Nitric acid digestion of tissues for liquid scintillation counting

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but in collaboration with a computation laboratory a program tailored t.o individual requirements could readily be constructed from the flow chart of Table 1. Once the program has been written and tested all that is required of the analyst, is to send the data from the analyzer to the comput,er staff and the results shoul~l bc available in the form of graphs or tables within an hour. At present the authors use a teleprinter to send data to the computer center, which keeps the program tapes. In many ways this is equivalent to having a computer in the laboratory since one can arrange for regular bookings on the computer, send the data just prior to the scheduled time, and receive the results minutes after the calculations are complete. The programs and running instructions are available from the Ferranti Ltd. library, The Melbourne Computer Centre, 34 Queens Road, Melbourne, Victoria, Australia, as programs S880 and S881. REFERENCES 1. SIJACKMAN, 2. IXGLIS,

D. A.

H.,

S., And.

STEIK, W. H., ASD MOORE, S., Anal. Biochrm. 7, If%177 11964).

Chena.

30,

1185

(1958).

R. D. B. FRASER A. S. INGLIS A. MILLER Division of Plot&n Chemistry, CSIRO, Wool Research Lnlmrntories, Pmkzdle N2 (Afelbowne), Victoria, A,ustraliu Receie>erl September 16, 1.963

Nitric

Acid Liquid

Digestion Scintillation

of

Tissues

for

Counting

We are interested in counting H3 and C’” in rat skin and insect cuticle. Skin is the less difficult, of these tissues to prepare. Nevertheless, the 30-mg samples could not be homogenized using a Servall Omnimixer with high-speed attachment, in water or 50% acetone or formamide or toluene. Digestion by Hyamine or formamide, with or without prior drying of the skin, was unsuccessful. Nitric acid at 38°C gave slow digestion, but at 70°C digestion was complete in a few minutes. This was also true for insect cuticle. Our problem therefore was to count radio-

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active samples in nitric acid. There is one report in the literature in which nitric acid was present during counting, but, only to the extent of 0.5%; this was acceptable for counting Caa5 (1). The scintillator solution throughout contained 1.0% PPO (2,5-diphenyloxazole), 0.05% dimethyl POPOP [ 1,4-bis-2- (4-methyl-5-phenyloxazolyl) benzene], and 5% naphthalene in a 5: 1 mixture of dioxane and ethylene glycol. Plastic counting vials were used, and samples counted in a Tri-Carb liquid scintillation counter. Addition of 1 ml of 100% HNO, to a standard in 10 ml of cocktail abolished counts; even with 10% HNO, the efficiencies were unaccept.ably low (e.g., 0.7% for H3-toluene and 10.470 for C?“-toluene), and a marked yellow color was found. The situation was improved by using a higher ratio of cocktail: with 18 ml of cocktail and 1 ml of 10% HNO, the efficiencies were 1.7% for H3-toluene and 24% for Cl”-toluene. Improvement in the efficiency was achieved by neutralizing the l-ml portion of 10% (1.6 M) nitric acid solution with an equal volume of 2 M NaOH. However, separation i&o t,wo phases was observed after about 15 min. This separation would not matter if one could be assured that the radioactive sample would partition into the organic phase along with the scintillator. However, we did not wish to assume anything about the partitioning behavior of our radioactivity, so we sought other methods of neutralizing the nitric acid, which would avoid phase separat,ion. It seemed probable that the volume of aqueous material (2 ml) was excessive for 10 ml of the cocktail. However 2 ml of water gave no separation, whereas even 0.5 ml of 1.6 M HNO, plus 0.5 ml of 2 M NaOH gave separation, although not until several hours after mixing. Furthermore 1 ml of the 1.6 M HNO, gave no separation whereas 1 ml of 2 M NaOH gave prompt separation. Clearly the NaOH was the problem. Use of a thixotropic gel (Cab-0-Sil) delayed separation for over 1 day. However, when H3-toluene was counted in this system, counts increased steadily for 6 hr. The increase between 1 and 10 hr was 10%. The original HN03-NaOH mixture showed similar increases. Attempts were made to use an organic base to neutralize the HNO,, but very poor efficiencies were always found. Hyamine as normally purchased is 1 M in methanol. Consequently 2 ml was used to neutralize 1 ml of the 1.6 M HNO,. With 10 ml of cocktail, an efficiency for H”-toluene of 0.45% was found. Aniline was then used, dissolved in cocktail (which also contained H3-toluene) so that 10 ml contained 2 mmoles. This with 1 ml of 1.6 M HNO, gave an efficiency for H3-toluene of 0.41%. o-Toluidene, similarly used, gave an efficiency of 0.42%. Since the above organic bases were ineffectual, inorganic compounds were examined. However, problems were encountered both with in-

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253

solubility and with separation. The maximum molarity obtainable for Na,P04 was 0.44 M, for Na,P,07 was less then 0.25 M, and for Na,B,07 was less then 0.7. With Na,PO,, even 0.5 ml of 0.44 M added to 10 ml of cocktail gave immediate crystallizat.ion. Finally the weak organic base Tris, i.e., tris (hydroxymethyl) aminomethane, was tried. A 2 M solution in water could be prepared. A mixture of 1 ml with 10 ml of cocktail and 1 ml of 1.6X HNO, did not show phase separation at room temperature, but separated promptly on cooling in an ice bath. Since the counting temperature is l”C, such a separation was not permissible. Increasing the cocktail to 18 ml did not prevent separation; but use of 18 ml of cocktail with 1 ml of only 1.5 M Tris and 1 ml of 1.6 M HNO, gave a stable colorless mixture even in the cold. The efficiency of this system was 3.9% for H” and 38% for C”. However, since it was necessary to dilute the digest of tissue (in concentrated HKOQ) lo-fold before taking 1 ml for counting, the over-all efficiencies were 0.39 and 3.8%, respectively. Attempts to improve over-all efficiencies by taking larger samples of the diluted digest were unsuccessful. Thus 18 ml of cocktail with 2 ml of a solution made up by diluting the 1 ml of concentrated HNO, digest with 1.5 M or with 0.75 ill Trie, led to separation into two phases. We also examined t.hc effect of reducing the water in the sample, and of using a lesser dilution. It was found that, if Tris was used to dilute the concentrated nitric digest, efficiency was not improved. Thus, t,aking 1 ml of a sample prepared by diluting 1 ml of concentrated HNO, to 10 vol with 0.75 M Tris, efficiency figures of 2.8% for H3 and 30% for C?” were found. A faint yellow color was discernible. It was not possible to USC2 ml of this diluted mat,erial, for separation resulted. Another possibility was t.0 dilute t.he nitric digest less, in order t,hat. a l-ml sampIe would represent a larger alicluot.. When the digest was diluted with an qua1 volume of 0.75 M Tris, and a l-ml sample added to 18 ml of cocktail, efficiencies of 0.21% for H3 and 2.7% for C” were found, which were too low to compcnsat,c for the large aliouot, represented. The final procedure was as follows, using skin for an example. The fresh shaved rat skin sample (about 30 mg wet weight) was placed in a test tube, 1 ml of colorless concentrated HNO, was added, and the tube was incubated in a water bath at 70-72°C until the skin was digested, e.g., 2-5 min. It was necessary to remove the sample promptly from the bath and dilute with 9 ml of water at once to avoid discoloration by nit,ric fumes. A l-ml sample was added to a counting vial containing 18 ml of cocktail, and then 1 ml of 1.5 M Tris was added, and the vial capped and shaken. Departures from this procedure may give colored samples.

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It seems probable that this procedure would offer advantages over that of Herberg (2) for other tissues as well as skin. His procedures involve digestion in Hyamine or alkali, shaking at 5@-60°C “for 24 hr or more:” decolorizing with H,O,, and dest,roying excess H,O,. Phase separation occurred in sonic cases. A major advantage of the present method is in avoiding i~cl~emilun~inesccnce,” which occurs with Hyamine digests in tritium counting (3). ACKNOWLEDGMENTS This work was supported in part by Grant RG 7804 of the National Institutes of Health. The skilled technical assistance is acknowledged of Mrs. C. E. Dannelley, who performed all of t,he cxperimcnts. REFERENCES 1.

&RN.~T,

2. HERBERG, 3. DULCINO, (1963).

B.

G., .~SD R. J., A7d. J., Bosco,

H., Anal. Chem. 34, 643 (1962). Chem. 32, 42 (1960). R., VERLY, W. G., AKD MAISIN, J. R., Clin.

JEFF.~Y,

Chim.

Acta

8, 58

R. D. O’BRIEN Department of Entomology Cornell University Ithaca, New York Received August 7, 1968

The

Quantitative Ethers

Microdetermination as Applied

to Nucleotide

of Triphenylmethyl Synthesis

The use of triphenylmethyl (trityl) or substituted trityl ethers (1) as protecting groups in the chemical synthesis of nucleotides (2) is derived from carbohydrate chemistry (3). When paper chromatograms of nucleotide trityl ethers were sprayed with the phosphate-detecting reagent of Hanes and Isherwood (4)) an intense but transient yellow color developed immediately at, the sites of these compounds, before the heating necessary for hydrolysis and complex formation revealed the typical phosphomolybdate blue (1, 5). The production of this yellow color has now been made the basis of a quantitative assay for the trityl group. The yellow color is due to the triphenylmethyl carboniurn ion, formed by acid hydrolysis of trityl ethers under relatively anhydrous conditions. The spectrum of t,he carbonium ion has been described by Gold and