I AIDS FOR ANALYTICAL CHEMISTS I
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Separation of Water from Biological and Environmental Samples for Tritium Analysis A. A. Moghissi, E. W. Bretthauer, and National Environmental Research Center-Las 891 14
E. H. Compton Vegas, U.S. Environmental Protection Agency, P. 0. Box 15027, Las Vegas, Nev.
Numerous investigations require the removal of water from biological and environmental samples for subsequent tritium analysis. Environmental tritium monitoring and biokinetics studies require separation of water from a variety of samples. In recent years, more emphasis has been placed on quantitative separation of water to be able to analyze the remaining part of the sample for nonaqueous tritium. By far the most commonly used separation technique for this purpose is distillation under various temperatures and pressures. In the case of tritium, if the distillation process is not conducted to completion, errors may be introduced as a result of the isotope effect. These errors increase as the temperature decreases ( 1 ) . If the distillation is conducted to complete dryness, other compounds which may be present decompose and contaminate the water, resulting in quenching and/or chemiluminescence in the subsequent liquid scintillation counting. In certain cases, because of the sensitivity of the sample to heat, distillation at low temperatures is required to avoid the decomposition of the sample. Low temperature distillation is, however, time consuming. All these disadvantages can be avoided when an appropriate azeotropic distillation technique is applied. Azeotropic distillation of water using various aromatic and aliphatic hydrocarbons is a standard technique in the oil industry ( 2 ) . Because of the compatibility of aromatic hydrocarbons with the liquid scintillation process, these hydrocarbons were considered as co-distillates and, because of reasons which will be discussed later in this paper, benzene was selected.
EXPERIMENTAL Standard glassware used in the oil industry was slightly modified to accommodate the requirements of the preparative scale. The modified system shown in Figure 1 consisted of adding a tube containing a desiccant t o the top of the condenser to avoid an exchange of the air moisture and the water originating from the sample. The system was designed to obtain 15-25 ml of water, a quantity required for liquid scintillation counting of environmental samples. Up to 250-gram samples were covered with dry thiophenefree benzene and distilled a t 69 "C. The optimum sample sizes and the required quantity of benzene is shown in Table I. The completion of the distillation process can be indicated by an increase in the boiling point from 69 to 80 "C. The completion of the distillation process utilized in this procedure, however, was indicated when the volume of the water in the moisture test receiver remained constant for 1 hr. The distillation time for the sample types shown in Table I ranged from 2 to 4 hr. Various types of samples were evaluated. In some cases, the specific activity of tritium was known. In other cases, the reproducibility of the procedure was evaluated by running replicates. Results of this procedure were compared with distillation at normal pressure and vacuum distillation a t room temperature. (1) W . M . Jones. J. Chem. Phys., 48 207 (1968). (2) "1969 Book of ASTM Standards," Part 18, 2nd ed., American ety for Testing and Materials, Philadelphia, Pa., p p 16-19.
Soci-
Rabbit tissues were prepared from an animal which was injected with tritiated water and sacrificed 24 hr later. Green chop and hay samples were in vitro labeled and therefore their tritium concentration was known. All samples were counted in a liquid scintillation counter as. previously described (3).
RESULTS AND DISCUSSION Table I1 shows the results of the experiments. The precision of the azeotrope distillation is obvious and the errors associated with the specific activity of tritium are well within expected limits. The accuracy of the procedure is demonstrated for the in vitro labeled samples-ie., urine and milk. In these cases, the results are in reasonable agreement with the known activity of the sample. The preparation of an in uiuo labeled biological sample with a known specific activity would have been tedious, if not impossible. A comparison with other techniques is, however, indicative of the accuracy of this procedure. In all cases, results obtained using azeotropic distillation are in general agreement with the results obtained using other procedures. The reason for the consistently higher specific activity of urine samples A and B using normal distillation is probably due to the presence of impurities in the distillate and subsequent chemiluminescence in the liquid scintillation system. Five approximately equal portions of water were removed from each of the sample types shown in Table I during the distillation process. Tritium analysis of these samples indicated that the isotope effect was less than 3%. The separation of water from inorganic material such as soil is usually possible only for that water which is relatively loosely bound. This is valid for azeotropic distillation as well as for any other distillation except for those carried out at temperatures substantially exceeding 100 "C. This is clearly demonstrated in an experiment carried out with copper sulfate. CuSO4 usually contains five water molecules, one of which is firmly bound. Azeotropic distillation removed an equivalent of 3.96 moles of water. The last mole of water could not be removed by this technique. The last water also could not be removed at 180 "C, indicating the stability of the binding. It is conceivable that soil may contain water which has similar binding properties and thus only a part of the water could be removed by azeotropic distillation. This disadvantage is, however, common to all distillation techniques unless very high temperatures are applied. The application of high temperatures, however, may cause the decomposition of organic compounds present in the soil and thus contaminate the water with impurities. Although a few experiments indicated that toluene and xylene can also be used for azeotropic distillation of water, (3) R Lieberman and A A Moghissi, Int J Appi Radiat Isotop 319 (1970)
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Table II. Results of Azeotropic Distillation (AD) of Water from Various Samples Expressed in nCi/l. and Compared with Distillation (ND) and Vacuum Distillation (VD) AD,
Sample type
Soil A Soil B
Animal tissue
Green chop A
Green choD B
1 Figure 1. Azeotropic distillation
HEATER
1
apparatus Milk A Milk B
Table I. Sample Sizes, and the Quantity of Benzene Required for Various Sample Types Sample type
Sample weight, grams
Benzene, mi
Urine A
Soil Hay Green chop Urine Animal and human tissue
200 50 30 20 30
1300 400 70 50 150
Urine B
neither one was considered. Xylene and toluene would have the advantage of a higher water content in the vapor of 40 and 20%, respectively, compared to a 9% water for benzene. The corresponding boiling points are 94.5, 85, and 69.4 "C, respectively. The lower boiling point of the benzene mixture was considered an advantage in preserving heat sensitive biological samples. Another advantage of benzene as compared to toluene and xylene was the stability of its hydrogen atoms for exchange with tritium present in water or other compounds of the sample. Experiments conducted in this laboratory indicated that less than 0.1% of the tritium atoms in water exchanged with benzene under experimental conditions of a normal azeotropic distillation. Although this advantage is to some extent valid for toluene and xylene, the presence of more mobile hydrogen atoms in their methyl groups was considered as a possible enhancement factor for hydrogen exchange. The operation and advantages of azeotropic distillation are numerous. Water extraction is as efficient as either normal or vacuum distillation. The separation time is usually short and comparable to the distillation a t normal
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Urine C Urine D
nCi/ml 107.0 107.5 128.7 129.5 130.0 129.7 128.7 129.0 4.06 4.12 4.1 1 4.1 1 106.9 110.1 110.3 109.1 113.1 112.8 113.6 109.9 110.6 112.6 0.840 0.846 9.17 9.14 9.29 9.18 8.02 7.89 14.35 14.90 3.66 3.42 82.2 81.3 81.9 81.6 82.3 81.9 81.9 81.8
ND, nCi/ml
VD,
Known,
nCi/ml
nCi/mi
109.1 109.2
0.841 0.846
0.850 0.850 8.94
9.20 9.13 15.64 16.02 3.23 3.09 82.9
pressure. The operation does not require constant observation, as subsequent to the removal of water, if the operation is not stopped, benzene is refluxed with no significant damage to the remaining sample. If the remaining part of the sample must be further processed, the bulk of benzene can be distilled off and the remainder removed either by vacuum distillation or by gentle heating of the sample in a steam bath. If the procedure is to be used for a large number of samples, substantial cost savings can be obtained by purification of the benzene for reuse. This purification technique consists simply of the addition of low level or background water to the benzene followed by an azeotrope distillation. Received for review October 30, 1972. Accepted February 9, 1973.
