The estimation of mercury in urine using di-beta-naphthylthiocarbazone

VOL.

CLINICA CHIMICA ACT.4

80

THE

ESTIMATION

OF

MERCURY

IN

URINE

1 (1956)

USING

DI-BETA-NAPHTHYLTHIOCARBAZONE

H. LEACH, Gvoup

Laboratory,

Caernarvon

E. G. EVANS

and Anglesey

AND

W.

R. C. CRIMMIN

General Hospital,

and Department

University College of North Wales, Bangor, Caernarvonshire

of Chemistry,

(Great Britain)

The use of di-beta-naphthylthiocarbazone (DBN) as a reagent for the estimation of heavy metals was first suggested by the work of SUPRUNOVICH1 who found it to be analogous to dithizone in its behaviour. HUBBARD 2 described the use of DBN for the calorimetric determination of urinary mercury. He found that the mercury-DBN complex was much more stable than the corresponding dithizone complex, which is light-sensitive. This method was modified by CHOLAK AND HUBBARD 3 who extended it to other biological fluids and tissues. This modified procedure has been successfully employed by the present authors but, during the routine estimation of urinary mercury during the past six years, certain points have emerged indicating that the method of CHOLAK AND HUBBARD may be simplified and that certain of their findings should be qualified to some extent. Briefly, their method involved the wet-oxidation of urine, decolorisation with hydroxylamine, and shaking out the digested urine with a chloroform solution of DBN. The chloroform layer, containing complexed mercury and copper, the latter always being present to a greater or lesser extent in normal urine, was then extracted with an acidified solution of sodium thiosulphate, which, according to CHOLAK AND HUBBARD, preferentially extracted the mercury, leaving the copper in the nonaqueous layer. The aqueous phase was then boiled up with potassium permanganate, decolorised and again extracted with DBN and the absorbancy measured. The present authors felt that for large scale estimations of urinary mercury efficient screening could be obtained by a simple reversion technique and so eliminate the time-consuming second extraction with its concomitant risk of loss of material. This reversion method involved reading the absorbancy of the first extraction with DBN and then adding acidified sodium thiosulphate solution, shaking out and reading the absorbancy of the now reverted reagent. If there was no difference in the optical densities it was assumed that mercury was absent. Any difference in the absorbancies would be due to mercury alone since the copper was not supposed to be extracted by thiosulphate. This reversion procedure was used successfully giving perfectly consistent results. Fig. I shows a plot of the differential optical densities against amounts of mercury. In the course of further experiments however, it was found that the sodium thiosulphate solution does not extract the mercury preferentially but in fact breaks up the copper complex also. The pH of the thiosulphate solution must be greater than 3 before the copper-DBN complex is unaffected and at such a high pH the mercury References p. 84

VOL.

ESTIMATION

1 (1956)

itself is not extracted any

experimental

into the aqueous

data

concerning

81

OF MERCURY IN URINE CHOLAK AND HUBBARD

phase.

the thiosulphate

extractions,

do not record

but the conditions

518 m/u

Fig. I. Curve a - 20 mg DBN/I. Molar E = 43,000 Curve b - 30 mg DBN/I. Molar E = 23.000

which they specify have been followed exactly. It may be noted at this point that the use of acidified sodium thiosulphate for the preferential extraction of mercury from

a

copper

mixture

of

complex

mercury

formed

The problem

with

of

towards

both

in urine

in amounts

ranging

examine

the

to which

the

CHOLAK

AND

mercury extent

maximum of which

interfere

in the estimate

HUBBARD.

in

are recorded

from

lf

read on a Unicam

consistent

reagent

the latter

must

interferes

were

in Table

of urinary

the very the

used.

and The

I, indicated

* but

results obtained

has a comparable interfere

the

because

amounts

of

results quite

of

clearly

it is present

copper this

by the

sensitivity

It was therefore

SP joo Spectrophotometer

decided

to

in excess

examination,

that copper

did not

15

0

1.5

40 20

49 0

40 0

40

at 518 my in I cm cells.

I Density

36 39 5 5

successful

mercury.

T.4BLE

20 20

is quite

less stable.

0.04 to 0.52 mg/litre.s

copper urine

dithizonates

is considerably

and copper,

found

samples

All densities

copper

then arose of explaining

method

of

and DBN

6.77 0.78 0.86 0.86 1.03 I.03 6.595 6.595

Reverted density 0.52 0.52 0.52 0.52

0.52 0.52

0.52 0.52

H. LEACH, E. G. EVANS, W. R. C. CRIMMIN

82

VOL.

1 (1956)

The results given in Table I were obtained using a solution containing 20 mg of DBN per litre. Sample results obtained on urines containing added mercury and copper are recorded in Table II. The reagent used by CHOLAK AND HUBBARD had been twice purified 8 and had a molecular extinction of 42,074 at 645 rnp. The yield of twice purified DBN is of the order of 200 mg per gram of crude product. Such a degree of purification would tend to make the cost of the reagent prohibitive. The present authors have found that for practical purposes there is no reason why the once purified product should not be used though there is some loss of sensitivity. Fig. I shows the plot obtained with the TABLE Hg ~g

Cu Pg

added

Urine

Demity

added

Reverted density

A 0 0

0.75 0.75

0.5’ O.jI O.j2

0.55

0.52

30

0.55

0.52 0.52 0.52

I5

0 0

15

40

0.55 0.78 0.78

0 IO IO Urine

II

30

0.58

B 0

0 0

25 ‘5 0

30

0.96 0.98

uriwe c 0

0.52 0.52 0.52

twice repurified reagent (a) and the once purified form (b). Though a higher concentration of once purified DBN is used the yield is much higher with consequent economy. Further, if large numbers of specimens are being examined, the reverted reagent can be re-used. If this is done the reverts are pooled and ethyl alcohol added toa concentration of 1%. It should be re-used on the same day. lMethod of purification of DBN

after

HUBBARD AND SCOTT @

Crude DBN is dissolved in purified chloroform (I g per roe ml). The chloroform solution is gently heated and washed with three portions of 25 ml of distilled water. The chloroform solution is filtered into a beaker and after evaporation to IO ml, 50 ml of chilled absolute alcohol are added. It is allowed to precipitate in the refrigerator for I hour, filtered at the pump and the precipitate washed with one lot of IO ml of alcohol. The residue is dried in air. The molar extinction is about 25,000 at 645 mp. For further purification redissolve in chloroform, evaporate to IO ml and reprecipitate with chilled alcohol as above. Method

of estimation

(a) Reagents 50%

&SO,

l2efcremes

p. 84

AnalaR KMnO, 50% aqueous hydroxylamine hydrochloride 0.1 N Na&O, Chloroform Di-/?-naphthylthiocarbazone (twice purified) 20 mg/litre in CHCl, ( once purified) 30 mg/litre

VOL.

1 (1956)

ESTIMATION

OF MERCURY

IN URINE

83

The chloroform is purified by shaking I litre of AnalaR chloroform with roe ml of r% hydroxylamine hydrochloride which has been neutralized with dilute ammonia, and filtering the chloroform through a thick paper to remove traces of water. Ethyl alcohol to a concentration of I O/eis then added. In the early stages of the work di-/I-naphthylthiocarbazone was synthesized according to the method of HUBBARD AND SCOTT. The reagent is now available from Eastman-Kodak (Eastman Organic Chemical P 4834). This requires purification as discussed above. (b) Procedure 50 ml of urine are digested with ro ml of 50% sulphuric acid and about 0.3 g of potassium permanganate under reflux. As the permanganate colour fades, further small quantities of permanganate are added until the colour persists for five minutes or until a total of about r.5 g has been added. (Compressed tablets of 0.3 g are very convenient.) The digest is cooled and 50% hydroxylamine added until the mixture is colourless. A further I ml of the hydroxylamine is added and the solution brought to the boil. It is then cooled and after dilution to about IOO ml extracted with 20 ml of the chloroform reagent. Should the reagent layer turn bright red, a further LO ml of reagent are added and the mixture reshaken. The separating funnel is now allowed to stand for a few minutes to allow the layers to separate fully and a portion of the reagent layer is drawn off for photometry. Care must be taken to avoid turbidity in the reagent and it is sometimes necessary to remove the reagent to a tube containing a little anhydrous sodium sulphate to remove traces of water. The density of the reagent layer is read in a suitable photometer at’ 515-520 m,u and then returned to the funnel. 5 ml of 0.1 N sodium thiosulphate are added and the mixture shaken vigorously to return the mercury to the aqueous phase. The reagent layer is again drawn ofI and the density read. The difference between the two densities is referred to the calibration curve. _A 50 ml portion of distilled water or better, mercury free urine is wet-ashed concurrently to provide a blank for traces of mercury which are always present in the potassium permanganate. The calibration curve is constructed with amounts of mercury from 5-40 pg in roo ml of water containing 5 ml 50% sulphuric acid and I ml of 50% hydroxylamine hydrochloride. These are each extracted with 20 ml of reagent. Densities are taken before and after thiosulphate reversion. The differences are plotted on the graph. For quantities of mercury between o and 5pg a dilute reagent containing 5 mg DBS per litre is used and the resultant extracts read in 4 cm ceels TABLE REC~VI~RY

EXPERIMENTSori

MERCURY

Mercury added pclg

III ADDED

To

Mercury

50 ml PORTIONS0s URINE

recovered .w

4.75 4.75 9.0 9.6 9.7 15.3 20.0 19.8 24.5 24.2 27.6

5 5

IO IO IO

15 20 20 25

25 28

SUMMARY

The use of an analogue of dithizone, di-p-naphthylthiocarbazone, for the determination of urinary mercury is discussed. Its use is satisfactory in the presence of amounts of copper greater than are found normally in urine. A simple and rapid method is described for the determination of mercury in urine without repeated transfer of the mercury. References p. 84

H. LEACH,

84 L’emploi

E. G. EVANS,

de la di-/3-naphtylthiocarbazone

cure dans l’urine, normalement

en prCsence

dans l’urine.

a Ctk examinit

de teneurs

Une

mkthode

VOL.

1 (1956)

pour le dosage

du mer-

W. R. C. CRIMMIN

en cuivre simple

plus fortes

et rapide

que celles prksentes

est d&rite.

ZUSAMMENFASSUNG

Durch

Verwendung

in Gegenwart der Harn genz

von

mit

durch

von

Di$-Naphtylthiocarbazon

vie1 Kupfer

quantitativ

Kaliumpermanganat

Hydroxylamin

verascht

und

ist es miiglich,

fehlerfrei

worden

Quecksilber

zu bestimmen.

ist, wird

der tiberschul3

Nachdem an Rea-

entfernt.

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MOWBOfi

REFERENCES

I. B. SUPRUNOVITCH, J. Gen. Chem. (U.S.S.R.), 8 (1938) 839. D. M. HUBBARD, Ind. Eng. Chem., Anal. Ed., 12 (1940) 768. J. CHOLAK AND D. M. HUBBARD, ibid., r8 (1946) 149. and Tentative Methods of Analysis, Association Oficial Agricultural Chemists, Oficial Association, Wahington, D.C., 6th Edn., 1945, p. 470. J. A. KOLMER, Clinical Diagnosis by Laboratory. Examinations, hppleton-Century-Crofts, New York, 1949, rgz. D. M. HUBBARD AND E. W. SCOTT, J. Am. Chem. Ser., 65 (1943) 2390. Received

July

The Inc.,

6th,r955