Journal of Minerals & Materials Characterization & Engineering, Vol. 7, No.3, pp 265-275, 2008 Printed in the USA. All rights reserved
Laser Irradiated Nucleation Reduction Strategy of AMHP (Ammonium
Magnesium Hydrogen Phosphate, In-Vivo Approach-1) Crystals in Gel
Medium and its Characterization Studies
G. Kanchana
, P. Sundaramoorthi
, G.P. Jeyanthi
Department of Bio-chemistry, Avinashilingam Deemed University, Coimbatore,
TamilNadu, India.
*Department of Physics, A.A.Govt. Arts College, Namakkal – India-637001.
Kidney stone consists of various organic, inorganic and semi organic compounds.
Mineral oxalate monohydrate and di-hydrate is the main inorganic constituent of kidney
stones. However, mechanisms leading to the formation of mineral oxalate kidney stones are
not clearly understood. In this field of study, there are several hypotheses including
nucleation, crystal growth and or aggregation of formation of AOMH (Ammonium oxalate
monohydrate) and AODH (Ammonium oxalate di-hydrate) crystals. The effect of some
urinary species such as ammonium oxalates, calcium, citrate, proteins and trace elements
were reported by the author. The kidney stone constituents are grown in the kidney
environments, the silica gel medium (SMS) which provides the necessary growth simulation
(in-vivo). In the artificial urinary stone growth process, identification of growth parameters
with in the different chemical environment was carried out and reported for the urinary
crystals such as CHP, SHP, BHP and MHP. In the present study, AMHP (Ammonium
magnesium hydrogen phosphate) crystals are grown in three different growth faces to attain
the total nucleation reduction. Extension of this research is that many characterization
studies have been carried out and the results are reported.
Key words: Kidney stone, ammonium oxalate, monohydrate, di-hydrate, silica gel, urinary
stone, ammonium magnesium hydrogen phosphate, nucleation reduction
266 G. Kanchana, P. Sundaramoorthi, G.P. Jeyanthi Vol.7, No.3
Most kidney stones consist of a complex 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 urinary stone matrix accounts for approximately 3% of the weight of a
calculus [3]. From the analysis, the matrix compound are soluble [4] and has been described
as a homogeneous or heterogeneous material composed of organic, inorganic and semi
organic compounds like protein, lipids, carbohydrates and cellular minerals etc. [5]. Proteins
are the major constituents of stone matrix and the principle macromolecule in the urine [6].
Urinary proteins with the potential to adjust crystallization of mineral oxalates and calcium
phosphate are Tamm-Horsfall protein, nepheromineralin, osteopontin, calprotectin, human
serum albumin and urinary prothrombin fragment [5]. The renal tubular epithelial cells of the
kidney chiefly produce most of the proteins. Other protein such as caprotectin, which is
produced by granulocytes are commonly released at the sites of inflammation has also been
of concern in stone formation. The bio-minerals contain hard minerals like Ca, Ba, Sr, Mg
and phosphates or its mixtures. The common and important constituents of all the stones are
calcium. Normally calcium is found at concentrations of 8.9-10.1 mg/ml in the plasma [7].
Hypermineraluria is a biological syndrome defined as the execration of more than
0.1mmol/kg/24 hours of major minerals in the urine. Hypermineraluria is the most common
metabolic abnormality in patients with nephrolithiasis [8]. Hypermineraluria raises urine
supersaturation with respect to the solid phase of mineral complex with phosphate thus
enhancing the probability of self-nucleation and growth in to clinically significant stones.
Urinary minerals excretion is continuously influenced by the 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]. More amounts of oxalates are
obtained from foods such as nuts, chocolate and dark green leafy vegetables [11]. The
concentration of citrate in plasma ranges from 0.05-0.03 mmoles/liter 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 forming
complex with 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 urate [16]. (c) Citrate converts glycoproteins to an active
disaggregated state probably enhancing their inhibitor activity against the crystallization of
calcium salts [17-18]. Due to the inhibitory role of citrate mentioned above, patients with
hypocitraturia would be at a higher risk of renal stones. This fact indicates that hypocitraturia
is an important factor for stone formation.
Vol.7, No.3 Laser Irradiated Nucleation Reduction Strategy of AMHP 267
The kinetic process of AOMH (Ammonium oxalate monohydrate) nucleation and
crystal growth requires super saturation [19], which can be obtained by excretion of the
reactants in the urine (Ammonium, calcium, trace element, 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 solid ion-ion bonds. Gradually,
clusters become crystal embryos. Above a critical size, embryos will grow into stable nuclei
and below the critical size, crystal embryos are too small and will reduce over all free energy
by dissolving. The size of the nuclei is usually 100A
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.
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 and mixed 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 which is known as a stock solution [22]. The gel densities of 1.03-1.06 grm/cc
were used. Simple test tubes of 25 mm diameter and 150 mm length was used. The
concentrations of orthophosphoric acid used in this experiment are 0.5N, 1N and 2N. The
concentration of supernatant solution (ammonium chloride and Mg (NO
O) varies form
0.5:0.5M to 2:2M [23-24]. One of the reactants, orthophosphoric acid was mixed with 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 was added slowly along the sides of the test tubes. The
mixture diffuses through the gel medium which contains orthophorphoric acid.
The chemical reaction takes places which leads to the growth of NH
The chemical reaction is
+ Mg
+ 2H
O => NH
MgPO4. 2H
O. + by products
268 G. Kanchana, P. Sundaramoorthi, G.P. Jeyanthi Vol.7, No.3
Table 1. Growth parameters of AMHP crystals (SDP).
gm /cc
Gel setting
Mg (NO
observed in
Types of crystal
observed/ Harvested
crystal size.
24 hours
34 hours
Many poly
Liesegang rings
are observed
Single crystals
14 hours
1 hour
28 hours
0.5 N
34 hours
48 hours
16 hours
1 hour
24 hours
Vol.7, No.3 Laser Irradiated Nucleation Reduction Strategy of AMHP 269
Fig.1 (a) Laser medium Fig (b) Laboratory medium Fig.1 (c) Sunlight medium
Fig.1. Growth of AMHP crystals in different environments (SDP)
Fig.2. Harvested AMHP crystal in SDP.
270 G. Kanchana, P. Sundaramoorthi, G.P. Jeyanthi Vol.7, No.3
AMHP crystals are grown in three different growth faces by applying various growth
parameters. Table-1 gives the growth parameters of AMHP crystals and the bold letters
shows the optimum growth parameters. Among them, the laser exposed growth medium
shows better nucleation reduction and no crystals were formed because of the inability to
attain super saturation. In sunlight exposed medium, partial nucleation was observed since
exposure of sunlight to the growth medium was only in day time that is 8 hours per day and
the growth period was 3 months.
3.1. FTIR Spectral Analysis of AMHP Crystal
AMHP-FTIR spectrum was recorded 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 range from 400 cm
to 4000 cm
. Fig-3 shows the
FTIR spectrum of AMHP crystal. The spectrum was interpreted with the earlier reported
values [25-27]. The absorption bonds, absorption frequencies and percentage of transmittance
are compared with the reported values and match the major constituents present in the AMHP
crystals. In this computation acid phosphate group frequency was 566.27 cm
, PO
frequency was 1117.2 cm
then NH in plane bending frequency was 1237 cm
and also
water molecule asymmetric, symmetric stretching was 3272 cm
1659 cm
Fig.3. FTIR spectrum of AMHP crystals
Vol.7, No.3 Laser Irradiated Nucleation Reduction Strategy of AMHP 271
3.3. Thermo Gravimetric (TGA and DTA) Analysis of AMHP Crystal
The TGA and DTA of AMHP crystal was carried out by STA 11500-PLTS
instrument. AMHP crystal of 2.439 mg sample was taken for the TGA process. The TGA
was started from room temperature to 1000
C by heating at a constant rate. Fig-4 shows the
TGA&DTA graph of AMHP crystal. The percentage of weight loss of the AMHP sample at
C was 0.5% alone due to moistures, further at 133
C weight loss was 25% due to water
molecule. Then above 364
C to still end (900
C) of the analysis the weight loss are 40.5%
due to hydroxyl and ammonium compounds.
Fig 4 Thermo gravimetric (TGA and DTA) analysis of AMHP cystals.
3.4. Etching Study of AMHP Crystal
A well-grown AMHP crystal was immersed in HCl solution at a desired
concentration. The dissolution of AMHP crystal depends upon on the etchant concentration,
temperature, crystal morphology, etching time etc. The etch pits are photographed. Fig.5
shows the etch pits of AMHP crystal [28-31]. The etch pit patterns observed are spirals,
dendrites, allies and straights.
3.5. Scanning Electron Microscopic Study of AMHP Crystal
A well-grown AMHP 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 was kept in lobe middle; the data
size was 640 µm x 480µm.The minor and major magnification of SEM was about 250 times.
SEM acceleration voltage was 25000 volts and the sample was kept in highly vacuum state.
272 G. Kanchana, P. Sundaramoorthi, G.P. Jeyanthi Vol.7, No.3
18200-µm work distance was maintained and monochromatic color modes were employed.
Fig.6. shows the SEM pattern of AMHP crystal [32-35].
Fig 5 Shows the etch pit pattern of AMHP crystals
Fig.6 SEM photo of AMHP crystal.
Vol.7, No.3 Laser Irradiated Nucleation Reduction Strategy of AMHP 273
3.6. X-ray Powder Diffraction
The XRPD results revealed that the grown crystal was in single phase of AMHP
crystal. The XRPD pattern and diffraction indices of the crystal are shown in Fig.7. Terror
programmer according to the values of 2θ in XRPD calculates the cell parameters. The unit
cell parameters of AMHP crystals are a =7.0672 Å, b=18.4739 Å, c=23.7069 Å and
the crystal system is Orthorhombic. The volume of the AMHP unit cell is
3092.87 (Å)
Fig 7 Powder XRD pattern of AMHP crystal
AMHP (Ammonium magnesium hydrogen phosphate) crystals were grown at room
temperature and exposure to sunlight and laser medium. It was found that AMHP crystal
nucleation rate have been reduced more in the laser exposed medium than the sunlight-
exposed medium which is due to variation of super saturations. FTIR-spectrum recorded the
functional group frequencies of AMHP crystal constituents. These results are compared with
the reported values. Chemical etchings are carried at room temperature which revealed the
grown crystal defects. SEM analyses are also done and it reveals the surface morphology of
AMHP crystal. The decomposition temperature and percentage of weight loss of the grown
crystal are recorded by TGA and DTA analysis. XRPD data gives the AMHP grown crystal
cell parameters and its structure.
274 G. Kanchana, P. Sundaramoorthi, G.P. Jeyanthi Vol.7, No.3
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