V O L U M E 25, NO. 6, J U N E 1 9 5 3
987
storage. The standard deviations indicate very good reproducibility. The coefficient of variation of this series is 2.2%. -4previous communication ( 2 ) demonstrated the precision and sensitivity attained when these cells are used for copper drterminations of human sera. SUMMARY
Absorption cells with a path length of 5 cm. but accommodating much smaller volunies than conventional cella have been constructed for the Beckman Model DU spectrophotometer. S o changes in the optical system of the instrument are required. With these cells marked gain in sensitivity and precision can be, achieved in analytical spectrophotometry. This is illustrated by data on the application of these cells to the determination of copper in biological material which could be ineasured with a co-
efficient of variation of 2.2Fo n hell thc total quantity to be determined was 1.8 micrograms. ACKNOW LEDGRIE\T
The author is indebted to Frederick Brech for help with the construction of the cells. and Thomas L. Coombs for technical assistance. This work \vas supported by the Charles F. Kettering Fountlation, Dayton, Ohio, and the Howard Hughes Foundation of Hollyv-ood, Calif. LlTERATURE C l T E D
(1) Gubler, C. J., Lahey,
AI. E., .ishenbrucker, H., C:artwvright, G. E.. and \Tintrobe, AI. XI., J . Biol. Chem., 196,200 (1962;. (2) Yallee, B. L., Metabolism, I, 420 (1962). RECEIVED for review November
17, 1952.
Acceiited February 12, 19>3.
Micromethod for Measurement of Carbon-14-Labeled Material JOHK H. PETERS
AND
HELMUT R . GUT3IANK
Radioisotope Unit, Veterans ’ddministration Hospital, and Department of Physiological Chemistry, L-niversity of Minnesota, Minneapolis, Minn. TOR
the routine assay of radioactive cat bon in biological
E materials or organic compounds the method of Lindenbaum, (1, 4 ) or a recent modification (8) has
Armstrong, and Schubert been shonn to be accurate and rrpioducilJk. .liter wet combustion of the sample, the radioactive carbon dioxide is absorbed in saturated barium hydroxide (4)or in 1% sodium hydroxide, followed by precipitation with barium chloride (8),and the radioactivity of the solid carbonate is meawred. In boih procedures the carbon content of the material is of such a magnitude that the weight of the radioactive barium carbonate falls within the range of “infinite thickness.” However, when only microquantities of material are available, as is often the case in the isolation of metabolites or in the tissue assay of small organs, it is advantageous to be able to measuie the iadioactivities of smaller samples than were employed in the original method (I, 4 ) . The present paper describes a simple and rapid modification of the procedure of Lindenbaum el al. ( 4 ) to samples which may range in carbon content from 0.5 to 3.0 mg. The barium cai honate precipitates which are counted have a thickness of 3 to 10 mg. per square centimeter. T o facilitate the handling of small amounts the size of the original apparatus has been reduced (Figure 1). The saturated barium hydroxide (4)has been re-
placed by a measured volume of approximately 0.25 aV IJarium hydroxide u-hich contains 2 grams of barium chloride for each 100 ml. of solution (6). This eliminates both the filtration of the saturated barium hydroside as it is introduced into the receiver and the centrifugation of the final mixture (4). I n addition, the use of barium chloride-barium h? droside solution minimizes the coprecipitation of barium hydroxide which is troublesome with saturated barium hydroxide ( 5 ) . Following combustion, the excess of barium hydroxide is titrated with standard acid to the phenolphthalein end point ( 2 ) . The weight, of the barium carbonate is calculated from the t,itration data, which obviates the necessity for the tedious quantitative collectioii of the radioactive precipitate (1, 8). .%iter collecting and xveighing, the radioact.ivity of the carlionate is measured. The radioactivity of the sample is calculated a s follo\vs: n e t couiits,’niiii. X Radioactivitj- of the sample = -vli-al sorption factor mg. of carhonatr titrated mg. of carbonate wighed ~
JVith the size of sample employed, R combustion time of 2 instead of 3 minut,es ( 4 ) has been found adequat,e. Sweeping of the apparatus following combustion with air freed of carbon dioxide (8) has been found unnc~essary. Combustion of the sample, absorption of carbon dioxide, and titration require only 20 minutes, whereas the improved macroprocedure takes 35 to 40 minutes (8). PROCEDURE:
Figure 1.
Conihiistiori Apparatus
The coml)ustion apparatus is show1 in Figure 1. The combustion tube A has a capacity of 12 to 13 nil. The receiver D is a 40-ml. borosilicate glass centrifuge tube with conical hottom, fitted with a two-hole rubber stopper through which pas.< both t,he delivery tube for the carbon dioxide and a one-way stopcock n-hich connects the apparatus to a vacuum pump. The rubber stopper in D which gives rise to leaks after some use may l)e eliminated by use of the ground glass unit E . The sample, which should contain 0.5 to 3.0 mg. of carbon, is weighed into the combustion tube if it is a solid. In the case of solutions, the sample is pipetted into the combustion tube with a micropipet, and the solvent is evaporated. Three hundred milligrams of a mixture of potassium iodate (2 arts by weight) and potassium dichromate (1 part) is added. !tandard barium hydroxide solution (4.00 to 5.00 ml.) is delivered into the receiver from a 10-ml. buret. The buret and the barium hydroxide reservoir are protected from atmospheric carbon dioxide by soda lime tubes. The apparatus is assembled, and all ground surfaces in direct contact with the combustion mixture are lubricated with phosphoric acid. Two milliliters of modified Van Slyke-Folch reagent ( 7 )is placed into reservoir B and run into the combustion
ANALYTICAL CHEMISTRY
988 Talde I.
Recovery of Carbon by Wet Combustion of Small Samples
Compound Combusted Blank
Benzoic acid 2-hminofluorene
2-Acetylaminofluorene 2-Benzoylaminofluorene
L
2-Chloro-7-acetylaminofluorene L-Arginine monohydrochloride Urea nitrate
Weight of Carbon, hig. Sample, JIg. (Calculated)
...
...
2.47 3.21 0.64 1.09 1,29 1.95 1.62 1.93 1.96 2.27 1.15 1.19 1.49 1.56 1.55 2.04 2.38 2.48 1.05 2.31 3.29 6.92 9.18
1,70 2.22
0.55 0.94 1.11 1.68 1 31 1,56 1,:s 1,83 0.97 1.00 1.25 1.31 1,08 1.43 1.66 1.73 0.36 0.79 1.12 0.68 0.90
Carbon Found 0.03 0.03 0.03 0.04 0.04 0.06 1.69 2.29 0.54 0.93 1.07 1.74 1.32 1.59 1. .57 1.78 0.95 0.99 1.28 1.27 1.04 1.41 1.63 1.70 0.35 0.79 1.11 0.69 0.94
% Recovery ...
99.4 103
98.2 98.9 96.5 103 101 102 99.4 97.5 97.9 99.0 103 97.5 96.3 98.6 98.2 98.3 97.2 100 99.2 101 104
tube through vacuum stopcock C. The apparatus is evacuated t o 20 to 25 mm. of mercury by means of the vacuum pump. The combustion mixture is then boiled gently for 2 minutes. After boiling has stopped, the receiver is surrounded by an ice bath and absorption of carbon dioxide is allowed to proceed for 15 minutes. Stopcock C is then opened to the air. The receiver is disconnected, and the delivery tube and receiver are rinsed with 5 to 6 ml. of boiled distilled water. The excess of barium hydroxide is titrated immediately with 0.1 N hydrochloric acid which has been previously standardized against the barium hydroxide solution. The weight of carbon in the sample is calculated from the barium hydroxide used. The receiver is stoppered and allowed to stand overnight a t 5 to 10" C. Figure 2 shows the apparatus for collecting and mounting the barium carbonate. The filter paper (Whatman To. 540, 2.4 cm.) and the planchet (Tracerlab E-20 cut down to a height of inch) are washed, dried, and weighed prior to collection. The filter paper is then placed into the modified filtration assembly (Tracerlab E-%$) shown in Figure 2, and the barium carbonate is collected with suction. The barium carbonate is washed with boiled water and acetone. K i t h suction still applied, the top of the asseinbly is removed, and the filter paper is transferred to the planchet, dried a t 103 to 105' C. for 20 minutes, cooled in a desic-
Table 11. Radioactivity Measurements of Carbon-14Labeled Materials
Figure 2. Apparatus for Collecting and 3Ionnting Carbon-14-Labeled Barium Carbonate for Counting cator, and reweighed. This method of collection has been found more satisfactory than the procedure outlined in the Tracerlab literature. The radioactivity of the carbonate is measured in a windodess gas flov counter (Nuclear hleasurements Corp. , Model PC-2). Correction is made for self-absorption according to the method of Henriques et al. ( 3 ) . RESULTS
Data on recovery of carbon from pure organic compounds are shown in Table I. I t ill be seen that blanks are low, an average oE 0.04 mg. of carbon having been obtained in six runs. The data indicate that barium hydroxide is suitable as absorbing medium in semimicro or microcombustions. Table IT summarizes the results of radioactivity measurements on a variety of labeled substances. Constant specific radioactivities (counts per minute per milligram of compound or dry tissue) were observed with variable amounts of the same sample. Combustion of the laheled material and subsequent absorption of the radioactive carbon dioxide n-ere therefore complete. When the quantities of laheled material are not weighable, sufficient carrier must be added to the combustion tube to raise the carbon content to the previously specified range. Addition of carrier (in the form of sodium carbonate) to the receiver instead of to the combustion tube results in low recoveries of the isotope. Alternating combustions of highly radioactive samples and of nonradioactive samples demonstrated absence of contamination due to traces of radioactive carbon dioside. The apparatus may, therefore, be used in a series of runs without dismantling and cleaning between combustions.
fFollowinr wet combustion) I
Specific Weight of Weight of Radioactivity, Material, RIg. Carbon, U g . Counts/Min./hIg. o 45 x 1 0 - 3 " 4 1 . 6 X 104 o 75 x 10-7 4 1 . 2 X 10' 1 . 4 9 x 10-3" 4 4 . 4 x 104 2-Aminofluorene1 . 8 2 x 10-30 9 . 6 x 104 9-C" 1.82 10-3" 9 . 4 x 104 1 . 8 2 X 10-30 1 0 . 0 x 104 1 . 8 2 x 10-25 1 2 x 104 P-Benzoylamino0.63 0 53 74 5 fluorene-9-C14 1.99 1.77 75.9 Muscle 1 .78 1.01 909 2.37 1.30 962 Feces 2.09 0.88 66 7 2.94 1.23 63 4 Crine 10.2 2.13 38.9 10.2 2.16 39.7 5.0 1.14 39.4 Carcass 1.26 0 . 65 31 6 2.52 1.21 34.5 2.70 1.26 32.8 3.24 1.52 32 5 a Carrier compound u-as added t o the combustion tube t o bring the carbon content within the range specified. b 1.0 ml. of,a 0.047 .W solution of sodium carbonate was added t o the receiver as carrier. Compound or Tissue 2-Benzoylaminofluorene-9-C 14
x
LITERATURE CITED
(1) Armstrong, W, D., and Schubert, J.,
.~N.
