Department of Chemistry, Annamalai University, Annamalai Nagar, TN 608002, IndiaIndia
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Introduction : One of the most common and painful diseases observed in the tropical regions is kidney stone formation. This can be due to various factors such as the amount of water consumption, climatic conditions, lifestyle and diet. Although, kidney stones are composed of calcium phosphate were only found to be 10%, it has been observed that the deposition of such stones have been increasing steadily for the past 30 years in females. Aim : To study the effects of diethylenetriaminepentaacetic acid (DTPA) on crystal growth and morphology of calcium oxalate. Materials and Methods : Calcium oxalate was synthesized from calcium chloride solution using oxalic acid in the presence of DTPA at different concentrations. The experiments were carried out at 60 °C. The samples were characterized using powder X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM) techniques. Results : The study revealed that in the absence of DTPA, samples exhibited flower like morphology by agglomeration of rod like basic blocks and the tendency for agglomeration decreases with increase in DTPA concentration. Conclusion : Interpretation of XRD pattern confirmed that whewellite with a monoclinic structure is the most favoured structure in the absence of DTPA where as calcium oxalate hydrate having orthorhombic structure is most favoured structure in the presence of DTPA
Keywords: Calcium oxalate; Kidney stone; Chelation therapy; Crystal growth; Fourier transform infrared spectroscopy; Scanning electron microscope; X-ray diffraction; DTPA; Calcium oxalate; Kidney stone; Chelation therapy; Crystal growth; Fourier transform infrared spectroscopy; Scanning electron microscope; X-ray diffraction; DTPA
Deposition of stones in kidney is one of the most common and painful diseases particularly in
tropical regions. Various factors such as consumption of water, its quality, climate, lifestyle and
diet can affect the formation and type of kidney stone. Majority of the kidney stones are com-
posed of calcium, which may be in the form of calcium oxalate (70-80%) or calcium phosphate
1-4
composition, kidney stones may also contain organic matrix accounting for 2-5% of the total
stone weight.
crystal phases, several studies have reported that the precipitate in the urinary tract consists of
COM, having a greater stone forming tendency than COD and that all papillary stones are
8,9 COM stones.8,9 The stone forming process is highly complex in-
volving nucleation, crystal growth, and aggregation of crystals in
an environment containing supersaturated crystal-forming ions
2+ 2− 3− 2+ 2− (e.g. Ca , C2O4 , PO4 , Mg , SO4 ) in the presence of pro-
moters and inhibitors.10 The acid-rich urinary proteins suppress
the crystallization of calcium oxalate even under supersaturated
conditions,11 thus preventing the formation of stones. Chelation
therapy is one of the recent techniques for the treatment of many
diseases including kidney stones.12 Preferential crystallization of
different forms of calcium oxalate has been reported by many
researchers by using certain synthetic and natural molecules,
13,14 15 such as polypeptides, sodium diisooctylsulfosuccinate, poly
(ethyleneglycol)-block-poly (methacrylic acid),16 renal epithe-
17,18 19 lial cells, poly (sodium 4-styrene-sulfonate), biopolymeric
20 21 additives,20 and protein isolated from bean seed coats.21
Reports by Shinichi et al suggest that the conventional
homogeneous precipitation results into monohydrate and granu-
lar calcium oxalate. However, hydrolysis of oxamic acid cata-
lyzed by enzyme (hydrolase) generates trihydrate and fibrous
calcium oxalate.22 Recent studies carried out on synthesis of
CaCO3 revealed that the crystalline structure of calcium carbon-
ate particles mainly depend on the precipitation condition, such
as pH, temperature and presence of sequestrants, etc.
The above survey revealed that effect of the presence
of calcium ions in water on the formation of calcium oxalate
while using oxalic acid has not been reported so far. In this pa-
per we describe the effects of diethylenetriaminepentaacetic acid
(DTPA), a chelating agent, on the morphology and structure of
calcium oxalate precipitated from calcium chloride solution us-
ing oxalic acid. The results indicate that morphology and struc-
ture of the resulted calcium oxalate vary with the conditions.
The architecture of the samples shows that DTPA has significant
influence on the morphology and crystalline structure of calcium
oxalate.
Analytical grade CaCl2, DPTA and Na2C2O4 were obtained from
Sigma-Aldrich chemical company, Bengaluru, KA, India. The
reagents were used as such. Double distilled water was used
for the preparation of aqueous solutions. Analytical grade 1:1
ammonia and acetic acid were used to adjust the pH whenever
necessary.
Experiments were carried out in a similar fashion as explained
by Vijaya et al.31 Precipitation was carried out from a 0.1 M
CaCl2 solution using 0.1 M oxalic acid at 60 °C. In a typical
synthesis, 50 ml of CaCl2 solution was taken in a round bottom
flask and 50 ml DTPA was added. The pH was adjusted to 7. The
solution was heated to 60 °C and 50 ml oxalic acid was added
h and the precipitate obtained was filtered using Watman No. 40
filter paper. It was dried at 40 °C and then kept in a desiccator.
The pH measurements were made using Elico pH me-
ter, model LI-120. FT-IR spectra were taken in the range 500-
-1 4000 cm-1 using Avatar-330 FTIR spectroscopy after KBr pel-
letization. Microscopic morphological structure measurements
were performed with Jeol JSM 5610 LV scanning electron mi-
croscope (SEM). The samples were coated with Au prior to ex-
amination. The powder X-ray diffraction (XRD) patterns were
recorded on an INEL Equinox 1000 Advanced XRD diffractom-
eter with Cu Ka radiation at k=1.5406 Å.
Figures 1a, 1b, 1c, 1d and 1e represents the XRD pattern of the
samples. Figure 1a depicts the XRD pattern of the blank sam-
ples; i.e. prepared in the absence of DTPA. The data confirms the
presence of calcium oxalate in the form of whewellite (calcium
oxalate hydrate) vide JCPDS card 78-6695. The cell parameters
a, b and c are 9.9780, 7.295 and 6.292 respectively which con-
23-30 firmed that the samples exhibit monoclinic structure.
The XRD pattern of sample prepared in the presence
of 10, 20, 30 and 40 ml DTPA are presented in Figure 1b, 1c,
1d and 1e respectively. The powder XRD pattern of samples
showed the existence of calcium oxalate in the form of mono-
clinic. The samples with 10 ml and 20 ml DTPA had same cell
parameters; a, b and c as 9.794, 14.74 and 6.306 respectively and
were matching with joint committee on powder diffraction stan-
dards (JCPDS) card 20-0231. The sample synthesized in pres-
ence of 30 ml DTPA had cell parameters; a, b and c as 9.796,
7.294 and 6.291 respectively and were in good agreement with
JCPDS card 20-0231. The cell parameters of the sample synthe-
sized in presence of 40 ml DTPA were different from the above
and were 9.976, 7.294 and 6.291 for a, b and c respectively and
were in good agreement with JCPDS card 20-0231.
Although, the positions of the peaks (°2θ) in the XRD
pattern of all the samples correspond to whewellite and match
very well with each other, the ratio of their intensities of the
samples prepared in the presence of DTPA varied much from
the blank sample. In the absence of DTPA, the two peaks around
°2θ~15 and 24.5 showed similar intensities. With 10 ml and 20
ml DTPA, the peak at °2θ~24.5 was more intense than the peak
around °2θ~15. With higher concentrations of DTPA (30 and 40
ml), the peak at °2θ~1.5 was more intense than the peak around
°2θ~24.5. This indicates that the crystalline nature of the sam-
ples varied with the environment and the degree of crystallinity
was affected much by the presence of DTPA.
Aslin et al32 have reported the physicochemical analysis of uri-
Figure 1a. XRD Pattern of Calcium Oxalate Sample Synthesized in the Absence of DTPA.
acterized functional groups and phases of the stones using X-ray
diffraction (XRD), Fourier transform Raman spectroscopy and
Fourier transform infrared spectroscopy (FT-IR). Their study re-
vealed that the majority of the stones were found to be calcium
oxalate monohydrate (COM) and mixed stones had a minor ex-
istence of struvite and uric acid.
The FTIR spectrum of the blank sample as well as with
10, 20, 30 and 40 ml DTPA are shown in Figure 2a, 2b, 2c, 2d
and 2e respectively. The characteristic bands assigned for sam-
ple without DTPA (Figure 2a) are 511 cm-1 to O-C-O in-plane
-1 bending , 660 cm-1 to weak band bending and wagging modes,
- 1 -1 774 cm (O-C=O ), 954 cm sym C-O stretch, 1319 sym C=O
stretch, 1603 C-O stretch, 1784cm , 3023 cm symmetric and
asymmetric O-H stretching.
The sample prepared in the presence of 10 ml DTPA ex-
-1 hibited frequencies at 511 cm-1 corresponding to O-C-O in-plane
-1 bending , 660 cm-1 weak band bending and wagging modes, 774
- 1 -1 cm (O-C=O ), 888 cm C-C stretch ( rocking mode of water),
-1 954 cm sym C-O stretch, 1307 sym C=O stretch, 1603 C-O
Figure 2a. FTIR of Calcium Oxalate Sample Synthesized in the Ab- sence of DTPA.
The bands observed for 20 ml DTPA (Figure 2c) are
-1 -1 511 cm-1 O-C-O in plane bending, 660 cm-1 weak band bending
- 1 -1 and wagging modes, 784 cm- 1 (O-C=O ), 888 cm-1 C-C stretch
-1 (rocking mode of water), 954 cm-1 sym C-O stretch, 1307 sym
-1 -1 -1 -1 C=O stretch, 3056 cm , 3250 cm , 3331 cm , 3433 cm , 3490
-1 cm-1 symmetric and asymmetric O-H stretching.
-1 Figure 2d shows the FTIR of the sample synthesized in
presence of 30 ml DTPA. Characteristic bands were observed at
-1 -1 511 cm O-C-O in-plane bending , 660 cm weak band bending
-1 -1 - 1 -1 and wagging modes, 772 cm (O-C=O ), 886 cm C-C stretch
-1 (rocking mode of water), 954 cm-1 sym C-O stretch , 1307 sym
-1 -1 -1 C=O stretch, 1613 C-O stretch, 3045 cm , 3250 cm , 3329 cm ,
-1 3488 cm-1 symmetric and asymmetric O-H stretching.
The calcium oxalate synthesized in presence of 50 ml
-1 DTPA exhibited FTIR bands at 511 cm O-C-O in plane-bend-
-1 ing , 660 cm weak band bending and wagging modes, 784 cm
Figure 2b. FTIR of Calcium Oxalate Sample Synthesized in the Pres- ence of 10 ml DTPA.
-1 3011 cm-1 symmetric and asymmetric O-H stretching.
It is obvious from the above data that in the absence of
DTPA, frequencies/bands corresponding to C-C stretch (rocking
mode of water) and sym C-O stretch at around 886 cm-1 and 954
-1 -1 cm-1 are absent. Similarly, the band around 1478 cm-1 is absent
in all samples prepared in the presence of DTPA except with 40
ml. This band can be attributed to in-plane bending of CO (ν).
-1 -1 Again the bands around 511 cm-1 and 660 cm-1 are distinct and
clear in the case of samples with 10, 20 and 30 ml DTPA. The
intensities of these peaks decreased in blank as well as in the 40
ml DTPA sample. Analysis of urinary stone constituents using
powder X-ray diffraction and FT-IR has been reported by Prag-
nya Bhatt and Parimal Paul.33 They have reported the presence
of the apatite phase with an uncertainty on whether it has formed
independently or the calcium oxalate monohydrate phase has
been transformed into these phases with time. Similarly, in our
study, in the case of 40 ml, there could be a possibility of the
formation of an apatite phase with the transformation of calcium
oxalate monohydrate phase with time.
Figure 3a and 3b show the SEM images of sample prepared in
Figure 3(a,b): SEM Omages of Calcium Oxalate Sample Synthesized in the Absence of DTPA.
Figure 2e. FTIR of Calcium Oxalate Sample Synthesized in the Presence of 40 ml DTPA.
the absence of DTPA. Flower like morphology formed by the
interlinking of petals ranging from about 5-10 μm length and
width of about 1 μm were observed under these conditions. The
images also showed indications of smaller units exhibiting ag-
glomeration at the centre of this structure or at the junction of
cross linking (Figure 3b). The morphology of sample prepared
-1 with 10 ml DTPA are depicted in Figure 3c and 3d. They showed
similarity with that of samples without DTPA. However, the ten-
dency of agglomeration or degree of cross linking was compara-
tively less. Unlike the blank sample (without DTPA), it could be
noticed from the SEM image (Figure 3b) that all the crystallites
did not show flower like morphology. Some of the crystallites
had only 4/6 petal like blocks attached to form the flower. In
the sample without DTPA, the numbers of blocks to form ag-
glomeration were too many. Some loose blocks were also seen
in this image.
The sample prepared with 20 ml DTPA exhibited
marked difference in the morphology. The flower like morphol-
ogy was quite fewer in number. Majority of the crystallites ex-
hibited block like structures (Figure 3e).
It could be observed from the magnified image (Figure
3f) that these blocks resulted from rod like structures. On in-
creasing the DTPA concentration to 30 ml, the flower like mor-
phology reappeared again (Figure 3g) along with the rod like
Figure 3(c,d): SEM Images of Calcium Oxalate Sample Synthesized in the
Presence of 10 ml DTPA.
Figure 3(g,h): SEM Images of Calcium Oxalate Sample Synthesized in
the Presence of 30 ml DTPA.
crystallites. However, with 30 ml DTPA, the flower like mor-
phology was equal in numbers with the rod like crystallites. The
rod like morphology showed tendency for sharp ends leading to
an ellipsoidal shape. The trend continued with increase in DTPA
concentration. With 40 ml DTPA, the dual morphology contin-
ued to exist. The number of ellipsoidal particles increased and
the flower like structures reduced in numbers.
On the basis of the above experimental results, the mechanism
of formation of this flower structure of calcium oxalate can be
explained as follows: The crystallization of calcium oxalate in
the absence of DTPA forms these basic blocks with rod like
structures. From the SEM images (Figure 3a, 3b) it could be
observed that there are no free building blocks in the images.
This shows that the agglomeration is uninhibited in the absence
of DTPA. Usually, the crystal growth is governed by both kinetic
and thermodynamic factors. The observed flower morphology
reflects the relative rates of growth of the crystal to different
directions in the absence of DTPA. It is expected that in the
absence of DTPA, more sites are available for further agglom-
eration or joining (Figure 3b). However, the presence of DTPA,
which forms a complex with calcium, inhibits this tendency and
the sites for growth could not go beyond a certain number. This
is clearly observable in Figure 3c, 3d and further images, where
the number of petals are few when compared to that of the blank.
The above mechanism of inhibition of agglomeration or joining
is further inhibited with the rise in concentration of DTPA and is
evident from the SEM images of calcium oxalate synthesized in
the presence of 30 and 40 ml DTPA.
Figure 3(e,f): SEM Images of Calcium Oxalate Sample Synthesized in the
Presence of 20 ml DTPA.
Figure 3(i,j): SEM Images of Calcium Oxalate Sample Synthesized in the
Presence of 40 ml DTPA.
The above observations indicate that DTPA strongly influences
the crystallization of calcium oxalate. The study revealed that
the crystallization process of calcium oxalate is entirely differ-
ent in presence of DTPA. Whewellite with monoclinic structure
is the most favoured polymorph in the absence of DTPA, where
as calcium oxalate hydrate with an orthorhombic structure is the
most favoured structure in the presence of DTPA. With increase
in DTPA concentrations, there is significant difference in the
morphology and the dual morphology was predominant. Simi-
larly, the unit structures showed an increased tendency of cross
linking in the absence of DTPA. These results could be helpful
to understand the effects of DTPA on crystal growth of calcium
oxalate based kidney stone and form a basis for the further stud-
ies on chelation therapy using DTPA and other chelating agents.
The authors declare that they have no conflicts of interest.