An Alternative Combustion Technique for Tritium Scintillation Counting SIR: Miniaturization of the Schoniger principle (I) for making certain radioisotopes available for scintillation counting was described by Gupta ( 2 ) . The technique offers the advantage of removing all quantitative procedures after combustion, as the isotope remains wholiy within the vial in which it will be counted. For material of low activity this is an added advantage. When used for tritium determinations, the method has revealed three weaknesses. First, firing the specimens by the technique described has been, in our hands, uncertain. Second, liquid nitrogen for freezing the water of combustion is not always readily available. Third, and in our view the most serious criticism of the method in its present form, is the potentially appreciable amount of water, and therefore tritium, which may be lost, when vials are opened to introduce the scintillator solution. Naked eye observation indicates that part of the water vapor condenses, as combustion proceeds, on the cool inner surface of the neck and the foil lining the cap of the container. Brief immersion in any refrigerant, even liquid nitrogen, will not trap this. Neither will it condense the water vapor in the vial atmosphere, which is at ambient temperature or, if freezing is carried out immediately after combustion, possibly above ambient temperature. The means proposed for avoiding these problems add to the reliability and possibly the accuracy of the method and thereby to confidence in it. The first difficulty can be overcome by using India ink to blacken the carrier paper (3), followed after the ink has dried by a spot of 2-3% nitrocellulose (celloidin) in any volatile solvent. It has been found more convenient, however, to impregnate a sheet of pure cellulose tissue paper (weight about 1 mg per cmz) with ink, dry it on aluminum foil lightly smeared with silicone oil to prevent adhesion, and then dip it into the nitrocellulose solution. When the solvent has evaporated, primers can be made by cutting the sheet into triangular slivers 2.5 mm wide at the base and approximately 8 mm long. About eight can be made from 1 cm2 of prepared tissue, each weighing less than 0.5 mg. The apex of a primer is wedged between the cup and the basket, the broad end of the primer projecting vertically above the basket's rim. When correctly done, ignition is rapid and certain. The second and third problems are interrelated and have been met by means of a deep freeze. First, allcombustion water is collected on the bottom of the vial, in which the specimen has been burned, and then it is frozen there. This ensures absolutely minimal water loss when the scintillator is added. To this end, vial holders of size suited to individual requirementh are made by cutting holes in foam polyurethane sheet of the type used for thermal insulation. The diameter of the holes must be such that the vials are a neat push fit. The thickness of the plastic should be as near as possible the height of the vials with their caps screwed on. A thinner (1) W. Schoniger, Mikrochim. Acta (Wien), 1955 (l), 123. 38, 1356 (1966). (2) 6. N. Gupta, ANAL.CHEM., (3) V. T. Oliverio, C . Denham, and J. D. Davidson, Anal. Biochem., 4, 188 (1962).
sheet, with the same face dimensions but without holes, is required for attachment to the holder during freezing. As soon as combustion is complete, the vials are pushed, bottom foremost, into the holder. After the baskets have cooled (several minutes), the holder with vials in place is inverted and stood on a warm surface at about 40-45 "Ce.g., the top of a drying oven. The condensed water on the vial neck and cap (now dependent and against the heat source) distills onto the cool bottom, which is in contact with the air at room temperature. After about 15 minutes the thin plastic sheet, which acts as the cover, is attached with rubber bands to the undermost-Le., the warm-side of the holder. The holder with vials and cover in position is placed at once in the deep freeze, the exposed bottoms of the vials still pointing upwards. The unprotected bottom of each vial rapidly cools, and the water already there freezes. At the same time, water vapor in the vial atmosphere condenses on this surface, thus allowing any residual water on the warm neck, shoulder, and cap to evaporate and be transferred to the bottom. Convection currents aid in scavenging the water from the dependent parts of the vial, including the basket. Water transfer can continue as long as there is a temperature gradient, but is complete before thermal equilibrium is restored. In our practice, vials are left in the freezer (- 85 "C or - 120 "C)for 3 to 4 hours or overnight, but a shorter time would probably suffice at these temperatures. Once distillation is complete, the cover is taken from the holder, and the vials are partially extruded, cap first, from their holes. They are then extracted individually for further treatment. Until their turn comes, vials remain in the deep freeze in an inverted position. As soon as a vial is removed, still inverted, the cap is quickly unscrewed and the basket is gently shaken out; under no circumstances should the basket come in contact with the frozen combustion water. The vial is now turned upright and the scintillator reagent added immediately. The sample is ready for counting, when the ice and any of the reagent components, which might have crystallized out, redissolve. It is hardly necessary to mention the need for gloves or towelling when handling the chilled vials, partly to prevent thawing and partly to avoid low temperature burns. The amount of water vapor lost by displacement, when the scintillator is introduced, is clearly a function of the temperature. At 20 "C each 25-ml vial contains approximately 0.4 mg of water vapor in its atmosphere-i.e., about 10% of the total combustion water produced by an average biological sample burned to completion. At -29 "C this drops to about 0.0125 mg or 0.3% of the total (4). It is apparent that a freezing unit working at -30 "C or lower would provide adequate cooling for most purposes. Lower temperatures, of course, would bestow the advantage of lessening the risk of premature thawing, even if not greatly influencing water vapor loss. In this regard it should be remembered, that (4) Smithsonian Tables, W. E. Forsythe, Ed., Lord Baltimore Press, Baltimore, Md., 1954. VOL. 40,
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the water vapor mass remains constant at any given temperature, and is independent of the total amount of combustion water produced (assuming the latter is sufficient to saturate the atmosphere). It follows that the proportion of water vapor in the vial rises as the total amount of water falls. If the higher temperature ranges are used for freezing, it would be wise to minimize the relative displacement loss, when very small samples are being assayed, by bringing the total weight of the charge to be burned to 6 mg with fuel (see below). The possibility of isotope being lost on the baskets, when they are shaken from the vials, has been investigated at count rates around 2000 cpm per burned specimen, and at a freezing temperature -85 "C. Provided baskets did not touch the ice in the vials, no increase over the background count was produced, when batches of 10 baskets were dropped directly into the scintillator solution. As no reason exists for thinking that baskets are preferentially dried, it is considered that the technique achieves virtually complete tritium trapping on the bottom of the vials. Because it is imperative that a gas-tight seal should exist between the foil of the cap and the vial neck, it is advisable to inspect the foil and the lip of the vial for imperfections before use. Pin hole openings in the former can be closed by routinely smearing the surface with heavy silicone oil; this has been shown to have no quenching effect. A smear around the lip also effects lubrication, and a better seal between glass and foil, when the cap is being tightly screwed home. In practice, baskets with shorter stems than those described by Gupta have practical and theoretical merits, First they are
more stable. Second they are cheaper, being made from about 9-cm wire. Third, the closer the burning specimen is to the bottom of the container, the longer it remains surrounded with oxygen undamped by the combustion products being formed. On trial the maximum amount of material (specimen, carrier cup, primer, and fuel) which could be burned was 7 mg, and for certain complete combustion, 6 mg. This figure is in agreement with that given by Kalberer and Rutschmann ( 5 ) using a 1-liter combustion flask--i.e., 1 mg per 4 ml of oxygen. As primer, carrier cup, and fuel amount to approximately 2 mg, samples should not exceed 4 mg dry weight. ACKNOWLEDGMENT
The author acknowledges with gratitude the every facility placed at his disposal in the Institut fur Krebsforschung der Universitat, Wien, by its Director, Professor Heinrich Wrba. T. H. VICKERS~ Krebsforschungsinstitut der Universitat 1090 Wien, Austria 1 Present address, Department of Pathology, University of Queensland, Medical School, Herston, Brisbane, 4006, Queensland, Australia
RECEIVED for review April 29, 1968. Accepted July 22, 1968. (5) F. Kalberer and J. Rutschmann, Biochemical Department,
Sandoz, Basel, supplied by Packard Instrument Co., Inc., U. S. A.,1967.
Automated Structure Elucidation of Several Kinds of Aliphatic and Alicyclic Compounds SIR: The automated data acquisition and computer-aided interpretation of spectra is rapidly expanding (1-3). For instance, the determination of the amino acid sequence in oligopeptides was accomplished by the computer interpretation of high resolution mass spectra (4, 5). Aliphatic saturated normal and monomethyl substituted hydrocarbons from C,to C ~were O also identified by computer-aided mass spectrometry (6). However, only one kind of spectrometer was tied into an electronic computer in every case. Here we wish to report an actual attempt to feed physical data, acquired via computers from WlS, NMR, IR, and UV, into a central computer which is programmed to structure elucidation based on a combination of all four types of data (Figure 1). The research up to date has been done with both aliphatic and alicyclic compounds containing less than fifteen carbons and only one oxygen, and with less than four sites of unsaturation. The whole system is composed of MS (JEOL, (1) D. H. Anderson and G. L. Covert, ANAL.CHEM.,39, 1288 (1967). (2) D. S. Erley, Presented at the Pittsburgh Conference on An-
alytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1968. (3) €3. P. Benz, Presented at 9th Experimental Nuclear Magnetic Resonance Conference, Pittsburgh, Pa., 1968. (4) M. Senn, R. Venkataraghavan,and F. W. McLafferty, J. Amer.
Chem. SOC.,88, 5593 (1966). (,5,) K. Biemann, C. Cone, B. R. Webseer, and G. P. Arsenault, ibid., 5598. (6) B. Petterson and R. Ryhage, ANAL,CHEM., 39, 790 (1967).
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ANALYTICAL CHEMISTRY
+ I Autcmaric
Figure 1. System for automated structure elucidation JMS-OISG, Mattauch-Herzog type), NMR (JOEL, JNM-C60H), IR (Shimadzu IR-27G) and UV (Shimadzu MPS-SOL) spectrometers, four smaller computers (slave computers 1, 2, 3, and 4; JEOL, Science Master EC-1, binary 16 bits, 4096 words, arithmetic time 200 psec), and one larger computer (master computer, JEOL, JRA-5, binary 16 bits, 4096 words, arithmetic time 20 psec) with an outer memory to automatically elucidate the molecular formulas and partial structures of these compounds. The slave computer 1 connected with a comparater microphotometer (JEOL, JMA-1) calculates the possible combina-
