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Development of an organically bound tritium standard
ARTICLE IN PRESS
Applied Radiation and Isotopes 61 (2004) 389–393
Development of an organically bound tritium standard A.V. Harms*, S.M. Jerome National Physical Laboratory, Queens Road, Teddington, Middlesex TW11 0LW, UK
Abstract High-concentration factors between seawater and marine organisms have been observed for organically bound tritium (OBT). The absence of an available OBT standard impedes the validation of an analytical method for environmental samples. This paper describes the secondary standardisation of tritiated thymidine, which was chosen to act as an OBT standard, using liquid scintillation counting. Traceability was provided by using internal standards of tritiated water (HTO). The assumption that the counting efficiencies for tritiated thymidine and HTO were identical was tested with separate quench curves. The rate of self-decomposition for tritiated thymidine, which resulted in an activity concentration of tritiated thymidine lower than the total tritium activity concentration, was determined. Crown Copyright r 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Organically bound tritium standard; Tritiated thymidine; Secondary standardisation; Liquid scintillation counting
1. Introduction Discharges of tritiated organic compounds into the Severn estuary/Bristol Channel are thought to be responsible for the significantly elevated tritium concentrations in aquatic organisms in the vicinity of the sewer outfall (Williams et al., 2001; Lambert, 2002). Concentration factors of up to 104 between seawater and marine organisms were observed as the tritium concentration in flounder (Platichthys flesus) was found to be in the order of 105 Bq kg1 (dry weight) (McCubbin et al., 2001). Almost all of the tritium found in these organisms is organically bound tritium (OBT). OBT may be defined as tritium bound to carbon in organic molecules, which excludes tritium bound to oxygen, nitrogen or sulphur in organic molecules, because these forms are thought to be more labile, and it would be difficult to separate/distinguish them from tritiated water (HTO) (Diabate! and Strack, 1993). A recent study suggests dose coefficients to be 4 1011 Sv Bq1 for HTO and 9 1011 Sv Bq1 for OBT, which are about twice the current ICRP values (Harrison et al., 2002). *Corresponding author. Tel.: +44-20-8943-8512; fax: +4420-8614-0488. E-mail address: [email protected] (A.V. Harms).
In order to determine HTO and OBT concentrations, environmental samples are generally first distilled or freeze-dried. Any tritium present in the condensate is designated as HTO. Subsequently, the residue is burned at 700–850 C in a special furnace with highly purified oxygen in the presence of a catalyst (e.g., Pt ceramic bead, CuO or CoO) (Lockyer and Lally, 1993; Pointurier et al., 2003). Any tritium collected in this step in a trap (generally as HTO) is designated as OBT. Alternatively, one sample is distilled or freeze-dried and another (wet) sample is combusted. In that case, OBT is taken to be the difference. However, in both cases the absence of an available OBT standard prevents the validation of the combustion step. In general, recoveries are thought to be high, but individual results may vary significantly (Pointurier et al., 2003). This paper describes the approach to a secondary standardisation of tritiated thymidine, which may act as an OBT standard. The nucleoside thymidine (C10H14N2O5), a building block of DNA consisting of the heterocyclic base thymine linked to the sugar 2deoxyribose, seems to fulfil the criteria of relevance, availability, non-volatility (a melting point of B186 C) and relative inertness to combustion. Tritiated thymidine is widely used in different immunological tests and for studies in cell proliferation.
0969-8043/$ - see front matter Crown Copyright r 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.03.012
ARTICLE IN PRESS 390
A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
The most convenient and practical route to standardise tritiated thymidine is liquid scintillation counting (LSC). Internal standards of HTO may be used to correct for a reduction in efficiency due to quenching effects and to provide traceability to a primary standard. Tritium emits beta particles with a maximum energy of only 18.6 keV and, consequently, chemical quench will strongly reduce its counting efficiency. In this work, HTO standards were obtained from a primary standardisation which involved the conversion of HTO into hydrogen gas (HT) and measurement of this HT with an internal proportional gas counter (Makepeace et al., 1996). It is assumed that the addition of the HTO spike to the sample will not increase the level of quenching. It is also assumed that for a given sample there is no difference between counting efficiencies for HTO and tritiated thymidine. Small but significant differences in efficiency for tritiated toluene and HTO using emulsion scintillators have been reported (Van der Laarse, 1967; Turner, 1969; Zarybnicky and Reich, 1979), but this may be explained by phase separation in the scintillation cocktail. Toluene will be extracted into the organic phase, which contains the scintillator molecules, while water will be present in the micelles formed by the surfactants in the emulsion scintillation cocktail (Zarybnicky and Reich, 1979). Although the mean range of tritium beta particles in water in general is thought to be (much) longer than the diameter of the formed micelles, there still may be a small loss of absolute efficiency due to self-absorption. These potential problems may be avoided by the use of a more homogeneous cocktail based on a mixture of toluene and ethanol, which was used to standardise tritiated toluene by comparison with HTO standards (Garfinkel et al., 1965). However, in this work it was decided to use a modern emulsion scintillator (Ultima Gold AB), because thymidine is soluble in both water and methanol but not in toluene (Zarybnicky and Reich, 1979). In order to check whether there was a difference in sensitivity towards quenching, quench curves for both HTO and tritiated thymidine were made by addition varying amounts of a chemical quenching solution to the sources. Tritiated thymidine, like most other tritiated compounds, may, however, not be stable during storage (Evans, 1992). The activity of a tritiated compound may not only decrease by the (natural) decay of the tritium nucleus, but also by decomposition of the tritiated compound itself (in HTO or tritiated organic fragments). This decomposition may be caused by either (i) chemical or microbiological effects or (ii) the decay of a neighbouring tritium nucleus by either direct interaction with the emitted beta particles or by interaction of free hydroxyl radicals created by the radiation. The rate of additional decomposition is related to the specific chemical form of the tritiated compound, the number
of tritium atoms in a molecule, the specific activity, the composition of the solution, and the storage temperature. In general, low specific activity sources, containing only one tritium label per molecule, dissolved in solutions containing ethanol and stored at 2–5 C are the most stable. Clearly, the rate of self-decomposition for any potential OBT standard needs to be known before provision of the standard to the user community. The decomposition of 4.25 TBq mg1 [6-3H]-thymidine in 10% ethanol under recommended storage conditions (2 C) is given as less than 0.5% per month (Evans, 1992). It was decided to check this value by applying a selective separation method between tritiated thymidine and HTO (evaporation) in order to analyse the extent of the decomposition of the former into the latter.
2. Materials and methods 2.1. Tritiated thymidine One millilitre of 37 MBq ml1 [6-3H]-thymidine in 10% ethanol was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). The quoted specific activity was 4.25 GBq mg1 with a quoted radiochemical purity of 97.0% (reference date 27 November 2001). After 7 months of storage at 4 C, a working solution of tritiated thymidine was prepared by gravimetric dilution (dilution factor 323.87) of the stock solution with 10% (w/w) ethanol containing 50 mg g–1 thymidine. The working solution was stored in a Schott bottle at 4 C. The remainder of stock solution was transferred to a flame-sealed ampoule and stored at 4 C. 2.2. Tritiated water In total, 1.5 g of a 95.1(10) kBq g1 HTO standard was used (reference date 1 July 2003). The method used to obtain this NPL standard were chemical reduction of the HTO to HT and subsequent measurement with internal proportional gas counting (Makepeace et al., 1996). 2.3. Nuclear data The half-life of tritium is assumed to be 4497(4) days (MacMahon, 2003). 2.4. Decomposition checks The decomposition checks were based on the difference between volatilities of HTO and tritiated thymidine, which has a melting point of B186 C. A known amount (approximately 1.2 g) of tritiated thymidine working solution was transferred onto a watch glass
ARTICLE IN PRESS A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
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(diameter of 6.5 cm). The watch glass was placed in a desiccator filled with 80 g of dried desiccant (silica with colour indicator), which was found to be capable to absorb at least 7 g of evaporated water in a day. After a day, the solvent (and the HTO fraction) was completely evaporated and absorbed by the desiccant, as was indicated by the colour change of the desiccant and the presence of a small white solid residue (thymidine) on the watch glass. Subsequently, the desiccator was opened and the white residue was redissolved in 1 g of water. The cover was replaced and the desiccator was left overnight. The next day, all water was evaporated and taken up by the desiccant. The desiccator was opened again and the white thymidine residue was redissolved again in 1 g of water. The resulting tritiated thymidine solution was carefully transferred to a LS vial and analysed for its tritium content. The topside of watch glass was carefully washed with a second portion of 1 g of water, and this solution was also transferred to a LS vial filled and analysed for its tritium content. The silica in the desiccator had 60 g of water added and the desiccator was closed. After standing for 3 days, 30 g of water could be collected from the wet silica. Four samples of 1 g were taken from this solution and analysed for their tritium content. A similar decomposition check was done for the tritiated thymidine stock solution. In this case, a known amount (approximately 25 mg) of tritiated thymidine stock solution and 1.0 g of 10% (w/w) ethanol containing 50 mg g–1 thymidine were mixed on a watch glass and treated as described above.
2.6. Quench curves
2.5. Liquid scintillation counting
where AT is the total tritium activity concentration in tritiated thymidine stock solution (Bq g1), GDF the gravimetric dilution factor between stock and working solution, mH the mass of the HTO spike (g), AH the tritium activity concentration in HTO spike (Bq g1), mT the mass of the sample of tritiated thymidine working solution (g), CT the count rate of the sample of tritiated thymidine working solution (cps), CBG the count rate of the background (cps), CST the count rate of the spiked sample (cps).
A Packard (Packard Instrument Co., Meriden, CT, USA) Tri-Carb model 2700 TR scintillation spectrometer (0–2000 keV range), 20-ml low-potassium glass vials and Ultima Gold AB (Packard) liquid scintillation cocktail were used. According to the supplier, Ultima Gold AB consist of diisopropyl naphthalene isomers (60–80%), 2,5-diphenyloxazole (0–1%), 1,4-bis (4-methyl-alpha-styryl)benzene (0–1%), ethoxylated alkylphenol (20–40%) and 2-(2-butoxyethoxy)ethanol (10–20%). Each vial contained 10 g of liquid scintillation cocktail and 1 g of aqueous phase (containing the tritium source and water) resulting in a total volume of approximately 11 ml for all samples. Subsequently, the vials were shaken thoroughly and placed in the counter to cool and dark-adapt. Quenching was measured using the tSIE parameter (transformed Spectral Index of the External standard), which has a range of 0–1000, where 0 indicates a completely quenched sample and 1000 an unquenched sample, respectively. All count rates were corrected for background.
Separate quench curves for both HTO and tritiated thymidine were made by adding varying amounts of up to 0.3 g of a 10% nitromethane in ethanol solution to vials containing either accurately known activities of HTO or accurately known amounts of tritiated thymidine working solution, respectively. The samples were counted for 10 min on the day of preparation. The samples were remeasured three times on three successive days. No significant difference in the count rate or the level of quenching as a function of time was observed. Therefore, the unweighted means of the efficiencies (for HTO), count rates per gram (for tritiated thymidine) and quench indicator parameters (tSIE) were used. 2.7. Internal standardisation Internal standards of HTO were used to standardise tritiated thymidine. The spiked samples were counted for 10 min. It was assumed that the addition of the HTO spike to the sample did not increase the level of quenching. This assumption was verified by comparing the value of the quench indicator parameter tSIE of the spiked samples with the tSIE value of the tritiated thymidine samples. They were found to be identical (71%). The total tritium activity concentration in the tritiated thymidine stock solution was calculated as follows: AT ¼
GDFmH AH CT CBG ; mT CST CT
ð1Þ
3. Results As can be seen in Figs. 1 and 2, where the absolute counting efficiency for HTO and the relative counting efficiency for tritiated thymidine are plotted as functions of the quenching indicator parameter tSIE, there seems to be no significant difference in the relative sensitivity towards quenching between HTO and tritiated thymidine. This strongly suggests that the assumption that the absolute counting efficiencies of HTO and tritiated thymidine are identical is correct.
ARTICLE IN PRESS A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
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Table 1 Uncertainty components for the tritiated thymidine activity concentration in the stock solution
50
Absolute eff (%)
45
Component
Relative uncertainty (%)
Internal uncertainty of the weighted mean of seven measurements (A) Tritium activity concentration in the HTO spike (B) Counting efficiency (B) Decay of tritium (B) Chemical form (B) Combined standard uncertainty
0.59
40 35 30 25 300
350
400
450
500
550
QIP (tSIE)
Fig. 1. The absolute counting efficiency for HTO as a function of the quench indicator parameter tSIE.
105
Rel. eff. (%)
95 85 75 65 55 300
350
400
450
500
550
QIP (tSIE)
Fig. 2. The relative counting efficiency for tritiated thymidine as a function of the quench indicator parameter tSIE.
Act. Conc. (MBq g-1)
40
38
36
34
32 300
350
400
450
500
QIP (tSIE)
Fig. 3. The total tritium activity concentration in tritiated thymidine stock solution as a function of the quench indicator parameter tSIE.
Fig. 3 shows the total tritium activity concentration in tritiated thymidine stock solution as a function of the level of quenching. The weighted mean of the results of seven measurements (each calculated with Eq. (1)) was used to determine the total tritium activity concentration in the tritiated thymidine stock solution. The uncertainty
0.96 0.2 0.05 1.0 1.6
budget for the tritiated thymidine activity concentration in the stock solution is given in Table 1. The result of the F -test (2.47) indicated that there was no significant difference between the internal and external uncertainty of the weighted mean of the seven measurements at a 95% confidence level. The total tritium activity concentration in the tritiated thymidine stock solution was 35.770.9 MBq g1 (reference date 1 July 2003). This is slightly higher than the indicative value reported by the supplier (i.e., 34 MBq g1 at the same reference date). The reported uncertainty is based on the standard uncertainty multiplied by a coverage factor of k ¼ 2; which provides a level of confidence of approximately 95%. The decomposition checks for both the stock and the working solution resulted in a recovery of 95% of the tritium in the thymidine fraction. Approximately 4% of the tritium was present in the water fraction (i.e., absorbed by the silica in the desiccator). Comparison of the obtained purity value of 95% with the quoted value of the supplier (97%) resulted in a self-decomposition rate of tritiated thymidine of 0.1% per month of storage at 4 C, which agreed with the value reported by Evans (1992). The activity concentration of tritiated thymidine in the stock solution was 33.971.1 MBq g1 (reference date 1 July 2003). The reported uncertainty is based on the standard uncertainty multiplied by a coverage factor of k ¼ 2; which provides a level of confidence of approximately 95%. The next stage of the project will be the provision of diluted low-level tritiated thymidine standards to the user community. Furthermore, an alternative approach, in which tritiated thymidine is converted to HTO using electrochemically generated Ag(II) ions, will be studied.
Acknowledgements The authors thank Julian Dean, Lena Johansson and Andy Pearce for advice and Florentyna Kapuscinska and Chris Gilligan for technical assistance. The authors
ARTICLE IN PRESS A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
thank the National Measurement System Policy Unit of the UK Department of Trade and Industry for their financial support.
References Diabat!e, S., Strack, S., 1993. Organically bound tritium. Health Phys. 65, 698–712. Evans, E.A., 1992. Guide to the Self-Decomposition of Radiochemicals. Amersham International plc, Little Chalfont, UK. Garfinkel, S.B., Mann, W.B., Medlock, R.W., Yura, O., 1965. The calibration of the National Bureau of Standards’ tritiated-toluene standard of radioactivity. Int. J. Appl. Radiat. Isot. 16, 27–33. Harrison, J.D., Khursheed, A., Lambert, B.E., 2002. Uncertainties in dose coefficients for intakes of tritiated water and organically bound forms of tritium by members of the public. Radiat. Prot. Dosimetry 98, 299–311. Lambert, B., 2002. Invited Editorial: Welsh tritium. J. Radiol. Prot. 21, 333–335. Lockyer, J.F., Lally, A.E., 1993. The determination of tritium, 14 C and 35S in milk and crop samples using a combustion technique. Sci. Total Environ. 130/131, 337–344.
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MacMahon, T.D., 2003. Evaluation of the half-life of 3H. NPL internal communication. Makepeace, J.L., Day, F.E., Gunther, . E., Unterweger, M.P., 1996. Intercomparison of measurement of tritiated water by internal proportional gas counting. Nucl. Instrum. Methods A 369, 458–462. McCubbin, D., Leonard, K.S., Bailey, T.A., Williams, J., Tossell, P., 2001. Incorporation of organic tritium (3H) by marine organisms and sediment in the Severn Estuary/ Bristol Channel (UK). Marine Pollut. Bull. 42, 852–863. Pointurier, F., Baglan, N., Alenic, G., Chiappini, R., 2003. Determination of organically bound tritium background level in biological samples from a wide area in the south-west of France. J. Environ. Radioactivity 68, 171–189. Turner, J.C., 1969. Tritium counting with the Triton X-100 scintillant. Int. J. Appl. Radiat. Isot. 20, 499–505. Van der Laarse, J.D., 1967. Experience with emulsion counting of tritium. Int. J. Appl. Radiat. Isot. 18, 485–491. Williams, J.L., Russ, R.M., McCubbin, D., Knowles, J.F., 2001. An overview of tritium behaviour in the Severn Estuary (UK). J. Radiol. Prot. 21, 337–344. Zarybnicky, V., Reich, M., 1979. Tritium counting efficiencies and time stabilities of samples with different amounts of aqueous salt solutions in a Triton X-100/toluene scintillant. Int. J. Appl. Radiat. Isot. 30, 729–737.
Applied Radiation and Isotopes 61 (2004) 389–393
Development of an organically bound tritium standard A.V. Harms*, S.M. Jerome National Physical Laboratory, Queens Road, Teddington, Middlesex TW11 0LW, UK
Abstract High-concentration factors between seawater and marine organisms have been observed for organically bound tritium (OBT). The absence of an available OBT standard impedes the validation of an analytical method for environmental samples. This paper describes the secondary standardisation of tritiated thymidine, which was chosen to act as an OBT standard, using liquid scintillation counting. Traceability was provided by using internal standards of tritiated water (HTO). The assumption that the counting efficiencies for tritiated thymidine and HTO were identical was tested with separate quench curves. The rate of self-decomposition for tritiated thymidine, which resulted in an activity concentration of tritiated thymidine lower than the total tritium activity concentration, was determined. Crown Copyright r 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Organically bound tritium standard; Tritiated thymidine; Secondary standardisation; Liquid scintillation counting
1. Introduction Discharges of tritiated organic compounds into the Severn estuary/Bristol Channel are thought to be responsible for the significantly elevated tritium concentrations in aquatic organisms in the vicinity of the sewer outfall (Williams et al., 2001; Lambert, 2002). Concentration factors of up to 104 between seawater and marine organisms were observed as the tritium concentration in flounder (Platichthys flesus) was found to be in the order of 105 Bq kg1 (dry weight) (McCubbin et al., 2001). Almost all of the tritium found in these organisms is organically bound tritium (OBT). OBT may be defined as tritium bound to carbon in organic molecules, which excludes tritium bound to oxygen, nitrogen or sulphur in organic molecules, because these forms are thought to be more labile, and it would be difficult to separate/distinguish them from tritiated water (HTO) (Diabate! and Strack, 1993). A recent study suggests dose coefficients to be 4 1011 Sv Bq1 for HTO and 9 1011 Sv Bq1 for OBT, which are about twice the current ICRP values (Harrison et al., 2002). *Corresponding author. Tel.: +44-20-8943-8512; fax: +4420-8614-0488. E-mail address: [email protected] (A.V. Harms).
In order to determine HTO and OBT concentrations, environmental samples are generally first distilled or freeze-dried. Any tritium present in the condensate is designated as HTO. Subsequently, the residue is burned at 700–850 C in a special furnace with highly purified oxygen in the presence of a catalyst (e.g., Pt ceramic bead, CuO or CoO) (Lockyer and Lally, 1993; Pointurier et al., 2003). Any tritium collected in this step in a trap (generally as HTO) is designated as OBT. Alternatively, one sample is distilled or freeze-dried and another (wet) sample is combusted. In that case, OBT is taken to be the difference. However, in both cases the absence of an available OBT standard prevents the validation of the combustion step. In general, recoveries are thought to be high, but individual results may vary significantly (Pointurier et al., 2003). This paper describes the approach to a secondary standardisation of tritiated thymidine, which may act as an OBT standard. The nucleoside thymidine (C10H14N2O5), a building block of DNA consisting of the heterocyclic base thymine linked to the sugar 2deoxyribose, seems to fulfil the criteria of relevance, availability, non-volatility (a melting point of B186 C) and relative inertness to combustion. Tritiated thymidine is widely used in different immunological tests and for studies in cell proliferation.
0969-8043/$ - see front matter Crown Copyright r 2004 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2004.03.012
ARTICLE IN PRESS 390
A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
The most convenient and practical route to standardise tritiated thymidine is liquid scintillation counting (LSC). Internal standards of HTO may be used to correct for a reduction in efficiency due to quenching effects and to provide traceability to a primary standard. Tritium emits beta particles with a maximum energy of only 18.6 keV and, consequently, chemical quench will strongly reduce its counting efficiency. In this work, HTO standards were obtained from a primary standardisation which involved the conversion of HTO into hydrogen gas (HT) and measurement of this HT with an internal proportional gas counter (Makepeace et al., 1996). It is assumed that the addition of the HTO spike to the sample will not increase the level of quenching. It is also assumed that for a given sample there is no difference between counting efficiencies for HTO and tritiated thymidine. Small but significant differences in efficiency for tritiated toluene and HTO using emulsion scintillators have been reported (Van der Laarse, 1967; Turner, 1969; Zarybnicky and Reich, 1979), but this may be explained by phase separation in the scintillation cocktail. Toluene will be extracted into the organic phase, which contains the scintillator molecules, while water will be present in the micelles formed by the surfactants in the emulsion scintillation cocktail (Zarybnicky and Reich, 1979). Although the mean range of tritium beta particles in water in general is thought to be (much) longer than the diameter of the formed micelles, there still may be a small loss of absolute efficiency due to self-absorption. These potential problems may be avoided by the use of a more homogeneous cocktail based on a mixture of toluene and ethanol, which was used to standardise tritiated toluene by comparison with HTO standards (Garfinkel et al., 1965). However, in this work it was decided to use a modern emulsion scintillator (Ultima Gold AB), because thymidine is soluble in both water and methanol but not in toluene (Zarybnicky and Reich, 1979). In order to check whether there was a difference in sensitivity towards quenching, quench curves for both HTO and tritiated thymidine were made by addition varying amounts of a chemical quenching solution to the sources. Tritiated thymidine, like most other tritiated compounds, may, however, not be stable during storage (Evans, 1992). The activity of a tritiated compound may not only decrease by the (natural) decay of the tritium nucleus, but also by decomposition of the tritiated compound itself (in HTO or tritiated organic fragments). This decomposition may be caused by either (i) chemical or microbiological effects or (ii) the decay of a neighbouring tritium nucleus by either direct interaction with the emitted beta particles or by interaction of free hydroxyl radicals created by the radiation. The rate of additional decomposition is related to the specific chemical form of the tritiated compound, the number
of tritium atoms in a molecule, the specific activity, the composition of the solution, and the storage temperature. In general, low specific activity sources, containing only one tritium label per molecule, dissolved in solutions containing ethanol and stored at 2–5 C are the most stable. Clearly, the rate of self-decomposition for any potential OBT standard needs to be known before provision of the standard to the user community. The decomposition of 4.25 TBq mg1 [6-3H]-thymidine in 10% ethanol under recommended storage conditions (2 C) is given as less than 0.5% per month (Evans, 1992). It was decided to check this value by applying a selective separation method between tritiated thymidine and HTO (evaporation) in order to analyse the extent of the decomposition of the former into the latter.
2. Materials and methods 2.1. Tritiated thymidine One millilitre of 37 MBq ml1 [6-3H]-thymidine in 10% ethanol was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). The quoted specific activity was 4.25 GBq mg1 with a quoted radiochemical purity of 97.0% (reference date 27 November 2001). After 7 months of storage at 4 C, a working solution of tritiated thymidine was prepared by gravimetric dilution (dilution factor 323.87) of the stock solution with 10% (w/w) ethanol containing 50 mg g–1 thymidine. The working solution was stored in a Schott bottle at 4 C. The remainder of stock solution was transferred to a flame-sealed ampoule and stored at 4 C. 2.2. Tritiated water In total, 1.5 g of a 95.1(10) kBq g1 HTO standard was used (reference date 1 July 2003). The method used to obtain this NPL standard were chemical reduction of the HTO to HT and subsequent measurement with internal proportional gas counting (Makepeace et al., 1996). 2.3. Nuclear data The half-life of tritium is assumed to be 4497(4) days (MacMahon, 2003). 2.4. Decomposition checks The decomposition checks were based on the difference between volatilities of HTO and tritiated thymidine, which has a melting point of B186 C. A known amount (approximately 1.2 g) of tritiated thymidine working solution was transferred onto a watch glass
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(diameter of 6.5 cm). The watch glass was placed in a desiccator filled with 80 g of dried desiccant (silica with colour indicator), which was found to be capable to absorb at least 7 g of evaporated water in a day. After a day, the solvent (and the HTO fraction) was completely evaporated and absorbed by the desiccant, as was indicated by the colour change of the desiccant and the presence of a small white solid residue (thymidine) on the watch glass. Subsequently, the desiccator was opened and the white residue was redissolved in 1 g of water. The cover was replaced and the desiccator was left overnight. The next day, all water was evaporated and taken up by the desiccant. The desiccator was opened again and the white thymidine residue was redissolved again in 1 g of water. The resulting tritiated thymidine solution was carefully transferred to a LS vial and analysed for its tritium content. The topside of watch glass was carefully washed with a second portion of 1 g of water, and this solution was also transferred to a LS vial filled and analysed for its tritium content. The silica in the desiccator had 60 g of water added and the desiccator was closed. After standing for 3 days, 30 g of water could be collected from the wet silica. Four samples of 1 g were taken from this solution and analysed for their tritium content. A similar decomposition check was done for the tritiated thymidine stock solution. In this case, a known amount (approximately 25 mg) of tritiated thymidine stock solution and 1.0 g of 10% (w/w) ethanol containing 50 mg g–1 thymidine were mixed on a watch glass and treated as described above.
2.6. Quench curves
2.5. Liquid scintillation counting
where AT is the total tritium activity concentration in tritiated thymidine stock solution (Bq g1), GDF the gravimetric dilution factor between stock and working solution, mH the mass of the HTO spike (g), AH the tritium activity concentration in HTO spike (Bq g1), mT the mass of the sample of tritiated thymidine working solution (g), CT the count rate of the sample of tritiated thymidine working solution (cps), CBG the count rate of the background (cps), CST the count rate of the spiked sample (cps).
A Packard (Packard Instrument Co., Meriden, CT, USA) Tri-Carb model 2700 TR scintillation spectrometer (0–2000 keV range), 20-ml low-potassium glass vials and Ultima Gold AB (Packard) liquid scintillation cocktail were used. According to the supplier, Ultima Gold AB consist of diisopropyl naphthalene isomers (60–80%), 2,5-diphenyloxazole (0–1%), 1,4-bis (4-methyl-alpha-styryl)benzene (0–1%), ethoxylated alkylphenol (20–40%) and 2-(2-butoxyethoxy)ethanol (10–20%). Each vial contained 10 g of liquid scintillation cocktail and 1 g of aqueous phase (containing the tritium source and water) resulting in a total volume of approximately 11 ml for all samples. Subsequently, the vials were shaken thoroughly and placed in the counter to cool and dark-adapt. Quenching was measured using the tSIE parameter (transformed Spectral Index of the External standard), which has a range of 0–1000, where 0 indicates a completely quenched sample and 1000 an unquenched sample, respectively. All count rates were corrected for background.
Separate quench curves for both HTO and tritiated thymidine were made by adding varying amounts of up to 0.3 g of a 10% nitromethane in ethanol solution to vials containing either accurately known activities of HTO or accurately known amounts of tritiated thymidine working solution, respectively. The samples were counted for 10 min on the day of preparation. The samples were remeasured three times on three successive days. No significant difference in the count rate or the level of quenching as a function of time was observed. Therefore, the unweighted means of the efficiencies (for HTO), count rates per gram (for tritiated thymidine) and quench indicator parameters (tSIE) were used. 2.7. Internal standardisation Internal standards of HTO were used to standardise tritiated thymidine. The spiked samples were counted for 10 min. It was assumed that the addition of the HTO spike to the sample did not increase the level of quenching. This assumption was verified by comparing the value of the quench indicator parameter tSIE of the spiked samples with the tSIE value of the tritiated thymidine samples. They were found to be identical (71%). The total tritium activity concentration in the tritiated thymidine stock solution was calculated as follows: AT ¼
GDFmH AH CT CBG ; mT CST CT
ð1Þ
3. Results As can be seen in Figs. 1 and 2, where the absolute counting efficiency for HTO and the relative counting efficiency for tritiated thymidine are plotted as functions of the quenching indicator parameter tSIE, there seems to be no significant difference in the relative sensitivity towards quenching between HTO and tritiated thymidine. This strongly suggests that the assumption that the absolute counting efficiencies of HTO and tritiated thymidine are identical is correct.
ARTICLE IN PRESS A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
392
Table 1 Uncertainty components for the tritiated thymidine activity concentration in the stock solution
50
Absolute eff (%)
45
Component
Relative uncertainty (%)
Internal uncertainty of the weighted mean of seven measurements (A) Tritium activity concentration in the HTO spike (B) Counting efficiency (B) Decay of tritium (B) Chemical form (B) Combined standard uncertainty
0.59
40 35 30 25 300
350
400
450
500
550
QIP (tSIE)
Fig. 1. The absolute counting efficiency for HTO as a function of the quench indicator parameter tSIE.
105
Rel. eff. (%)
95 85 75 65 55 300
350
400
450
500
550
QIP (tSIE)
Fig. 2. The relative counting efficiency for tritiated thymidine as a function of the quench indicator parameter tSIE.
Act. Conc. (MBq g-1)
40
38
36
34
32 300
350
400
450
500
QIP (tSIE)
Fig. 3. The total tritium activity concentration in tritiated thymidine stock solution as a function of the quench indicator parameter tSIE.
Fig. 3 shows the total tritium activity concentration in tritiated thymidine stock solution as a function of the level of quenching. The weighted mean of the results of seven measurements (each calculated with Eq. (1)) was used to determine the total tritium activity concentration in the tritiated thymidine stock solution. The uncertainty
0.96 0.2 0.05 1.0 1.6
budget for the tritiated thymidine activity concentration in the stock solution is given in Table 1. The result of the F -test (2.47) indicated that there was no significant difference between the internal and external uncertainty of the weighted mean of the seven measurements at a 95% confidence level. The total tritium activity concentration in the tritiated thymidine stock solution was 35.770.9 MBq g1 (reference date 1 July 2003). This is slightly higher than the indicative value reported by the supplier (i.e., 34 MBq g1 at the same reference date). The reported uncertainty is based on the standard uncertainty multiplied by a coverage factor of k ¼ 2; which provides a level of confidence of approximately 95%. The decomposition checks for both the stock and the working solution resulted in a recovery of 95% of the tritium in the thymidine fraction. Approximately 4% of the tritium was present in the water fraction (i.e., absorbed by the silica in the desiccator). Comparison of the obtained purity value of 95% with the quoted value of the supplier (97%) resulted in a self-decomposition rate of tritiated thymidine of 0.1% per month of storage at 4 C, which agreed with the value reported by Evans (1992). The activity concentration of tritiated thymidine in the stock solution was 33.971.1 MBq g1 (reference date 1 July 2003). The reported uncertainty is based on the standard uncertainty multiplied by a coverage factor of k ¼ 2; which provides a level of confidence of approximately 95%. The next stage of the project will be the provision of diluted low-level tritiated thymidine standards to the user community. Furthermore, an alternative approach, in which tritiated thymidine is converted to HTO using electrochemically generated Ag(II) ions, will be studied.
Acknowledgements The authors thank Julian Dean, Lena Johansson and Andy Pearce for advice and Florentyna Kapuscinska and Chris Gilligan for technical assistance. The authors
ARTICLE IN PRESS A.V. Harms, S.M. Jerome / Applied Radiation and Isotopes 61 (2004) 389–393
thank the National Measurement System Policy Unit of the UK Department of Trade and Industry for their financial support.
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