CALCIUM DETERMINATION BY FLAME PHOTOMETRY;

METHODS FOR SERUM, URINE, AND OTHER FLUIDS

BY J. W. SEVERINGHAUS AND J. W. l?ERREBEE

(Prom The Mary Imogene Bassett Hospital,* Cooperstown, New York)

(Received for publication, May 29, 1950)

The convenience and accuracy of flame photometric determinations of

sodium and potassium in biological fluids (1, 2) have suggested the de-
sirability of extending these advantages to determinations of calcium.
Since calcium emission lines are much less intense than those of sodium
and potassium (3), the samples of diluted serum (1: 50) customarily used
for sodium and potassium determinat.ions are not adequate for the meas-

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urement of calcium concentrations. Serum dilutions of the order of 1: 10
are adequate provided their viscosity is reduced by precipitation of the
proteins with trichloroacetic acid. At the wave-length used for calcium
measurements, a correction is necessary for the effect of sodium and po-
tassium upon calcium emission and upon flame background. The correc-
tion may be easily derived. The resulting determinations of calcium agree
well with those obtained by the Van Slyke-Sendroy technique and in our
hands are somewhat more rapid and accurate.

Method
Preparation of Serum-l.00 cc. of serum and 5.00 cc. of distilled water
were accurately pipetted into 15 cc. conical centrifuge tubes fitted with
glass stoppers and mixed. 4.00 cc. of 10 per cent trichloroacetic acid were
added, the stopper inserted, and the solution mixed by inverting ten times,
10 minutes were allowed for protein precipitation. The tubes were then
centrifuged at 2000 r.p.m. for 10 minutes. About 3 cc. of supernatant
were decanted or pipetted into the 5 cc. atomizer beakers.
Urine and Other Fluids-Samples of urine and other fluids for calcium
measurements were prepared in a manner similar to that for serum. The
final concentration of trichloroacetic acid was 4 per cent and the dilution
of the sample was such as to bring the concentration of calcium into the
0.1 to 1.0 m.eq. per liter range. Aliquots of urine and other fluids must
be taken from well mixed specimens, since sediments often contain large
amounts of calcium. This calcium is brought into solution in 4 per cent
trichloroacetic acid (4).
No&Filter paper should be avoided at all stages in the preparation of
+ AflZated with Columbia University.
621
622 CALCIUM BY FLAME PHOTOMETRY

the samples, since acid solutions will extract a measurable amount of

calcium from most “ash free” filter papers (4).
Prepartction of Standards--A stock solution of 10.00 m.eq. of calcium
per liter was prepared by dissolving 0.5004 gm. of CaC03 (Iceland spar)
in 10 cc. of concentrated HCl and diluting it to 1 liter with distilled water.
The usual working standard made from this solution contained 0.5 m.eq.
of calcium, 14.0 m.eq. of sodium, and 0.5 m.eq. of potassium (as chlorides)
per liter of 4 per cent trichloroacetic acid solution, corresponding essentially
to a 1: 10 dilution of serum in 4 per cent trichloroacetic acid. For re-
covery experiments, a standard with 1.0 m.eq. of calcium per liter was
prepared. The reading for the calcium blank was determined with a

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solution containing 14.0 m.eq. of sodium and 0.5 m.eq. of potassium per
liter of 4 per cent trichloroacetic acid.
All preparations of 10 per cent trichloroacetic acid solution we have
measured have contained 0.01 to 0.02 m.eq. of calcium per liter. When
diluted to 4 per cent solution, the actual concentration is 0.004 to 0.008
m.eq. per liter, which contributes 0.04 to 0.08 m.eq. per liter to the read-
ing of calcium, an error of 0.8 to 1.6 per cent of normal serum concentra-
tion. The effect of this contribution is cancelled by including 4 per cent,
trichloroacetic acid in the standards. Trichloroacetic acid at this con-
centration has no measurable effect on the flame emission of sodium or
calcium.
Instrument-The instrument used was a Beckman quartz spectrophotom-
eter with a flame attachment. Light from the flame was measured on
the transmission scale of the instrument, since the units on this scale are
related linearly to the intensity of the light. The settings of the instru-
ment are somewhat arbitrary, the following serving only as a guide.
Calibration curves may vary somewhat with different settings and different
instruments and should be checked for each instrument.
Oxygen-22 inches of water pressure.
Gas---Adjusted to give medium small cones of blue flame, about 0.3 to
0.4 cm. in height. Shorter cones tended to cause an unstable reading.
The propane pressure gage read about 1.2 cm. but was not used in flame
adjustment.
Air-20 pounds per sq. in.
Wave-Length-The 556 rnp calcium oxide line is the most satisfactory
for calcium measurement.
Sensitivity-Adjusted to give readings on the transmission scale of about
70; about four turns of the sensitivity knob from the counter-clockwise
limit with switch position 0.1.
Phototube-The ultraviolet tube was used at 556 mp; it is more sensitive
than the red phototube.
 J. W. SEVERINGHAUS AND J. W. FERREBEE 623

Slit Width-O.5 mm. Wider slits admit a higher proportion of back-

,round light. Narrower slits admit less calcium light than is needed for
ccurate measurement. Slits wider than 1.2 mm. admit the intense 589
1~ sodium light.
Measurement-The reading on the transmission scale was recorded for
listilled water, for the sodium- and potassium-containing calcium blank,
or the working standard, and for the unknown, followed by repeated
eadings of the standard and unknown to determine the average difference
between the two readings. After three pairs of agreeing readings were
btained, the calcium blank and distilled water readings were checked.
Vhen necessary, data for a sodium and potassium correction were ob-

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ained by reading the transmission at 589 rnp and at 767 rnp of the standard
nd unknown solutions. For this purpose the slit,was decreased to about
8.2 mm., the sensitivity was increased if necessary to 1.0, and the red
lhotocell was used.
E$ect of Sodium and Potassium upon Calcium Measurements at 666 rnp
-Sodium and potassium add to the flame background and also increase the
ntensity of the basic calcium emission at 556 rnp. Milliequivalents of
odium and potassium are approximately interchangeable in producing
10th of these effects. The effect on basic calcium emission is plateau-
ke, being about 9 per cent for sodium plus potassium concentrations
‘etween 1.0 and 24.0 m.eq. per liter in the solution being nebulieed. In-
lusion of sodium or potassium in concentrations greater than 1.0 m.eq.
ter liter in blanks, standards, and unknown solutions cancels out this
‘lateau-like effect.
The effect upon flame background is progressive, but if the concentra-
ions of sodium and potassium in the unknown and standard solutions are
easonably similar, as in the case of most serum determinations, no correc-
ion for background effect is necessary. A sodium variation (from 140
l.eq. per liter) of 10 per cent will introduce no more than 2.5 per cent
rror in serum calcium determination when the recommended sodium- and
otassium-containing blank and standard solutions are used as points of
:ference. A potassium variation (from 5 m.eq. per liter) of ~5 m.eq.
er liter will introduce no more than 0.5 per cent error. Accurate calcium
measurement in solutions other than serum requires that the sodium and
otassium background light in the unknown be computed to provide a
&urn blank for the unknown. The method requires measurement of
odium and potassium concentration as described below.
Calculation of Sodium Concentration-The sodium concentration of the
nknown solution is determined from the 589 rnp reading by reference to
‘ig. 1. This curve has been derived by plotting, as fractions of the trans-
mission reading (T) of the standard at 589 mp, the transmission reading of
624 CALCIUM BY FLAME PHOTOMETRY

a series of solutions of known sodium concentration. From the observed

ratio of the T (unknown) to T (standard), the sodium concentration of
the unknown solution is read from the graph.
Calculation of Potassium Concentration-The potassium concentration of
the unknown is determined from the 767 rnp readings by reference to Fig. 2.
These curves have been derived in a manner similar to that used for sodium
concentration, by recording at 767 rnp the transmission of a series of solu-
tions with known sodium and potassium concentrations. For both Fig. 1
and Fig. 2 the readings of the calcium standard, or calcium blank, which
contain 14.0 m.eq. of sodium and 0.5 m.eq. of potassium per liter, are

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1.0 - RATIO OF
. T (UNKNOWN-)
T ( STANDAR 0 )

SODIUM CALIBRATION CURVE

A=589 MU

FIG. 1. Sodium calibration curve for determining the concentration of sodium

in unknown solutions. The transmission readings of a series of sodium solutions
are plotted as ratios in which the transmission reading of a 140 m.eq. per liter stand-
ard read in 1: 10 dilution is given the value of 1 in the denominator. (Potassium,
calcium, and magnesium in concentrations less than 10 times those found in serum
have no measurable effect on sodium emission.)

taken as the reference standard and plotted as 1.00. From the previously
calculated sodium concentration and the observed ratio of T (unknown)
to T (standard), the potassium concentration may be read from the graph.
Computation of Cc&urn Blank Reading for Unknown-The calcium
blank reading for the unknown is the reading of background light due to
sodium and potassium in the unknown. Fig. 3 is employed to calculate
this reading as a fraction of the standard calcium blank reading. Fig. 3
is obtained by measuring at 556 rnp solutions containing no calcium but
varying amounts of sodium and potassium. Again the transmission read-
ing of the sodium- and potassium-containing calcium blank solution is
plotted as 1.00.
 J. W. SEVERINGHAUS AND J. W. FERREBEE 625

Location of the sodium and potassium concentrations of the unknown

on these curves indicates a factor on the abscissa. This factor when
multiplied by the 556 rnp reading of the calcium blank gives the corrected
calcium blank for the unknown. See Table I for an example of the calcu-
lation.
15
1 RATIO OF
- T(uNKNOWN)
. T (STANDA RD)

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POTASSIUM CALIBRATION CURVES
A; 767MU
140 MEQ SODIUM PER LITER

POTASSIUM CONCENTRATION - MEQ/L

FIG. 2. Potassium calibration curves for determining the concentration of potas-

sium in unknown solutions. The curves for solutions of potassium containing vary-
ing amounts of sodium are given, since potassium emission is increased by sodium.
In plotting the transmission ratios on the ordinate, the transmission reading of a
standard containing 140 m.eq. of sodium and 5 m.eq. of potassium per liter, read in
1:lO dilution, was given the value 1. (Calcium and magnesium in concentrations
less than 10 times those found in serum have no measurable effect on potassium
emission.)

Calculation of Calcium Concentration-Since calcium light at 556 rnp is a

linear function of calcium concentration (5), an unknown calcium con-
centration may be measured by employing a single calcium standard and
a calcium blank to determine the slope of the line relating concentration
and intensity of emitted light. A simple proportion then relates the
concentrations and the transmission readings (corrected by subtracting
calcium blanks) of the unknown and standard solutions.
Ca (unknown) = T (unknown)
Ca (standard) T (standard)
626 CALCIUM BY FLAME PHOTOMETRY

or, with a 1: 10 dilution of both the 5.00 m.eq. per liter calcium standard
and the unknown solution,
5.00 x T (unknown)
Ca (unknown) = m.eq. per liter
T (standard)

I40
SODIUM
MEQ/i

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100

100 MEQ POTASSIUM PER LITER

CORRECTION FACTOR
FOR CALCIUM BLANK
0
0 0.5 I.0 1.5
Fro. 3. Background correction curves for determining the appropriate calcium
blank for unknown solutions of calcium containing varying amounts of sodium and
potassium. The curves are constructed by recording in 1:lO dilution the transmis-
sion readings at 556 rnl.c of a series of solutions containing known amounts of so-
dium and potassium and plotting these readings on the abscissa as ratios in which
the reading of the standard calcium blank in the denominator is given the value 1.0.

Example with data given in Table I:

(29.1 - 19.4 - 2.3) 7.4
Ca (unknown) = 5.00 X (66.0 _ lg.4 _ 6.7) = 5.00 X G = 0.923 m.eq. per liter

Determination of Calcium by Van Slylce Gasometric Technique-The modi-

fied method of Sendroy was used (4). Calcium was precipitated from 1
cc. of serum plus 5 cc. of water by adding 1 cc. of saturated ammonium
oxalate. The solution was mixed, allowed to stand 16 hours, and centri-
fuged 5 minutes, at 2600 r.p.m. All but 0.2 to 0.3 cc. of supernatant was
aspirated with a pipette having an upturned tip. The precipitate was
 J. TV. SEVERINGHAUS AND J. W. FERREBEE 627

washed three times with 3 cc. of fresh 2 per cent ammonia water, with use
of 1 cc. to wash down the wall and 2 cc. to promote mixing by running it
rapidly down the slanted tube wall. Centrifuging and aspirating were
done between washes as above. COz was released from the oxalate with
ceric sulfate.
TABLE I
Calculation of Corrected Calcium Blank
Reading on transmission scale

At 556 mfi At 589 mg At 161 ~JI

--
19.4 Distilled water

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0 1.0
26.1 24.0 29.1 Calcium blank (40/, trichloroacetic acid
containing 14 m.eq. sodium and 0.5 m.eq.
potassium per liter)
66.0 24.0 29.1 Calcium standard (4% trichloroacetic acid
containing 14 m.eq. sodium, 0.5 m.eq.
potassium, and 0.5 m.eq. calcium per
liter)
29.1 8.6 91.3 1:lO dilution of unknown urine (and tri-
chloroacetic acid, final concentration
4%)
-
(1) The ratio of the readings of the unknown solution and the calcium blank
at 589 rnp = 8.6/24.0 = 0.36. (2) From Fig. 1, the sodium concentration is 25
m.eq. per liter. (3) The ratio of the readings of the unknown solution and the
calcium blank at 767 n+ = (91.3 - 1.0)/(29.1 - 1.0) = 3.2. (4) From Fig. 2, the
potassium concentration is 17 m.eq. per liter. (5) From Fig. 3, the correction fac-
tor for the calcium blank is 0.35. (6) The standard calcium blank at 556 rnp =
26.1 - 19.4 = 6.7. The calcium blank of the unknown solution = 6.7 X 0.35 =
2.34.

Results
Table II is a record of twenty-three serum calcium measurements be-
fore and after addition of known amounts of calcium. The average re-
covery of about 1 per cent more than was added corresponds well with the
expected effect on calcium concentration introduced by the volume dis-
placement incident to protein precipitation.’
i Protein constitutes 6 per cent by weight of normal serum, which is 4.5 per cent
by volume, assuming a specific volume of protein of 0.75 (6). In a 1:lO dilution,
this volume is 0.45 per cent of the total. If this ls all precipitated and centrifuged
into the bottom of the tube, this space is no longer available for calcium (and other
solutes), which are thereby displaced upward, increasing the supernatant calcium
concentration by 0.45 per cent. Experimentally, working with 1:5 dilutions, Sendroy
(4) gives the magnitude of this effect as 2.5 per cent, which in 1:lO dilution is 1.25
per cent. The recovery experiments presented in Table II indicated a value of
628 CALCIUM BY FLAME PHOTOMETRY

TABLE II
Recovery of Calcium Added to Serum

Serum No. Calcium Plus 4.00 m.eq. RWY.Wly

per liter
-
m&q. )W 1. meq. per 1.
4.93 8.98 4.05
5.00 9.15 4.15
5.03 9.15 4.12
4.70 8.65 3.95
4.84 8.87 4.03
4.97 9.05 4.08
4.43 8.50 4.07
5.36 9.39 4.03

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.-
Average. ............ 4.06 = +1.339r,
S.d.m., 4 ............ 0.057
Plus 5.00 m.eq.
per liter

6 4.64 9.75 5.11

7 5.86 10.88 5.02
9 5.13 10.15 5.02
10 5.42 10.45 5.03
11 5.36 10.38 5.02
12 4.90 9.93 5.03
13 5.40 10.37 4.97
14 5.23 10.32 5.09
15 4.87 9.88 5.01
15 4.95 9.99 5.04
17 4.74 9.75 5.01
18 4.78 9.83 5.05
18 4.79 9.79 5.00
20 4.68 9.78 5.10
20 4.69 9.76 5.07

Average. ............ 5.040 = +0.8%

Combined average re
covery error ....... +0.97s
S.d.m., c.. .......... 0.038

In Table III the data obtained in calcium determination by flame

analysis and by gasometry on the same sera are compared. In this series,
about 1 per cent. Since these values are about 2 to 3 times the effect predicted by
the volume of the protein, it is assumed that the precipitating agent is bound with
the protein, increasing the volume of the precipitate. The amount of calcium bound
or occluded in the precipitate is not known. Until a more accurate estimate of the
magnitude of the volume displacement effect is obtained, a correction of -1 per cent
is applied to calcium determinations on protein-free preparations.
 J. W. SEVERINGHAUS AND J. W. FERREBEE 629

the results with the flame photometer have all been lowered 1 per cent to
correct for volume displacement. The average of differences between the
methods was 0.2 per cent and is not significant.
Many of the sera were measured several times by flame photometry
and by gasometry. Gasometric determinations were usually done in du-

TABLE III
Comparison of Results by Gasometry and Flame Photometry

Serum No. Gasometric calcium Difference

-
I- me*. per 1. per cent

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18 4.72 4.70 Jro.02 -l-o.4
19 4.61 4.53 +0.03 +0.6
19 4.62 4.64 -0.02 -0.4
19 4.70 4.70 0.00 0.0
21 4.48 4.55 -0.07 -1.4
22 4.81 4.71 +0.10 -l-2.0
23 4.71 4.72 -0.01 -0.2
23 4.71 4.68 +0.03 +0.6
23 4.73 4.72 +0.01 +0.2
23 4.73 4.72 +0.01 +0.2
24 4.71 4.73 -0.02 -0.4
25 4.83 4.79 +0.04 +o.s
--___- - -
Average. ....... fO.O1 +0.2
S.d. ........... 0.04 0.8

All flame photometer determinations were averages of multiple readings on a

single serum preparation and have been corrected for volume displacement effect
by -1 per cent.

TABLE IV
Reproducibility of Calcium Determinations by Flame Photometry

No. of determinations done on 17 sera measured

more than once.. _. 50
Maximum difference between any two readings on
same serum. . 0.15 m.eq. per liter
S.d.m.. _. _. _. _. _. 0.026 m.eq. per liter = 0.5%

plicate or triplicate. Table IV demonstrates the reproducibility of the

two methods. The largest difference between duplicates in 50 flame
analyses of seventeen sera was 0.15 m.eq. per liter. The standard devia-
tion from the mean was 0.5 per cent. None of the determinations carried
out on the flame photometer by the above method was excluded for any
reason. In the Van Slyke series, because of the more complicated pro-
G30 CALCIUM BY FLAME PHOTOMETRY

cedures, occasional large errors occur and duplicates differing by more than
2 per cent were not included.
DISCUSSION

The results obtained with the above procedures for calcium measurement
suggest that flame photometric determinations in our hands are slightly
more accurate and reproducible than are gasometric determinations.
The accuracy of the method depends primarily on volumetric technique.
The variability of flame measurement may be reduced by repeated read-
ings to no more than 1 per cent.
SUMMA4RY

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Calcium in serum, urine, and other fluids may be rapidly and easily
measured by flame photometry. The accuracy of the method is equal to
that of the gasometric determination of calcium oxalate. In the case of
serum, proteins are precipitated in 4 per cent trichloroacetic acid, and the
supernatant solution, effectively a 1: 10 dilution of the serum electrolytes,
is nebulized into a constant flame. The intensity of the flame light at
556 mp is compared with that produced by standard solutions containing
calcium, sodium, potassium, and trichloroacetic acid. A correction for the
effect of variations in sodium and potassium concentration in unknown
solutions of urine and biologic fluids may be made. For serum calcium
determinations, the usual stability of serum sodium and potassium con-
centration makes correction for sodium and potassium variations of limited
value.

The writers wish to acknowledge their indebtedness to Mr. John Rowe,

Harvard Medical School, for his assistance in developing the above method
for calcium determination.
BIBLIOGRAPHY

1. Hald, P. M., J. Biol. Chem., 167,499 (1947).

2. Overman, R. R., and Davis, A. K., J. Biol. Chem., 168, 641 (1947).
3. Gilbert, P. T., Jr., Hawes, R. C., and Beckman, A. O., Anal. Chem., 22,772 (EGO).
4. Sendroy, J., Jr., J. Biol. Chem., 162, 539, 557 (1944).
5. Gilbert, P. T, Jr., Determination of sodium and calcium by flame photometry,
Beckman Reprint R-26, South Pasadena (1948).
6. Cohn, E. J., and Edsall, J. T., Proteins, amino acids and peptides, American
Chemical Society monograph series, New York (1943).
 CALCIUM DETERMINATION BY
FLAME PHOTOMETRY; METHODS
FOR SERUM, URINE, AND OTHER
FLUIDS
J. W. Severinghaus and J. W. Ferrebee
J. Biol. Chem. 1950, 187:621-630.

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