Vol. 52, No. 6
Printed in U.S.A.
T H E AMERICAN JOURNAL OF CLINICAL PATHOLOGY
Copyright © 1969 by The Williams & Wilkins Co.
A COMPARISON OF X-RAY DIFFRACTION AND INFRARED TECHNICS FOR
IDENTIFYING KIDNEY STONES
SIDNEY S. POLLACK, P H . D . , AND GERALD L. CARLSON, P H . D .
Mellon Institute, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15218
ABSTRACT
Pollack, Sidney S., and Carlson, Gerald L.: A comparison of x-ray diffraction
and infrared technics for identifying kidney stones. Am. J. Clin. Path., 52: 656660, 1969. Mixtures of the common constituents of kidney stones were analyzed
by x-ray diffraction and infrared absorption. The roentgenograph^ technic was
more sensitive for detecting the minor constituent in eight mixtures and infrared
was more sensitive in two. When the minor constituent makes up less than 10%
of the sample, probably neither technic will identify it.
Infrared absorption and x-ray diffraction
are two instrumental technics which have
been especially useful in the study of kidney
stones. These enable the identification of the
compounds present and thus provide more
information than an elemental analysis. In
addition, these technics permit analysis of
much smaller samples than those required
for conventional analysis. Kidney and
urinary stones range from milligrams to a
kilogram,3 are usually mixtures of crystalline
compounds, and most commonly contain
calcium oxalate hydrates and hydroxyapatite. Although no way of preventing the
initial kidney stone is known, identification
of stones may help to prevent their recurrence.
Recent investigators4"7 have used x-ray
analysis in the routine identification of renal
stones. Beischer2 and Weissman and colleagues9 analyzed stones using infrared
analysis, whereas Beeler and associates1
compared the results from wet chemical
methods with x-ray and infrared procedures.
When crystals are large enough, probably the
most sensitive method of identification is
the microscopic technic. Study of the differences between various portions of a stone
or comparison of the nucleus with the rest of
the stone usually requires a combination of
Received March 10, 1969; accepted for publication July 24, 1969.
This study was supported in part by National
Institutes of Health General Research Grant FR558001-5.
all these technics or the microscopic and one
of the others. Microscopic identification can
be performed on crystals about 1 n or larger,
whereas x-ray diffraction or infrared absorption permits identification of crystals millimeters or angstroms in size. X-ray diffraction
is most useful in the study of crystalline
materials because it may be used to distinguish between various hydrates or polymorphs, whereas infrared absorption may be
used to identify noncrystalline compounds as
well as crystalline ones.
Inasmuch as both x-ray diffraction and
infrared absorption are useful in the study
of urinary stones, the object of this study
was to compare the sensitivity of the two
technics in routine analysis. Each stone was
ground and treated as one sample. No
attempt was made to isolate a nucleus of the
stone. The analytic procedures, which are
described later, were chosen because of their
suitability for routine analysis and are not
necessarily the most sensitive ones which
could be used. Approximately 35 urinary
stones which had been previously analyzed
by the x-ray diffraction technic were selected
and analyzed by infrared analysis. The
mixtures were chosen to represent the
varieties of stones that had been analyzed
previously by the x-ray technic; emphasis
was, however, placed on the more common
constituents of stones.
The x-ray and infrared analyses of the 35
urinary stones were in agreement as to the
identification of the major constituents of
656
Dec. 1969
IDENTIFYING KIDNEY
the stones but, in some instances, one of the
technics did not detect a minor component
revealed by the other. In addition to unidentifiable compounds found only by one
technic, infrared did not detect small
amounts of MgNH 4 P0 4 -6H 2 0 mixed with
hydroxyapatite and x-ray did not detect
small amounts of hydroxyapatite mixed with
CaC 2 0 4 -H 2 0. In order to test the minimal
detection limits of the most common compounds found in urinary stones, a series of
synthetic mixtures was prepared and analyzed by both technics. Commercially
available reagent grade chemicals were used
in the mixtures. Only the monohydrate of
calcium oxalate was used since the dihydrate
is not easily obtained.
The most common crystalline compounds
which have been identified in urinary
stones are calcium oxalate monohydrate
(CaC 2 04-H 2 0), calcium oxalate dihydrate
(CaC 2 04-2H 2 0), calcium hydroxyapatite*
[Ca 6 (P0 4 )30H], magnesium ammonium phosphate hexahydrate
(MgNH 4 P04-6H 2 0),
uric acid (C5N4H4O3), uric acid dihydrate
(C6N4H4O3 • 2H 2 0), calcium hydrogen phosphate
dihydrate (CaHP0 4 • 2H 2 0), lcystine [SCH 2 CH(NH 2 )-COOH], and ammonium acid urate (NH4C5H3O3N4). Other
compounds occasionally found in stones are
listed by Parsons 6 and Herring.4
Prien and Frondel,7 Parsons, 6 and Morriss
and Beeler6 reported that stones containing
pure CaC 2 0 4 • H 2 0 were the most common
single component stones and that mixtures
of the calcium oxalate hydrates, either pure
or mixed with calcium hydroxyapatite, were
found in over 60 % of the stones analyzed.
EXPERIMENTAL
ROENTGENOGRAPHIC
IDENTIFICATION
Roentgenographic identification was performed by means of the Debye-Scherrer
technic which can analyze samples weighing
only several milligrams and is one of the
most common technics used in x-ray diffraction laboratories. The powdered sample was
poured into a thin-walled glass capillary, 0.5
* No attempt has been made to determine
whether or not the hydroxyapatite contains carbonate.
STONES
657
mm. in diameter, mounted in a Philips 11.46cm. diameter camera, and exposed to nickelfiltered copper x-radiation for 2 hr. When
large samples are available, the common
diffractometer technic can also be used,
provided that the samples are ground to a
fine powder and preferred orientation does
not occur. When large numbers of samples
are to be analyzed and the samples are not
too small, a Guinier-DeWolff camera may be
useful.
Probably the most difficult compound to
identify in a mixture is hydroxyapatite.
Hydroxyapatite crystals from urinary stones
are usually poorly crystallized, on the order
of several hundred angstroms, and, therefore, give weak diffuse lines while the other
crystalline materials produce sharp lines. If
the analyst is not aware of this, the hydroxyapatite may be missed completely or grossly
underestimated. We have found that a pattern which appears to contain only weak
calcium oxalate hydrate or MgNH 4 P0 4 6H 2 0 phosphate lines usually also contains
hydroxyapatite. Since the strongest fines of
the latter have the same d spacings as some
of the other compounds, we always compare
the Debye-Scherrer photographs of the unknowns to those of pure compounds to make
sure that the relative intensities as well as the
d spacings show a proper match. An example
of this, pointed out by Morriss and Beeler,5
is mixtures of MgNH 4 P04-6H 2 0 and
hydroxyapatite. The two strongest hydroxyapatite lines, 2.S and 3.45, overlap the 2.S0
and 3.4S lines of MgNH4P0 4 -6H 2 0. Most
hydroxyapatite from kidney stones contains
the lines and relative intensities fisted in
Table 1.
In view of the weak patterns of kidney
stone hydroxyapatite, x-ray patterns of three
synthetic preparations were also prepared.
Two synthetic materials were also poorly
crystalline, whereas the third, Fisher tribasic
calcium phosphate whose formula was listed
as Ca 6 (P04) 3 (OH), gave a pattern which
contained sharper, stronger lines (Fig. 1).
Measured by internal standard technic, the
3.4 A d spacing (002) of one kidney stone had
a relative intensity only 45% as intense as
that of the Fisher sample. The three peaks
which appear in the Fisher sample between
65S
POLLACK AND CARLSON
2.S0 and 2.63 A occur as a broad band with a
o
maximum at 2.78 A in kidney stones. The
total intensity in this broad band is also
about 45 % of the intensity found in the three
lines of the more crystalline material. Since
the diffraction pattern varies considerably, it
would be difficult to develop a quantitative
hydroxyapatite analysis based on x-ray
diffraction alone. The poorly crystalline
hydroxyapatite in kidney stones has a diffraction pattern similar to that shown for
dentine by Trautz and co-workers.8
EXPERIMENTAL INFRARED IDENTIFICATION
Infrared spectra were obtained using the
KBr pellet technic. Normally 2 mg. of the
TABLE 1
I N T E N S I T Y AND d SPACINGS O F H Y D R O X Y A P A T I T E
Vol. 52
sample are mixed with ~ 2 0 0 mg. of KBr
powder and the resulting mixture is pressed
into a disk. Samples smaller than 2 mg. can
be analyzed by using micro-pelleting technics
and beam-condensing optics in the infrared
instrument.
Spectra were obtained on a Beckman IR-9
infrared spectrophotometer which covers the
range 4000 to 400 cm."1 (2 to 25 y). There is
some benefit in using one of the newer extended range instruments rather than the
older instruments which cover only the 2 to
15 n region. For example, in detecting
C*(OH)(P0 4 )« in MgNH<P(V6H 2 0, all of
the bands due to Ca 6 (OH)(P0 4 )3 in the 2 to
15 n regions are masked by the MgNEUPCV
6H 2 0 spectrum, whereas in the 15 to 25 M
region the spectra are sufficiently different to
allow the detection of Ca6(OH)(P04)3-
FROM K I D N E Y S T O N E S
d
Line No.
1
2
3
4
5
6
7
8
9
Peak Intensity
3.45
2.80
2.70
2.63
2.45
1.94
1.90
1.84
1.72
4
10
1
1
2
2
1
2
1
34
30 26
26 COPPER Ka
F I G . 1. Diffractometer traces of three hydroxyapatite samples: A, kidney stone;
B, s y n t h e t i c ; C, Fisher's chemical reagent. To compare intensities, the peaks on A
and B should be multiplied by 2 since the trace of C was made using a larger scale
factor. Minor tracing irregularities have been eliminated.
58
54
50
46
42
38
RESULTS AND DISCUSSION
Binary mixtures containing weight ratios
of 80:20 and 90:10 of the more common kidney stone constituents were prepared and
analyzed by both infrared and x-ray diffraction technics. The results of these analyses
are shown in Table 2.
In eight of the 16 samples the minor
component of the mixture was more easily
detected by the x-ray technic; in two, infrared was more sensitive, and in six, the two
Dec. 1969
technics were about equal. One case where
infrared was more sensitive (90% CaC 2 0 4 H 2 O:10% hydroxyapatite) is an important
one because these are two of the more comTABLE 2
EASE
OP
659
IDENTIFYING KIDNEY STONES
DETECTION
OF
MINOE
COMPONENT
IN BINARY MIXTURES*
Detectability of
Minor Component
Detectability of
Minor Component
Mixture
X-Ray
Infrared
90%CaC 2 O 4 H 2 O
10% uric acid
Not definitive
Not definitive
90%CaC 2 O 4 H 2 O
10% MgNH 4 P0 4 -6H 2 0
Not definitive
Not detectable
90% MgNH 4 P0 4 -6H 2 0
10% Ca 5 (P0 4 ) 3 (OH)
Difficult
Not definitive
90% MgNH
M g N H 4 P0
P 0 4 -6H
- 6 H 20
10% C a C 2 0 4 H 2 0
Difficult
Not definitive
Mixture
X-Ray
80% Ca 6 (P0 4 ) 3 (OH)
20% CaC 2 0 4 H 2 0
80% Ca 6 (P0 4 ) 3 (OH)
20% MgNH 4 P0 4 -6H 2 0
80% Ca 6 (P0 4 ) 3 (OH)
20% uric acid
Infrared
Easy
E
asy
Easy
Easy
Not definitive
Easy
Difficult
Easy
Not definitive
Easy
Easy
80% MgNH 4 P0 4 -6H 2 0
20% Ca 5 (P0 4 ) 8 (OH)
Difficult
Not definitive
80% MgNH 4 P0 4 -6H 2 0
20% C a C 2 0 4 H 2 0
Easy
Not definitive
80% CaC 2 0 4 -II 2 0
20% Ca 6 (P0 4 ) 3 (OH)
Easy
Easy
90% Ca 6 (P0 4 ),(OH)
10% CaC 2 0 4 -H 2 0
Not detectable
Not definitive
90% CaC 2 0 4 H 2 0
10% Ca 5 (P0 4 ) 3 OH
Not definitive
Easy
90% Ca 6 (P0 4 ) 3 (OH)
10% MgNH 4 P0 4 -6H 2 0
Difficult
Not detectable
90% Ca 5 (P0 4 ) 3 (OH)
10% uric acid
Not definitive
Not definitive
80% CaC 2 0 4 -H 2 0
20% uric acid
80% CaC 2 0 4 -H 2 0
20% MgNH 4 P0 4 -6H 2 0
* T h e ease of detection of the minor constituent
has been divided into four categories: 1, easy; 2,
difficult (a definite identification was possible
b u t hard to m a k e ) ; 3, not definitive (only the
strongest infrared band or x-ray diffraction line
appeared on the p a t t e r n and a positive identification could not be m a d e ) ; and 4, not detectable
(not even the strongest band or line was present
on the p a t t e r n ) .
mon compounds in urinary stones. Results
discussed earlier indicate that roentgenographic analysis is even less sensitive than is
shown in Table 2 for kidney stone hydroxyapatite. Hydroxyapatite in kidney stones is
more poorly crystalline than the synthetic
material and diffracts with appreciably less
intensity in the peaks. If all kidney stone
hydroxyapatite diffracts with only 45% of
the intensity of well-crystallized material as
we have found, then the minimal detectable
amount of hydroxyapatite using x-ray diffraction is probably somewhere between 20
and 30%, depending on the mixture. A
component present as less than 10% of a
sample will probably not be detected by
x-ray diffraction or infrared; therefore, when
large stones are available it may be desirable
to run wet chemical in addition to instrumental analysis. For samples weighing only
milligrams, either x-ray or infrared analysis
can reveal the major constituents of the
samples. X-ray diffraction is generally more
sensitive in detecting the minor constituents
in routine analysis; however, when a more
complete analysis is required, infrared,
microscopic, or other technics may be
needed.
Acknowledgment.
D r . Robert S. Bowman
granted permission to prepare x-ray p a t t e r n s of
synthetic hydroxyapatite.
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POLLACK AND CARLSON
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