Complexometric Titration of Urinary Calcium and
Magnesium
CLAUDE L. YARBRO and ROBERT L. GOLBY
Deparfrnenf o f Biochemistry and Nufrifion, University o f Norfh Carolina, Chapel Hill,
b Urinary calcium is determined by
addition of an excess of standard
ethylene glycol bis-(0-aminoethyl
ether)-N,N’-tetraacetic acid and back-
titrating with calcium, using Calcon as
an indicator. Calcium plus magnesium
may b e determined by addition of an
excess of (ethylenedinitri1o)tetraacetic
acid, followed by back-titrating with
calcium using Eriochrome as an indi-
cator. The phosphate and citrate
normally present in urine do not inter-
fere. Protein when present in amounts
greater than 1 mg. per ml. interferes;
it may be removed b y precipitating
with trichloroacetic acid. A single
determination using this method re-
quires 5 minutes as compared with
2 to 24 hours for other methods.
T HIS rapid, accurate method for the
determination of calcium in urine
saves considerable time over methods
based on the isolation of calcium oxalate
(1, 3, 8). Wilson (9) determined
calcium in urine by titration with di-
sodium (ethylenedinitri1o)tetraacetate
(EDTA), using ammonium purpurate
(murexide) as an indicator after first
removing phosphate. This indicator
has certain disadvantages: The pink to
purple color change is difficult to judge
and, even where photometric titrations
are used (6), the rapid destruction of the
dye is a cause of concern. Although
calcium may be determined in the pres-
ence of phosphate by back-titration
procedures utilizing an excess of EDTA
( d ) , the presence of magnesium inter-
feres with such procedures, even though
it is first precipitated as the hydroxide
(excess EDTA will dissolve magnesium
hydroxide). However, EDTA can be
used for the determination of calcium
plus magnesium in urine by back-
titration.
Hildebrand and Reilley (6) found that
1 - (2 - hydroxy - 1 - naphthylazo) - 2-
naphthol-4-sulfonic acid (Calcon) gives
a very sharp color change from pink to
blue when calcium is titrated a t p H 12.5
with EDTA. Golby, Hildebrand, and
Reilley (4) used this indicator and re-
agent to titrate calcium in serum di-
rectly. Although Calcon overcomes the
objections associated with murexide as
an indicator, the presence of magnesium
in urine still constitutes a problem.
504 ANALYTICAL CHEMISTRY
Schmid and Reilley ( 7 ) used ethylene
glycol bis-(p-aminoethyl ether)-AV,N’-
tetraacetic acid (EGTA) to determine
calcium in the presence of magnesium
by a coulometric titration. Because
this reaction seemed to be selective for
calcium it appeared that ethylene glycol
bis-(p-aminoethyl ether)-N,N’-tetra-
acetic acid could determine calcium in
urine in the presence of both magnesium
and phosphate by back-titration.
DETERMINATION OF CALCIUM
Reagents and Apparatus. Stand-
ard calcium, 0.100OOM. Dry pri-
mary standard grade calcium carbo-
nate to a constant weight, and dis-
solve in the minimal quantity of liL‘
hydrochloric acid with heating to
ensure complete solution and evolu-
tion of carbon dioxide. Dilute to the
appropriate volume.
Ethylene glycol bis-(8-aminoethyl
ether)-N,N’-tetraacetic acid, 0.0lAf
(EGTA, available from Geigy Industrial
Chemicals as CHEL DE). Dissolve
approximately 0.85 gram in the minimal
quantity of 2M sodium hydroxide and
dilute to 250 ml. Standardize against
0.1OOOOM calcium, using Calcon as an
I
indicator.
Sodium hydroxide, 26f.
Potassium cvanide. 0.7M.
Calcium indicator.’ Dissolve 150 mg.
of Calcon (J. T. Baker Chemical Co.)
in 100 ml. of methanol.
Syringe microburet (Micro-Metric
Instrument Co.), calibrated to deliver
0.5 ~ 1per
. scale division.
Procedure. To 1 ml. of urine
(calcium concentration, 1 t o 8 milli-
moles per liter) in a 30-ml. beaker, add
1 ml. of 0.7M potassium cyanide from
a buret. Pipet in succession 1 ml. of
standard 0.01M EGTA and 2 ml. of
2M sodium hydroxide into the above
mixture, and dilute to approximately
15 ml. with distilled mater. Add 5
drops of calcium indicator just prior to
titration to obtain a blue to green color,
depending upon the amount of pigment
in the urine. Titrate the solution to the
first appearance of pink color, using
standard 0.1M calcium solution deliv-
ered from the syringe microburet.
Stir the solution continuously during
the titration with an electric stirrer
fitted with a small paddle.
DETERMINATION OF CALCIUM PLUS
MAGNESIUM
Reagents and Apparatus.
ard calcium, 0 . l O O O O M .
N. C.
(Ethylenedinitri1o)tetraacetic acid,
0.015M. Dissolve approximately 1.1
grams of disodium (ethylenedinitri1o)-
tetraacetate dihydrate (J. T. Baker
Chemical Co.) in 200 ml. of water and
standardize against 0.1N calcium, using
Calcon as an indicator.
Potassium cyanide, 0.7M.
-4mmonia buffer. Dissolve 67.5
grams of ammonium chloride in 570 ml.
of concentrated ammonium hydroxide
and make up to 1 liter with distilled
water.
Magnesium plus calcium indicator.
Dissolve 1 gram of Eriochrome Black T
(Matheson, Coleman & Bell) in 25 ml. of
distilled water, add 1 ml. of 0.5M
sodium carbonate, and dilute to 100 ml.
with isopropyl alcohol. Filter through
a Buchner funnel and store in the cold.
Syringe microburet.
Procedure. This procedure is
essentially a modification of the Wil-
son method (9). To l ml. of urine
(Ca + Mg concentration 3 to 10 milli-
moles per liter) in a 30-ml. beaker, add
1 ml. 0.7M potassium cyanide from a
buret, and then in succession add 1 ml.
of standard 0.0151M EDTA and 2 ml.
of ammonia buffer. Dilute the solution
to approximately 15 ml. with distilled
water. Add 2 drops of magnesium plus
calcium indicator to give a blue to green
color, depending upon the amount of
pigment in the urine. Titrate the
solution to the first appearance of wine-
red color, using standard 0.1M calcium
solution as described above.
Both procedures may be adapted for
use Tvith a 5-ml. buret calibrated in 0.02
or 0.01 ml. If this adaptation is used,
the standard calcium solution should
be 0.0100011.f and a quantity of EDTA
(for Ca + hIg) or ethylene glycol
bis-(p-aminoethyl ether)-A-,h”-tetra-
acetic acid (for Ca) should be added to
the urine R-hich will give a back-titra-
tion of 1 to 3 ml.
RESULTS
Water Solutions. The method was
first tested on n-ater solutions con-
taining known amounts of calcium and
magnesium. The total concentration
of calcium plus magnesium was of the
order of O.OlM, with a calcium to mag-
nesium molar ratio varying from 0.1 to 9
(Table I). The maximum deviation
of calcium found from that taken was
3.6%, with an average of 2.3%. For
calcium plus magnesium the maximum
Stand- deviation was 1.1%, with an average
of 0.5%. Where the molar ratio of
calcium to magnesium is greater than
2 , the reliability of determination of Table I. Determination of Calcium and Calcium plus Magnesium in Water Solutions
magnesium by difference decreases rap-
idly. hk Ca Ca + Mg Found,
.1k
Taken, Taken, Found, Dev., Found,
Recoveries of Calcium and Mag- bimolesj mmoles/ mmoles/ mmoles/ Dev., mmolesj Dev.,
nesium f r o m Urine. The recovery of L. 1. 1. 70 1. % 1. 70
calcium added t o urine ranged from 98 1.01 8.65 8.91 1-3.0 9.68 $0.2 0.77 -23.7
t o 104%, with a n average of l O l % , 2.03 i.69 7.91 +2.9 9.73 +O.l 1.82 -10.3
a n d for magnesium, t h e recoveries 3.05 6.73 6.86 +1.9 9.78 0.0 2.92 -4.3
5.09 4.80 4.03 +2.7 9.87 -0.2 4.94 -2.0
ranged from 95 t o 1047,, with a n i.13 2.88 2.84 -1.4 9.92 -0.9 7.08 -0.7
average of 100% (Table 11). 8.14 1.92 1.99 "3.6 9.95 -1.1 7.96 -2.2
Tests f o r Interference in Urine. 9.16 0.96 0.97 +1.0 10.02 -1.0 9.05 -1.2
T h e effect of added citrate and phos-
phate on t h e determination of calcium
a n d calcium plus magnesium in urine Table II. Recovery of Added Calcium and Magnesium from Urine
was tested (Table 111). Addition of
phosphate up t o 0.05 millimole per ml. Added,
Mg
Added,
Ca
Found, Found,
big
Ca + Recovered hlg Recovered
________
of urine had no effect upon the deter- Sam- mmoles/ nimoles/ mmoles/ Mmoles/ mmoles/ mmoles/
mination of calcium plus magnesium. CI c-
ple L. 1. 1. L. 1. /a 1. /C
However, added phosphate a t this 11 0.00 0.00 5.44 8.68 ... ... ...
upper Icvel (0.05 millimole per ml. of 0.00 9.61 15.17 18.44 9.73 ioi:2
urine) produced interference in the 10.18 0.00 5.40 18.92 ... 10124 10h:5
determination of calcium, in one case 5.09 4.80 10.24 18.58 4.80 lb0:O 5.06 99.4
12 0.00 0.00 6.59 10.65 ... . . . .
giving a value 13% below that obtained 0.00 9.61 16.34 20.34 9.75 ioi:4 , . . ...
n itli the urine alone. Because the level 10.18 0.00 6.68 20.59 ... ... 0.94 97.6
of phosphate occurring in urine is in the 5 09 4.80 11.60 20 51 5 01 104 4 5 06 99 4
range of 12 to 30 millimoles per liter 13 0 00 0 00 2 92 4 91
0 00 9 61 12 56 14 76 9 61 100 3
and the amount of added phosphate 10 18 0 00 2 96 15 19 10 28 101 0
n hich caused interference was approsi- 5 09 4 80 7 84 14 56 4 72 98 3 4 83 95 3
niately t n ice that normally found in 14 0 00 0 00 3 31 4 93
urine, no interference may be expected 0 00 9 61 12 81 14 89 9 50 98 8
10 18 0 00 3 27 15 40 10 4i 102.8
from the phosphate normally occurring 5 09 4 80 8 27 15 04 4 95 103 1 5 31 104 3
in urine. Levels of added citrate u p
to 0.01 millimole per ml. of urine had no
effect on either determination. Table 111. Effect of Added Phosphate and Citrate on Determination o f Calcium and
As protein (usually albumin) may ap- Magnesium in Urine
pear in the urine in various pathological
conditions, the effect of added serum Phosphate Citrate Calcium __ Calcium +
Magnesium
albumin upon the determination of cal- Added, Added, Found, Dev., Found, Dev.,
cium and of cnlciuni plus magnesium in Sample Xfmoles/L. Mmoles/L. mmoles/l. 70 mmoles/l. %
urine was evaluated. Although Golby, 11 0 00 0 00 5 43 8 68
Hildebrand, and Reilley (4) have deter- 20 00 0 00 5 50 +24 8 86 $2 1
50 00 0 00 5 43 00 8 87 1-2 2
mined calcium in serum by direct 0 00 3 00 5 36 -13 8 77 +10
titration without removing proteins, 0.00 10.00 5.35 - 1.5 8 70 +o. 2
10 mg. serum albumin per ml. of urine
12 0.00 0.00 6.59 10 6.5
in these back-titration procedures made
the determination of calcium or mag-
20,oo
50.00
0.00
0.00
6.85
6.77
+-
+ 3.9
2.7
10.86
10,75
+i.g
$0.9
nesium plus calcium impossible. At 0.00 3.00 6.55 - 0.6 10.68 $0.3
protein levels of 1 mg. per nil. of urine 0.00 10.00 6.60 + 0.2 10.55 -0.9
or less, no interference was noted. I n 13 0.00 0.00 2.92 4.91 ...
the present procedure, the protein is
apparently competing with the indi-
20.00
50.00
0.00
0.00
2.98
2.83
+' 2.0
- 3.1
4.91
5.05
0.0
$2.8
cator for the calcium titrant; the pro- 0.00 3.00 2.89 - 1.0 4.72 -3.9
tein was removed from a n y urine sample 0.00 10.00 2.86 - 2.1 4.62 -5.9
which gave a n immediate positive test 14 0.00 0.00 3.31 4.93
for protein when acidified Kith acetic 20.00
50.00
0.00
0.00
3.34
2.88
+' 0.9
-13.0
5.03
4.91
+2:0
-0.4
acid and heated. The following pro-
cedure for its removal may be used: 0.00 3.00 3.11 - 6.0 5.04 $2.2
0.00 10.00 3.24 - 2.1 5.10 +3.4
To 5 ml. of urine in a 15-ml. centri-
fuge tube, add 5 ml. of 5y0 trichloro-
acetic acid, mix well, and allow to Table IV. Comparison of Proposed Method with Other Methods
stand for several minutes. After centrif- Method of Shohl
ugation a t 1500 r.p.m. for 10 minutes, Present Method Method of Wilson (9) and pedley (8),
take 2-ml. aliquots of the supernatant
solution (equivalent to 1 ml. of urine)
Ca,
mmoles/l.
Ca big,
mmoles/l.
+ Ca,
mmolesjl.
Ca +hfg,
mmoles/l.
Ca,
?rfmoles/L.
for analysis. h-eutralize the trichloro- 12 6.59 10.6-5 6.86 10.22 6.57
acetic acid present with 0.2 ml. of 2-11 17 8.94 15.45 8.79 15.91 8.80
sodium hydroxide and analyze the re 19 4.09 7.55 3.85 7.72 4.14
sulting mixture by the above procedures. 20 5.18 8.39 4.92 8.46 5.16
21 8.15 11.88 7.73 11.33 7.41
22 6.25 11.71 6.13 11.70 6.06
I n other tests on the methods pre- 23 3.51 7.94 3.62 7.72 3.89
sented, trace metals (principally iron) ~~

VOL. 30, NO. 4, APRIL 1958 505

present in urine interfered slightly.
This interference was removed by the
addition of cyanide as described in the
experimental procedure.
Comparison w i t h Other Methods.
Calcium was determined in seven
different samples of urine by t h e
present method, by t h e complex-
ometric method of Wlson (9),and by
the oxalate precipitation method of
Shohl and Pedley (8). I n Table IV,
the vaIues for calcium obtained by the
present procedure compare favorably
with the other methods, as do the values
for calcium plus magnesium with the
Wilson procedure (9). The method of
Sholil and Pedley requires approxi-
niately 24 hours for completion and the
procedure of Kilson require 2 t o 3 hours.
Precision of Method. To calculate
the standard deviation of these pro-
cedures, calciuni and calcium plus
magnesium were determined on 20
Infrared identification of Disaccharides
JONATHAN W. WHITE, Jr., C. R.
Eastern Regional Research laboratory, Philadelphia, Pa.
b The value of infrared spectra for
the identification of amorphous di-
saccharides and their acetates, b y
comparison with spectra of known
disaccharides and their acetates, i s
demonstrated. Infrared spectra of
ten amorphous disaccharides of D-
glucose, of D-glucose and D-fructose,
and of their 0-octaacetates are pre-
sented over the range of 650 to
1500 cm.J Potassium bromide disks
were used. All spectra differ in
sufficient detail to allow differentia-
tion among closely related disaccha-
rides.
A LTHOUGH chromatographic analysis
has been of inestimable value in
separation of mixtures of sugars, their
identification by chromatographic evi-
dence alone is not conclusive. Rela-
tive rates of migration on paper or
column, together with color reactions
v i t h spray reagents, only contribute
to identification. Physical properties
of isolated samples or derivatives are
also required. Heretofore, crystalliza-
tion of the sugar or derivative has been
considered imperative before an identi-
fication is unequivocal.
Sugar samples isolated by chroniato-
graphic means may be so small that i t
is practically impossible to crystallize
506 ANALYTICAL CHEMISTRY
individual aliquots of the same urine.
The magnesium content of the urine
was obtained by difference between the
calcium and the calcium plus mag-
nesium determination. The results of
this experiment were (in millimoles
per liter): Ca = 8.41 =t0.032; Ca + (4) Golby, R. L., Hildebrand, G. P.,
hIg = 14.44 =t0.033; AIg = 6.03 =k
0.177.
Twenty triplicate titrations of urinary
calcium where the amount of calcium
varied from 2.23 to 8.94 millimoles per
liter agreed within i 2.9%.
ACKNOWLEDGMENT
The authors n-ish to acknowledge the
technical assistance of Thomas Austin.
They also express their appreciation to
Geigy Industrial Chemicals for supply-
ing saniples of EGTA used in this ex-
periment.
EDDY, JEANNE PETTY, and NANCY HOBAN
them. Hence, melting point, x-ray
diffraction, and crystallographic study
are eliminated as tools for identification.
Determination of optical rotation does
not require crystalline samples, but
values may become only approxinia-
tions as sample weights decline into
the lower milligram range.
The infrared absorption spectrum of
a compound is increasingly used for
its identification and analysis. As a
unique physical property that is not
primarily dependent on crystallization,
it offers a most useful criterion for iden-
tification of disaccharides.
Infrared spectra of all types of car-
bohydrates have been published. I n
the first of a series of papers on this
subject, Barker and his colleagues (5)
stated that determinations of infrared
spectra offer poll-erful means for the
comparison of supposedly identical
samples of carbohydrates. They ex-
amined the spectra of a variety of car-
bohydrates and their derivatives, but
have not published them in sufficient
detail for comparison purposes. They
noted that spectra in the 730- to 960-
cm.-' range allow assignment to the
CY- or p-series of D-glucopyranoses,
Khistler and House (20) have also re-
ported that certain regions of absorp-
tion are characteristic of the configura-
tion of the anomeric carbon.
LITERATURE CITED
(1) Berger, E., Clin. Chem. 1 , 249 (1955).
(2) Brunisholz, G., Genton, M., Plattner.
E., Helv. Chim. $eta 36, 782 (1953),
(3) Fiske, C. H., Logan, M. A , , J . B i d .
Chem. 93, 211 (1931).
Reilley, C. N., J . Lab. Clin. M e d .
50. 498 11957).
(5) Hildebrand, G.' P., Reilley, C. S . ,
A h A L . CHEK 29, 258 (1957).
(6) Horner, I\-.H., J . Lab. Clin.M e d . 45,
951 i
4.51 (1955).
14.5.5)
7 ) sei;;
((7) Schmid, R. W., Reilley, C. K., AXAL.
CHEU.29, 264 (1957).
(8) Shohl, A. T., Pedley,
Pt F. G., J . Bio2.
Chem. 50. ,537 t(1922).
L H L L I.
(9) W&on, A,' A , , ' J . Comp. Pathol.
Therap. 63, 294 (1953).
RECEIVED for review July 29, 1957. Ac-
cepted November 26, 1957. Supported in
part by research grant (PHS8-248) from
the National Institute of ilrthritis and
Metabolic Diseases, Sational Institutes
of Health, Public Health Service.
Kuhn (13) has published the spectra
of 79 carbohydrates and derivatives
over the range 8.0 to 15.0 microns. Of
these, only t n o are of sugars included
here. Kuhn's spectra were determined
with an amount of snniple in the beam
too small to permit maximum utility
of the curres for comparison purposes.
Infrared spectra of a large number of
sugar acetatrs and related compounds
have been determined a t the National
Bureau of Standards (12). Crystalline
materials were used but the spectra
were all determined in solution.
The pressed-disk technique is useful
when water-soluble materials are ex-
amined. It also prrmits the use, where
necessary, of less-than-milligram sam-
ples ( 1 ) . Some difficulties have been
experienced in comparing disk spectra
with those obtained from mulls (d, 3, 8).
Barker et al. (6) have ascribed the
changes they previously (3)noted during
aging of disks containing certain carbo-
hydrates to the relatively high moisture
content of potassium bromide disks.
They did not find any such changes in
any of the three disaccharides included
in their study. The mlue of infrared
spectra in identification of larger mole-
cules such as trisaccharides was cited
by Whiffen (19), n h o noted that iden-
tification by melting point and rotation