Anal. Chem. 1997, 69, 536-541
Packed-Column Supercritical Fluid Chromatography: Quantitative Determination of Uranium without Liquid Waste Generation V. Martin-Daguet,† P. Gasnier,‡ and M. Caude*,§
Centre d’Etude de Saclay, Commissariat a` l’Energie Atomique, DCC/DPE/SPEA/SAIS, Bat391, 91191 Gif sur Yvette, France, Service Laboratoire, Etablissement de Marcoule, Coge´ ma, BP 170, 30205 Bagnols sur Ce` ze Cedex, France, and Laboratoire de Chimie Analytique (associe´ au CNRS 437), Ecole Supe´ rieure de Physique et de Chimie Industrielles de la Ville de Paris, 10 rue Vauquelin, 75231 Paris Cedex 05, France
A new procedure for the determination of uranium by packed-column supercritical fluid chromatography is proposed. A nonfluorinated chelating agent selective for copper and uranium, the 2,6-diacetylpyridine bis(benzoylhydrazone), has been chosen. We have studied its chromatographic properties on different stationary phases and the influence of the methanol content in the carbon dioxide mobile phase. The separation of the metal compounds was conducted with and without solvent injection. A calibration curve was obtained for uranium in the range of 52-323 ng injected. The accuracy of the method is 0.5%, the repeatability 4%. The same studies were performed with a new compound, diacetyl-2,6 pyridine bis(4-tert-butyl benzoylhydrazone). An increase in retention and efficiency was then observed. It is a well-known fact that the use of organic solvents presents two major dificulties, first, personnel exposures to toxic and highly flammable vapors and second, environmental disposal problems.1 In analysis laboratories working either in the nuclear domain or with highly toxic compounds, the first concern is easily managed by the sytematic use of gloveboxes. On the other hand, the second has proven to be more critical. Here, to overcome these two major difficulties, we look into the study of alternative techniques to put in place of a widespread analytical procedure, that is, liquid phase chromatography. In this technique, the mobile phase, whose flow can reach several milliliters per minute, is contaminated by the toxic sample introduced in the chromatographic system, thus generating large amounts of waste. In chromatography, only two practical solutions can be considered. The first is to use microchromatography, in order to reduce drastically the mobile phase consumption.2 However even these small amounts of liquid waste remain difficult to reprocess because the toxic mixtures are often chemically complex, highly flammable, or corrosive. The second solution, which is presented in this study, is to use supercritical fluid chromatography (SFC).3 The mobile phase can then be CO2, which is the most commonly used supercritical fluid. It is †
Commissariat a` l’Energie Atomique. Coge´ma. § Ecole Supe ´rieure de Physique et de Chimie Industrielles de la Ville de Paris. (1) Hawthorne, S. B. Anal. Chem. 1990, 62, 633A-642A. (2) Rosset, R.; Caude, M.; Jardy A. Chromatographies en phases liquide et supercritique; Masson: Paris, 1991; pp 200, 815. ‡
536 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
nontoxic, nonflammable, nonpolluting, and relatively inexpensive4 but has the weakness of low polarity. The addition of a small quantity of a polar organic solvent such as methanol easily gets round this disadvantage. Compared to liquid phase chromatography, the amount of solvent used is then severely reduced, and compared to microchromatography, the wastes produced are chemically very simple as they consist of only CO2 and methanol. Moreover, these wastes are gaseous, owing to the slow expansion of the supercritical mobile phase out of the chromatographic system in an experimental disposal described here. This gas is easily treated by simple techniques such as filtration, thus facilitating disposal problems. We have chosen to apply SFC to a radioactive metal cation, uranyl. Because ionic compounds cannot be dissolved in supercritical carbon dioxide, it is necessary to complex the metal cations with an organic chelating agent prior to the injection in the SFC system. 2,6-diacetylpyridine bis(benzoylhydrazone)21-23 (H2DIB), shown in Figure 1, has been chosen here. The chromatographic properties of the uranyl complex UO2DIB are studied on various (3) Eckert, C. A.; Van Alsten, J. G.; Stoicos, T. Environ. Sci. Technol. 1986, 20, 319-325. (4) Liu, Y.; Lopez-Avilla, V.; Alcaraz, M.; Beckert, W. F.; Heithmar, E. M. J. Chromatogr. Sci. 1993, 31, 310-316. (5) Lin, Y.; Brauer, R. D.; Laintz, K. E.; Wai, C. M. Anal. Chem. 1993, 65, 2549-2551. (6) Laintz, K .E.; Wai, C. M.;Yonker, C. R.; Smith, R. D. Anal. Chem. 1992, 64, 2875-2878 (7) Laintz, K. E; Wai, C. M. Anal. Chem. 1992, 64, 2475. (8) Wang, J.; Marshall, W. D. Anal. Chem. 1994, 66, 1658-1663. (9) Mannien, P.; Riekkola, M. L. J. High Resolut. Chromatogr. 1991, 14, 210211. (10) Laintz, K. E.; Shieh, G. M.; Wai, C. M. J. Chromatogr. Sci. 1992, 30, 120123. (11) Yu, J. J.; Wai, C. M. Anal. Chem. 1991, 63, 842. (12) Laintz, K. E.; Yu, J. J.; Wai, C. M. Anal. Chem. 1992, 64, 311-315. (13) Carey, J. M.; Vela, N. P.; Caruso, J. A. J. Chromatogr. 1994, 622, 329340. (14) Ashraf-Khorassani, M.; Taylor, L. T. J. Chromatogr. Sci. 1989, 27, 329. (15) Wenclawiak, B.; Bickmann, F. Fresenius Z. Anal. Chem. 1984, 319, 305. (16) Bickmann, F.; Wenclawiak, B. Fresenius Z. Anal. Chem. 1985, 320, 261. (17) Jinno, K.; Mae, H.; Fujimoto, C. J. High Resolut. Chromatogr. 1990, 13, 13-17. (18) Ashraf-Khorassani, M.; Hellgeth, J. W.; Taylor, L. T. Anal. Chem. 1987, 59, 2077-2081. (19) Laintz, K. E.; Meguro, J.; Iso, S.; Tachikawa, E. J. High Resolut. Chromatogr. 1993, 16, 372. (20) Musikas, C. Sep. Sci. Technol. 1988, 23, 1211. (21) Casoli, A.; Mangia, A.; Predieri, G. Anal. Chem. 1985, 5, 561. (22) Casoli, A.; Mangia, A.; Mori, G.; Predieri, G. Anal.Chim. Acta 1986, 186, 283. (23) Casoli, A.; Mangia, A.; Predieri, G. Anal. Chim. Acta 1977, 24, L5-L6. S0003-2700(96)00163-1 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Structure of H2DIB and H2terBuDIB.
alkyl stationary phases. UO2DIB is then separated from H2DIB and from the copper complex CuDIB. We have also prepared a new, more hydrophobic, chelating agent, H2terBuDIB (Figure 1). The modification of the chromatographic properties induced by the presence of the tert-butyl groups is discussed. EXPERIMENTAL SECTION Chemicals. The chelating agent H2DIB was synthesized from the Aldrich products 2,6-diacetylpyridine and benzoylhydrazide, as reported by Pelizzi26 and recrystallized from an acetone/ methanol mixture (20:80) (v/v). H2terBuDIB was prepared from 4-tert-butylbenzhydrazide (Lancaster, Bischheim, France) with the same experimental conditions as H2DIB. Uranium solutions (CEA, Paris, France) were obtained from a 400 g/L standard solution in 3 M nitric acid and copper from a 1 g/L standard solution in 5% nitric acid (Johnson Matthey GmbH, Karlsruhe, Germany). Metal complexes were dissolved in ethyl acetate (Merck, Darmstadt, Germany) and prepared by liquid/liquid extraction from an aqueous nitric phase adjusted by dilution to pH 3. The two phases (50 mL each) were shaken for 2 min and then separated. A 10,000 µg/mL uranium standard solution used to evaluate the method (PLU2-3Y, Spex Plasma, Longumeau, France). All products were characterized by direct chemical ionization mass spectrometry (Delsi-Nermag, R3010, Choisy Le Roi, France) and UV/visible spectrometry. The UV/visible spectra of H2terBuDIB, UO2terBuDIB, and CuterBuDIB, are similar to those of H2DIB, UO2DIB, and CuDIB, respectively.22 Apparatus. A homemade supercritical fluid chromatographic system has been designed in order to minimize the number of devices located inside the glovebox. (Figure 2). Only the apparatus in contact with the toxic sample (the injection valve, the analytical column, the oven, the detector cell, the outlet pressure gauge, the back-pressure regulation valve, and the mobile phase expansion disposal) were thus installed inside the enclosure. Packed-column SFC is preferred to capillary SFC as it allows easy pressure monitoring with variable restrictors and enables one to work at high mobile phase flow rate entailing fast analysis.2 Moreover, in our opinion, packed columns are easier to handle than capillary columns and the obtained results are often more reproducible. Carbon dioxide purchased from Air Liquide (B20 or B50, N48 grade, Paris, France) was pumped with a monopiston pump (Model 305, Gilson Medical, Villiers-Le-Bel, France). A Lauda RM6 cryostat (Prolabo, Fontenay sous bois, France) was used to cool the CO2 pump head at -7 °C. A 307 Gilson Medical pump equipped with a 5 mL head size was employed to add the methanol (24) Bonilla-Alvarez, M.; Palmieri, M.; Davis, D.; Fritz, J. Talanta 1987, 5, 473. (25) Main, M. V.; Fritz, J. S. Anal. Chem. 1989, 61, 1272. (26) Nardelli, M.; Pelizzi, C.; Pelizzi, G. Trans. Met. Chem. 1977, 2, 35.
Figure 2. Experimental setup: (1) polar modifier, (2) gaz cylinder, (3) cryostat, (4) pump, (5) dynamic mixer, pulse dampener, inlet pressure gauge, (6a and 6b) UV-visible diode array detector, (7) analytical column, (8) injection valve, (9) oven, (10) outlet pressure valve, (11) pressure regulation valve, (12) mobile phase expansion device (a) pressure regulation valve, (b) tubing (5 m, 1.59 mm), (c) tubing covered by ice (3 m, 3.18 mm), (d) tubing (2 m, 6.35 mm), (e) cylinder (0.5 m, 60 mm), (f) tubing(0.5 m, 6.35 mm), and (g) air paper filter detail], (13) glovebox ventilation air intake, and (14) glovebox ventilation outake filters.
(Merck). Carbon dioxide and CH3OH were mixed at room temperature in a 1.5 mL dynamic mixer (Gilson, Model 311c). A Croco-sil oven (C.I.L., Sainte Foy La Grande, France) was used to control the temperature of the analytical column. The UV/ visible diode array detector (DAD 440, Kontron, Montigny-LeBretonneux, France) equipped with a high-pressure cell (1 cm path length, 8 µL internal volume) was divided into two parts in order to keep all control knobs outside of the box. Only the optical part (cell, diode, lamp, etc.) was thus situated inside the enclosure. Back-pressure regulation was realized with a Tescom valve (Model 26-1729-24-082, Tescom Corp., Elk River, MN). The column pressure drop was measured with two pressure gauges: the inlet pressure P1 (Gilson) and the outlet pressure P2 (Model 944, Chromatem Touzart et Matignon, Courtaboeuf, France). All pressure values mentioned in this paper are the outlet values P2. The expansion of the mobile phase out of the back-pressure regulator was ensured by passing it through tubes of increasing diameter, as shown. The endothermic expansion of the mobile phase occurred mainly within the tube c. In this way, heating the pressure regulation valve was not necessary, entailing a more simple device. The mobile phase, even if containing volatile polar modifier, was entirely gaseous in the cylinder. This disposal was connected to the glovebox ventilation system. This system is mainly made of two paper filters (Model 3202.04, Camfil, La Garenne Colombes, France) especially designed for gloveboxes in order to hold back uranium. One filter was located inside the box, the other outside. The gazeous waste obtained from the slow expansion of the mobile phase could then pass through the paper air filters without wetting them. The uranium complex was stopped by the first paper filter. Nevertheless, both paper filters Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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were periodically changed and incinerated. It is important to notice that the use of a high-pressure SFC system inside a glovebox entailed some safety precautions. The pressure inside the box was continuously monitored by a manometer connected to an alarm (ACV, Gif sur Yvette, France). An oil pressure regulation safety valve was located on the box roof to prevent any rapid increase of pressure inside the box. Samples were injected through a Rheodyne 8125 valve equipped with a 5 or 10 µL loop (Rheodyne, Cotati, CA). For injections with a solvent elimination technique, a 7010 Rheodyne valve was added to the 8125 valve already mentioned by Rocca.29 Liquid sample was introduced onto the precolumn. The sampling solvent was then evaporated by percolating nitrogen gas. The mobile phase flowed through the precolumn, sweeping the sample onto the analytical column. The nitrogen used was from Air Liquide (N48 grade). Seven stationary phases were studied: Spherisorb ODS2, Nova Pack C18, Inertsil ODS2, Zorbax RX-C18, Capcell-Pack C18, Asahipac C18 and PRP-1 (Interchim, Montluc¸ on, France). All columns are 25 cm long and have a 0.46 cm internal diameter, except for the Nova Pack column, which is 30 cm long with a 0.39 cm internal diameter. The precolumn used for solvent venting is 1.25 cm long and 4 mm wide (RX-C18, Zorbax, Interchim, Montluc¸ on, France). The variability of the parameters (k′, h, As) was obtained by evaluating the dead and retention times (( 0.2 min). The variability of the number of plates was calculated graphically and thus depends somewhat on the printer paper speed. RESULTS AND DISCUSSION Complexing Agents. Relatively few publications deal with supercritical fluid extraction or chromatography of metal chelates. Extraction of various metal cations such as lanthanides, copper, or zinc from aqueous media or solid materials has been shown recently.5-8 The main problem is the choice of the chelating agent. The molecular complex formed must be nonpolar and exhibit high thermal and chemical stability. Derivatives of dithiocarbamic acid9-12 and β-diketone13 have been used in capillary SFC with varying success. Diethyldithiocarbamic acid was found to have a low solubility in supercritical carbon dioxide. Sample decomposition and irreversible retention were then observed. These problems were solved by the substitution of the ethyl groups of diethyldithiocarbamic acid for isobutyl groups9 or fluorine.10-12 Substituting fluorine for hydrogen generally enhances the volatility and the thermal stability of the resulting metal chelate.7,11 This increase of stability accompanied by an enhanced solubility in supercritical CO2 is then responsible for the improved chromatographic behavior. In packed-column SFC, separations of porphyrins,14 oxinates,15,16 and β-diketonates17,18 have been reported. For most of these chromatographic separations, dissociation of the metal complexes and peak tailing were observed. As in capillary SFC, these problems seem to be solved by the use of a fluorinated chelating agents such as thenoyltrifluoroacetone.19 It is important to notice that we have added a new criterion relating to the elementary composition of the complexing agent. The organic wastes do have to be reprocessed without generating harmful residues. In order to satisfy this waste disposal rule, we have restricted our choice of reagents (polar modifier, chelating agent, injection solvent, etc.) to compounds containing only carbon, hydrogen, oxygen, and nitrogen atoms. When held up 538
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by the filtration system, theses compounds can be destroyed by standard organic waste incineration procedures without generating toxic gases (e.g., HCl, HF) or solid residues (e.g., P2O5 for tributyl phosphate).20 2,6-Diacetylpyridine bis(benzoylhydrazone) was selected as its properties respect our entire list of criteria. H2DIB and its watersoluble derivatives24,25 have previously been studied for liquid chromatography. H2DIB is particularly suitable for the determination of uranium in the presence of other actinides (e.g., plutonium, neptunium) because of its selectivity toward the uranyl cation. It is a pentadentate chelating agent forming four fivemembered rings around the uranyl cation.21 This characteristic enhances the stability of the UO2DIB complex, obviating the need for chelating agent addition in the mobile phase. The only other cation that can interfere with uranyl is copper. Chromatographic Properties of UO2DIB. We first chose to pay attention to the chromatographic properties of the uranyl chelate UO2DIB. We evaluated in particular the performances of the stationary phases in order to combine the optimum UO2DIB peak efficiency with the minimum analysis time and methanol consumption. We first carried out experiments on organic polymeric stationary phases (PRP-1, Asahipac C18). A very high retention was then observed. For instance, a capacity factor of 300 was calculated with the PRP-1 stationary phase even for a mobile phase containing up to 20% (v/v) methanol. This retention could be related to the aromatic π-π interaction between the phenyl groups of the metal complex and the aromatic structure of the copolymer. We have chosen to use columns packed with deactivated C18grafted high-purity silica beads. These phases were deactivated either by dimethylsilane or trimethylsilane (Spherisorb ODS2, Nova-Pack C18, Inertsil ODS2, Zorbax RX-C18) or by silicon coating27,28 (Capcell-Pack C18). The variation of the capacity factor of UO2DIB vs the methanol content in the supercritical carbon dioxide is shown in Figure 3. With the Spherisorb ODS2 stationary phase, the retention time of the chelate appeared to be very high. Moreover UO2DIB was eluted as a tailing peak (k′ ) 20 and asymmetry factor equal to 5.5, for a methanol content equal to 5% v/v). Both phenomena were assumed to be due to interactions between the metal chelate and the residual silanol groups of the stationary phase. Compared to it, the Nova Pack C18, Inertsil ODS2, Zorbax RX C18, and Capcell Pack C18 stationary phases provided much better results in terms of retention (Table 1). Unfortunately the elution peak efficiency remains very low whatever the nature of stationary phase. For instance with the Capcell-Pack C18, a methanol content in the CO2 as low as 0.5% (v/v) was sufficient to elute within 5 min the uranyl chelate whereas at least 4% and 15 min were necessary for the Spherisorb ODS2 stationary phase. For the four mentioned stationary phases a leveling effect of the capacity factor can be observed for a methanol content greater than 5% (v/v). This was attributed to the covering of the residual silanol groups on the surface of the C18-grafted silica beads by the polar modifier. In order to confirm this hypothesis, we compared the retention of the uranyl chelate to that of a nonpolar polyaromatic compound, benz[a]anthracene, assumed to interact weakly with the residual (27) Fujimoto, C.; Suzuki, M.; Jinno, K. Chromatographia 1992, 34, (3/4), 121124. (28) Ohtsu, Y.; Shirota, O.; Ogawa, T.; Tanaka, I.; Ohta, T.; Nakaya, O.; Fujiyamma, Y. Chromatographia 1987, 24, 351-356.
Table 2. Capacity Factors of Benz[a]anthracene and UO2DIB with Different Stationary Phasesa k′ stationary phase
benz[a]anthracene
Nova-Pack C18 Spherisorb ODS2 Capcell-Pack C18 Zorbax Rx C18 Inertsil ODS2
4.47 ( 0.38 4.92 ( 0.38 6.5 ( 0.39 6.89 ( 0.43 9.47 ( 0.54
UO2DIB 0.55 ( 0.16b 200 ( 0.24c 6.4 ( 0.4b 20.00 ( 0.1.0c 0.56 ( 0.14b 1.28 ( 0.17c 0.58 ( 0.14b 1.47 ( 0.18c 0.59 ( 0.14b 1.74 ( 0.19c
a Conditions for UO DIB: eluent, MeOH/CO (v/v). b 10-90. c 52 2 95. P2, 265 bar; flow rate, 7 mL/min at 0 °C, temperature, 45 °C; detection at 345 nm. Conditions for benz[a]anthracene: eluent, CO2; P2, 265 bar, flow rate, 7 mL/min at 0 °C; temperature, 45 °C; detection at 230 nm; number of injections, 2.
Figure 3. Capacity factor variations of UO2DIB vs the methanol content in carbon dioxide (v/v) with different stationary phases: (O) Spherisorb ODS2, (×) Nova Pack C18, (2) Inertsil ODS2, (9) Zorbax RX C18, ([) Capcell-Pack C18. Eluent, P2 265 bars; flow rate, 7mL/ min at 0 °C; temperature, 45 °C. Detection at 345 nm. Table 1. Measured Capacity Factors, Efficiencies, Reduced Plate Heights, and Asymmetry Factors of the Uranyl Complex Elution Peak for Different Stationary Phasesa stationary phase Nova-Pack C18 Zorbax Rx C18 Inertsil ODS2 Capcell-Pack C18
k′UO
2DIB
3.8 ( 0.34 2.7( 0.24 2.8( 0.24 2.2 ( 0.22
h
As
175 ( 35 90 ( 24 96 ( 25 40 ( 18
6.3 ( 1 1.4 ( 0.3 5.7 ( 0.8 1.1 ( 0.3
a Conditions: eluent, (97:3) CO /MeOH (v/v), P , 275 bar, temper2 2 ature, 45 °C; flow rate, 8 mL/min at 0 °C for Zorbax RX C18, 7 mL/ min for the others, particle diameter, 7 µm for Zorbax RX C18, 5 µm for the others, number of injections, 2.
silanol groups. The results are shown in Table 2. This comparison has been accomplished with methanol contents of 10 and 5% (v/v) in the mobile phase. At 10% methanol content, the retention of benz[a]anthracene and UO2DIB was proportional only for the four stationary phases previously cited. The retention of the uranyl chelate on the Spherisorb ODS2 stationary phase was very high compared to the PAH retention because of the presence of the residual silanol groups. With a methanol content of 5% in the supercritical CO2, the proportionality was only observed for Inertsil ODS2, Zorbax RX-C18 and Capcell-Pack C18 stationary phases. For the Nova Pack C18 stationary phase, it appeared that a methanol content between 5 and 10% is needed to cover substantially the silanol groups entailing a low retention of the uranyl complex.
Figure 4. Capacity factor variations vs the methanol content in carbon dioxide (v/v) on Capcell Pack C18 stationary phase: (b) H2DIB, (O) H2terBuDIB, (2) UO2DIB, (4) UO2terBuDIB, (9) CuDIB, and (0) CuterBuDIB. Plain lines are for the compounds substituted by tert-butyl groups. Eluent, P2 265 bars; flow rate, 7 mL/min at 0 °C; temperature 45 °C. Detection at 330 nm.
From Figure 3 and Table 1, it appears that the silicon-coated C18-grafted silica (Capcell-Pack) provides the best results in terms of retention time, methanol consumption, peak efficiency, and symmetry. This stationary phase has been chosen to study the chromatographic properties of the chelating agent H2DIB and the copper complex CuDIB. Chromatographic Properties of H2DIB and CuDIB. The chromatographic properties of H2DIB have been studied vs the methanol content in the supercritical carbon dioxide. (Figure 4). We have found that UO2DIB and H2DIB were completely resolved, the retention time of ligand being much shorter than that of the uranyl chelate. In spite of a satisfactory resolution value (1.3 under Analytical Chemistry, Vol. 69, No. 3, February 1, 1997
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Table 3. Influence of Solvent Evaporation Prior to the Injection on Peak Number of Plates, Reduced Plate Height, and Resolution between Copper and Uraniuma injection “with solvent” complex
N
h
Rs
UO2DIB CuDIB UO2terBuDIB CuterBuDIB
1.250 ( 170 2.000 ( 280 5.000 ( 1.000 4.600 ( 900
40 ( 6 25 ( 4 10 ( 3 11 ( 3
1.3 ( 0.3 2.4 ( 0.3
“solventless” injection
UO2DIB CuDIB UO2terBuDIB CuterBuDIB
N
h
Rs
10.750 ( 1.700 7.600 ( 1.300 10.500 ( 1.700 9.400 ( 1.600
4.7 ( 1.5 6.6 ( 1.7 4.7 ( 1.5 5.3 ( 1.3
2.6 ( 0.3 3.5 ( 0.3
a Chromatographic conditions: stationary phases, precolumn Zorbax RX-C18; analytical column, Capcell-Pack C18; eluent, (97.5-2.5) CO2/ MeOH (v/v), P2, 300 bars, temperature, 45 °C; flow rate, 7 mL/min at 0 °C; detection at 330 nm; number of injections, 2.
the experimental conditions mentioned in Table 3) the reduced plate height of the uranyl and copper complexes were very high (h ) 40). In order to improve the chromatogrphic properties, two other analytical procedures were explored. The first one was to remove the injection solvent. It is well-known that liquid solvents having higher solvating power than carbon dioxide can cause peak splitting and band broadening when used for injection into the SFC system.29,30 As a matter of fact, part of the solute moves through the column with the injection solvent that is only slightly retained and according to the circumstances peak distortions, such as leading peak and double peak, can be observed. The improved injection process consisting in evaporating the injection solvent was realized with UO2DIB and CuDIB. The results are given in Table 3. Solventless injections improved the peak efficiencies by a factor of 8.5. The low solubility of the compounds in the supercritical mobile phase compared with the high solvating power of the injection solvent (ethyl acetate) could be partly responsible for the peak tailing as the solute may not has been tightly focused on the head of the column prior to the elution. Chromatographic Properties of H2terBuDIB, UO2terBuDIB, and CuterBuDIB Complexes. As solventless injections are not very easy to handle in a glovebox, we explored another way to improve the chromatographic properties of the complexes. A new chelating agent, H2terBuDIB, has been synthesized with the intention of lowering its polarity because of the presence of tert-butyl groups on both benzene rings (Figure 1). Of course, its chelating properties were not modified as the functional groups involved in the chelating mechanism remained unchanged. Both chelate solubility in the mobile phase and interactions between the solutes and the stationary phase were expected to be enhanced. The variations of H2terDIB, UO2terBuDIB, and CuterBuDIB capacity factors vs the methanol content in the mobile phase are shown in Figure 4. An increase in retention times compared to the previous compounds was observed because of the enhance(29) Cretier, G.; Madjalani, R.; Rocca, J. L. Chromatographia 1990, 11/12, 645650. (30) Kischner, C. H.; Taylor, L. T. J. High Resolut. Chromatogr. 1993, 73-84.
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Figure 5. Separation of UO2terBuDIB and CuterBuDIB with packed SFC. Stationary phase, Capcell-Pack C18; eluent, (3-97) MeOH/ CO2 (v/v); P2, 265 bars; flow rate, 7 mL/min at 0 °C; temperature, 45 °C; injection volume, 10 µL. Quantities injected: 150 ng for uranium and 250 ng for copper. Detection at 330 nm.
ment of lipophilic interactions between the solutes and the stationary phase. The resolution between CuterBuDIB and UO2terBuDIB was improved by a factor of 2 (Table 3). This was mainly due to the increase of the peak efficiencies (h measured on the UO2terBuDIB and CuterBuDIB elution peak is equal to ∼10). A chromatogram is presented in Figure 5. The substitution of tert-butyl groups on the benzene rings has evidently enhanced the solubility of the compounds in the supercritical mobile phase. However, we must underline that peak efficency did not increase as much. The efficiency of the UO2terBuDIB peak is only half that of the UO2DIB peak in the case of solid injection. Solventless injections were then conducted in the same experimental conditions as for the original compounds in order to further improve the peak shape. An increase of elution peak efficiency measured on the UO2terBuDIB and CuterBuDIB was then observed (Table 3). These results demonstrate that the injection process is mainly responsible for the efficiency of the complex elution peak. Uranium Determination. We preferred to privilege the methanol consumption and the time of analysis to the uranium detection limit. Consequently, the analytical potential of this procedure has been evaluated by injecting ethyl acetate solutions of UO2DIB. A linear dependence of the peak area vs the injected quantity of chelate is obtained in the 52-323 ng range. The solution corresponding to the 323 ng measurement was obtained by liquid/liquid extraction with the complexing agent. The aqueous solution used for this extraction was the 400 g/L uranium standard nitric acidsolution after adequate dilution. The other UO2DIB quantities were obtained by diluting the concentrated organic solution with ethyl acetate. The chelate was eluted with a mobile phase of 3% methanol/CO2 (v/v). The experimental data fitted the equation y ) 1.77 × 10-2x, where y is the peak area in arbitrary units and x is the amount injected in nanograms. To evaluate the method, we have analyzed a sample obtained by liquid/liquid extraction of the 10.000 µL/mL standard solution diluted 241 times beforehand. A total of 207 ng of uranium was injected. The measure was repeated five times. The calibration
curve gave 208 ng as a result with a repeatability equal to 4%. The accuracy of the method is then 0.5%. This result show that packed-column SFC can provide fast and reliable uranium determination. CONCLUSION The investigation of copper and uranyl H2DIB and H2terBuDIB nonfluorinated chelate separation leads to the conclusion that packed-column supercritical fluid chromatography is a valuable alternative to the existing quantitative methods for determining uranium. The progressive expansion of the CO2/methanol mobile phase enables a gas phase to form which can be treated with a simple paper filter to retain the uranium. In this respect, our setup allows us to analyze toxic metals in a clean manner without contamination of the atmosphere with metal particles. The stability of the metal complexes and their high solubility in carbon dioxide/methanol mixtures allow a uranyl determination in less than two min. In this way, a very low amount of solvent, ∼500 µL, is needed for each analysis in SFC, compared to the 10 mL needed for reverse phase liquid chromatography.21 This is an important feature because although no liquid waste is generated, it remains essential for environmental reasons to lower
organic solvent consumption. The volume of solvent used under the SFC conditions could be even further reduced on a micropacked column (e.g., only 3 µL of methanol by analysis on a packed fused-silica column of 0.32 mm internal diameter). In this respect, the investigation reported here further confirms the high potential of the packed-column SFC technique for fast quantitative metal chelate determination. Other complexing agents will have to be studied in the future to analyze other highly toxic metal cations (cadmium, lead, arsenic, plutonium, neptunium, etc.). Of course these compounds will have to contain only carbon, hydrogen, oxygen, and nitrogen atoms in order to allow destruction by standard organic waste incineration procedures without generating harmful residues. ACKNOWLEDGMENT We gratefully acknowledge the support of this research by Cogema. Received for review February 20, 1996. Accepted October 2, 1996.X AC960163Z X
Abstract published in Advance ACS Abstracts, December 15, 1996.
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