by flame photometry
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J. clin. Path. (1961), 14, 463
A new principle applied to the determinationof calcium in biological materials
by flame photometryJ. K. FAWCETT AND V. WYNN
From the Surgical Unit, St. Mary's Hospital, London
SYNOPSIS The effect of magnesium sulphate in releasing calcium emission from interference byphosphate and sulphate has been investigated.
Samples were diluted in 10 mM MgSO4, 2 mM NaCl, giving final calcium concentrations ofabout 0 05 to 0-10 mM. In this diluent, galvanometer readings were proportional to calcium con-centrations up to 04 mM. The magnesium sulphate released calcium emission from depression byphosphate and sulphate. The excess sodium chloride eliminated enhancement of calcium emissionby added sodium and potassium in the sample. Subtraction of background readings excluded directinterference.A 3 % correction was made for the effect of the viscosity of 1: 50 plasma dilutions. Satisfactory
recoveries of added calcium were obtained from plasma, urine, and faeces using the diluent describedabove. Results on urine and faeces correlated closely with those obtained by an EDTA titrationmethod. Results on plasma were consistently 2% higher by flame photometry than by EDTAtitration.
Other methods of calcium determination, depending on the use of radiation buffers or standardaddition, were found to be unsatisfactory because of variable interference by phosphate at differentcalcium levels.
The measurement of calcium in biological materialsby flame photometry is more difficult than that ofsodium and potassium because of its lower emissionenergy and more serious interference by contaminat-ing substances. The first difficulty can be overcomeby using a high flame temperature, a prism mono-chromator, and a sensitive detector. Instrumentsincorporating these features permit the use of theweak calcium line at 422-7 m,u where interference isless than using the calcium oxide bands at 554 m,uand 622 m,u. Even at 422-7 m,u, however, there arethree important sources of interference as follows:
(I) Background (direct) interference: using a prismmonochromator, this is due mainly to hetero-chromatic spectral radiation by sodium and potas-sium, only a fraction being due to scattered light.(2) Enhancement or depression of calcium emission(indirect interference): the emission by calcium itselfis enhanced by sodium and potassium and depressedby certain anions, notably phosphate and sulphate.
Received for publication 21 November 1960.
(3) Alteration of the physical properties of thesolution because the quantity and dispersal of thespray reaching the flame may be influenced bydifferences in viscosity and surface tension due toprotein and other organic compounds.Many procedures for measuring calcium by flame
photometry involve prior separation of calcium frominterfering substances by precipitation of calciumoxalate (Powell, 1953; Chen and Toribara, 1953;Butterworth, 1957; Woollen and Walker, 1959) or bymeans of a cation exchange resin (Brabson andWilhide, 1954; Denson, 1954; Jackson and Irwin,1957). These preliminary steps are time-consumingand have errors of their own, thus combining dis-advantages of other methods for calcium deter-mination with those of flame photometry, whilelosing the simplicity of the latter.Apart from the separation of calcium from inter-
fering substances, many methods have been intro-duced to control their effects, but none has beengenerally accepted. The complexity of interferingeffects makes the calculation of correction factors
J. K. Fawcett and V. Wynn
(Severinghaus and Ferrebee, 1950) impracticable.Synthetic standards (Chen and Toribara, 1953;MacIntyre, 1954; Teloh, 1958), with a compositionsimilar to that of the solutions analysed, cannot beused when the composition of the materials is widelyvariable. Internal standards (Baker, 1955) are of onlylimited value because few sources of interferenceaffect calcium emission and that of the internalstandard proportionately.Background correction (Vallee, 1954; Margoshes
and Vallee, 1956) is made by subtracting readingstaken at wavelengths near the analytical wavelengthfrom those taken at the analytical wavelength. Thisaccounts for direct interference, but indirect inter-ference effects present much greater difficulty.
Radiation buffers (Severinghaus and Ferrebee,1950; Maclntyre, 1957), which incorporate an excessof interfering ions, can control sources of indirectinterference which have no increased effect abovecertain concentrations of the interfering substance orwhich have a plateau region over a restricted range.
In the determination of magnesium, anion inter-ference has been controlled (West and Cooke, 1960;Fawcett and Wynn, 1961) by adding a large excess ofethylene diamine tetra-acetic acid (EDTA). West andCooke (1960) found that EDTA could also be appliedin this way to the determination of calcium butthat the excess had to be very much greater, 160-foldinstead of 10-fold. They used the disodium salt ofEDTA, from which the high background emission isa serious disadvantage, particularly with instrumentsnot equipped for automatic spectral scanning.The standard addition method (Rothe and
Sapirstein, 1955) may be used when the relationshipbetween concentration and emission is linear, andsources of indirect interference have the same pro-portional effect upon standard calcium added todiluted samples as upon the calcium already present.The original concentration is thus calculated fromthe equation:calcium in sample emission by samplecalcium added increase in emission due to
calcium addedWe investigated the use of radiation buffers and
the standard addition procedure but, as reportedbelow, neither was found satisfactory for our pur-pose.
Mitchell and Robertson (1936) reported that highconcentrations of strontium released calcium emis-sion from the inhibitory effect of aluminium. Willis(1960), measuring calcium by atomic absorptionspectroscopy, used strontium chloride to controlphosphate interference, and we tested its use for thesame purpose in flame photometry. We also exploredthe potentialities of other compounds and achieved
the best results with magnesium sulphate, which isreadily available in a calcium-free state.
Since we completed our investigation on a methodof releasing calcium emission from anion inter-ference, Dinnin (1960) has reported results of aninvestigation on alkaline earth and rare earthelements, and iron, yttrium, and scandium asreleasing agents, and he has proposed an explanationfor their mode of action. The results of our investi-gation are reported below.
INSTRUMENTAL The Unicam SP 900 flame spectro-photometer was used. It has a Meker type burner utilizingacetylene and compressed air. The latter conveys theatomized solution from a separate spray chamber. Theinstrument is equipped with a fused silica prism mono-chromator and a photomultiplier detector. A slit width of0-08 mm., corresponding to a nominal band width of 1 mp,was selected for all experiments. Air pressure was 30lb./in.2 and acetylene pressure was about 12 cm. of water,except when the effect of altering flame conditions wasbeing tested.
PREPARATION OF SAMPLES Samples were diluted to givea final calcium concentration of about 0-05 to 0 10 mM.Plasma samples were obtained from heparinized bloodand diluted 1 : 50. Twenty-four-hour urine samples werepreserved with 10 ml. of hydrochloric acid and diluted1: 50 or 1: 100, according to the expected calciumconcentration. Faeces were prepared by homogenizingwith water and transferring portions of about 2 g. tonickel crucibles of 50 mm. diameter. The contents weredried at 1050 and ashed overnight in the uncoveredcrucibles at 475 to 5250. If magnesium, as well as calcium,was to be determined, ashing was at 420 to 450 becauseof loss of magnesium at higher temperatures (Fawcettand Wynn, 1961). The ash was dissolved by carefulboiling with I ml. of I 0 N. hydrochloric acid, transferredquantitatively and diluted to 100 ml. This solution wasfurther diluted 1: 50 to 1: 10, according to the expectedcalcium concentration.
GALVANOMETER READINGS When special diluting solu-tions were used, standard calcium solutions were dilutedin the same diluent as the samples, and this diluent wasalso used for zero-setting the galvanometer. Blank andstandard readings were checked between every two orthree readings of unknowns. Each dilution was read atleast three times, and more often if any reading differedby more than I % from the mean.
INTERFERENCE BY SODIUM AND POTASSIUM The effects ofsodium were observed by adding various concentrationsof spectroscopically pure sodium chloride to deionizedwater and to solutions containing 0 05 mM and 0-10 mMcalcium chloride.Sodium interference was of two kinds: direct inter-
ference due to spectral radiation by sodium in the regionof the calcium line, and indirect interference due to
Determination of calcium by flame photometry
enhancement of calcium emission by sodium. It wasfound that direct interference by sodium was proportionalto its own concentration and represented about 0-04% ofthe emission from equimolecular concentrations ofcalcium, under the conditions of the investigation. TenmM sodium chloride gave identical readings at 422-7 mju,418 mz, and 428 mju, and readings at the latter two wave-lengths were unaffected by calcium emission.
In contrast to the direct interference, the enhancementinterference at any stated sodium level was directly pro-portional to calcium concentration. As the sodium con-centration was raised, the percentage enhancement roseto a maximum of 9% at 1 mM, remaining at this per-centage for sodium concentrations at least as high as10 mM. This is shown in Fig. 1.The interference effects of potassium were similar to
those of sodium, and