The effect of the atomiser on the estimation of magnesium by atomic absorption spectrophotometry

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CLINICA CHIMICA ACTA

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COMMUNICATIONS

The effect of the atomiser on the estimation of magnesium by atomic absorption spectrophotometry Although atomic absorption spectrophotometry is a relatively new method it is apparent that differences are found, both in the reported “normal range” for serum magnesium’, and in the type and extent of various interfering factor+8. Previously our method’ gave a mean normal serum magnesium of 1.74 f 0.11 mequiv/l in IOO blood donors. Recently our burner, atomiser and lamp were replaced by the Zeiss FA-I atomic absorption attachment. Because of the likelihood of different interfering factors with the new system, all interference studies were repeated and a further series of normal blood samples was measured. In this communication our results are reported. Instrumental

data

The burner is supplied with oxygen at 0.3 kg/cm2 and acetylene at 150 mm H,O and the sample is aspirated directly into the flame. Standard instrumental settings are: wavelength (established by maximum absorption) 285.2 rnp, slit-width 0.05 mm, recorder gain position o. The hollow-cathode lamp is supplied with IO mA a.c. By raising the acetylene pressure from IOOmm H,O to 200 mm H,O sensitivity was increased. The limit was not reached even at 200 mm HzO, but pressure was not increased further due to white coloration at the base of the flame. Raising the oxygen pressure reduced sensitivity slightly. Raising the height of the burner and lengthening the cannula both decreased absorption. Organic solvents in the aspirated solution increased absorption, the range investigated being limited by flame instability. Chloride, sulphate and phosphate ions added as acids had no significant effect on absorption. The addition of increasing concentrations of sodium and potassium ions (as chlorides), either alone or in combination, led to an initial decrease and subsequent increase in absorption. This effect was masked in the presence of calcium, which itself significantly increased absorption over the O-IO mequiv/l range. At higher calcium concentrations the further increase in absorption was minimal and it was decided to add calcium (IO mequiv/l) to all standards and samples. At this concentration of calcium increasing concentrations of sodium and potassium had no significant effect. The stock standard solution was prepared from spectrographically standardised magnesium crystals*. Standard curves (Fig, I) cover the range 0-0.4 mequiv/l and are linear, differing marginally from day to day, since the burner has to be removed for cleaning and the optimum position re-established. Direct I : IO dilution of serum with water gave results 12% too high. Magnesium * Johnson Matthey

and Co., Ltd.,

London. Cbn. Chim. Acta,

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(1966)

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was therefore measured after precipitation of protein with trichloroacetic acid (final TCA concentration in samples and standards 4%). Mean recovery of magnesium was 99.2%. Serum from eighty blood donors was measured for magnesium concentration twice in duplicate. The mean magnesium concentration (Fig. z) was 1.61 mequiv/l (SD. f 0.08) with no significant age or sex difference. With urine deproteinisation is not required and magnesium concentration was estimated after direct I: 25 or I : 50 dilution. Rilean recovery of added magnesium from seven urines each estimated in triplicate was 99.60/b. 30 0.3

STANLMRD CURVES oxygen pRswn a3 kg/cm= Acetylene pawn 150 mmH@

0

0.1 0.2 a3 Magnesium concentration

MptlO

mcquidl +4%TCA

a4 mcquiv/l

Fig. I. Typical working standard curves, with tion. Fig. 2. Distribution population.

Ca

0.3

BY ATOMIC ABSORPTION mean end SD. lbl?QO6 mequiv/l

1.4 Serum

w L 1.6 magnesium

I

f 1.8

t

mquiv/l

TCA for serum and without TCA for urine estima-

of serum magnesium concentrations

in 80 blood donors, representing a normal

Comments Our main aim was to improve sensitivity of measurement as much as possible. With this instrument the light-path passes through the flame at a fixed height above the burner. Higher acetylene pressure increased flame temperature and also enlarged the size of the flame thus altering the relative position of the light-path. It is difhcult to estimate which change had the greater influence on sensitivity. In contrast to emission photometry, flame temperature is relatively unimportant in absorption techniquess. Only the ground-state atoms absorb energy at 285.2 rnp and increasing flame temperature increases the number of excited atoms without significantly reducing the large majority of atoms which are in the ground state. However with the direct aspirating system, a hotter flame results in more efficient atomisation which increases the number of ground-state atoms dispersed in the Aame2. Since raising the height of the burner decreased sensitivity we concluded that the increase caused by higher acetylene pressure was due more to the flame temperature effects than to any change in the light-path/flame-zone relationship. Changes in acetylene pressure had no effect on the flow rate of sample. Lengthening the cannula by 7.5 cm with tubing lowered the flow rate of sample from 2.7 to 1.2 ml/min, and caused a 30% decrease in absorbance presumably because less solute reached the flame per unit of time. On the other hand sensitivity was not enhanced, but actually lessened, when the flow rate of sample was increased by raising oxygen pressure, possibly because the increase in relative oxygen content of the

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flame inhibited the dissociation of magnesium oxide into magnesium atoms2. TCA improved sensitivity without altering flow rate. Organic solvents, known to increase sensitivity in both spray-chamber and direct aspiration systemsg, exerted their characteristic effect despite a decrease in flow rate of sample, by increasing flame temperature, thus producing more efficient vaporisation of spray droplets@. The sensitivity of different instruments can be compared by quoting the aqueous concentration of ion required to give 1% absorption. Willis quotes a figure of 0.01 mg/l for magnesiuma. Our corresponding value is 0.10 mg/l at an acetylene pressure of 150 mm H,O. The addition of calcium and TCA increased sensitivity further but serum dilutions greater than I : IO were unpractical due to the comparatively low sensitivity of the instrument. The burner appears to be responsible for this limitation. The use of high concentrations of one ion to overcome other chemical interferences, either by competition or repression of ionisations is well known. The absence of the sodium and potassium interference in the presence of added calcium is probably a reflection of the above mechanisms, resulting in fewer interfering monovalent cations. The considerably increased absorption which calcium itself causes, over and above any effect on other interfering factors, may also be due to its effect on the dissociation of magnesium compounds in the flame. In our experience this has occurred with both direct and indirect atomising systems. It can be seen (Table I) that there are wide variations in the interferences and protein effects found by different investigators. Similar variations are known in emission studieslo. The dilution factor and also the relative efficiencies of different burners and atomisers contribute to these variations. The mean normal serum magnesium concentration obtained with the present system (significantly lower than found previouslyl: P < o.ooo5) nevertheless falls within the lower range of normals reported by othersl. On considering Table I it is not surprising that reports of mean normal magnesium concentrations in serum vary. We consider that the common use of the word “atomiser” is misleading. Since it is the heat of the flame which finally atomisess, while the atomiser merely supplies to the flame part of the solution as a fine spray, we suggest that the term “aspirator” would be more appropriate, especially in direct systems such as the Zeiss and Beckman burners. TABLE PATTERN

I OF

INTERFERENCES

Authors

Year of publication

Allan p David3 Dawson and Heaton Horn and Latners Sunderman and Carroll6 Jones and Thomas’ Willis8 Stewart et al.’ Present report N = no effect

1958 1960 1961 1963 196.5 1965 1965 1963 1965

Type of flame Air-AC.* Air-AC. Air-AC. Air-propane Air-H, Air-propane Air-AC. Air-AC. O,-AC.

I = increased absorption

Interferences-Effect

on magnesium absorption

Protein

Phosphate

Sulphate

Na

K

Ca

-

N D N N D N N N

N D

N

N

N

N N N D N N D-+1

N N N

N N

I N N D+I

r N I I

I N D I D I

D = decreased absorption

N N D N

*AC. = acetylene

C&n. Chim. Ada,

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In conclusion, our experience emphasises the need for each new investigator using atomic absorption to examine the effect of many possible interfering factors with his own instrument; potentially these can be widely variable, depending on the type of burner and aspirator used. De#wtment of Medicine, Uhversity of St. Andrew, Queen’s ~~~~ge, ~~~~ee ~G~eutBritain) I 2 3 4 5 6 7 8 9 IO

LAURA

W.

FLEMING

w. K. STEWART

W. K. STEWART, F. HUTCHINSON AND L. W. FLEMING, J. Lab. Clin. Med., 61 (1963) 858. J. E. ALLAN, Analyst, 83 (1958) 466. D. J. DAVID, Analyst, 85 (1960) 495. J. B. DAWSON AND F. W. HEATON,BGX~~~. J., 80 (196~) 99. D. B. HORN AND A. L. LATNER, Clin. Chim. Acta, 8 (1963) 974. F. W. SUNDERMAN AND J. E. CARROLL, Am. J. C&n. Pathol., 43 (1965) 302. D. I. H. JONES AND T. A. THor+fas, Hilger J., g (1965) 39. J. B. WILLIS, CEk. Chem., I I (1965) 251. J. E. ALLAN, Spectrochim. Acta, 18 (1962) 605. A. M. VAN LEEUWEN, J. J. VAN DAATSELAAR AND L. A. DE VRIES, Lancet, ii (1963) 94.

Received November 8th, 1965

Association Waldenstriim

de I’lgM

avec I’albumine

dans certains

cas de maladie de

Les immuno~lectrophor~ses de s&rums de huit malades atteints de macroglobulinemie de Waldenstrom ont montre des particular&% supplementaires a celles deja connues permettant le diagnostic de I’affection. Ces particularites concernent le trait de precipitation anticorps-antigene de l’albumine qui dessine une double ou une triple courbure, les arcs cathodiyues &ant en regard du precipite d’IgM (Figs. I et 3).

Fig. 1. Immunotilectrophoreses avec un immunserum anti-humain normal: (a) serum G(maladie de Waldenstrom) dilue 1/2; (b) serum normal.

Des chromato~aphies sur gel de Sephadex G 200 suivies ~immuno~le~trophor&es ont 4th effect&es avec ces serums. Elles ont apporte la preuve que fes protCines lourdes eluees dans le premier pit: /LlipoprotCine, ar,-macroglobuline et IgM sont accompagnees d’une quantite appreciable d’albumine donnant un trait de precipitation specifique distinct des autres traits (Fig. 2). Les ultracentrifugations analytiques de ces mCmes huit serums, effect&es en CZiE. Chim. Acta, 14 (1966) 134-136