Journal of Minerals & Materials Characterization & Engineering, Vol. 7, No.1, pp 49-57, 2007
jmmce.org Printed in the USA. All rights reserved
49
Nucleation Reduction Strategy of (Brushite) CHP Crystals in SMS
Media and Its Characterization Studies
G. Kanchana
1
, P. Sundaramoorthi
2
, R. Santhi
3
, S. Kalainathan
4
, G.P. Jeyanthi
1
1
Department of Bio-Chemistry, Avinasliilingam University, Coimbatore, India.
2
Department of physics, Aringar Anna Government Arts and Science College,
Namakkal. (E-mail: [e-mail])
3
Department of Physics, MEC, Mallasamudram (W), Namakkal, India.
4
Department of physics, VIT, Vellore, India
Abstract
Kidney stone consist of various organic, inorganic and semi organic compounds.
Mineral oxalate monohydrate and di-hydrate is the main inorganic constituent of kidney
stones. However, the mechanisms for the formation of calcium oxalate kidney stone are
not clearly understood. In this field of study there are several hypothesis including
nucleation, crystal growth and or aggregation of formation of COMH, AOMH
(Ammonium oxalate monohydrate), CODH, and AODH (Ammonium oxalate di-hydrate)
crystals. The author has reported the effect of some urinary species such as ammonium
oxalates, calcium, citrate, proteins and trace mineral. The kidney stone constituents are
grown in the kidney environments, the silica gel medium (SMS) provides the necessary
growth simulation (in-vivo). In the artificial urinary stone preparation (growth) or
crystal growth, growth parameter identification with in the different chemical
environments is carried out. In the present study, CHP (calcium hydrogen phosphate)
crystals are grown in three different growth faces to attain the total nucleation
reductions. As an extension of this research, many characterization studies have been
carried out, and the results are compared and reported.
Key words: Renal stones, MHP, calculi, surface morphology, growth parameters, trace
elements, SDP, AMHP, Brushite.
1. INTRODUCTION
Most kidney stones consist of an organic matrix with bio-minerals. The organic
matrix has a composition that remains constant regardless of the type of crystals that
make up the stone [1, 2]. The kidney stone accounts for approximately 4% of the weight
of a calculus [3]. Most of the current knowledge of matrix composition emerges from the
analysis of the 30 % of matrix substances that are soluble [4]. The matrix has been
described as a heterogeneous material composed of inorganic minerals, proteins, lipids,
carbohydrates and cellular components [5]. Proteins are the major constituents of stone

50 G. Kanchana, P. Sundaramoorthi, R. Santhi, S. Kalainathan and G.P. Jeyanthi Vol.7, No.1
matrices and are the principal macromolecules in urine [6]. Urinary proteins with the
potential to adjust crystallization of calcium oxalate and calcium phosphate are Tamm-
Horsfall protein, nephrocalcin, osteopontin, calprotectin, human serum albumin and
urinary prothrombin fragment [5]. Most of these proteins are produced by the kidney,
chiefly by the renal tubular epithelial cells [5]. Other protein such as calprotectin, is
produced by granulocytes and are commonly released at the sites of inflammation, has
also been of concern in stone formation [5]. The bio-mineral contains hard minerals like
Ca, Ba, Sr, Mg and phosphates or its mixtures. The most common and important human
body element of all the stones is calcium. Naturally calcium is found with concentrations
of 8.9-10.1 mg/ml in the plasma [7]. Hypercalciuria is a biological syndrome defined as
the excretion of calcium in the urine of more than 0.1 mmol/kg/24 hours of major
minerals dietary manipulation. Hypercalciuria is the most common metabolic
abnormality in patients with nephrolithiasis [8]. In general hypermineralueria raises urine
super saturation with respect to the solid phase of mineral complex with phosphate,
enhancing the probability of self-nucleation and growth into clinically significant stones.
Urinary mineral excretion is continuously influenced by dietary intakes of
calcium, sodium, protein, carbohydrates, alcohol, ammonium, trace element and
potassium [9]. A mineral has been shown to bind to oxalate to form mineral oxalate
monohydrate. Thus, mineral has been shown to affect the concentration of oxalate. In
addition, oxalate is a major component of urinary stones and its urinary concentration
plays an important role in stone formation. Even a small increase in urinary oxalate has a
significant impact on mineral oxalate saturation. Although primary hyperoxaluria is
relatively uncommon, patients with mineral oxalate stones have some degree of
hyperoxaluria [10]. So, reducing the oxalate concentration would be helpful to most stone
patients. More amounts of oxalate can be obtained from foods such as nuts, chocolate,
and dark green leafy vegetables [11]. Citrate concentrations of plasma range from 0.05-
0.03 mmol/l and it exits as an alkaline citrate [12]. Citrate inhibits crystallization of
mineral oxalate and mineral phosphate by several mechanisms. (a) It decreases urinary
saturation of mineral salts by complexion minerals and reducing ionic minerals
concentration [13]. (b) Citrate directly inhibits spontaneous precipitation of mineral
oxalate [14], agglomeration of mineral oxalate [15], crystal growth of mineral phosphate
[13] and heterogeneous nucleation of mineral oxalate by monosodium ureate [16]. (c)
Citrate converts glycoprotein to an active disaggregated state probably by enhancing their
inhibitor activity against the crystallization of calcium salts [17-18]. Due to the
prohibitive role of citrate mentioned above, patients with hypocitraturia would be at a
higher risk of developing renal stones. This fact indicates that hypocitraturia is an
important factor for stone formation.
The kinetic process of COMH (calcium oxalate monohydrate) nucleation and
crystal growth requires super saturation [19], which can be obtained by excretion of the
reactants in the urine (calcium, oxalate and water). Few molecules are combined together
to form clusters. In the early step, clusters do not show a high degree of internal ordering
[20] the longer time they exist, however, their degree of ordering increases by replacing
internal salvation bonds by solid ion-ion bonds [21]. Gradually, clusters become crystal
embryos [21]. Above a critical size, embryos will grow into stable nuclei, and below

Vol.7, No.1 Nucleation Reduction Strategy of Brushite Crystals 51
some critical size, crystal embryos are too small and will reduce over all free energy by
dissolving [21]. The size of the nuclei is usually 100Å or less [21]. Once crystal nucleus
has reached its critical size and super saturation ratio remains above one, over all free
energy is decreased by adding new crystal components to the nucleus (self/spontaneous
growth). This process is technically called as crystal growth [37].
Proteins (macromolecules) influence nucleation, crystal growth and aggregation
of COM within the urinary system. It is reported that macromolecules can modify COM
crystal habit [5, 38]. Crystal growth is slow in some directions since macromolecules
adsorb on specific directions and prevent them from lattice ions [5.23and 24]. Face (-101)
of COM crystal is more active, presents more closely packed Ca
2+
atoms and has
significantly more adsorptive characteristics for many macromolecules [5,39]. Specific
adsorption of macromolecules can also bind non-specifically covering the crystal thin
with plate–like square edges. Macromolecules can also bind non-specifically covering the
crystal surfaces and retarding crystal growth. Khan [40] reported that adsorbed
macromolecules could promote heterogeneous nucleation by attracting calcium ions to
their calcium-binding domains with all the standard faces [40].
2. MATERIALS AND METHODS
The silica gel also known as water glass was used in the present work as an
intermediate growth medium. SMS (ARG-sodium meta silicate powder) was added to the
double distilled water, in the ratio of 1:1, mixed and stirred well and kept undisturbed
for few days to allow sedimentation. Then the clear top solution was filtered and stored in
a light protected glass container. This is known as a stock solution [22]. The gel densities
of 1.03-1.06 gm/cc
3
were used. Simple test tubes of 25mm diameter and 150mm length
were used. The concentrations of orthophosphoric acid used in these experiments were
0.5N, 1N and 2N.The supernatant solution of calcium chloride concentration varied form
0.5M to 2M [23, 24]. One of the reactant orthophosphoric acids were mixed in the gel
solution. The gel solution was taken as one third of its volume of the test tubes and after
the gel set, the supernatant solution of calcium chloride was added slowly along the sides
of the test tubes. The mixtures diffuse through the gel medium, which contains
orthophosphoric acid. The chemical reaction takes places, which leads to the growth of
CHP crystal.
The chemical reaction is
Ca
2+
+ HPO
42-
+ 2H
2
O => CaHPO
4
. 2H
2
O + Waste

52 G. Kanchana, P. Sundaramoorthi, R. Santhi, S. Kalainathan and G.P. Jeyanthi Vol.7, No.1
Fig-1 Fig-2 Fig-3 Fig-4
Fig-1 Growth of crystal in room temperature
Fig-2 Growth of crystal in sunlight medium
Fig-3 Growth of crystal in laser light medium
Fig-4 Grown Brushite crystal (Harvested)
Table 1. Growth parameters of CHP crystals (SDP)
SMS
gel
dens
ity
gm
/cc
3
Ortho
phospho
ric acid
concentr
ation
Gel+
H
3
PO
4
pH
value
Gel setting
time
Supernatant
Concentration
CaCl
2
(M)
Nucleatio
n
observed
in hours
Gro
wth
perio
d
days
Types of crystal
observed/
Harvested crystal
size.
1.05
0.5N
6.5
6.9
7.2
24 hrs
10 min
34 hrs
2.
-do-
-do-
16
20
100
70
Many poly
crystals,
Dendrite
crystals,
Liesegang rings
are observed
Single crystals
1N
6.5
7.0
7.5
14 hrs
1 hour
28 hrs
-do-
-do-
-do-
10
06
64
80
1.04
0.5 N
6.4
6.9
7.3
34 hrs
1hr
48 hrs
-
do
-
-do-
-do-
10
12
38
60
1N
6.5
6.8
7.3
16 hrs
1 hr
24 hrs
-do-
-do-
-do-
24
10
64
65

Vol.7, No.1 Nucleation Reduction Strategy of Brushite Crystals 53
3. RESULT AND DISCUSSION
3.1. FTIR spectral analysis of CHP crystals
CHP-FTIR spectrum was recorded by using SHIMADZU FTIR-435 instrument.
The FTIR spectrometer have KBr pellets sample holder and KBr detector. The KBr pellet
samples were used and the absorption frequencies start in the range from 600cm
-1
to
4000cm
-1
. The spectrum was interpreted with the earlier reported value [25-27]. The
absorption bonds, absorption frequencies and percentage of transmittance were compared
with the reported values. The values are tabulated in Table 2.
Table 2. Comparative table of FTIR-CHP crystal.
S.No. Bonds/Vibrations Reported
values cm
-1
Observed
values cm
-1
% of
transmittance
01.
02.
03.
04.
05.
06.
07.
Calcium with Hydrogen
bond
H-O-H Symmetric
stretching bond
O-H out of plane bond
P-O-P asymmetric
stretching bond
PO
4
bond
(H-O-) P=O bond (strong
absorption) acid
phosphates
Week absorption
HPO4
2-
3477
1620
662, 781
987
874
794
1000 to 1100
665
577
525
2375
1722
3492.8
1649.0
663.5
792.7
985.6
873.7
792.7
1134
1060.8
663.5
576.7
526.5
2345.3
1649
9
13
17
14
7
13
14
5
6
17
9
7
22
13
3.2. Atomic Absorption Spectroscopy of CHP Crystals (AAS)
AAS spectrum was recorded by Magnesium flame technique. The spectrum was
recorded as concentration versus absorption. Fig.5 shows the AAS spectrum. The
characterization concentration used here was 7.245 mg/l. The characterization
concentration and residuals are tabulated in Table 3.

54 G. Kanchana, P. Sundaramoorthi, R. Santhi, S. Kalainathan and G.P. Jeyanthi Vol.7, No.1
Fig. 5. AAS of Brushite crystal.
Here three standard concentrations were used and the corresponding observations
were tabulated. Two samples were prepared; one gave the results in out of calibration
range, and another one gave reliable data. Following procedure was used to calculate the
percentage of composition present in the CHP crystal.
Table 3. Concentration and residuals of CHP crystals.
S.No Calculated
concentration mg / l
Residuals
1.
2.
3.
4.
0.703
97.414
207.150
489.464
-0.703
2.588
-7.150
10.536
3.3. Thermo Gravimetric (TGA and DTA) Analysis of CHP Crystals
The TGA and DTA of CHP crystals were carried out by STA 11500-PLTS
instruments. The CHP crystal of 2.439 mg sample was taken to the TGA process. The
TGA was started from room temperature to 900
0
C by heating at a constant rate. The
percentage of weight of sample remaining present in the CHP sample at a particular
temperature was tabulated in Table 4.
3.4. Etching Study of CHP Crystals
A well-grown CHP crystal was immersed in HCl solution at a desired
concentration. The dissolution of CHP crystal depends upon on the etchant
concentration, temperature, crystal morphology, etching time etc. The etch pits were
photographed. Fig.6 shows the etch pits of CHP crystal [28-31]. The etch pit patterns
were observed as spirals, dendrites, allies and straights.

Vol.7, No.1 Nucleation Reduction Strategy of Brushite Crystals 55
Table 4. Thermal decomposition of CHP crystals.
Points TGA DTA in
0
C
Temperature
% of CHP crystal present
1
2
3
4
5
6
7
8
35
95.96
126.32
181.86
205.23
425.87
491.69
-
100
99.71
95.63
86.86
81.02
77.68
74.45
-
93.01
122.41
146.42
185.33
200.53
257.11
453.03
3.5. Scanning Electron Microscopic Study of CHP Crystals
A well-grown CHP single crystal was selected for the investigation of surface
morphology of the grown crystal by using SEM. The SEM photograph was obtained in
the version S-300-I instrument. The sample named as VCA-600 kept in lobe middle; the
data size was 640x480 µm. The magnification of SEM was about 250 times. SEM
acceleration voltage was 25000volts and the sample was kept in highly vaccum state.
18200-micrometer work distance was maintained and monochromatic color modes were
employed. Fig.7 shows the SEM pattern and surface morphologies of CHP crystal [32-
35].
Fig-6 Etch pit pattern of CHP crystal Fig-7 SEM pattern of CHP crystal
3.6. X-Ray Diffraction
The XRD results revealed that the grown crystal was single phase of (CHP)
brushite crystal. The XRD pattern and diffraction indices of the crystal were recorded.
The unit cell parameters are calculated and the cell parameters of CHP crystals are a
=7.0672 Å, b=18.4739 Å, c=23.7069 Å and α=β=γ=90
o
.The crystal system is found to
be orthorhombic.

56 G. Kanchana, P. Sundaramoorthi, R. Santhi, S. Kalainathan and G.P. Jeyanthi Vol.7, No.1
4. CONCLUSION
The CHP crystals were grown at room temperature and exposed to sunlight and
laser medium. It was found that, CHP crystal nucleation rate was reduced more in the
laser exposed medium than the sunlight-exposed medium, which is due to variation of
super saturation. FTIR-spectrum recorded the functional group frequencies of CHP
crystal constituents. These results were recorded and compared with the reported values.
Chemical etching was done at room temperature, which revealed the grown crystal
defects. SEM analysis was also done and it reveals the surface morphology of CHP
crystal. The decomposition temperature and percentage of weight loss of the grown
crystal was recorded by TGA and DTA analysis. XRD data gives the CHP grown crystal
cell parameters and its structure.
REFERENCES
[1] S.R.Khan,P.O.Whalen, and P.A.Glenton,J.Cryst.Growth 134,211(1993).
[2] W.H.Boyce,Am.J.Med. 45 (5), 673 (1968).
[3] H.G.Tiselius,Clin.Chim.Acta.122, 409 (1982).
[4] R.W.Marshall and W.G.Robertson,Clin.Chim.Acta.72, 253 (1976).
[5] S.R.Khan,Urol.Int 59, 59 (1997).
[6] R.L.Ryall,A.M.F.Stapleton,in calcium oxalate in Biological system,Eds.S.R.Khan
(CRC press) (1995).
[7] C.G.Duarte and F.G.Knox, in text book of renal pathophysiology, (Harper and Row
,(1978).
[8] F.L.Coe,J.H.Parks, and J.R.Asplin,N.Engl.J.Med.327,1141(1992)
[9] Maurice Audran and Erick Legrand,Joint Bone spine 67 (6),509 (2000).
[10] Y.Ogawa,T.miyazato, and T.Hataano,World j.Surgery 24 (10),1154 (2000).
[11] D.S.Goldfarb and F.L.Coe, Am.Fam.Physicican.60 (8) 2269 (2000).
[12] D.P.Simpson,Am.J.Physiol.224(3), F223 (1983).
[13] J.L.Mayer andL.Hsmith,Invest.Urol 13, 36 (1975).
[14] M.J.Nicar,K.Hill and C.Y.C.pack,J.Bone ,Miner.Res.2 (3), 215 (1987)
[15] D.J.Kok,S.E.Papapoulus,and O.L.M.Bijvoet,Lancet41,1056 (1986).
[16] C.Y.C. Pakand Paterson, Arch.Intern.Med.146, 863 (1986).
[17] B.Hess,Miner.Electr.Metab 20,393 (1996)
[18] C.Y.C.Pak,Hypocitraturia; a critical review and furture direction,from 8
th
European symposium on Urolithiasis-Parma,Italy,June 9-12 (1999).
[19] A.Mersmann,Crystallization technology,Hand book,2
nd
Edi.
Marcel Dekker,Inc.(2000).
[20] V.A.Garten and R.B.Head,Phil.Mag.14, 1243 (1966).
[21] D.J.Kok,The role of crystallization process in mineral oxlates urolithasis,
Ph.D thesis,Univerasity of Leiden (1991).
[22] Henisch, H.K. etal,J Electro Chemical. Soc.,112 (1965) 627.
[23] A.E.Alexander, and P.Honson, Colloid Science ,Clarendon Press, Oxford,(1949).
[24] W.Eitel, In:Physical chemistry of Silicate University of Chicago Press,(1954).
[25] Yean. Chin etal ’Application of IR Spectroscopy to analysis of uninary calculi J.
Urol. Vol 86, (1961) 838-854.

Vol.7, No.1 Nucleation Reduction Strategy of Brushite Crystals 57
[26] C.M. Corns ”Infrared analysis of real Calculi – a comparison with conventional
Techniques”Ann. Clin. Bio-Cherm, Vol 20, (1983) 20-35.
[27] A.Hesse , D. Bach,. Stone analysis by IR spectroscopy, Clinical and laboratory
Aspects, Edi.Alan Rose, University Park Press, Baltimore, (1982), 87-105.
[28] J.J.Gilman, J.,Johnsion and G.W.Sears , J.Appl.Physics., 29,(1958) 749.
[29] J.J. Gilman., etal, , J.Appl.Physics, 27,(1956)1018.
[30] J.C.Fisher, in: Dissolutions and Mechanical Properties of Crystals, John Wiley and
sons, NewYork (1957).
[31] J.B. New Kirk., In:Director observation of Imperfection in crystals, Interscience
Publisers, New York (1962).
[32] K. Taukamot, J. Cryst. Growth, 61, (1983) 99.
[33] H.C.Gates, Thirty years of progress in Surface Science, in: Crystal growth and
characterization,(Edi),North Holland (1975).
[34] H.Bethage etal, Electron Microscopy in Solid State Physics, Elsever, Amsterdom
(1987)
[35] N. Albon etal, in: Growth and Perfection of Crystals, Wiley, New York, P.44 (1958).
[36] P.Sundaramoorthi, S.Kalainathan, Asian journal of chemistry,19-4, (2007).
[37] B.D.Hess and D.J.kok, Nucleation, Growth and aggregation of stone- Forming
crystals, In Kidney stones: Medical and surgical management eds.F.L.Coe et al
(Lippincott-Raen.publishers), (1996).
[38] L.Addadi,A.Berman,J.Moradian-Oldk,and S.Weiner,Connect Tiss.Res.21,127-135
(1989).
[39] S.Deganello, Calcif.Tiss.Int.48, 421 (1991).
[40] S.R.khan, Urol.Int.59, 59 (1997).