and 9.9 nm.
Substituting calcium by zinc in the apatite lattice slightly decreases
the crystallite dimensions (27.6 nm and 7.6 nm). However, an in-
crease in crystallite sizes (54.2 nm and 42.2 nm) was observed for
the sample HA calcined at 900  C, in good agreement with the
evolution of the XRD patterns. Comparison of the length and the
width values suggests that the precipitated crystals are elongated
along the c-axis (L002 > L310). A similar tendency was observed for
compounds synthesized in similar conditions [20,21]. Contrarily,
the lacunar apatites prepared by dry method reveal a different
behavior: the specimen Ap-Ca exhibits high values as well as an
isotropic attitude with respect to the crystallite dimensions
(L002 ¼ 117.0 nm and L310 ¼ 126.4 nm). Besides, an important drop
in the crystals length (62.6 nm) happened when zinc species were
incorporated in the apatite lattice.
The structural refinements were done using the Rietveld
method with the Fullprof program integrated in WINPLOTR soft-
ware [12,22]. The starting model for Rietveld refinement was taken
from Ref. [19]. 34 structural and non-structural parameters were
allowed to vary in order to adjust the refinement. Fig. 2 shows the
Rietveld refinement patterns for (a) K2Pb4Ca4(PO4)6 and (b) the
calcined hydroxyapatite (Ca10(PO4)6 (OH)2).
The structural parameters and their estimated standard
4 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225

Table 2
Crystallographic characteristics obtained from Rietveld refinements of lacunar
apatite Ap-Ca, Ap-Zn and calcined hydroxyapatite HA900.

Formula K2Pb4Ca4(PO4)6
Lattice parameters Space group: P 63/m a (Å) ¼ 9.6710(1), c (Å) ¼ 7.1593(1),
g ¼ 120 ( ),
V (Å3) ¼ 579.89(1)
R-Factors % RF ¼ 8.66, % RB ¼ 5.83, c ¼ 3.25
Site x y z Occupancy

Pb(1)/Ca(1) 6h 0.2563(2)
0.2566(2) 0.25 1.81/1.19
Pb(2)/Ca(2)/K 4f 0.6667 0.3333 0.499(1) 0.19/0.81/1
P 6h 0.377(1) 0.409(1) 0.75 3
O(1) 6h 0.495(1) 0.348(1) 0.75 3
O(2) 12i 0.268(1)0.3576(9) 0.571(1) 6
O(3) 6h 0.491(2) 0.591(2) 0.75 3
Formula K2Pb6Zn2(PO4)6
Lattice parameters Space group: P 63/m a (Å) ¼ 9.8531(6) c (Å) ¼ 7.3194(5),
g ¼ 120 ( ),
V (Å3) ¼ 615.40(7)
R-Factors % RF ¼ 1.81, % RB ¼ 3.99, c ¼ 4.50
Site x y z Occupancy

Pb(1)/Zn(1) 6h 0.252(4)0.2499(12) 0.25 2.058/0.942

Pb(2)/Zn(2)/K 4f 0.66667 0.33333 0.5145(18) 0.942/0.058/1
P 6h 0.376(1) 0.408(2) 0.75 3
O(1) 6h 0.477(1) 0.328(1) 0.75 3
O(2) 12i 0.273(1) 0.368(2) 0.576(2) 6
O(3) 6h 0.476(2) 0.584(2) 0.75 3
Formula Ca10(PO4)6(OH)2
Lattice parameters Space group: P 63/m a (Å) ¼ 9.4228(4) c (Å) ¼ 6.8828(3),
g ¼ 120 ( ),
V (Å3) ¼ 529.24(4)
R-Factors % RF ¼ 2.5, % RB ¼ 4.68, c ¼ 1.86
Site x y z Occupancy

Fig. 2. Final Rietveld plot of (a) lacunar apatite Ap-Ca (K2Pb4Ca4(PO4)6) and (b) Ca1 4f 0.33333 0.66667 0 4
calcined hydroxyapatite HA900 (Ca10(PO4)6(OH)2). The solid line with dots illustrates Ca2 6h 0.25 0 0.25 6
the observed data and the calculated pattern (solid line). The vertical markers show P 6h 0.391(3) 0.374(3) 0.25 6
calculated positions of Bragg reflections. The lower curve is the difference diagram. O1 6h 0.335(6) 0.5 0.25 6
O2 6h 0.578(7) 0.457(7) 0.25 6
O3 12i 0.3333 0.25 0.073(6) 12
O4 2a 0.0000 0.0000 0.25000 2
deviations for K2Pb4Ca4(PO4)6, K2Pb6Zn2(PO4)6 and
Ca10(PO4)6(OH)2 are given in Table 2; a list of selected bond lengths
and angles is given in Supplemental files (SF-Table I).
P63/m space group was adopted by the lacunar apatites (Ap-Ca, be provided by the Y (F, Cl, OH) anions that occupy the large
Ap-Zn) as well as the calcined hydroxyapatite (HA-900). As an tunnels encompassing 63 axes. Whenever the Y anions are removed
example, the structure of K2Pb6Zn2(PO4)6 is built up from [PO4]3- from the lattice, the apatite network collapses and rises a mixture
tetrahedra and Pb(1)2þ/Zn(1)2þ of sixfold coordination cavities (6h of phases or a new structural type, except in the cases where the
positions) creating tunnels along c-axis. The tunnels are connected host channels are occupied by cations having lone pairs such as
by cations of mixed sites (4f), half occupied by Pb(2)2þ/Zn(2)2þ and Pb2þ or Bi3þ. This structural stabilization has been attributed to the
half by Kþ alkali cations. Fig. 3 shows a structural view comparing orientation of the lone pairs 6s2 of lead within the tunnels.
the lacunar apatite (Ap-Zn) and the calcined hydroxyapatite The stability of Me10(XO4)62 lacunar apatites is linked to the lone
(HA900). While the tunnels are empty along c-axis for Ap-Zn, they pair ns2 like in Tlþ, Pb2þ or Bi3þ [25e27]. This stabilization is due to
are fully occupied by the anions OH in HA900. the orientation of the lone pairs 6s2 within the tunnels. In the lead
The Pb2þ(2)/Zn þ(2)/Kþ-O bonds in the mixed site Me(4f) are lacunar apatites, the stereo activity of Pb2þ cations is manifested
ionic in character, the average distances of Me(2)-O9 vary from only in the 6h sites but not in the mixed sites (Kþ, Pb2þ and Ca2þ).
2.581 to 2.679 Å. Pb2þ (1)/Zn2þ (1)-O in the mixed site Me(6h) The interatomic distances for PeO vary between 1.51 Å and 1.55 Å
shows a more covalent character and the average distances of and the average of OePeO angles is 108.5 , as indicated in Sup-
Me(1)-O6, varies from 2.23 to 2.79 Å (SF-Table I); this can be plemental files (SF-Table I). The average distances are similar and
explained by the distorting effect of the lone pair electrons. agree well with those reported previously [11,19].
The small tunnel (mixed site Me(2)) is occupied half by Kþ and
half by Pb2þ (2)/Zn2þ (2) cations forming (K/Pb/Zn)O9 polyhedra. 3.2. Infrared spectroscopy and chemical analysis
Each polyhedron is linked to 3 PO3 4 tetrahedra via corners and to
three others via edges (Fig. 3). The bigger tunnel in the other site is The synthesized samples were examined by FTIR spectroscopy
surrounded by lead-zinc cations Me(1). These cations are inserted (Fig. 4). The precipitated powders showed spectra characteristics of
into six fold sites that constitute the walls of the tunnels; each hydroxyapatite environments, as it can be observed by the main
vibration bands of PO3 4 species n3 (1000-1100 cm1), n1
polyhedron is linked to 4PO3
4 tetrahedra via corners and to one (961 cm ), n4 (550-600 cm ), n2 (472 cm1) as well as the OeH
1 1
PO3
4 via edge. Mathew et al. [23] and Engel et al. [24] showed that sharp bands (Elongation mode centered at 3570 cm1 and libra-
the stabilization of the structural apatite seems to be directly tion mode centered at 632 cm1). The OH bands are well illustrated
related to the presence of a minimum electronic density within its in the calcined apatite HA900. The samples Ap-Ca and Ap-Zn ob-
large tunnels. In the normal apatites these negative charges could tained by the dry method indicated also the typical apatitic bands.
 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225
Fig. 3. Polyhedral view in the ab-plane of the crystal structure of lacunar apatite Ap-Zn
and calcined hydroxyapatite HA900. The large tunnel in the case of the lacunar spec-
imen illustrates the metal and oxygen arrangement around (6h) sites connected to the

5PO34 tetrahedra. In the case of simple HA900 this tunnel is occupied by OH anions.
Fig. 4. FTIR spectra of lacunar apatites Ap-Ca and Ap-Zn and the hydroxyapatites HA,
Zn-HA and HA900.
However, we noticed the absence of the OH vibrations, attesting of
the lacunar character of the Ap-Ca and Ap-Zn specimens. Besides,
there was a shift of PO3
4 bands towards low wavenumbers for the
lacunar apatites compared to those of precipitated samples. This
shift is due to the difference in PeO distances, as given in the
supplementary files. Not only that, but we also noticed that the unit
cells parameter of the hydroxyapatites are much smaller than those
of the lacunar ones (Table 2).
The chemical analyses (Table 1) show that the sample HA is a
slightly calcium-deficient hydroxyapatite and that fact agrees with
FTIR results, while Zn-HA exhibits an atomic ratio ([Me]/[P] close to
stoichiometry. The content of the elements in the samples obtained
by the dry method (Ap-Ca and Ap-Zn) was determined using the
Rietveld refinement of the occupation parameters, leading to a
5
stoichiometric composition (atomic ratio [Me]/[P] of 1.67).
The powders synthesized by double decomposition exhibit
specific surface area (SSA) values within the range 8e120 m2/g, as
shown for samples prepared under similar experimental conditions
[14,28]. Zinc incorporation in the apatite matrix lowered the crys-
tals dimension and raised the SSA value of the yield sample (Zn-
HA), compared to the mother phase (HA). However, calcination of
HA powder drastically decreased the SSA (down to 90%), without
destroying the original framework. These findings agree with the
evolution of the crystallinity degree with operative conditions, as
confirmed by XRD diagrams (Fig. 1). The influence of calcination
temperature on the surface area is more pronounced in the samples
Ap-Ca and Ap-Zn prepared by dry method; the values found are less
than 1 m2/g.
3.3. Adsorption
The kinetic study aimed at determining the contact time needed
to reach the thermodynamic equilibrium conditions. Fig. 5 shows
the variation of the amount of BSA adsorbed per unit of mass of the
apatite as a function of contact time. Examination of the data
indicated that a contact time of 20 min was sufficient to attain a
stationary concentration for the sample HA, while the yield plateau
was reached after 6 h for the specimen Ap-Ca. Therefore, the
adsorption tests were conducted using incubation periods of 2 h
and 8 h for the powders prepared by the wet and dry methods,
respectively. Those times ensure a constant concentration of the
adsorbate in the liquid phase even after 24 h of incubation. The
obtained results indicate a rapid kinetic for the precipitated hy-
droxyapatite (HA) and a relatively slow process for the sample
obtained by solid reaction (Ap-Ca). The quick adsorption of the
former indicates a strong surface affinity for the biomolecules,
compared to the sample Ap-Ca. Slow kinetic processes reported for
phosphate materials tested with a variety of adsorbates were
assigned to the existence of repulsive electrostatic interactions
between the adsorbent and the adsorbate as confirmed by elec-
trokinetic measurement [29].
The adsorption experiments conducted by varying the adsor-
bate initial concentration (0e3.5 mg/mL) reveal that the uptake
rate ranged from 5 to 95% for the precipitated samples, while the
apatites formed through solid state reaction had a lower loading
capacity (1e24%). This fact suggests that the latter specimens are
Fig. 5. Adsorption kinetics of BSA onto apatite samples HA and Ap-Ca: 1 mM potas-
sium nitrate, initial concentration Co ¼ 2.5 mg/mL, pHads ¼ 7.1 and temperature
T ¼ 25  C.
6 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225

elsewhere (20,29).
The results demonstrate that the synthesis conditions affect
significantly the physicochemical characteristics of the prepared
solids, which consequently impact on their surface reactivity. As
can be seen, the samples precipitated in aqueous solutions at
relatively low temperature exhibited higher binding capacities per
unit of mass, compared to the solids calcined at high temperature.
The thermal treatment of HA crystals prepared at 80  C reduced the
adsorption capacity of its calcined powder (HA900). The maximum
coverage sequence noticed for the Hydroxyapatites (Zn-
HA > HA > HA900) is in accordance with the evolution of their XRD
patterns and microstructure. Thus, the most active apatite surface
(Zn-HA) exhibited the lowest crystallinity index, the smallest
crystal dimensions and the highest surface specific area.
When the uptake data are normalized by the specific surface
area, the plot of the maximum amount adsorbed as a function of
the BET specific surface area of the solids showed a different trend
(Fig. 7). Hence, the precipitated apatites (HA and Zn-HA) with large
surface values showed less sorption efficiency, while their capacity
Fig. 6. Adsorption isotherms of BSA (0e3.5 mg/mL) onto the prepared apatites in was slightly improved as the temperature of treatment increased
1 mM potassium nitrate at room temperature (T ¼ 25  C) and pH in the range 7.1e7.4: (HA900). This effect is particularly noticeable for the samples pre-
evolution of the amount of BSA adsorbed as a function of its equilibrium concentration.
pared by solid state reaction (Ap-Ca followed by Ap-Zn) and having
values of SSA less than 1 m2/g that exhibited the largest monolayer
less reactive toward BSA molecules. The experimental data of the
coverage. These findings suggest, in addition to SSA as an important
compounds investigated under similar experimental conditions
parameter, the existence of specific interactions and additional
(concentration 0e3.5 mg/mL, room temperature, pH 7.1e7.4, same
factors that contribute to the phenomena occurring at apatite
solid-to-solution ratio) are accessible as Supplemental files (SF-
surface upon contact with the surrounding medium. Complemen-
Table II).
tary work should be undertaken with simple and active adsorbates
Fig. 6 represents the variation of BSA adsorption by the solids as
for a better understanding of the surface properties and the inter-
a function of the concentration remaining in solution after reaching
facial phenomena of lacunar apatite having empty tunnels and low
the equilibrium. The shape of the obtained isotherms is of Langmuir
specific surface area.
type. The quantity adsorbed increased quickly and reached plateaus
It is proven in this work that various parameters i.e., crystallinity
at relatively low protein concentrations, which is indicative of a
index, surface defects, size dimensions and specific surface area
high affinity between the adsorbent and the adsorbate.
The amount adsorbed was normalized by the mass unit of the
solids to allow comparison of the different materials. The adsorp-
tion parameters (N and K) were determined [18,20] by the
commonly used linearized form of the Langmuir equation [Ce/
Q ¼ Ce/N þ 1/KN], where Q (mg/g) is the adsorbed amount, Ce (mg/
L) is the equilibrium concentration, N (mg/g) is the maximum
sorption capacity of the solid, and K (mL/mg) is the binding coef-
ficient. The conformity of the adsorption data to the Langmuir
isotherm was tested by plotting Ce/Q as a function of Ce, which give
a linear tendency with correlation coefficients of about 99%. The
linearization (of the Langmuir equation) obtained for the investi-
gated samples in the domain of concentration explored are shown
in Supplemental files (SF - Fig. 1).
The adsorption parameters determined by linear regression are
reported in Table 3. The maximum amount adsorbed (N) was
normalized to the unit of mass and to the surface area of the solids,
to point out the factors of particular importance in their behavior
upon contact with the biological medium. The evolution of the
affinity constant was not considered in the discussion due to the
lack of enough experimental points at low coverage and to the
Fig. 7. Maximum amount adsorbed (N) of BSA as a function of solids specific surface
irreversible aspect of the process at apatite interface as reported area.

Table 3
Adsorption parameters of the prepared apatites: affinity constant (K) and maximum amount adsorbed (N) normalized to the unit of mass and to the surface area of the solids.

Adsorbent Langmuir Parameters

N ± DN (mg/g) K ± DK (ml/mg) N ± DN (mg/m2) K ± DK (ml/mg) R2

Ap-Zn 8.9 ± 0.6 1.5 ± 0.1 14.3 ± 1.0 1.5 ± 0.1 0.98
Ap-Ca 30.0 ± 2.1 2.9 ± 0.2 43.5 ± 3.1 2.9 ± 0.3 0.99
HA900 47.4 ± 3.3 6.6 ± 0.5 4.1 ± 0.3 5.3 ± 0.4 0.99
HA 87.7 ± 6.2 8.8 ± 0.6 1.1 ± 0.1 8.5 ± 0.6 0.99
Zn-HA 172.4 ± 12.1 4.8 ± 0.3 1.5 ± 0.1 3.8 ± 0.3 0.99
 S. Louihi et al. / Journal of Molecular Structure 1202 (2020) 127225
play a key role in the interactions process. The adsorption of bio-
molecules at the apatite surface is of key importance in the field of
biomaterials and the control of the specific properties of the apatite
mineral (solubility, reactivity, resorption …) may contribute to
improve its biological function as a component of calcified tissues.
Furthermore, the information gained in this study regarding the
driving forces at the mineral surface with regards to its chemical
and biological environments can also be beneficial for the devel-
opment of effective sorbents for potential use in waste water
purification.
4. Conclusion
In this work we reported on the synthesis, physicochemical
characterization and surface properties of various lacunar- and
hydroxy-phosphorus apatites. The XRD studies showed that all
examined compounds crystallize in the hexagonal system with P63/
m (No 176) as space group. Using Rietveld refinements, we have
shown that while the Y sites are empty in the lacunar apatites, in
the case of hydroxyapatites those sites are fully occupied by the
anions OH. We have shown that the majority of Pb2þ cations are
located in the (6h) sites of the lacunar apatite.
The interaction of the prepared compounds with a model pro-
tein (BSA) revealed a fast kinetic process for the precipitated
samples, compared to the crystals obtained by solid chemical re-
action. However, all the obtained isotherms were Langmuirian in
shape. The difference in the adsorption parameters of the materials
was discussed considering the surface characteristics of the speci-
mens. It can be concluded that the degree of crystallinity, surface
defects and crystals size, as well as surface areas do, to some extent,
contribute to the surface properties of apatite materials.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
rifien des Phosphates
The authors are grateful to the Office Che
in the Moroccan Kingdom (OCP group), the University Hassan 1st
for their support. A part of this work was supported through the
R&D Initiative “Appel
a projets autour des phosphates APPHOS”
sponsored by OCP (Project ID: MAT-BAR-01/2017); the authors
would like to thank OCP Foundation, R&D OCP, UM6P, CNRST and
MESRSFC-Morocco. The authors are also grateful to the Swedish
Research Council for the financial grant (SRL (MENA) # 348-
2014e4287), Sweden.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.127225.
References
[1] M. Anderson, K. Greenwood, G. Taylor, K. Poeppelmeier, B-cation arrange-
ments in double perovskites, Prog. Solid State Chem. 22 (1993) 197e233,
https://doi.org/10.1016/0079-6786(93)90004-B.
[2] M.G. Brik, A. Suchocki, A. Kamin  ska, Lattice parameters and stability of the
spinel compounds in relation to the ionic radii and electronegativities of
constituting chemical elements, Inorg. Chem. 53 (2014) 5088e5099, https://
doi.org/10.1021/ic500200a.
[3] P. Loveland, An introduction to forensic geoscience, Soil Use Manag. 28 (2012),
https://doi.org/10.1111/j.1475-2743.2012.00419.x, 647e647.
[4] T.J. White, D. ZhiLi, Structural derivation and crystal chemistry of apatites,
Acta Crystallogr. B 59 (2003) 1e16, https://doi.org/10.1107/
7
s0108768102019894.
[5] J.C. Elliott, Structure and Chemistry of the Apatites and Other Calcium Or-
thophosphates, Elsevier Science, Amsterdam, 1994.
[6] R. Ternane, M. Ferid, M. Trabelsi-Ayedi, B. Piriou, Vibrational spectra of lead
alkali apatites Pb8M2(PO4)6 with M ¼ Ag and Na, Spectrochim. Acta Part A
Mol. Biomol. Spectrosc. 55 (1999) 1793e1797, https://doi.org/10.1016/S1386-
1425(99)00003-7.
[7] C. Rey, C. Combes, C. Drouet, D. Grossin, G. Bertrand, J. Soulie , Bioactive cal-
cium phosphate compounds: physical chemistry, in: P. Ducheyne (Ed.),
Comprehensive Biomaterials II, Elsevier, Oxford, 2017, pp. 244e290, https://
doi.org/10.1016/B978-0-12-803581-8.10171-7.

[8] M. Supov 
a, Substituted hydroxyapatites for biomedical applications: a review,
Ceram. Int. 41 (2015) 9203e9231, https://doi.org/10.1016/
j.ceramint.2015.03.316.
[9] M. Azrour, M. Azdouz, B. Manoun, R. Essehli, S. Benmokhtar, L. Bih, L. El
Ammari, A. Ezzahi, A. Ider, A.A. Hou, Rietveld refinements and vibrational
spectroscopic studies of Na1-xKxPb4(PO4)3 lacunar apatites (0  x  1), J. Phys.
Chem. Solids 72 (2011) 1199e1205, https://doi.org/10.1016/
j.jpcs.2011.06.013.
[10] M. El Koumiri, S. Oishi, S. Sato, L. El Ammari, B. Elouadi, The crystal structure of
the lacunar apatite NaPb4(PO4)3, Mater. Res. Bull. 35 (2000) 503e513, https://
doi.org/10.1016/S0025-5408(00)00254-3.
[11] J. Rodriguez-Carvajal, FULLPROF 2000: A Rietveld Refinement and Pattern
on Brillouin (CEA-CNRS), France,
Matching Analysis Program 2, Laboratoire Le
2008. Version: April.
[12] A. Boultif, D. Loueer, D. Loue €r, Indexing of powder diffraction patterns for low-
symmetry lattices by the successive dichotomy method, J. Appl. Crystallogr.
24 (1991) 987e993, https://doi.org/10.1107/S0021889891006441.
[13] S. Lahrich, M.A. Elmhammedi, B. Manoun, Y. Tamraoui, F. Mirinioui, M. Azrour,
P. Lazor, Elaboration, Rietveld refinements and vibrational spectroscopic
study of Na1-xKxCaPb3(PO4)3 lacunar apatites (0  x  1), Spectrochim. Acta
Part A Mol. Biomol. Spectrosc. 145 (2015) 493e499, https://doi.org/10.1016/
j.saa.2015.02.083.
[14] M. Iafisco, J.M. Delgado-Lo pez, Apatite : Synthesis, Structural Characterization,
and Biomedical Applications, Nova Science Publishers Inc, New York, 2014.
[15] C. Rey, C. Combes, C. Drouet, H. Sfihi, Chemical diversity of apatites, Adv. Sci.
Technol. 49 (2006) 27e36. https://doi.org/10.4028/www.scientific.net/AST.49.
27.
[16] A. Barroug au, J. Lemaitre, P.G. Rouxhet, Lysozyme on apatites: a model of
protein adsorption controlled by electrostatic interactions, Colloids Surface.
37 (1989) 339e355, https://doi.org/10.1016/0166-6622(89)80130-1.
[17] G. Charlot, Chimie analytique quantitative: Me thodes se lectionnees d’analyse
chimique des e le
ments, sixth ed., vol. 2, Masson et Cie, Paris, 1974.
[18] S. Ouizat, A. Barroug, A. Legrouri, C. Rey, Adsorption of bovine serum albumin
on poorly crystalline apatite: influence of maturation, Mater. Res. Bull. 34
(1999) 2279e2289, https://doi.org/10.1016/S0025-5408(00)00167-7.
[19] S. Lahrich, B. Manoun, M.A. El Mhammedi, Synthesis, characterization and
electrochemical investigation of NaPb4xCax(PO4)3 (0  x  1.5) in capturing
cadmium (II), S. Afr. J. Chem. Eng. 23 (2017) 98e106, https://doi.org/10.1016/
J.SAJCE.2017.04.001.
[20] F. Errassifi, S. Sarda, A. Barroug, A. Legrouri, H. Sfihi, C. Rey, Infrared, Raman
and NMR investigations of risedronate adsorption on nanocrystalline apatites,
J. Colloid Interface Sci. 420 (2014) 101e111, https://doi.org/10.1016/
j.jcis.2014.01.017.
[21] J. Gomez-Morales, M. Iafisco, J.M. Delgado-Lo  pez, S. Sarda, C. Drouet, Progress
on the preparation of nanocrystalline apatites and surface characterization:
overview of fundamental and applied aspects, Prog. Cryst. Growth Charact.
Mater. 59 (2013) 1e46, https://doi.org/10.1016/J.PCRYSGROW.2012.11.001.
[22] T. Roisnel, J. Rodríquez-Carvajal, WinPLOTR: a windows tool for powder
diffraction pattern analysis, Mater. Sci. Forum 378e381 (2001) 118e123.
https://doi.org/10.4028/www.scientific.net/MSF, 378-381.118.
[23] M. Mathew, W.E. Brown, M. Austin, T. Negas, Lead alkali apatites without
hexad anion: the crystal structure of Pb8K2(PO4)6, J. Solid State Chem. 35
(1980) 69e76, https://doi.org/10.1016/0022-4596(80)90464-8.
[24] G. Engel, W. GOtz, R. Eger, A
propos des apatites silicate es contenant du
bismuth. Oxidapatites inhabituelles, Z. Anorg. Allg. Chem. 449 (1979)
127e134, https://doi.org/10.1002/zaac.19794490113.
[25] A. Verbaere, R. Marchand, M. Tournoux, Localisation du doublet solitaire dans
les compose s oxyge
n e
s cristallise
s du thallium I, J. Solid State Chem. 23 (1978)
383e390, https://doi.org/10.1016/0022-4596(78)90088-9.
[26] W.H. Baur, The geometry of polyhedral distortions. Predictive relationships
for the phosphate group, Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst.
Chem. 30 (1974) 1195e1215, https://doi.org/10.1107/S0567740874004560.
[27] C. Colbeau-Justin, G. Wallez, A.M. Xuriguera, A. Elfakir, S. Jaulmes, M. Quarton,
Structure determination of Tl3Li (MoO4)2. Stereochemical activity of Tl(I) lone
pair, Eur. J. Solid State Inorg. Chem. 34 (1997) 1097e1106.
[28] A. Barroug, J. Lemaître, P.G. Rouxhet, Influence of crystallite size on the surface
properties of calcium-deficient hydroxyapatites, J. Alloy. Comp. 188 (1992)
152e156, https://doi.org/10.1016/0925-8388(92)90664-U.
[29] A. Barroug, E. Lernoux, J. Lemaitre, P.G. Rouxhet, Adsorption of catalase on
hydroxyapatite, J. Colloid Interface Sci. 208 (1998) 147e152, https://doi.org/
10.1006/JCIS.1998.5759.