The determination of metals in blood serum by atomic absorption spectroscopy—III

SpectrochimicaActa, 1960, Vol. 10, pp.651 to 568. PergamonPressLtd. Printed in NorthernIreland

The determination of metals in blood serum by atomic absorption spectroscopy-III Sodium

and potassium

J. B. WILLIS Division of Chemical Physics, CSIRO, Chemical Research Melbourne, Australia (Received 30 January

Laboratories

1960)

Abstract-The sodium and potassium content of blood serum can be determined on samples of less than 0.1 ml by atomic absorption measurements in an air-coal gas or air-acetylene flame. The results agree with those obtained by flame photometry.

Introduction IN PARTS I and II of this series of papers [l, 21 it was shown that the method of atomic absorption spectroscopy [3, 41 could be applied to the rapid and accurate determination of calcium and magnesium in blood serum. In the present paper it is shown that sodium and potassium may also be estimated by this technique, and that the same solution of serum may be used if necessary for determination of both sodium and potassium or of potassium, calcium and magnesium.

Apparatus The apparatus was that described in Part I. Hollow-cathode discharge tubes to give the resonance lines of sodium and potassium were constructed by depositing a layer of sodium or potassium nitrite in an aluminium hollow cathode, but Philips discharge lamps were found equally satisfactory, providing they were under-run in order to minimize pressure broadening of the resonance lines (Na, 5890, 5896 A; K, 7665 A). Replacement of the 1P 28 photomultiplier by a 1P 22 was found advantageous in measurements at 7665 A. Flame photometric measurements were carried out with an “EEL” flame photometer using an air-coal gas flame.

Experimental

procedure

Freshly distilled water taken from a Pyrex-glass still was stored in Polythene containers. It showed no measurable sodium or potassium absorption in the lo-cm flame. Standard solutions were made up by dilution from stock solutions containing 1000 p.p.m. of the metals in the form of their chlorides. All solutions were stored in Polythene bottles. Determination

of sodium

Sodium chloride is apparently completely atomized in the flame, as measurements showed that the absorption of a solution sprayed at a given rate into a [l] [21 [3] [4]

J. J. A. B.

B. WILLIS, S~ectrochim.Acta 16, 259 (1960). B. W1~~1s,i3~ectrochim.Acta 16, 273 (1960). WALSH,S~~C~-K~V~WL Acta 7, 108 (1955). J. RUSSELL, J. P. SHELTON and A. WALSH, Spectrochi~.

551

Acta 8, 317 (1957).

J. R. WILLIS WELS almost independent of the type of flame and of the height, in t#he flame at which the absor@ion was measured. Since sodium occurs at relatively high concentrations in serum (-3500 p.p.m.) and the atomic absorption method is so sensitIive, it is desirable to have a convenient means of reducing the sensitivity to a level at which sodium and potassium can be measured in the same solution and at which sodium. concentrations are high

flame

0

(b)0 (cl0

100 500

50 250 N%

150 750

2 3 I( IO

p.p.m.

Fig. 1. Typical calibration curves for sodium: (a) IO-cm bmner, 5890 A. (b) IO-cm burner at right angles to light path, 5890 A. (c) lo-cm burner, 3302 A.

enough to make risk of accidental contamination negligible. By the methods described below, solutions containing up to 1000 p.p.m. of sodium could be measured: (1) Use of the IO-cm burner with the 5890 if resonance line el~abledmeasurenlel~t of soWions of O-10 p.p.m. (2) By turning the IO-cm burner through SO” the light path was reduced to about 0.5 em and solutions containing up to about 200 p.p.m. could be measured, using the 5890 A line. (3) Still higher concentrations (up to about 1000 p.p.m.) could be measured using the IO-cm burner in the ordinary way but using the second resonance doublet of sodium (4 2P,,,3 2&‘I,2t 4 2PI,2-3 2XIiz; 3302.3, 3302.9 8) for which the oscillator strengths are only O-014 of t,hose for the first resonance doublet at 5S90, 5596 if [5]. Typical calibration curves for the three methods are shown in Fig. 1. [5] A. FILXPOVand W. FROKOFIEF,2. Phystk 56, 458 (1929).

The determination

of met,& in blood serum by atomic absorption spectroscopy-III

The other components of the serum were found not to interfere with the determination of sodium, so that standards containing sodium alone could be used. Table 1 shows that, by use of the three different techniques described above, dilutions of serum from 1: 10 to 1: 500 could be satisfactorily measured in either the air-gas or air-acetylene flame. Table 2 shows the reproducibility of the results obtained by method (2) on six replicate solutions of one serum. While Table

Dilution

)

1 :lO 1 :20 1 : 50 1 : 500

1. Sodium determination: effect of dilution (Xa, mg/lOO ml)

Flame length

Resonance line

(cm)

(8)

10 10 -0.5 10

Serum 1 Air-gas

3302 3302 5890 5890

~

and flame type

Air-acetylene

313 309 303 306

319 311 308 306

Serum 2A -i-li,,s -1

Air-acetylene 341 335 342 343

340 342 344 341

Table

2. Results of replicate determinations of sodium in serum (Serum 2A, 0.125 ml diluted to 5 ml, method 2) Air-gas (mg/lOO ml) ~__~__ 344 349 346 344 348 348 Mean S.d.

346.5 2.2

Air-acetylene (mg/lOO ml) ____

i

: ~

~

/ I

336 341 339 334 340 338 338.0 2.6

these figures include any volumetric errors in making up the solutions they do not show errors in the calibration procedure. Table 3 shows the results of recovery experiments using method (2), while agreement between the results of atomic absorption measurements and flame photometric measurements is shown for a series of sera in Table 4. Determination

of potassium

In contrast to sodium, the degree atomization of potassium in the flame depends markedly on the type of flame used. In general a cool, fairly rich flame is necessary to obtain optimum sensitivity in absorption, as can be seen from Fig. 2, which shows typical calibration curves for (a) an air-coal gas flame, measured just above the zone of unburnt gas, (b) a rich air-acetylene flame, measured at the tip of this zone, and (c) a lean air-acetylene flame, measured several millimetres above this zone. 553 3

J. B.

Table 3.

Serum

Sodium determination: recovery experiments (method 2) Cone. of Na ’ in serum soln. 1 Na added

Flame

(p.p.m.) 1

2A 40 41

WILLIS

Air-gas Air-gas Air-acetylene Air-acetylene Air-gas Air-acetylene Air-gas Air-gas

Total Na (p.p.m.)

~ (p.p.m.)

47.9 47.9 47.8 47.8 51.8 52.2 46.5 34.5

~

1

67.9 87.9 67.8 87.8 91.8 92.2 76.5 62.5

20.0 40.0 20.0 40.0 40.0 40.0 30.0 28.0

Recovery

~ Recovered ~

(p.p.m.) __68.4 89.0 67.2 86.1 95.0 95.7 77.6 61.0

1

!

/ i

(%) 100.7 101.3 99.1 98.1 103.5 103.8 101.4 97.6

~ ~ ~ Av.

Table 4.

Sodium determination: comparison of atomic absorption and flame photometry

100.7

(method 2)

(Na, mg/lOO ml) Serum

Atomic absorption

Flame photometry

Difference __~_.

1 2A 40 41 42 43 44 45 46

310 345 340 353 291 338 324 328 349

309 341 332 345 284 332 321 352

(%)

-

to.3 ~1.8 +2.4 $2.3 t2.5 72.7 -2.4 12.2 -0.9

Investigation of the effect on potassium absorption of the presence of sodium, calcium, magnesium and phosphorus in the proportions in which they occur in serum showed that’ the only measurable effect was a slight enhancement by sodium (Fig. 2). This enhancement was greatest for the lean air-acetylene flame and least for the air-coal gas flame. Though small, it was sufficient to lead to erroneous results if measurements were made on directly diluted serum relative to standards containing potassium alone. Such measurements yielded results which were approximately the same as those obtained by flame photometry, but which varied slightly with dilut’ion and with the type of flame used. Recovery of added potassium varied from 90 to 110 per cent. When a large excess of sodium (1000 p.p.m.) was added to both the serum solutions and standard solutions, results were obtained which were independent of both degree of dilution and type of flame (Table 5), and recovery of added potassium then approximated closely to 100 per cent. Table 6 shows the results of reproducibility t’ests on replicate solutions. 554

The determination of metals in blood serum by atomic absorption spectroscopy-III

06

0.5

0.4

x .C c” s 03 z .o ‘i 0 02

0.1

0

2.5

50

K,

75

Fig. 2. Typical calibration curves for potassium: __ 65 p.p.m.

Table 5.

I

P.p.m

K alone.

----

-

K + Na,

Potassium determination: effect of dilution and flame type (serum 50) (K, mg/lOO ml) Flame

Dilution

____ 1 : 25 1 : 40 1 : 70 1 : 17 (in 1

:

1

:

(in water) (in water) (in water) water with Na 1000 p.p.m.) 25 (in water with Ka 1000 p.p.m.) 50 1000 (inp.p.m.) water wit.h Na

Reference solutions Air-gas

I

__-

I-

~ K alone I K alone K alone K + Na 1000 p.p.m.

~

K + Na 1000 p.p.m. K + Na 1000 p.p.m.

1

15.1

I / I

555

15.4 16.0 17.3 15.4

i I

15.5

)

Air-acetylene (rich)

Air-acetylene (lean)

17.0 17.6 18.7 15.4

17.4 18.2 20.3 15.6

15.1

15.4

15.0

15.5

J. B. WILI.IS

The potassium content of serum may be measured on solutions prepared by direct dilution with water provided that the standards contain sodium at about’ the same concentration at which it occurs in the serum solution [(method (a)]. AlternaCvely, excess sodium may be added to both the serum and reference solutions, and a convenient way of doing this is to dilute the serum with a solution of the disodium salt of ethylene diaminetetracetic acid as was done in Parts I Table 6. Results of replicate determinations of potassium in serum (Serum 2A, 0.5 ml diluted to 10 ml with water*) Air-acetylene

Air-gas

Mean s.d.

(rich)

14.80 14.80 14.65 14.68 15.06

15.68 15.80 15.61 15.40 15.80

14.80 0.16

15.66 0.17

* The difference between the results obtained with the two flames is due to the fact that the reference solutions did not contain any sodium.

and II for the determination of calcium and magnesium [(method (b)]. Table 7 shows t’he results of recovery experiments t’o demonstrate the effectiveness of these two techniques. Table 8 shows the potassium contents of a number of sera measured by the atomic absorption technique and by flame photometry. It will be seen that the agreement between methods (a) t and (b)f is better in absorption than in emission and that on the average the atomic absorption results are slightly lower than those obtained by flame photometry. However, in view of the very high sensitivit.y of the flame photometer reading to small changes in air pressure and t,he difficulty of holding the air pressure perfectly constant, it would be unwise to place too much t With many flame photometers an enhancement of the potassium emission by sodium has been noted [S-S]. This interference is reported to be negligible with the “EEL” flame photometer at the concentrations found in dilute serum solutions [9], and the makers of the instrument state that there is no need to add sodium to the reference solutions in serum potassium determinations [lo]. However, the writer has sometimes found a slight enhancement of the potassium emission by sodium, so that t,he use of sodium-containing standards was considered advisable. The extent of the sodium interference on the emission of potassium serns to be critically dependent on the type of fuel and is least for a low-tetnpernture flame such as air-butane. $ The filter supplied with the “EEL” instrument transmits so little light at 5890 _% that potassium concentrations in the O-10 p.p.m. range can be measured even in the presence of large amounts (1000 p.p.m.) of sodium. [Cl J. fh’ECTOR, Amd. L’hem. 27, 1432 (1955). [7] R. VALENCIA, Bull. sot. chim. bid. 38, 1071 (19.56). [S] H. Ter,ori, C’linicnl FZnwwPhotonretrypp. 51-53. Charles C. Thomas, In] M. PUFFELES and S. E. NESSIM, anrrlyst 32, 467 (1937). [lo] Method Sheet 1X. Evans Elect~roselo&m Ltd., J,ondon.

Springfield,

Ill. (1959).

The determination of metals in blood serum by at,omio absorption spectroscopy---III Table 7.

Potassium determinations:

’

j Serum

Cone. of K in serum

Flame

i

SOltl.

/

~-

I 40

Air-gas Air-acetylene Air-acetylene Air-gas Air-acetylene Air-acetylene Air-gas Air-aeetyiene Air-acetylene

41

50

(p.p.m.)

--

~ , K added

(rich) (lean) (rich) (lean)

Total K

Recovered

@p.m.) I_ (p’p’m’) __t-__-._

(p.p.m.)

Method (a) 2-00 / 3-82 2.00 3.75 2.00 3.85 2.40 3.89 2.40 3.87 2.40 I 3.87 1.50 : 8.55

1.82 1.75 1.85 1.49 1.47 1.47 7.05 7.00 7.15

(rich) (lean)

recovery experiments

1.50

1.50

i I

3.54 3.78 3.92 3.95 3.78 3.95 8.46 8.54 8.67

8.50 8*65

Recovery

i

(%)

II

100.8 101.8 101.5 97,7 102.1 98.9 100.5 100.2

”

j I i Av.

Air-gas Air-acetylene Air-acetylene Air-gas Air-acetylene Air-acetylene Air-gas Air-acetylene Air--acetylene

40

41

50

Method 2.00 ’ 2.00 2.00 2.40 2.40 / 240 4-00 4.00 4.00 i

1.86 1-79 l-93 1.56 1.49 1.62 2.41 2.43 2.37

(rich) (lean} (rich) (lean) (rich) (lean)

(b) 3.86 3.79 3.93 3.95 3.89 4.02 6.41 6.43 6.37

3.84 3.80 4.00 3.99 3.87 4.16 6.33 6.24 6.15

/ Table 8. Pot~assium ~let~~in~tion:

Serum

2A 40 42 43 44 45 46 47 48 49 50

/ j 1 ~ ;

1 / /

100.4 99.5 100.3 101.8 101.0

99.5 /

103.5 98.8 97.0 96.5

/ / I ___ i Av. 99.8

comparison of atomic absorption a,nd flame photometry (K, mg/lOO ml)

j/ Atomic absorption ! (b) :_..‘“‘: 15.1 -Pj___FP-14.4 13.1 21.5 22.1 18.9 19.6 18.2 19.0 15.1 15.6 15.8

100.5

13.5 22.0 22.7 18.9 20-2 18.6 19.4 15.4 16-2 14.6

/ Flame photometry j

:

/ t 1 ’ 1

(a)

(b)

15.1 13.0 22.5 23.0 19.7 19.5 18.7 19.0 15.7 16.8 15.8

15-l 14.2 22.1 23.2 19*3 20.7 19.5 20.0 16.0 17.2 16.6

Method (a) 0.5 ml serum diluted to 25 ml with water. Reference solutions: K + K?;a65 p.p.m. Method (b) 0.5 ml serum diluted to 25 ml with EDTA solution (10,000 ppm.). Reference solutions: K i- EDTA 10,000 p.p.m. 557

J. B. WILLIS

reliance on the flame-photometer absorption method. Determination

result,s in assessing the accuracy

of the atomic

of several metals in the same solution

If it is desired to measure the sodium and pot’assium contents of serum on the same solution, this may be done by diluting the serum twenty- to fifty-fold wit,h water and measuring sodium by method (2) or (3) and potassium by method (a). Potassium, calcium and magnesium may all be measured on a solution prepared by diluting the serum twenty-fold with EDTA solution (10,000 p.p.m.) and measuring the absorptions relative to standards containing the same concentration of EDTA.

Conclusions The atomic absorption method applied to the determinatfion of sodium and potassium in blood serum gives results which agree with those obtained with the flame photometer. The amount of serum required and the time t’aken are about the same in the two methods. Thus, if calcium and magnesium estimations are being carried out with an at’omic absorption spectrophotometer, sodium and potassium determinations may with advantage be made on the same instrument. Acknowledgements-The writer is indebted to Dr. T. F. NEALES, of the Botany Department, University of Melbourne, for the use of the flame photometer, and to Dr. SARA WEIDEN for the samples of serum.

558