International Journal of Applied Radiation and Isotopes, 1975, Vol. 26, pp. 465 469. Pcrgamon Press. Printed in Northern Ireland

The Analysis of “H and 14CLabelled Compounds in the Form of Doubly Labelled Methane P. POVINEC Department of Nuclear Physics, Comenius University, Bratislava, Czechoslovakia (Received2 1 January 1974;

in retiedform 25 November 1974)

A new method of simultaneous analysis of sH and 1% labelled compounds is described. A doubly labelled sample is burned to form HTO and r4C0, which are used as reactants in the presence of zinc dust and a ruthenium catalyst for preparation of doubly labelled methane. sH and 1% activities of methane are counted simultaneously by means of an internal proportional counter. The method is very sensitive and may be used even for naturally labelled samples. The same apparatus may be used for preparation and counting of singly or doubly labelled samples.

1. INTRODUCTION and radiocarbon are the most often used radioisotopes for labelling compounds in tracer experiments. As they are pure soft beta emitters, with maximum energy of 18.6 and 155 keV respectively, internal methods of counting should be applied. Mostly liquidscintillation and proportional counters are used for low-level sH and 1% radioactivity measurements. The liquid-scintillation method is more convenient and generally preferred in tracer experiments with sH and 14C. When it is necessary to measure very low activities and it is impossible to incorporate a sample into a liquid scintillator because of strong quenching effects or chemiluminiscence, the proportional counting technique is usually applied. In many applications of radioactive tracers it is advantageous to make simultaneous measurements of 3H and r4C.(l) Gas counters usually count the 3H and 1% activities separately,(1*2) which is tedious and laborious, while liquidscintillation counters provide more economical simultaneous sH and 14C counting in one sample.(1*3) TYKVA(~*~) has described a method of simultaneous counting of SH and W in organic compounds by means of a proportional TRITIUM

counter C,A,.

*

filled This

with

a

method

mixture

of

has

proved

60,

and to

be

useful for simultaneous 3H and 14C assay in organic substances and on paper and thin layer chromatograms. A similar method has been described by JoRDAN,(~*‘) using argon plus 10 % CH, &O,

as the counting

as labelled

constituents,

gas, and H, or in the

case

of

singly labelled compounds, or H, and eH, in the case of double labelling. In this case the composition of the gas filling is the variable that causes a shift in the counter plateau. If an external calibration with a gamma-ray source is used, a precision as high as 0.2 % can be reached even in this case. SIMON et al.(a) have presented a comprehensive study of methods for the analysis of radioactive and stable isotopes of hydrogen, carbon, nitrogen and oxygen. They have reached a high factor of merit for tritium counting with propane as the counting gas. Using the 60-keV gamma rays of 241Am they have performed a very precise counter calibration for simultaneous 3H and 14C counting. The common disadvantage of these methods is the using of CO, as the counter filling. It is

* Symbols I% and c^ indicate labelling with sH and 14C, respectively. 4.65

466

P. Povinec

well known that CO, is very sensitive to electronegative impurities and therefore it has to be purified from them. In some cases of analysis of biological samples it is difficult to obtain a suitable counter plateau. The second disadvantage of these methods is the separate preparation of 3Handr4Clabelled gases and their subsequent mixing with an inactive counting gas* In the present paper a new method of analysis of aH and 14C labelled compounds is described using doubly labelled methane as the gas filling of a proportional counter. This method has been originally developed for simultaneous sH and 14C counting in atmospheric water vapours and carbon dioxide,(s) but can be very easily adopted for purposes of tracer experiments with labelled compounds. 2. EXPERIMENTAL The experimental equipment for the simultaneous analysis of sH and r4C labelled compounds consists of an apparatus for water and carbon dioxide production, methane preparation, and an apparatus for pulse registration with a low-background proportional counter. Methane Preparation Organic samples labelled with *H and 14C are combusted in a quartz tube in the presence of The apparatus for water and carbon oxygen. dioxide production is shown in Fig. 1. The sample is placed in a quartz boat in the first part of the quartz tube. The sample is heated with a mobile electric furnace F, to working temperature of 800%. In the second part of the quartz tube, copper oxide and silver wool are placed, and are heated with an electric furnace F, to a temperature of 750°C. (11) Water formed during the combustion is trapped in traps T, and T, cooled to -80°C with a mixture of acetone and dry ice. Carbon dioxide is purified by passing through copper and silver furnaces Fs and F, where traces of nitrogen oxides and halogens are removed. The temperatures of these furnaces are 550 and 3OO”C, respectively. Carbon dioxide is then trapped in traps Ts and T4 with liquid nitrogen. After cooled to -196% combustion, gases which could not condense at - 196°C are pumped away by a rotary pump.

1

F4

I

F3

FXG. 1. A schematic diagram of the apparatus for water and carbon dioxide production. Fr is a mobile furnace which can attain 800%; F, is a furnace filled with copper oxide and silver wool which can attain 750%; F, is a furnace filled with copper which can attain 550%; F, is a furnace filled with silver wool which can attain 300%; T,,T, are traps for water collection at -80%; T,,T4 are traps for carbon dioxide collection at - 196%. Carbon dioxide is then transfered from T, to T, and it is ready for production of methane. The sample of water is transferred from the volume calibrated traps T, and T, to a glass ampoule and it is sealed off. Water, labelled with sH, and carbon dioxide, labelled with 14C, obtained in this way are used for methane synthesis in a reaction vessel in the presence of zinc dust and a ruthenium catalyst. The doubly labelled methane is formed in the reactor according to the reaction:u2) 2l%,O + 60,

+ 4Zn -+ &I,

+ 4ZnO

and, after purification, is used as the gas filling of a proportional counter. The procedure for methane preparation and purification has been described elsewhere.03) Proportional

counter and electronics

Doubly labelled methane is used as the gas filling of a proportional counter. The IOWbackground proportional counter with built-in anticoincidence counter has been used for simultaneous sH and 14C activity measurement. The counter is of the Oeschger type,(14) using a gold-coated polyethylene foil as the inner counter and the copper tube for the ring anticoincidence counter.(iO)

The am&-is of W and 1% labelled compounds in the form of doubly labelled methane

467

A three-channel, fully transistorized electronic apparatus with an automatic print-out facility is used for pulse registration. In the first channel pulses from both sH and 14C disintegrations are registered. Inthe secondchannel the discrimination threshold is above the tritium spectrum, therefore only pulses from 14C disintegrations are registered. The third channel serves for the ring counter and it is used for the blocking of coincidence pulses between the inner and the ring counter. 3. RESULTS

AND

DISCUSSION t

Doubly labelled methane synthesis For the synthesis of doubly labelled methane it is necessary to use both radioactive components and therefore this requiresaknowledge of the relative ratio of these components in the reactor. An ideal solution, from the point of view of minimizing isotopic fractionation during methane synthesis, would be the use of stoichiometric amounts of H,O and CO,. It was necessary therefore to investigate the methane yield as a function of excess water in the reactor and to check the possibility of isotopic fractionation. The experimental dependence of the efficiency of the carbon-dioxide conversion to methane, as a function of excess water in the reactor, is shown in Fig. 2 for different initial pressures of carbon dioxide in the reactor. It can be seen that a higher initial pressure of CO, requires lower excess water and vice versa, if the reasonable conversion efficiency would be obtained. With increasing of water excess at the constant initial pressure of CO, the conversion efficiency increases up to the maximum level of 99%. A high hydrogen excess in the reactor is not advantageous because this could cause an isotopic fractionation and higher losses of methane during purification. The best way to reach a high conversion efficiency is therefore to use a high total gas pressure in the reactor. At l-5 atmosphere of CO, pressure and 10% excess water, the total yield of methane, influenced by the conversion efficiency and losses during methane purification, was 98%. If the stoichiometric amount of water and carbon dioxide were used, the total yield dropped to 85%. At 5 % excess water the total yield was 90 %. 8

: v

80 t

I

0

Water

exces

,

‘%

FIG. 2. Variation of conversionefficiency from excess water in the reactor, for different initial pressures of carbon dioxide for 1 - 1 atmosphere2, 2 - 1.5 atmosphere2, and 3 - 2 atmosphere2. It is advantageous therefore to use from 5 to 10 % of excess water. Under these conditions the reaction was fully completed, and only traces of unconverted carbon dioxide were found. We did not find any effect due to isotopic fractionation. As the total methane yield is determined by comparing the initial amount of carbon dioxide with the final amount of methane prepared, it was necessary for the determination of the conversion efficiency, to find the losses of methane during purification after extraction from the reactor. The estimation of methane losses was made experimentally by purification of methane from the known amount of hydrogen. It was found that the admixture of hydrogen in the range from 5 to 20% causes 1 to 5 % loss of methane. The conversion efficiency obtained in this was was checked by calculation of the conversion efficiency from the amount of unreacted carbon dioxide. The two values were approximately the same. Simultaneous

sH and 14C counting

Several samples of doubly labelled methane were prepared and counted in the proportional counter. The method was tested by preparing

468

P. Povinec TABLE 1

Sample

Experimental values sH in pCi/ml Ha0 14C in pCi/gC

Theoretical values

sH standard

1.60 f 0.04 pCi/ml I-Is0

1.59 f 0.06

14C standard 3H + 14C standard naturally labelled wood naturally labelled grass

644

1.56 f 0.10 0.85 f 0.12 0.72 f 0.12

& 0.06 pCi/g C

and counting samples of known activity. The experimental results were within standard errors with the theoretical values. The tritium standard ER-1, from the Institute for Research,

Production and Application of Radioisotopes, Prague, which had an activity of l-60 pCi/ml of water, and the 14C standard, from the National Bureau of Standards, Washington D.C., which had an activity of 6.44 pCi/g of carbon,(ls) were used for the counter calibration. The background methane filling was prepared from dead water, below 3 pCi/l of H,O, and dead carbon dioxide, prepared from anthracene. The counter was filled with a singly labelled methane standard, aH or 14C, and the detection efficiencies in the first and the second channel were determined. At the discrimination levels of 3 and 60 mV in the first channel and 63 and 450 mV in the second channel the absolute efficiencies for tritium in the first channel was 85% and zero in the second channel. The radiocarbon detection efficiency in the first channel was 16 % and 76 % in the second channel. The total detection efficiencies were 85 % for tritium and 92 % for radiocarbon. The unknown counting rates of sH and 14C were calculated as follows :

where N,, N, represent the net counting rates measured in the first and the second channel, and cl, ct are the relative detection efficiencies in the first and the second channel, c, = 0.174 and c, = O-826. Typical experimental data for simultaneous sH and 14C counting of doubly labelled methane

6.38 640 8.25 8.02

rt f f f

0.18 0.25 0.38 0.27

samples are listed in Table 1. It can be seen that a very high detection sensitivity has been reached. 4. CONCLUSIONS The procedure described for simultaneous 3H and 14C preparation and counting, using doubly labelled methane is very sensitive and can be applied even for naturally labelled samples. The same procedure and apparatus may be used for preparation and counting of singly, Cl?I, or dH4, and doubly labelled samples. The composition of the gas filling is the same for the singly or doubly labelled samples. Using this method it is possible to obtain a constant composition of the gas filling for arbitrary types of samples, and the same shape of energy spectra for different concentrations of sH and 1% in the samples. As methane has very good counting properties it need not be mixed with an inactive counting gas, therefore a higher sensitivity may be reached. In the case of the sample materials where the H to C ratio is low, like sugar, and the sH activity is also low, it is necessary to oxidize larger amount of sample (a few grams). The further advantage of this method is that it does not require a separate preparation of 3H and 14C labelled gas fillings. The method has proved to be very useful also for radioecological studies of SH and 1% in the environment.(s*ls) Acknowledgements-The author is deeply grateful to Prof. D. LAL and the staffs of 8H and 14C labs of the Tata Institute of Fundamental Research, Bombay, for valuable discussions and for supplying us with The chemicals for the methane preparations. collaboration with the staff of the Department of Nuclear

Physics is highly acknowledged.

The analysis of oH and 1°C labelled compounds in theform of doubly labelled methane

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