586
ANALYTICAL CHEMISTRY
Table I.
Sugar Content I-ersus -4bsorbance in hnalyses of a Smyrna Type Tobacco
Sample Wt.,
Grams
15.83 7.26 8.01 13.5R 16.46 7.14
lloisture.
Glucose,
79.2 51.8 46.4 68.7 56.3 29.1
1.203 4.298 1 917 2.790 2.877 5.731
7%
%
___
wt, cn,, Gram 0.0405 0.0801 0.0402 0,0970 0.1202 0.1086
Abior.l~aiice . K t . CII. 0 970 1.061 1.107 0,098 1.002 1,002 ~.
DISCUSSION
The precision of the modification, if absorbances greatei than 0.1 are used, is the same as that obtainable in other specxtrophotometric measurements-namely, between 0.5 and 1.0% of the copper present. The results obtained by use of the procedure for some 200 sugai analyses are shown in Figure 1, in which the ratio of the observed absorbance to the weight of copper is plotted against observed absorbance. The data for this figure were obtained by cat r\-ing through the usual heating and weighing procedures, followed by dissolving the copper as previously described. The sratter of points falling below 0.1 absorbance is largely caused by inability to determine precisely the absorbance of the solutions examined. Figure 1 reflects errors in Lveights, composition of precipitate. ctc., as well as errors in the absorbance measurements. The rveight of copper, equivalent to the cuprous oxide i n the
1Iuiison arid Walker method, is roughly twice the weight of glu(‘ose in the aliquot analyzed. By following the procedure described above, one obtains copper-ethylenediamine solutions whose absorbances are essentially equal to the weight of copper precipitated from the Fehling’s solution. I t is possible to avoid absorbances below 0.10 if the weight of the sample of tobacco is cahosen such that the sugar present in the aliquot used for the cuprous oxide precipitation exceeds 50 mg. Sample results given in Table I of some sugar analyses of a Smyrna-type tobacco illustrate the relation between sugar content and absorbance. The percentages of glucose were calculated using one eighth of the recorded sample weights. as this was the size of aliquot analyzed. Glucose percentages are given on a “wet” basis. The choice of wave length for observation and the particular dilution values used were based on a detailed study of the copperethylenediamine system for analytical use by Harris (5) in thip lalmratory. LITERATURE CITED
(1) . h o c . Offic. Agr. Chemists, ”Official Xethods of Analysis,” 7th ed., p. 506, 29.38, 1980. (2) Darkis, F. R., Hackney, E. J.,and Gross, P. >I., Ind. Eng. Chenz., 39, 1631 (1947). (3) Harris, J. T., RIA. thesis, Duke University, Durham, X. C.. 1949. (4) Hogness, T. R., Zscheile, F. P., and Sidwell, A. E., J . P h y s . Chern., 41, 379 (1937). ( 5 ) Nunson, L. S., and Walker. P. H., J . A m . Chem. Soc., 28, 663 (1906): 29, 541 (1907). RECEIVED for review Ailgrist 2 1 , 19.53. Arcepted November 5 , 1953
Determination of Radioactive Gold in Biological Tissue LOUIS C. WEISS, ARTHUR W. STEERS, and HOWARD M. BOLLINGER U. S. Department of Health, Education, and Welfare, Food and Drug Administration, Lor TUDIES
by Xolan, Jones, and Keil (?)on theparametrialinjection
S of colloidal radioactive gold (gold-198) in monkeys and later
in dogs necessitated the development of a method for recovering the gold from gross amounts of various tissues. Block (1, Z), Jamieson ( b ) ,and Natelson (6) have reported methods of estimating inactive gold (gold-197) in biological materials by colorimetric procedures. Dunn ( 3 ) has proposed a method for determining radiogold by measuring the activity with a Geiger-Muller counter after electroplating together with added inactive carrier gold. The foregoing all employed wet ashing techniques. Ely ( 4 ) estimated the amount of radiogold by use of a Geiger-Muller counter on the dry ash of a small sample of tiswe. These methods are applicable only where small amounts (up to 10 grams) of tissue are to be analyzed. When larger animals are involved in the experiments, these methods are not feasible because of the difficulty of reducing large samples of tissue. I t was not considered practical to attempt to grind and subdivide large organs or masses of excised tissue because of the problems involved in obtaining a homogeneous sample and in decontaminating the grinding equipment. I n addition, the total radioactivity of some samples may be so low that the entire gold content must be recovered in order to obtain a satisfactory counting rate. Dry ashing of tissue in amounts in exceSs of about 25 grams is inconvenient and time-consuming
Angeles, Calif,
poiated liith the digests. This entire procsedure consumed a great deal of time and required very careful manipulation. I t was then decided to attempt to recover the gold from a hydrochloric acid hydrolyzate of tissue by treatment with activated carbon. The literature contains many references to the use of carbon in the recovery of gold from very dilute solutions. 1Ioqt of these are concerned with the treatment of mine or plating-works waste and some propose to treat sea water for its gold content. It was felt that this approach was especially promising inasmuch as the bulk of the organic material could be filtered off in solution and only a relatively small amount of carbon would remain to be ashed. This proved to be the case. rlfter preliminary experiments had indicated the practicability of the method, three series of recovery experiments were carried out a t intervals as radiogold was available. The gold was received in the form of a colloidal suspension of the metal. I n the first series the colloid was dissolved in aqua regia, diluted to a known volume with 10% hydrochloric acid, and added in varying amounts to samples of beef or pork liver. In the second series the colloid was diluted with distilled water and added to the samples in this form. I n the third series the gold was added as the colloid in some cases and as the chloroaurate in others. APPARATUS
Counting Plates. Ordinary 40-mm. watch glasses must be EXPERIMENTAL
Methods of wet ashing using sulfuric acid and nitric acid were investigated. Successful recovery of added radiogold was obtained, but careful scrubbing of all evolved gases and other volatile substances was necessary. Substantial amounts of gold were found in the scrubber solutions and distilled fat and were ieinror-
carefully selected for uniform depth and shape. This precaution is necessary because the “geometry” of the counter plate-GeigerMuller tube assembly must be as reproducible as practicable. Before re-use, the plates should be warmed in aqua regia, rinsed, dipped in alkaline potassium cyanide solution, and allowed to stand in air for about half an hour before final rinsing and drying. .4 check for residual radioactivity should then be made with the counting apparatus or a survey meter.
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4 Table I.
Recovery of Added Radiogold (AuI9*) from Biological Tissue
Sarnple
Wt.,
So. 17" 18" 19s
Grams 84 100 100
ion
20: 21
100 100
50
ion
in0 100 100 100 100 100 1 on 100 100 100 50 10
38 39 GO fil 62
63 in:!
9n
on
103"
90
104'1 10: 1OG inin
u .iu198
-
10 10
10
587
Au'gi Added,
Act i \.a ted Carbon Added. Grains 2 .i
Mg.
20 2.5 20 20 20
2 . .j 2 . .5 2.5 2.3 2 . .i 2 .3 2.5 2 3 2 5 2 , .i 2 .3
2.5 0 1 0 2 5 0 2 5 1 5 2 5 0 2 5
51.2
1 . .i
2.5
n
6.5
2 5
2 6 c7
Au 19s .idded, Microcuries 25.6 2.56 76.8 25.6
1 .i 0 : 0 5
j
2.5
n
Au 198 Found, Microcuries 25 8 2.46 73.9 25 1 51.2
o 202 202 213 212 5-26 342 I055 208 97.7 208 208 206 208 210
RESULTS Recovery.
='c
100 96.1 96.2 98.0 95.1 100 100 I 0.i
104 104 107 t n4
103 96 7 1O R 103
102 103 in4
140
73 5
0 0 0 0 0
data the microcurie factor is calculated by which the counts per minute of the samples are translated into microcuries.
144
143 75 1 20.7
added as chloroaurate .
Geiger-Muller Counter. .4thin-window GLI tube was mounted i n a heavy lead chamber. The counter plates were admitted to the chamber by means of a slidirig tray which uniformly positioned them under the tube. Counting was done bj- a L I o d ~ l GS 4 scaler, manufactured by Technical Associates, Glendalr, Calif. This instrument has a scale of 64. Pipets. Retested 1-ml. Mohr-type (graduated) pipets u c r ~ used to deliver sample aliquots onto the counting plates. Decaontamination and testing procedures similar to those descri1)rd tor the counting plates must be followed prior to re-use. PROCEDURE
M-eigh the sample, cut into approximately 0.5-inch cubes, and 1)lace in an Erlenmeyer flask of appropriate size to accommodate :it least five times the volume of the sample. Add 1 ml. of an aqua regia solution containing 2.5 mg. of inactive gold. Add a volume of concentrated hydrochloric acid equal to the volume of the sample and, in any case, sufficient to cover the tissue. Weigh the flask and place in a boiling water bath for 3 hours, agitating from time to time.Cool to about 60 C., dilute vAh an equal volume of water, and add 2.5 grams of activated carbon (Korit A or equivalent). Return the flask to the boiling XTater bath and continue heating foi an additional half hour with frequent swirling. Cool to room temperature and filter through a rapid, fluted filter paper. !T'ash the residue on the paper with 10% hydrochloric acid until the xvashings are nearly colorless. When the filter has drained completely, transfer it to a 100-ml. 1)orosilicate or 96% silica glass evaporating dish and place in a cold muffle furnace. Heat the furnace to 600" C. and ash until little or no carbon remains. Leach the residue repeatedly vith small portions of aqua regia, warming the dish with each portion. If sufficient radioactivity is present to permit the counting of a small aliquot of the sample, the leachings can be transferred t o a volumetric flask of suitable size and diluted to volume with 10% hydrochloric acid. The small amount of insoluble residue nred not be filtered off, since i t will rapidly settle to the bottom of the1 flask. Place an accurately measured aliquot, of appropriate size to give the desired counting rate, on a 40-mm. counting plate. The aliquot should be placed on the plate in multiples of 0.3-ml. volumes or less. Each portion should be gently evaporated to dryness before addition of the following one so that the aliquot i. drposited in a minimum area. Determine the radioactivity a i t h it Geiger-Muller counter. If the entire amount of radiogold is needed for counting, leaching should be done with four or five 0.5-ml. portions of aqua regia and each one filtered with suction through a micro filtei tube with a fritted glass disk into a 15-ml. centrifuge cone. Successive 0.30-ml. portions of this solution are evaporated on the counting plate until the entire quantity has been deposited. The radiogold solutions used for injection are standardized by diluting, "plating," and counting i n the same way. From these
In the range from 0.2 to 1000 microcuries of added radiogold the foregoing procedure gave 96 to 107% recoveries. Table I contains the data obtained in three series of recover?. experiments. DISCUSSION
The aliquots of sample solution were evaporated in the center of the counter plates so as to produce as uniform circular areas as practicable, This is essential to obtain reproducible results. An excessive concentration of carrier gold was avoided and aliquots of sample solutions were selected to minimize the self-absorption of the carrier gold and inert salt deposits. The addition of carrier gold may not be necessary under certain conditions: when large samples of tissue are tieing handled and the gold-198 is present as the colloid, or when, regardless of the sample size or the state of the gold-198, act'ivated carbon i$ present. S o advantage results from the addition of more than the 2.5 mg. of carrier gold specified. Good recoveries were obtained without the addition of activated carbon with both large and small samples when the radiogold \vas present in the colloidal date, but recoveries were poor n-hen the radiogold was added as the chloroaurate. Filtrations were slow without the carbon in the case of the large samples. The carbon apparently serves a t least two purposes. I t acts not only to reduce the ionic gold and probably to adsorb it but also as a filter aid. Time has not been available to investigate the cause of the rather consistent recoveries of over 100% in the series of samples 50-63. The specific activity of the radiogold was not furnished by the supplier and no attempt was made to determine it. Residue.; after ashing were very small and in general were not treighed. I n a series of eight determinations where the aah was weighed, the range was from 11 to 58 mg. per 100 grams of tissue. The method has been applied to the analypis of various organs 2nd injection sites in rhesus monkeys and dogs. S o difficulties ir-ere encountered with samples ranging from 0.3 to 532 grams and from lung tissue to bone and cartilage. The method offers the advantages of extreme simplicity of technique, minimum equipment requirements, and wide range of sample aize. The accuracy is adequate for most biological invedgations. ACKNOWLEDGMENT
Thi3 work was done in the laboratory of and with the aid of funds supplied by the Albert Soiland Cancer Foundation of Loe Angeles, Calif. The authors are indebted to Russell H. Sei]], physicist for the Albert Soiland Cancer Foundation, for valuable assistance in the radioactivity measurements for these studies. Permission to engage in this work was granted the aut'hors by the U. S. Food and Drug Administrat'ion. LITERATURE CITED B u c h a n a n , 0. H., J . Biol. Chem., 136, 379 (1940). Block, K. D., and B u c h a n a n , 0. H.. J . Lab. Clin. Men'., 28, 118 (1942). D u n n , R. W., Ibid., 33, 1169-76 (1948). E l y , J. O., J . Franklin rnst.. 230, 125 (1940). Jamieson, A. R., a n d W'atson, R. S.. Analyst, 63, 702 (1938). S a t e l s o n , S., and Zuckerman, J. L., ANAL.(?HEM., 23, 653-5 (1951). S o l a n , J. F., Jones, E. G., and Neil, R. H.. Am. J . Roentgen. R a d i u m Therapy .Tiidear X e d . , 69, 805-12 (1953).
(1) Block.
(2) (3) (4) (8)
(G)
(7)
W. D., and
R E C B I Y Efor D review March 30, 1953. Accepted December 16, 1953. Presented hefore the Division of Analytical Chemistry a t the 123rd Meeting of tlie . \ X E R I C . A N CHEMICAL SOCIETY. Los hngeles, Calif., March 1953.
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