ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979
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Salting-Out Chromatography Applied to Separation and Analysis of Mixtures of Thioureas and Thioacetamide by High Performance Liquid Chromatography Akio Hashirnoto Water Quality Research Section, Kitakyushu Municipal Institute of Environmental Health Science, 1-2-1 Shin-ike, Tobataku, Kitakyushu City, 804, Japan
Salting-out chromatography was applied to the separation of thiourea, ethylenethiourea, 1,3-dimethyl-2-thiourea, I-allyl2-thiourea, 1,3-diethyl-2-thiourea, and thioacetamide by high performance liquid chromatography. The best results were obtained when the flow rate is 18 mL/h, and the column temperature 60 ‘C, using 1.0-1.1 M of ammonium sulfate aqueous solution as a mobile phase. This method was applied to the analysis of field soil samples spiked with thiourea. A recovery of 99.3 f 2.66% ( n = 5 ) was obtained at the level of 160 ng/mL. The limits of detection were estimated to be 2.7 ng.
Thiourea (TU), one of the most widely used nitrification inhibitors in a compound fertilizer, has been reported to be a potential carcinogen in rats (1-4). In 1976 in Japan, 2714 tons of fertilizer containing T U was produced ( 5 ) . Because of t h e widespread usage of t h e fertilizer, data were needed t o see whether the compound would be a residue problem in t h e environment. T o obtain such data, a new method of analysis had t o be developed. Several methods for t h e determination of T U such as colorimetric procedures (6-9), a n d chromatographic procedures, Le., paper chromatography ( I O ) , thin-layer chromatography on silica gel ( 2 1 , I 2 ) , on alumina ( 1 3 ) , and column chromatography on ion-exchange resins (I4-16), etc. have been reported. However, these methods have not succeeded in complete separation of thioureas and thioacetamide, and also are insufficient for monitoring trace amounts of T U in t h e environment. In this paper, application of salting-out chromatography t o t h e separation of a mixture of thioureas [thiourea (TU), ethylene-thiourea (ETU), 1,3-dimethyl-2-thiourea (DMTU), 1-allyl-2-thiourea (ATU), and 1,3-diethyl-2-thiourea (DETU)], and thioacetamide (TAA) in water by HPLC, and application of this procedure t o the determination of T U spiked to field soil samples are described. T h e method is simple, rapid, and sensitive.
EXPERIMENTAL Apparatus. Chromatographic separations were carried out with a chromatographic system consisting of a Hitachi model 635 pumping system (Hitachi, Ltd., Tokyo, Japan), a Hitachi sampling injection valve (catalogue no. 645-0435) and a Hitachi Wavelength Tunable Effluent Monitor (catalogue no. 634-0514). Temperature of the column was controlled with a Hitachi Circulating Water Bath Temperature Controller (catalogue no. 635-0203). Retention time and peak area were measured by a Hitachi Model J211A Digital Integrator. The columns were 2.6 mm i.d. X 500 mm length stainless steel with a water jacket. Ultraviolet spectra were measured with a Shimadzu Multi-purpose Recording spectrophotometer Model MPS 5000 (Shimadzu, Ltd., Kyoto, Japan). Reagents and Materials. All reagents used were of analytical grade. Ethylene thiourea (2-imidazolidine thione), 1,3-dimethyl-2-thiourea, 1,3-diethyl-2-thiourea,and thioacetamide were supplied by Tokyokasei, Ltd., Tokyo, Japan. Thiourea was 0003-2700/79/0351-0385$01 OO/O
supplied by Kishida Chemical Ltd., Osaka, Japan. 1-Allyl-2thiourea was supplied by Kanto Chemical Co., Tokyo, Japan. A Hitachi custom cation-exchange resin with most probable pore size 17.5 pm (catalogue no. 2610) was supplied by Hitachi Ltd., Tokyo, Japan. Preparation of Mobile Phase. The ammonium sulfate aqueous solution used as the mobile phase was filtered with a Toyo No. 5C filter paper and the filtrate was degassed ultrasonically for 2 h. Packing of Cation-Exchange Resin. ‘The cation-exchange resin was packed with the conventional method, wet packing at a pressure of 100 kg/cm2, suspending in 1.0 M ammonium sulfate solution and equilibrating for 30 min. Preparation of Standard Solution. Thioureas except for ETU and TAA were weighed accurately to 100.0 mg and were dissolved in water purified by ion-exchange and distillation. ETU was weighed and dissolved in 20 mL of methanol, subsequently in water purified by ion exchange and distillation. Each solution was made up to 50 mL with water in a calibrated flask, and then these mother solutions were diluted to appropriate concentrations. Procedure. A 5-pL aliquot of an aqueous solution containing the mixture of thioureas and thioacetamide (40 pg/mL each) was injected by syringe with sampling injection valve in the separation experiments, and also in the sensitivity and precision section; 100 pL was injected with a loop type sampling system. In the separation section, three factors were varied: (1) the column temperature from 23 to 60 “C; (2) the flow rate of mobile phase from 18 to 42 mL/h; and (3) the concentration of ammonium sulfate from 0.05 to 1.5 M. The eluate was monitored at 240 nm. Extraction Procedure for Field Soil Sample. To 5 g of field soil sample spiked with 4.0 pg of T U was added 20 mL of pure water and stirred well with a magnetic mixer, then filtered with a dried 5C Toyo filter paper. Twenty pg of 1,3-dimethylthiourea as an internal standard was added to the filtrate and the total volume was made up to 25 mL in a calibrated flask. A 100-pL aliquot of the sample was injected into HPLC.
RESULTS A N D DISCUSSION T h e ultraviolet absorption curve for each compound in aqueous solution is shown in Figure 1. Absorption maximum and the molar absorptivity are 237 n m (1.02 x lo4), 234 n m (1.05 X lo‘)), 236 n m (1.06 X lo4),234 n m (1.37 X lo4),239 n m (1.05 X lo4), and 264 n m (1.11 X lo4) for T U , D M T U , DETU, ETU, ATU, and TAA, respectively. T o detect all the compounds tested, the absorption at 240 n m was selected for monitoring t h e eluate throughout t h e experiments. T o evaluate some characteristics of salting-out chromatography using HPLC, the usual formulas for capacity factor k’, the height equivalent to a theoretical plate H E T P , enthalpy AH, a n d salting-out constant K , ( I 7 ) ,were used. T h e limits of detection were estimated as twice the signal-to-noise ratio. Effect of Concentration of Ammonium Sulfate. T h e logarithms of h’ are plotted against t h e concentration of ammonium sulfate in Figure 2. These plots follow fairly closely Equation 1. logh’= logh’o+KsM
(1)
where k b is t h e capacity factor with water as mobile phase, k’ is t h e capacity factor with t h e aqueous salt solution of ’? 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 51, NO. 3, MARCH 1979 70
1
r
2 p
Figure 1. Ultraviolet absorption curve for each compound in water 1, TU, 2, TAA; 3, ETU, 4, DMTU; 5, ATU, and 6, DETU Concentration is 8 pg/mL each 4
0
8
16
24
Retertion ti-e
32
40
48
11n
Figure 3. Typical chromatogram of compounds. Numbering of compounds as in Figure 1. S = solvent. Conditions as in Figure 2. Mobile phase is 1.1 M ammonium sulfate
0.5 :.5
1 . 5 :,
1 , '? CCnC?ntT2tlOn
Of
('.L':'zSOd
Figure 2. log k ' v s . concentration of ammonium sulfate. -0-UDMTU, -HATU, - -A-ADMTU, -A-A- ETU, -0-0- TAA, -0-0- TU. Conditions: column, 500 mm X 2.6 mm, Hitachi cation-exchange resin #2610; flow rate, 18 mL/h (570 psi); column temperature, 60 OC; concentration, 200 ng for each compound with 5-pL injection; sensitivity, 0.16 AUFS
Table I. Salting-Out Constants' and A H compound
log k " ,
K,
A H kcalimol
TU TAA ETU DMTU ATU DETU
-0.071 0.037 0.049 0.097 0.20 0.32
0.038 0.18 0.24 0.29 0.27
1.88 2.33 2.82 2.65 2.87 3.22
0.45
a The constants listed were calculated from the mean value of at least two measurements.
molarity M , and K , is the salting-out constant. These constants are listed in Table I. Increasing the concentration of ammonium sulfate from 0.05 t o 1.5 M , the h'value for each compound, except for T U , increased, i.e., the higher the concentration of ammonium sulfate, the stronger are the compounds, except for T U , absorbed on the column. T h e salting-out constant K,, calculated from the slopes of Figure 2, shows the relative magnitude of the salting-out effect of ammonium sulfate on each compound. The K , is largest for DETU, DMTU, ATU, ETU, TAA, and smallest for TU, which is little affected by the concentration of ammonium sulfate as is the K , value shown. I n order to obtain some insight into the nature of an interaction in this system, enthalpies of transfer from the stationary phase t o the mobile phase were determined, AH was obtained by plotting the logarithm h'against 1/ T , and the values are given in Table I. The AH is largest for DETU.
ATU, DMTU, E T U , TAA, and is smallest for T U . T h e magnitude of the AH of this system is similar to that of usual absorption chromatography (18). The magnitude of K, of each compound is proportional to t h a t of the steric effect of Nsubstituted functional group of thioureas, Le., 1,3-diethyl of DETU is bulkiest among the compounds tested and 1,3-dimethyl of DMTU, 1-allyl of ATU, ethyl of ETU, and hydrogen of T U is smallest. These facts give a good explanation for the elution sequence of the compounds studied, Le., the elution sequence is determined by h b and K , in the salting-out chromatography. Effect of Flow Rate on Column Efficiency. T h e logarithm of H E T P , calculated from the data of D E T U on the last peak of the chromatogram, was plotted against the logarithm of flow rate. The relationship between H E T P and c follows closely the equation, H = Au" (19). T h e calculated n value is 0.935, and this value is higher than that of the other chromatographic systems reported ( I 8 , Z O ) . T h e result means t h a t the resin used is not suitable for higher speed operation than t h a t described in this paper in the column system of salting-out chromatography, because the high flow rate of the mobile phase causes the decrease of column efficiency. As was the result shown, the H E T P of the column is improved about twofold from 0.671 a t 42 m L / h t o 0.313 a t 18 m L / h . Effect of Column Temperature on Column Efficiency. T h e H E T P , calculated from the data of DETU, was improved about fourfold from 1.28 a t 23 " C to 0.29 a t 60 "C. In conclusion, when the concentration of ammonium sulfate, the flow rate, and the column temperature were 1.0-1.1 M , 18 m L / h , and 60 "C, respectively, the complete separation of five thioureas and TAA was obtained as shown in Figure 3. FVith the chromatographic conditions described, the relationship between peak height and concentration of each compound is linear within the range of 0 to 300 ng with 5-pL injections. T h e relative standard deviation calculated from a series of 10 replicate determinations a t the level of 200 ng of each compound, in the form of standard solution, was 2.670, 2.470, 3.4%, 2.470, 2.670, and 2.2% for T U , TAA, E T U , DMTU, ATU. and DETU, with 5-pL injections, respectively.
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applied to industrial waste, river water, seawater, and secondary treated sewage effluent, and none of the samples showed interfering peaks; T U was not detected.
ACKNOWLEDGMENT T h e author thanks T. Akiyama, Research Director of the Kitakyushu Municipal Institute of Environmental Health Sciences, for his valuable advice and encouragement. He also thanks the authority of the institute, for permission to publish this paper.
2
LITERATURE CITED
1 9
It
2q
1
0
1
iieteition t i r e
-
I E
I'
Tin
i
I6
H. J. Morris, J . Nafl. Cancer Inst., 7, 159 (1946). H. D. Purves and F. W. Grissbach, Br. J . Exp. Pafhol., 27, 24 (1946). H. D. Purves, B r . J . Exp. Patbol., 27, 294 (1946). A. Rosin and M. Bachmilewitz, Cancer Res., 14, 494 (1954). M. Watanabe, Hiryo Nenkan (in Japanese), 1978, p 337. R. C. Hoseney and K . F. Finny, Anal. Chem., 36, 2145 (1964). Association of Official Analytical Chemists, "Official Methods of Analysis", 11th ed., Washington D.C., 1970, p 350, 29.099, E. T. Raktzes, Anal. Chim. Acta, 78, 495 (1975). S. Goto, 0. Ogawa, I . Asakawa, and C. Oshima, Nippon Kogyo Kaishi, 88, 1067 (1972). W. P. Mckinley and R. Yasin, J. Assoc. Off. Anal. Chem., 43, 829 (1960). J. H. Onley and G. Yip, J . Assoc. Off. Anal. Chem., 54, 165 (1971). M. B. Devani. C. J. Shishoo, and B. K. Dadia, J . Chromatogr.. 105, 186 (1975). I . M. Kovina and N. K . Rozhkova, Dokl. Akad. Nauk. Uzb. SSR, 25, 25 (1968). K. Seiffarth and W. Aldelt, Plaste Kautsch., 15, 818 (1968). E. N. Boitsov, Yu. I. Mushkin, and V. M. Kavlik, Zavod. Lab., 35, 790 (1969). N. Nornura, D. Shiho, K. Osuga, and M. Yarnato, J . Chromatogr., 42, 226 (1969). R. N. Sargent and W. Riernan 111, J . Phys. Chem., 61, 354 (1957). K. Fujita, Y . Arikawa. and S. Ganno, Nippon Kagaku Kaishi, 3, 463 (1975). J. L. Waters, J. N. Little, and D. F. Horgan, J , Chromatogr. Scl., 7, 293 (1969). L. R. Snyder. J . Chromatogr. Sci,, 7, 352 (1969).
24
Figure 4.
HPLC of ( A ) blank o f field soil and (B) field soil spiked TU at t h e level of 160 ng/mL. (1) TU, ( 2 ) DMTU as internal standard. Conditions as in Figure 3. S a m p l e v o l u m e injected: 1 0 0 pL, U V monitored: 2 3 7 nm, AUFS: 0.08
T h e limits of detection were estimated to be 1.5 ng, 8.8 ng, 2.3 ng, 3.7 ng, 3.7 ng, and 9.1 ng for T U , TAA, E T U , DMTU, ATU, and D E T U , for a standard solution, with 100-pL inject ions, respectively. Analysis of TU Spiked to Field Soil Sample. Typical chromatograms of field soil samples are shown in Figure 4A and 4B. Control field soil samples showed no interfering peaks (Figure 4A). A recovery of 99.3 f 2.66% (mean f rel. stand. dev., n = 5) was obtained when T U was spiked to field soil samples at the level of 160 ng/mL. The limits of detection were estimated to be 2.7 ng in this case. The method was also
RECED-ED for review March 25, 1977. Resubmitted September 7. 1978. Accepted November 8, 1978. This work was financially supported by the Environment Agency of Japan.
High Gas Temperature Furnace for Species Determination of Organometallic Compounds with a High Pressure Liquid Chromatograph and a Zeeman Atomic Absorption Spectrometer Hideaki Koizumi, * ' Ralph D. McLaughlin, and Tetsuo Hadeishi Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720
Species determination of organometallic compounds is nowadays one of the most important subjects in analytical chemistry. This is because the toxicity of metals depends upon the binding conditions in the compound ( I ) . Speciation of organometallic compounds is also important in physiology and biology. Up to now, more than 1000 enzymes and coenzymes have been found a n d about one third of them are "metalloenzymes" or "metal-substrate complexes" (2). It is a well known fact t h a t commercial gasoline contains alkyllead compounds. In the United States, leaded gasoline contains around 0.19'0 of alkyllead, and commercial unleaded gasoline is defined as gasoline having not more than 0.05 g Pb/gal (13.16 pg/mL) ( 3 ) This paper describes the use of a special furnace Zeeman atomic absorption (ZAA) combination as a detector for a high pressure liquid chromatograph (HPLC) for the determination of organometallic lead com-
A new furnace has been constructed that allows atomic absorption detection of volatile organometallic compounds. The operation of this furnace is demonstrated by analyzing the eluent of a high pressure liquid chromatograph utilizing Zeeman atomic absorption spectrometry. The content of tetraethyllead in National Bureau of Standards gasoline standards was determined. Data are presented on the ability of this furnace to suppress interference with cadmium and lead determinations by MgCI,, CuCI,, and CaCI,. I t was found that two orders of magnitude more interferent can be tolerated. The determination of lead in automotive exhaust is also described.
'Permanent address, Naka Works, Hitachi Ltd., Katsuta, Ibaraki
312, Japan.
0003-2700/79/035 1-0387$01 O O / O
C
1979 American Chemical Society
