DOI: 10.1007/s10967-007-0608-0

Journal of Radioanalytical and Nuclear Chemistry, Vol. 272, No.3 (2007) 483–489

A new technique for tritium labeling of complex technical mixture of PCB congeners A. A. Kim,* G. T. Djuraeva, P. V. Zinovev, I. I. Sadikov, A. A. Rylov Group of Biological Radiochemistry, Department of Analysis and Information, Institute of Nuclear Physics, Uzbekistan Academy of Sciences, Ulugbek, Tashkent 702132, Uzbekistan (Received June 30, 2006)

The method of labeling by thermally activated tritium of technical mixture of PCB congeners (Sovol) has been developed. Influence of labeling procedure on mixture of PCB congeners was investigated and optimum conditions of producing of tritium labeled complex mixture of PCB congeners were determined. The system purification of tritium labeled Sovol by thin layer chromatography was developed. Purified tritium labeled Sovol was analyzed by gas chromatography. The developed technique can be used for producing of tritium labeled similar complex mixtures of congeners of other organic compounds.

Introduction Polychlorinated biphenyls (PCBs) are one of the most investigated industrial ecotoxicants. The quantitative analysis of multi-component mixtures of PCB congeners like Aroclors is complicated enough because of presence in analyzed samples of some hundreds of PCB congeners. The EPA methods allow to determine only about 20 toxic PCB congeners. In the USSR the complex mixture of PCB congeners (from three- up to hepta-chlorobiphenyls) under the trade mark “Sovol” was produced. The most part of Sovol was used by transformer factories for producing of electric transformers.1 The part of produced Sovol also was used as additives to pesticides and herbicides. Hence, in the environment at the territory of former USSR PCBs were introduced as Sovol within several decades. For the investigation of the biodegradation of Sovol the application of labeled preparation is the most effective. Earlier in ecological investigations biphenyls labeled by 14C were used and also separate isomers of polychlorinated biphenyls.2 Synthesis of biphenyls labeled by 14C is difficult and expensive. Application of tritium label can considerably reduce the price of producing labeled preparation. For research of PCBs biodegradation it seems to be most effectively to use completely tritium labeled Sovol, as use only one labeled PCB congener cannot give the complete real information about the process of the biodegradation of Sovol. The development of the method of labeling by thermally activated tritium such a complex mixture of PCB congeners as Sovol, allows producing of other complex mixtures of organic compounds labeled by thermally activated tritium, like polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polybrominated biphenyls. Earlier we had been developed techniques of labeling by

thermally activated tritium of lot of pesticides and the 2,3,7,8,-tetrachlorodibenzo-p-dioxin,3,4 allowing to receive preparations with high specific activity. The aim of this study was to develop a method of labeling by thermally activated tritium of complex mixture of PCB congeners, Sovol. We have investigated the influence of labeling procedure on PCB congeners. The optimum conditions of obtaining of tritium labeled Sovol were determined. The system of purification of tritium labeled PCBs by thin layer chromatography on silica gel in hexane was developed. Purified tritium labeled PCBs have been analyzed by gas chromatography. We have investigated the opportunity to receive simultaneously fractions of tritium labeled transformed PCB congeners. Experimental Tritium labeling of Sovol The labeling of Sovol by thermally activated tritium was carried out on special apparatus for tritium labeling.5 Solution of Sovol (300 mg) in hexane was sprayed on the inner surface of a reaction flask and dried at room temperature up to complete removal of hexane. After that reaction flask was cooled in liquid nitrogen and pumped to vacuum up to 1×10–3 mm Hg. Labeling was carried out by the cycles. Each cycle consists of filling of system with gaseous tritium up to pressure 5×10–3 mm Hg and activation of tritium by heated tungsten spiral at 2000 K during 3 minutes. Between cycles the reaction flask was defrosted, Sovol was dissolved in hexane and dried again. After each cycle the aliquots of Sovol were measured with a scintillation counter in toluene scintillator. After the end of labeling, the Sovol was eluted from a reaction flask by hexane and dried on the rotational evaporator for removal of labile and unbound tritium.

* E-mail: [email protected] 0236–5731/USD 20.00 © 2007 Akadémiai Kiadó, Budapest

Akadémiai Kiadó, Budapest Springer, Dordrecht

A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

For gathering of sublimated Sovol fractions in vacuum system the additional vacuum trap cooled by liquid nitrogen has been added. Before labeling the trap was thoroughly washed by hexane. After end of labeling Sovol was eluted from the trap by hexane and dried on a rotational evaporator to remove labile and unbound tritium. Purification of tritium labeled Sovol The purification of tritium labeled Sovol from byproducts was carried out with the help of thin layer chromatography (TLC) on plates with silica gel in hexane. Spot of PCBs was cut out from the plate and PCBs was eluted by hexane. Sublimated fraction of PCBs was analyzed TLC in the same system. Fractions was identified by autoradiography, cut out from a plate and eluted by hexane. The radioactivity was measured with a scintillation counter in toluene scintillator. Gas chromatography with mass spectrometry (GC/MS) analysis of tritium labeled Sovol Purified tritium labeled PCBs fractions were analyzed by gas chromatography (GC) on Agilent Technologies АТ 6890 gas chromatograph with the µECD (microdetector of electronic capture). Conditions: capillary column DB 608, length 30 m, internal diameter 0.53 mm, thickness of a film 0.5 µm. The flow rate was 1.5 ml/min, the gas carrier was nitrogen. The initial temperature of the thermostat was 150 °C during 2 minutes, then with a speed of 10 °С/min up to 250 °C for 43 minutes before the end of chromatography. Injector temperature was 280 °C, a mode splitless (without division of a stream). The temperature of the µECD detector was 300 °С, with a mode of a constant flow rate 45 ml/min, with nitrogen gas carrier. The purified fraction of PCBs was additionally analyzed on a chromatograph with mass-selective detector (Hewlett-Packard HP5972). Conditions: capillary column HP5MS, length 30 m, diameter 0.25 mm, a stationary liquid phase 5% phenylmethylsilicon. Temperature of thermostat was 150 °C for 3 minutes, further 10 °C/min up to 280 °C. Identification of PCB isomers was carried out with the use of spectra libraries, Willey 275 and Pfleger Toxilab. Results and discussion Purification of Sovol by TLC The task of purification of tritium labeled multicomponent mixture of PCB congeners is not trivial. The use of high pressure liquid chromatography (HPLC) which is very effective for purification of homogeneous

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compound, in our case cannot give optimum results. In our case the task includes not only purification of tritium labeled Sovol from the degradation products and transformation, but also preservation of all initial components. We had been used thin layer chromatography for purification. The application of TLC on silica gel seems to be optimal for purification as chromatography analysis of PCBs on silica gel is widely enough applied.6–8 We had been tested three TLC systems in which Rf of the basic spot of PCBs varies from 0.14 up to 0.78. We have found that at TLC on silica gel in hexane the Rf of the basic spot of PCBs was 0.78; in 40% ammonia the Rf was 0.14, and in system hexane : acetone : water (2 : 2 : 1) 0.6. For further purification we applied silica gel in hexane because at given Rf the by-products going ahead and behind of the spot of PCBs (Fig. 1) are well separated. The influence of the procedure of labeling on Sovol Sovol represents the viscous liquid oily substance containing of some tens of PCB congeners with a various degree of chlorination. Since tritium labeling occurs under vacuum at 1.10–3 mm Hg, we investigated the volatility of Sovol under this condition. We found significant reduction of Sovol mass in the course of labeling. From 300 mg of Sovol introduced into the reaction flask we revealed only 100 mg of tritium labeled Sovol. For investigation of this process we introduced an additional vacuum trap cooled by liquid nitrogen in the system for labeling. We found that part of PCB congeners is sublimated and collected on the vacuum trap cooled by liquid nitrogen. Most likely that significant sublimation of PCB congeners occurs during defrosting of reaction flask when Sovol is in liquid state under vacuum at room temperature. The data received allow to make conclusion that the increase of time of labeling of Sovol will results an increase of the process of PCB congeners sublimation. Method of labeling by thermally activated tritium possesses the factors destroying a part of target substance, intense light and thermal streams, and also the reaction of direct substitution of hydrogen by tritium. That is why it is necessary to select conditions of labeling at which destruction of target substance occurs in lesser degree. The degree of specific activity of tritium labeled preparation is determined by the time of interaction of thermally activated tritium atoms with the target substance. We investigated the dependence of specific activity of tritium labeled Sovol from the duration of the labeling procedure. The labeling of Sovol was carried out during 25 cycles. We found that the specific activity of tritium labeled Sovol was increased at the increase of number of cycles of labeling (Fig. 2).

A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

GC/MS analysis of fractions of PCBs Sovol eluted from the reaction flask was purified by TLC on silica gel in hexane. We cut out the spots of PCBs fractions from TLC plates, then eluted PCBs fractions by hexane : acetone (1 : 1), dried and determined the weight of fractions. We determined the radioactivity of all PCBs fractions. Data are presented in Table 1. In

the reaction flask the output of tritium labeled Sovol with specific activity of 3.08 MBq/mg was 55%. We washed the vacuum trap by the several portions of hexane : acetone (1 : 1). We collected 14 mg of tritium labeled PCBs. After purification of tritium labeled PCBs by TLC on silica gel we found the appearance of an additional fraction with Rf = 0.3. Data are presented in Table 2.

Fig. 1. TLC chromatogram of tritium labeled Sovol purified on silica gel; a – visualization Sovol in the ultraviolet; b – autoradiograph of the chromatogram; 1 – ammonia; 2 – hexane; 3 – hexane : acetone : water (2 : 2 : 1). At the left side on each track – non labeled PCBs, on the right side – tritium labeled PCBs

Fig. 2. Dependence of degree of specific activity of tritium labeled PCBs from time of labeling

Table 1. TLC analysis of PCBs fractions from reaction flask Number of fraction Fraction 1 Fraction 2 (Sovol) Fraction 3

Rf of the fraction 0 (start) 0.78 1.0 (front)

Weight of fraction, % from total 9.2 mg (7.97%) 64.1 mg (55.55%) 42.1 mg (36.48%)

Total radioactivity of the fraction, MBq 40.75 197.42 41.67

Specific radioactivity, MBq/mg 4.43 3.08 0.99

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A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

Table 2. TLC analysis of PCBs fractions from vacuum trap Number of fraction Fraction 1 Fraction 2 Fraction 3 (Sovol) Fraction 4

Rf of the fraction 0 (start) 0.3 0.78 1 (front)

Weight of fraction, % from total 3 mg (21.43%) 3 mg (21.43%) 5 mg (35.71%) 3 mg (21.43%)

Thus, we found that fraction of Sovol, sublimated on the vacuum trap has higher specific activity, than fraction of Sovol in the reaction flask. In the vacuum trap the output of tritium labeled Sovol with specific activity of 3.65 MBq/mg was 44.49%. In the PCBs fraction eluted from the vacuum trap there is a well defined additional fraction. Obviously, it is connected to partial degradation of PCBs and sublimation of products of degradation. We analyzed all four PCBs fractions by GC on chromatograph АТ 6890 (“Agilent Technology”) with the µECD (Fig. 3). The GC analysis has shown that the purified by TLC fraction of Sovol practically does not differ from the initial Sovol. We practically not revealed difference in spectra purified by TLC Sovol from the reaction flask and from the vacuum trap. The additional

Radioactivity of the fraction, MBq 15.72 1.65 18.25 5.4

Specific radioactivity, MBq/mg 5.24 0.55 3.65 1.80

transformed fractions of PCBs, appearing after labeling, differ in concentration of congeners and on Rf on TLC. The specific activity of labeled fractions of Sovol in the reaction flask and the vacuum trap considerably differ. The received data allow assuming, that at labeling of PCBs there is a sublimation of the transformed products. In first two fractions (Fig. 3a and b) we found the increase of concentration of low chlorinated congeners that can be the evidence of the effect of dehalogenation during treatment by thermally activated tritium. In the fourth fraction (Fig. 3d) increased contents of congeners was observed with high degree of chlorination. Since in the reaction flask tritium labeling occurs only in the surface layer, it is the most probable that a significant part of labeled PCBs are sublimated on the vacuum trap.

Fig. 3. GC analysis of fractions of tritium labeled PCBs removed from the trap; a – fraction 1 (start)

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A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

Fig. 3. GC analysis of fractions of tritium labeled PCBs removed from the trap; b – fraction 2

Fig. 3. GC analysis of fractions of tritium labeled PCBs removed from the trap; c – fraction 3 (Sovol)

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A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

Fig. 3. GC analysis of fractions of tritium labeled PCBs removed from the trap; d – fraction 4 (front)

Fig. 4. GC/MS analysis of purified tritium labeled Sovol and identification of congeners

This supposition can be confirmed by higher specific activity of PCB fraction from vacuum trap. The investigated effect can be used for receiving of tritium labeled Sovol with higher specific activity and for simultaneous production of tritium labeled mixtures of

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PCB congeners with initial and transformed compositions. Thus, for optimization of the method a change of the circuit of labeling is necessary, providing the introduction of an additional vacuum trap cooled by liquid nitrogen for labeling.

A. A. KIM et al.: A NEW TECHNIQUE FOR TRITIUM LABELING OF COMPLEX TECHNICAL MIXTURE

Table 3. Identification of tritium labeled PCB congeners of Sovol Number of peak 1 2 3 4 5 6 7 8 9 10 11 12 13

Retention time (RT) 1.05 5.68 6.06 6.58 7.16 7.54 7.77 8.40 8.86 9.58 9.71 10.09 10.41

Peak area, % 16.331 0.59 0.29 1.18 6.10 2.31 1.55 14.54 12.99 15.26 1.56 11.09 4.40

14

10.69

0.63

15

10.92

6.86

16 17 18

11.07 11.15 11.32

0.20 0.17 1.65

19 20

11.46 11.72

0.24 0.69

21

11.94

0.79

22

12.48

0.56

Library/ID Ethanone, 1-oxiranyl-(CAS) $$ 2-B(2-Tetradecyloxy)-ethanol 2,4,4’-Trichlorobiphenyl 2,4,4’-Trichlorobiphenyl 2,4,4’-Trichlorobiphenyl 2,2’,5,5’-Tetrachlorobiphenyl 2,2’,5,5’-Tetrachlorobiphenyl 2,2’,5,5’-Tetrachlorobiphenyl 2,2’,5,5’-Tetrachlorobiphenyl 2,2’,4,5,5’-Pentachlorobiphenyl 2,2’,4,5,5’-Pentachlorobiphenyl 2,2’,4,5,5’-Pentachlorobiphenyl 2,2’,4,5,5’-Pentachlorobiphenyl 2,2’,3,4,4’,5’-Hexachlorobiphenyl 2,2’,4,4’,5,5’-Hexachlorobiphenyl 2,2’,4,4’,5,5’-Hexachlorobiphenyl 2,2’,3,4,4’,5’-Hexachlorobiphenyl 2,2’,3,4,4’,5’-Hexachlorobiphenyl 2,2’,4,4’,5,5’-Hexachlorobiphenyl Heptachlorobiphenyl Heptachlorobiphenyl 2,2’,3,4,4’,5’-Hexachlorobiphenyl 2,2’,4,4’,5,5’-Hexachlorobiphenyl Heptachlorobiphenyl 2,2’,4,4’,5,5’-Hexachlorobiphenyl 2,2’,3,4,4’,5’-Hexachlorobiphenyl 1,1’-Biphenyl,2,2’,3,4,4’,5’,6-he 1,1’-Biphenyl,2,2’,3,3’4,6,6’-he 1,1’-Biphenyl,2,2’,3,4’,5,5’,6-he Heptachlorobiphenyl

Purified fraction of Sovol has been additionally analyzed by GC/MS on gas chromatograph with Hewlett-Packard HP5972 mass-selective detector (Fig. 4). We found full identity (both qualitative and quantitative) of spectra of Sovol before and after tritium labeling. Data of GC/MS analysis of Sovol are presented in Table 3. This data allow us to suppose that the sublimation of all PCB congeners of Sovol occurs uniformly under vacuum at 1.10–3 mm Hg. Conclusions Method of labeling by thermally activated tritium allows obtaining the tritium labeled multi-component mixture of PCB congeners, Sovol. The method developed of tritium labeling of Sovol also allows obtaining transformed fraction of mixture of PCB congeners. This technique can be used for production of tritium labeled similar complex mixtures of congeners of other organic compounds, like PCDDs and PCDFs.

References 1. U. A. TREGER, V. N. ROZANOV, Polychlorinated biphenyls. Series: Supertoxicants of XX Century, Moscow, 2000, p. 64. 2. IAEA, International Atomic Energy Agency Annual Report, 1999, p. 53. 3. A. A. KIST, A. G. KIST, A. A. KIM, A. N. IDRISOVA, V. N. SIDOROV, YU. YA. KIT, J. Radioanal. Nucl. Chem., 195 (1995) 219. 4. III All-Union Conf. on the Problem of Physiologically Active Compounds Labeled with Radioactive and Stable Isotopes, Zvenigorod, 18-21 March 1991, Moscow, 1991, p. 3. 5. V. N. SIDOROV, UY. V. POLYAK, V. V. ROSHKE, P. A. LAKTIONOV, A. G. KIST, Russian Patent No. SU 1823961 A3. 6. H. C. THREEMAN, Environ. Monit. Assess., 4 (1984) 389. 7. V. LEONI, Chromatogr., 62 (1971) 63. 8. V. CONTARDI, R. CAPELLI, Y. ZANICCHI, Analyst, 108 (1983) 510.

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