Anal. Chem. 1999, 71, 345-352
Sequential Injection Renewable Separation Column Instrument for Automated Sorbent Extraction Separations of Radionuclides Oleg Egorov, Matthew J. O’Hara, and Jay W. Grate*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352 Jaromir Ruzicka
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195
A novel approach to the automation of sorbent extraction separations is described. An advanced sequential injection renewable separation column (SI-RSC) instrument was designed and optimized for automatically packing a separation column on-line, using it for a separation, and then disposing of the used separation material. Column packing/separation material disposal steps are accomplished fluidically using a frit restriction. Using a 250-µL renewable column, procedures were developed for the separation and analysis of 90Sr, 241Am, and 99Tc in aged nuclear waste samples employing Sr-resin, TRU-resin, and TEVA-resin sorbent extraction materials (Eichrom Industries). The separation performance of the RSC was identical to that of a freshly packed conventional column of similar size. With a renewable column, it is not necessary to elute all species from the column, carryover on the column from one separation to the next is eliminated, the same instrument can be used for multiple separations requiring different sorbent extraction media without physical changes to the instrument (sorbent extraction medium is selected in software), and the sorbent extraction material can be recovered for analysis of noneluted species. Using a system of pumps and valves operating under computer control, flow injection (FI) and sequential injection (SI) techniques provide the means to automate various sample handling and separation steps on a microscale.1-6 Typically, though not always, these methods are used as part of an analytical system with a flow-through detector. These capabilities have led to the use of FIA in serial assays for many application areas, including environmental measurements, agriculture, biochemistry, pharmaceu(1) Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1986, 179, 1-58. (2) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; Wiley-Interscience: New York, 1988; Vol. 62, p 498. (3) Ruzicka, J. Anal. Chim. Acta 1992, 261, 3-10. (4) Fang, Z. Flow Injection Separation and Preconcentration; VCH: Weinheim, 1993. (5) Ruzicka, J. Analyst 1994, 119, 1925-1934. (6) Clark, G. D.; Whitman, D. A.; Christian, G. D.; Ruzicka, J. Crit. Rev. Anal. Chem. 1990, 21, 357-375. 10.1021/ac980623j CCC: $18.00 Published on Web 12/09/1998
© 1999 American Chemical Society
ticals, and clinical analysis.2 With the design of new flow cells,7 FIA has recently been extended to manipulation of microgram quantities of suspended solids and even to whole living cells.5 FI and SI have thus progressed from simple methodology for serial assays to a wide range of automated solution-handling methods. The manipulation and observation of surface-derivatized beads in FI and SI systems for chemical analysis has been pioneered by Ruzicka and co-workers.5,7-12 The principle of this approach is based on the introduction of a well-defined volume of a bead suspension into the flow system and consequent temporary capture of the bead material using a jet ring flow cell.5,7 The captured beads form a microbed within the observation field of a suitable detector. Changes in the bed properties (e.g., absorbance, reflectance, fluorescence, etc.) induced upon the introduction of the sample solution provide the basis for an analytical measurement. After use, the microbed is swept away, and a new microbed can be packed. This process renews the interactive surface for the next measurement. Features and advantages of the renewable surface technique have been demonstrated in a number of analytical applications including competitive immunoassays,8,12 bioligand interaction assays,11 nonaqueous titrations,13 a renewable enzymatic amperometric sensor,10 and a colorimetric fiber-optic sensor system.9 Although these techniques were developed primarily for capturing and observing the beads, the methodology creates the possibility of generating a separation column bed that can be automatically packed and replaced on-line.5 Recent work by Dockendorff and co-workers focused on the separation and analysis of theophylline using a 5-µL renewable solid-phase extraction column with downstream UV-visible detection.14 The (7) Ruzicka, J.; Pollema, C. H.; Scudder, K. M. Anal. Chem. 1993, 65, 35663570. (8) Pollema, C. H.; Ruzicka, J. Anal. Chem. 1994, 66, 1825-1831. (9) Egorov, O.; Ruzicka, J. Analyst 1995, 120, 1959-1962. (10) Mayer, M.; Ruzicka, J. Anal. Chem. 1996, 68, 3808-3814. (11) Ruzicka, J.; Ivaska, A. Anal. Chem. 1997, 69, 5024-5030. (12) Willumsen, B.; Christian, G.; Ruzicka, J. Anal. Chem. 1997, 68, 334823489. (13) Holman, D. A.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 69, 17631765. (14) Dockendorff, B.; Holman, D. A.; Christian, G. D.; Ruzicka, J. Analyst, in press.
Analytical Chemistry, Vol. 71, No. 2, January 15, 1999 345
renewable column was implemented using a machined flow cell equipped with an actuated rod acting as a mechanical restriction with leaky tolerances.13,14 With the rod in the flow path, beads were stopped while allowing fluid to pass. Mechanically withdrawing the rod from the flow path allowed the beads to be flushed out of the system.14 Thus far, this is the only application of the renewable surface technique to solid-phase extraction with postcolumn detection. Separation columns in a fixed format have been extensively used for analyte preconcentration and analyte/matrix separation both in FI2,4 and, more recently, in SI formats.15,16 The full potential of the renewable surface technique in the field of analytical separations remains to be explored. We set out to advance the renewable surface technique with emphasis on column separations and to apply the technique to automated sorbent extraction radiochemical separations under development in our laboratories.9,15-20 We developed a sequential injection renewable separation column (SI-RSC) instrument to automatically pack the sorbent column on-line, use it for a separation, and discard the used separation material. Radionuclide separation procedures for 90Sr, 99Tc, and 241Am were performed in the SI-RSC format using recently developed strontium selectiveSr-resin,21-23 technetium-selective TEVA-resin,24 and actinideselective TRU-resin.25 (These separation materials are comprised of selective extractants immobilized on inert polymeric support, and their applications to the analysis of nuclear waste solutions have been described.22-24,26,27) The new methodology is characterized in detail and applied to the analysis of aged nuclear waste samples from U.S. Department of Energy sites. In this study, we have demonstrated that renewable separations can be reliably performed on larger column sizes (e.g., 250 µL) and that the technique offers a number of advantages, including elimination of sample carryover from one separation procedure to the next, the option to leave strongly retained species that are not of interest in the analysis on the column material (since it will be disposed of rather than reused), the ability to use the same apparatus for separations requiring different separation materials without manually swapping columns, and the option to recover the separation material to analyze for strongly retained species. EXPERIMENTAL SECTION Sequential Injection System. A FIAlab 3000 (Alitea USA, Medina, WA) sequential injection system (Figure 1) was config(15) Grate, J. W.; Strebin, R. S.; Janata, J.; Egorov, O.; Ruzicka, J. Anal. Chem. 1996, 68, 333-340. (16) Egorov, O.; O’Hara, M. J.; Ruzicka, J.; Grate, J. W. Anal. Chem. 1998, 70, 977-984. (17) Grate, J. W.; Egorov, O. Anal. Chem. 1998, 70, 3920-3929. (18) Egorov, O.; Ruzicka, J.; Grate, J. W.; Janata, J. Int. Top. Meet. Nucl. Hazard. Waste Manage., SPECTRUM ’96, Seattle, WA, 1996. (19) Egorov, O.; Grate, J. W.; Ruzicka, J. J. Radioanal. Nucl. Chem. 1998, 234, 231-235. (20) Grate, J. W.; Fadeff, S. K.; Egorov, O., submitted for publication. (21) Horwitz, E. P.; Chiarizia, R.; Dietz, M. L. Solvent Extr. Ion Exch. 1992, 10, 313-336. (22) Horwitz, E. P.; Dietz, M. L.; Fisher, D. E. Anal. Chem. 1991, 63, 522-525. (23) Horwitz, E. P.; Dietz, M. L.; Chiarizia, R. J. Radioanal. Nucl. Chem. Art. 1992, 161, 575-583. (24) Horwitz, E. P.; Dietz, M. L.; Chiarizia, R.; Diamond, H.; Maxwell, S. L.; Nelson, M. R. Anal. Chim. Acta 1995, 310, 63-78. (25) Horwitz, E. P.; Chiarizia, R.; Dietz, M. L.; Diamond, H.; Nelson, D. M. Anal. Chim. Acta 1993, 281, 361-372. (26) Dietz, M. L.; Horwitz, E. P. LC-GC 1993, 11, 424-436. (27) Maxwell, S. L. Radioact. Radiochem. 1997, 8, 36-44.
346 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
Figure 1. Schematic representation of the SI-RSC instrument. C, water carrier; SP, syringe pump; HC, holding coil; MPV, multiposition valve; W, waste lines; RS, R, S, reagents, sorbent slurries, samples; SDL, slurry delivery line; TL, sample/reagents transport line; TPV A, four-port, two-position valve A; CB, renewable separation column body; TPV B, four-port, two-position valve B; FR, frit restriction; D, detector or fraction collector. Solid lines on TPV A and TPV B show port connections corresponding to valve position 1. Dashed lines on TPV A and TPV B show port connections corresponding to valve position 2. See text for more details.
ured with a 24 000-step digital syringe pump (syringe volume 10 mL) and a 10-port multiposition Cheminert valve (Valco Instrument Co., Houston, TX). A second auxiliary 10-port multiposition valve (not shown in Figure 1) was used as needed to accommodate all the reagent solutions required when performing analyses of nuclear waste samples. The central port of the auxiliary valve was connected to one of the ports of the main multiposition valve. The holding coil was constructed from 1.6-mm-i.d. FEP Teflon tubing (Upchurch Scientific, Oak Harbor, WA) of 6-m length (calculated volume 12 mL). Transport and reagent lines were made of 0.8mm-i.d. FEP Teflon tubing (Upchurch). A slurry delivery line was made of 35-cm-long × 1.6-mm-i.d. FEP tubing (Upchurch). The body of a renewable separation column was constructed of 2.12mm-i.d. PTFE tubing (Valco). The length of the column body used in the analysis experiments was 7.1 cm (calculated volume 250 µL). The renewable separation column apparatus was constructed using two four-port, two-position valves (Figure 1, valves A and B) with 1/4-28 threaded fitting details and 0.75-mm port diameter (Cheminert, Valco). The column body was placed between the ports of the two-position valves using flat-bottom flangeless fittings (Upchurch) as shown in Figure 1. A 5-mm-diameter, 25-µm-pore size polypropylene frit disk (Alltech Associates, Inc., Deerfield, IL) was used to retain sorbent beads within the renewable separation column instrument. The frit disk was placed directly into the port of the two-position valve B (Figure 1) and held in place by a flat-bottom chromatographic fitting. A conventional chromatographic column (75 × 2 mm, calculated volume 236 µL) was constructed of parts from the OmegaChrom column system (Upchurch) and frits from the Quick-Snap column system (IsoLab, Inc., Akron, OH). Bromthymol blue dye injection studies were performed using a SPD10AV UV-visible detector (Shimadzu). The SI instrument was controlled via a serial cable using FIAlab software (Alitea USA) running on a lap-top PC. Radioactivity Measurements. The on-line flow-through liquid scintillation detector, a Radiomatic A515A (Packard Instrument Co., Meriden, CT), was configured with a 0.5-mL flow cell and operated as described previously.15 The detector integration time
(time to accumulate counts for each data point reported) was 6 s. Off-line liquid scintillation measurements were performed with a Tri Carb 2550 TR/AB liquid scintillation spectrometer (Packard Instrument Co., Meriden, CT). R-Spectroscopy was performed using ULTRA (EG&G Ortec, Oak Ridge, TN) ion-implanted detectors housed in model 576A spectrometers (Ortec). Samples for R-spectroscopy were prepared using a neodymium fluoride microprecipitation procedure.28 Materials and Standards. All chemicals used were of analytical grade. Deionized water (MilliQ-Plus, 18 MΩ) was used as a carrier solution without degassing. Low-viscosity liquid scintillation cocktail Ultima-Flo M (Packard) was used for on-line radioactivity measurements. Ultima Gold (Packard) cocktail was used for all static liquid scintillation measurements. Nitric acid solutions of 99Tc(VII), 239Pu, 241Am, and 90Sr/90Y were prepared by dilution of standard stock solutions obtained from an in-house standards laboratory. Plutonium was maintained in the tetravalent state by making nitric acid sample solutions 0.05 M in NaNO2. Sorbent extraction materials (Sr-resin, 50-100-µm particle size; TRU-resin, 50-100-µm particle size; and TEVA-resin, 20-50-µm particle size) were obtained from Eichrom Industries, Inc. (Darien IL). The sorbent slurries were 0.074 g/mL of Sr-resin in 3 M HNO3; 0.142 g/mL of TEVA-resin in 4 M HNO3, and 0.076 g/mL of TRU-resin in 0.1 M HNO3. The sorbent beads were maintained in suspension by periodic gentle stirring (just prior to column packing) using a miniature magnetic stirrer (Variomag, Florida Scientific Services, Inc., South Daytona, FL). Nuclear Waste Samples and Reference Methods. The types of the nuclear waste samples used in this study included vitrified glass and a salt cake slurry. The samples were obtained as processed diluted solutions after hot cell sample preparation steps. The analysis of 99Tc was carried out on a dissolved salt cake sample derived from the underground storage tanks at the Hanford site. Sample preparation and treatment steps for salt cake samples have been described previously.16 To ensure that all the Tc was present as pertechnetate, the samples were oxidized using potassium permanganate and hydrogen peroxide treatment.16 Pulverized vitrified high-level nuclear waste glass samples, nominally 0.3 g, were fused in a mixture of 2 g of KOH and 0.2 g of KNO3 at 550 °C for 60 min in a hot cell. The fusion melt was dissolved in water followed by a crucible rinse with 5 mL of concentrated HNO3. The samples were then diluted to a 100-mL volume with deionized water and removed from the hot cell. Prior to analysis using our SI-RSC technique, aliquots of the nuclear waste samples were analyzed using standard manual radiochemical analysis procedures adopted at the Radiochemical Processing Laboratory (RPL) at Pacific Northwest National Laboratory. The manual method for 90Sr analysis in high-level nuclear waste samples was based on Sr-resin separation chemistry, and β-counting has been described earlier.15 R-Spectrometric determination of the Am isotopes following the TRU-resin separation has been described by Kaye et al.28 The manual procedure for 99Tc analysis was based on a combination of cation-exchange and solvent extraction separation steps and β-counting as described in our previous publication.16 (28) Kaye, J. H.; Strebin, R. S.; Orr, R. D. J. Radioanal. Nucl. Chem. 1995, 194, 191-196.
Figure 2. Schematic represention of renewable separation column configuration using a two-position valve, TPV, modified with a frit restriction, FR, directly connected to the bottom of the column body, CB. (A) System shown during column packing operation. (B) System shown during disposal of separation material. Valve dimensions are not to scale.
CAUTION! Highly radioactive solutions used in this work present severe radiological hazards. RESULTS AND DISCUSSION Operational Principles and Design of the SI-RSC Instrument. Simple fluidic procedures and a frit restriction were used to develop a technique for packing and disposing of a renewable separation column. The method is illustrated in Figure 2, where the downstream end of a tube serving as the column body is connected to the inlet port of a four-port, two-position valve. A frit restriction is placed in one outlet port of this valve. A second outlet port is unrestricted, while the fourth port is capped. When the flow of a bead slurry through the column is directed through the frit-restricted outlet, the beads are stopped by the frit, and a separation bed forms. To dispose of this bed, the position of the two-position valve is changed so that fluid flow proceeds through the unrestricted outlet of the column. This technique works best if the fluid flow is reversed briefly before changing the outlet pathway, backing up the separation bed slightly before directing it through the unrestricted outlet. The SI-RSC instrument shown schematically in Figure 1 was set up to automatically execute the following procedures: (1) introduce a slurry of sorbent extraction material to the renewable column; (2) retain the sorbent beads in the form of a uniformly packed sorbent bed of well-defined geometry; (3) introduce sample and reagent solutions to the renewable separation column and transport the separated analyte fractions to a detector or fraction collector; and (4) expel the spent sorbent beads from the system. Solutions or slurries selected with the multiposition valve (MPV) are first pulled into the holding coil (HC) with the syringe pump (SP). After the multiposition valve is repositioned, they are pushed out to the remainder of the system. A wide-bore holding coil (1.6 mm i.d.) facilitated the manipulation of large solution and slurry volumes and permitted high flow rates without outgassing when Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
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pulling solutions into the holding coil. A 100-µL air segment between these solutions (or slurries) and the carrier fluid prevented dispersion, as described previously.16,20 Slurries were dispensed out to the column via the slurry delivery line (SDL) of the same diameter as the holding coil (1.6 mm i.d.), while sample and reagent were dispensed via the transport line (TL) of narrower diameter (0.8 mm i.d.). The use of a wide-bore slurry delivery line simplified and increased the reliability of the column packing/ disposal procedures. A four-port, two-position valve (TPV A) at the top of the column selects which of these two lines connects to the column and defines the top of the sorbent bed. During the column packing procedure, two-position valve A is positioned to connect the slurry delivery line to the column (valve position 2; the corresponding port connections are shown with dashed lines in Figure 1). Two-position valve B is positioned to send the fluid flow through the frit restriction (valve position 1; port connections shown with solid lines in Figure 1). The sorbent particles are retained by the frit restriction and gradually accumulate in a uniformly packed bed within the column body. Since an excess of the sorbent slurry is used, the sorbent bed extends temporarily into the slurry delivery line. After packing of the column, two-position valve A is switched so that sample and reagent solutions are delivered to the sorbent column via a narrowbore transport line (valve position 1; port connections are shown with solid lines in Figure 1). The sorbent bed is then precisely defined by the volume between the top of the column body at port 4 of two-position valve A and the frit in port 1 of two-position valve B. The portion of the sorbent bed in the body of the valve is only a few microliters and is thus negligible compared to the volume in the column body (typically 250 µL in the present study). To dispose of the sorbent bed, flow reversal steps are used to pull the sorbent beads back away from the frit, and two-position valve B is switched to expel the beads directly to waste via the unrestricted port 3. The use of excess slurry in the above packing procedure eliminates any dead volume just above the packed bed. In addition, the sorbent bed volume defined by this procedure is not critically dependent on variations in slurry. Detailed Implementation of the SI-RSC Technique. Several parameters and procedures were investigated in order to develop practical working protocols for reliable renewable columns. The behavior of bead slurries in the flow system varies with the slurry concentration, the solvent composition, the bead size, and the particular extractant loaded on the beads. Optimal slurry compositions must compromise between higher slurry concentrations desired to obtain fast packing speeds and lower slurry concentrations necessary to avoid clogging at the valve ports and to pack the column evenly. The following bead concentrations provided reliable behavior: 0.074 g/mL of 50-100-µm particle size Sr-resin, 0.076 g/mL of 50-100-µm particle size TRU-resin, and 0.142 g/mL of 20-50-µm particle size TEVA-resin. Under these conditions, sorbent beds with volumes ranging from under 50 to 500 µL were reliably and reproducibly packed and expelled using all three selected sorbent materials. A 250-µL column (71- × 2.1-mm column body) was selected for all subsequent experiments as a compromise between the bed volume, packing/disposal speed, and sorbent consumption. With these parameters established, automated protocols for column packing and sorbent bed disposal were developed. The 348 Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
Table 1. Automated Protocol for Renewable Separation Column Packing step no.
event (flow rate)
1
switch two-position valves A and B to position 2 and 1,a respectively aspirate 100 µL of air into the holding coil (15 mL/min) aspirate 635 µL of carrier into syringe (35 mL/min) aspirate 700 µL of sorbent slurry into the holding coil (3 mL/min) dispense 700 µL of sorbent slurry to column body via the slurry delivery line (3 mL/min) repeat steps 4 and 5 as necessaryb dispense 635 µL to the slurry delivery line (3 mL/min); pause 12 s switch two-position valve A to position 1 aspirate 100 µL of air into the holding coil (15 mL/min) aspirate 750 µL of carrier into syringe (35 mL/min) dispense 750 µL through the slurry delivery line to waste (6 mL/min) aspirate 100 µL of air into the holding coil (15 mL/min) aspirate 750 µL of 50% ethanol solution into the holding coil (15 mL/min) aspirate 1.8 mL of carrier into the syringe (35 mL/min) empty syringe through the slurry delivery line to waste (10 mL/min)
2 3 4 5 6 7 8 9 10 11 12 13 14 15
a Position 1 connects port 1 to port 2, while position 2 connects port 1 to port 4. b Steps 4 and 5 were executed three times to pack Sr-resin or TRU-resin columns, and two times to pack TEVA-resin column.
protocol for column packing is given in Table 1. Sorbent slurries were aspirated into the holding coil and delivered to the renewable column in two or three 700-µL increments (Table 1). Larger slurry volumes had two disadvantages. First, excessive settling of the sorbent particles in the holding coil was observed for larger volumes. Second, larger volumes exposed longer lengths of the holding coil to the sorbent slurry, where potential extractant losses (clearly evident in the case of Sr-resin) deposit on the tubing walls. The excess sorbent material present in the slurry delivery line after column packing (at the end of step 7) was expelled to waste via valve A by sending a 100-µL air segment followed by 750 µL of water carrier through the slurry delivery line (Table 1, steps 10 and 11). The air segment was found to be effective in sweeping out any residual sorbent particles left in the wide-bore tubing. A final series of ethanol wash steps (Table 1, steps 12-15) was employed to remove any extractant that may have been deposited on tubing walls exposed to the slurry. The time required to automatically pack a 250 µL sorbent column was under 4.5 min, including the wash steps. Table 2 lists the automated procedure developed for the sorbent bed disposal. An initial flow reversal step (Table 2, step 2) was used to loosen the compacted sorbent bed and partially disperse it into the slurry delivery line. Additional flow reversal steps (Table 2, steps 6 and 10) were used to ensure complete removal of any residual sorbent particles present in the system (e.g., packed against the frit material). Using the described procedures, the reliability of the frit restriction and its useful frit life was superb. No frit clogging was evident after 57 packing/ separation/disposal cycles. Moreover, our instrument design provides for a straightforward renewal of the frit, if necessary, simply by disconnecting port 1 of valve B (see Figure 1) and replacing the frit. After column disposal, ethanol wash steps (Table 2, steps 15 and 16) were employed to remove any potential coating
Table 2. Automated Protocol for Sorbent Bed Disposal step no.
event (flow rate)
1 2
switch two-position valves A and B to position 2 aspirate 350 µL through the column body and slurry delivery line (10 mL/min) aspirate 100 µL of air into the holding coil (15 mL/min) aspirate 4.25 mL of carrier into syringe (35 mL/min) switch two-way valve B to position 1 aspirate 100 µL through the column body and packing line (10 mL/min) switch diverter valve B to position 2 dispense 900 µL through the column body to waste (10 mL/min) switch diverter valve B to position 1 aspirate 100 µL through the column body (10 mL/min) switch diverter valve B to position 2 aspirate 100 µL of air into the holding coil (15 mL/min) dispense 1.5 mL through the slurry line and column body to waste (10 mL/min) switch diverter valve B to position 1 aspirate 100 µL of air into the holding coil (15 mL/min) aspirate 1.2 mL of 50% ethanol solution into the holding coil (10 mL/min) dispense 2.6 mL through the slurry delivery line and column body to waste (10 mL/min)
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Table 3. Automated Protocol for
90Sr
Separationa
step no.
description and reagent (flow rate)
1 2
pack Sr-resin column condition column: 1.5 mL of 8 M HNO3-0.12 M HF (1 mL/min) load sample/wash column: 150 µL of 8 M HNO3-0.12 M HF/100 µL of sample/6 mL of 8 M HNO3-0.12 M HF (0.5 or 1 mL/min)b elute Sr: 5 mL of 0.05 M HNO3-0.12 M HF (0.5 mL/min)c expel Sr-resin sorbent
3 4 5 a
In steps 2-4, an air segment is pulled into the holding coil prior to the indicated solution or solution sequence. b The sample zone is sandwiched between 8 M HNO3-0.12 M HF zones. Aspiration sequence: 6 mL of eluent/100 µL of sample/150 µL of eluent. c The flow rate in step 4 was 1 mL/min for the analysis of nuclear waste samples.
of the RSC tubing walls with the organic extractants. The time required for the disposal of a 250-µL sorbent bed was under 3.5 min. Reproducibility and CarryoversSeparation and Analysis of 90Sr Standards. We selected the sorbent extraction separation of 90Sr using Sr-resin as an initial model chemistry to evaluate the performance of the SI-RSC instrument. An on-line liquid scintillation detector was used to observe eluting peaks. Strontium separation chemistry using Sr-resin has been characterized in detail previously21 and recently transferred to an automated SI format using stationary columns.15,20 The sample is loaded on the column in strong nitric acid solution (>3 M concentration), where Sr ions are strongly and nearly selectively retained. The matrix and most interfering radioactive ions, including the 90Y daughter, show no retention and are removed with a strong nitric acid wash. Strontium is then eluted using a dilute nitric acid solution (∼0.05 M). Our protocol for implementing this separation with the SIRSC technique is given in Table 3. In this procedure, the Sr sample need not be in strong acid. The Sr sample zone is sandwiched between strong acid zones when it is pulled into the holding coil, and dispersion ensures that it is diluted at least 3-fold by the strong
Figure 3. Detector traces corresponding to a triplicate analysis of a high-activity 90Sr/90Y standard, obtained each time using an automatically repacked 250-µL renewable column, overlaid with a detector trace obtained using a conventional 236-µL chromatographic column. Inset traces correspond to immediate reagent blank runs obtained using a conventional column (A) and a renewable column (B). Notations along x-axis: (a) sample load/column wash in strong acid, step 3, Table 3; (b) reagent aspiration step; and (c) strontium elution in weak acid, step 4, Table 3. Separation flow rate, 0.5 mL/ min; liquid scintillation cocktail flow rate, 1 mL/min.
acid. (Dispersion was measured by dye injection experiments with UV-visible detection.) In this manner, the sample is loaded on the column in sufficiently high nitric acid concentration for strong strontium retention. We used 8 M HNO3-0.12 M HF as a column wash to ensure removal of the tetravalent actinides.20 The experimental procedure listed in Table 3 was applied to a high-activity 90Sr/90Y standard (2.14 × 105 90Sr dpm/mL in 2 M HNO3) in triplicate: the separation flow rate was 0.5 mL/min, the cocktail flow rate was 1 mL/min, and the detection cycle was initiated at the beginning of the sample load step (step 3, Table 3). The corresponding detector traces for the triplicate runs are all included in Figure 3. The traces overlap quite closely, showing the reproducibility of the procedure using freshly packed columns each time. The same separation/detection procedure was also performed using a standard column of a comparable size (see Experimental Section) instead of the RSC apparatus. The corresponding detector trace is included in Figure 3, and it overlaps with the traces using the renewable column. Thus, the 90Sr separation can be performed as efficiently using a renewable column as when using a conventional fixed column. The detector traces indicate that the analyte recoveries and elution peak widths are virtually identical in both cases. For triplicate SI-RSC runs on standards, both net peak area and peak maximum counts were reproducible within a 2σ counting error (3% and 9%, respectively). The separated Sr fractions from the standard runs were collected and counted off-line to estimate the on-line detection efficiency (Ed) and separation recovery (Es).15 The 90Sr separation recovery, Es, was 92 ( 2%, and the on-line detection efficiency, Ed, was 62 ( 3%. The effective efficiency of the automated SI-RSC separation/ detection procedure,15 Ee ) Es Ed, was 57 ( 3%. Analytical Chemistry, Vol. 71, No. 2, January 15, 1999
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It has been previously demonstrated that the reuse of Sr-resin generally requires additional column wash steps in order to reduce strontium carryover into the subsequent analysis.15,20 A reagent blank run using a conventional chromatographic column performed immediately after the separation of a high-activity 90Sr standard indicated carryover of ∼7% (Figure 3 inset, trace A). However, no carryover was evident if the reagent blank run following the analysis of the high-activity standard was performed on the automatically repacked column (detector trace B, Figure 3 inset). No carryover was detected when using a more sensitive off-line liquid scintillation analysis of a collected Sr fraction (less than 0.07% carryover). Consequently, one of the primary benefits of using the SI-RSC technique is that analyte carryover into succeeding analyses due to retention on column material is eliminated. Lengthy column cleanup steps and blank runs, which are sometimes required when reusing sorbent extraction columns,15,20,29 are unnecessary when using the SI-RSC technique. In addition, this procedure is consistent with current manual practices, where new columns are used for each Sr analysis. Thus, the RSC technique may find more ready acceptance as an automation method in the user community than automated techniques where columns are reused. Species Left on the Column after Elution of the Analytes Separation of 241Am from Nuclear Waste Samples. Determination of 241Am in nuclear waste samples using R-spectroscopy requires that Am is separated from the stable matrix, highly radioactive fission products, and potential radioactive interferences (e.g., 238Pu). Recently, Horwitz et al. described a sorbent extraction methodology based on an actinide-selective extractant (0.75 M solution of octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide in tri-n-butyl phosphate) immobilized on an inert polymeric support (TRU-resin, Eichrom Industries, Inc.).25 Americium separation on TRU-resin can be carried out by loading the sample in nitric acid, washing the column with additional nitric acid to remove the matrix and interferences (e.g., fission products), and eluting the Am(III) with hydrochloric acid.25,28,30 Other tetra- and hexavalent actinides (e.g., Pu, Th, and U) will remain on the column and can be removed from the column by using ammonium hydrogen oxalate (bioxalate) as a complexing eluent.25,31 The chemistry associated with actinide separations on TRU-resin have recently been investigated in a flow injection format.17 We set out to automate the Am separation using TRU-resin in SI-RSC format. The procedure given in Table 4 was applied to a dissolved vitrified glass nuclear waste sample prepared in 2 M HNO3-0.05 M NaNO2. The sample was spiked with 5.0 × 105 dpm/mL of 241Am and 239Pu. On-line radioactivity detection was used to monitor the separation. Duplicate experiments were performed, and the detector traces are shown in Figure 4. Peaks A and B in 2 M nitric acid correspond to the elution of unretained (peak A, 90Sr and 137Cs) and slightly retained (peak B, 90Y and 99Tc) highly abundant fission products. Peak C corresponds to the elution of trivalent actinides (Am, Cm) and lanthanides using (29) Aldstadt, J. H.; Kuo, J. M.; Smith, L. L.; Erickson, M. D. Anal. Chim. Acta 1996, 319, 135-143. (30) Goldstein, S. J.; Hensley, C. A.; Armenta, C. E.; Peters, R. J. Anal. Chem. 1997, 69, 809-812. (31) Horwitz, E. P.; Dietz, M. L.; Diamond, H.; LaRosa, J. J.; Fairman, W. D. Anal. Chim. Acta 1990, 238, 263-271.
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Table 4. Automated Protocol for Am Separation step no.
description and reagent (flow rate)
1 2 3
pack TRU-resin column condition column: 1.5 mL of 2 M HNO3 (1 mL/min) load sample/wash column: 100 µL of sample/ 6 mL of 2 M HNO3 (1 mL/min) elute Am: 4 mL of 3 M HCl (1 mL/min) elute actinides: 4 mL of 0.1 M ammonium bioxalate (1 mL/min)a expel TRU-resin sorbent
4 5 6
a Bioxalate elution step 5 was omitted in the analysis of nuclear waste samples.
Figure 4. Detector traces illustrating Am separation procedure on a renewable 250-µL TRU-resin column applied to a nuclear waste sample spiked with 5.0 × 105 dpm/mL of 241Am and 239Pu (sample volume 100 µL). The separation was performed in duplicate each time using a freshly packed column. Notations along x-axis: (a) sample load/column wash in 2 M HNO3, step 3, Table 4; (b) reagent aspiration; (c) trivalent actinides elution in 3 M HCl, step 4, Table 4; and (d) actinides elution with bioxalate, step 5, Table 4. Separation flow rate, 1 mL/min; liquid scintillation cocktail flow rate, 1 mL/min.
3 M HCl. Peak D corresponds to the elution of tetravalent and hexavalent actinides (Pu, Th, U) using 0.1 M ammonium bioxalate. Results in Figure 4 illustrate that the Am separation using the SI-RSC technique can be performed efficiently (sharp elution peaks) and reproducibly. The ammonium bioxalate elution step is required if the TRUresin column is to be reused for subsequent Am separations. However, using the SI-RSC technique, the sorbent column can be automatically repacked after the Am elution step. In this case, there is no need to elute the actinides still present on the column (Table 4, step 5). Additional column wash steps to reduce potential carryover into subsequent analysis are also eliminated. The separation time is reduced, and mixed waste generation is minimized. We applied this separation approach toward the analysis of nuclear waste samples described below. Random Access Radionuclide Separation SystemsAnalysis of 90Sr and 241Am in Nuclear Waste. Because multiple reagent solutions and sorbent slurries can be easily accommodated by the SI instrument, the SI-RSC technique provides a capability to select sorbent materials, reagent solutions, and samples required
Table 5. Automated Protocol for
description: reagent (flow rate)
1 2
pack TEVA-resin column condition column: 1.5 mL of 0.05 M HNO3-0.12 M HF (0.5 mL/min) load sample: 400 µL of sample solution (0.5 mL/min) wash column: 5 mL of 0.05 M HNO3-0.12 M HF (0.5 mL/min) wash column: 1.5 mL of DI water (0.5 mL/min) expel TEVA-resin sorbent and collect in a vial
5 6
for a given analysis via the computer keyboard. In this manner, multiple analytical procedures using different sorbents can be performed automatically on a single sample solution. To demonstrate this capability, we performed sequential separation and analysis of 90Sr and 241Am on a single nuclear waste sample. Because a large number of sample solutions, reagents, and sorbent slurries were required in our analysis procedures, an auxiliary multiposition valve was added to the SI instrument (see Experimental Section). This configuration is shown schematically in Figure 5. Nuclear waste samples and sorbent slurries were set up in advance using the main multiposition valve. The second multiposition valve was used to accommodate multiple eluent and reagent solutions. The following sample solutions (2 mL volume) were prepared and used in the analysis of 90Sr and 241Am: (1) reagent blank (2 M HNO3), (2) dissolved vitrified glass waste sample (2 M HNO3-0.05 M NaNO2 solution), and (3) an identical nuclear waste sample with known added amounts of 90Sr (4.12 × 105 dpm/mL) and 241Am (5.63 × 103 dpm/mL). First, the Sr analysis on a single sample solution was carried out in triplicate according to the Sr analysis procedure in Table 3. A flow rate of 1 mL/min was used during the sample load and column wash step (Sr elution flow rate remained at 0.5 mL/min). The quantification was performed using on-line radioactivity detection (cocktail flow rate 1 mL/min). To minimize the amount of radioactivity sent to the detector, only the Sr fractions were analyzed. Next, the Am separation was carried out in triplicate on the same sample using the protocol outlined in Table 4. The separated Am fractions were collected in vials and later analyzed off-line by R-spectroscopy. This sequence of triplicate analyses for both 90Sr and 241Am was repeated for each sample solution. The analytical results for both analytes were calculated using the standard addition formula, A ) NAs/(Ns - N), where A is the sample activity (dpm/mL), As is the added standard activity (dpm/mL), N is the blank subtracted (net) detector count rate for the unspiked sample, and Ns is the net count rate for the spiked sample. The analysis results ((2σ counting error) for 90Sr (5. 25 × 105 ( 2.52 × 104 dpm/mL) were in excellent agreement with the value (5.20 × 105 ( 1.04 × 104 dpm/mL) determined by standard analysis procedures (see Experimental Section). The
Separation
step no.
3 4
Figure 5. Schematic diagram of the SI-RSC instrument using two multiposition valves for sequential separation of 90Sr and 241Am on a single nuclear waste sample (random access instrument). Abbreviations as in Figure 2.
99Tc
estimated effective efficiency, Ee, for the 90Sr analysis in the nuclear waste sample was 58% and is in agreement with Ee ) 57% obtained from the analysis of 90Sr standard solution (as described in the earlier section). The result for the analysis of 241Am was 6.27 × 103 ( 2.65 × 102 dpm/mL. This is in satisfactory agreement with the reported value of 5.92 × 103 ( 2.14 × 102 dpm/mL. R-Spectroscopy of the collected Am fractions revealed no detectable Pu activity, indicating that the procedure provided the required separation factor for the analysis of Am isotopes in the vitrified glass waste sample. Recovery and Analysis of the Sorbent BedsAnalysis of 99Tc in Nuclear Waste. The separation of 99Tc(VII) from other radioactive and stable ions using TEVA-resin has been described previously for manual,32-35 automated FI,36 and automated SI formats.16 The separation is based on the nearly selective retention of the pertechnetate ion from dilute acid solutions.24 Pertechnetate can be eluted using 6-12 M nitric acid. However, high-molarity acid is generally an undesirable matrix for analysis by liquid scintillation spectrometry.34 Additional sample treatment steps (e.g., evaporation) are necessary to eliminate nitric acid quenching. An alternative approach lies in direct counting of the sorbent bed, without eluting the Tc.32,33 We were interested in demonstrating the utility of the SI RSC technique to automate this analytical technique. The automated protocol listed in Table 5 was developed to pack and condition TEVA-resin column, load the sample, and perform the column wash that removes stable matrix and radioactive interferences (e.g., 137Cs and 90Sr/90Y). We employed 0.05 M HNO3-0.12 M HF wash reagent to eliminate retention of the teravalent Pu.16 Note that the same reagent was used to perform Sr elution in the previous section. Residual nitric acid present on the column after the column wash step was removed using 1.5 mL of water (Table 5, step 5). After the sample load and column wash steps (Table 5), the TEVA-resin sorbent was expelled from the system. The sorbent slurry in water (∼3 mL) was collected into a vial, mixed with 15 mL of scintillation cocktail, and analyzed off-line by liquid scintillation spectrometry. The following samples (1-mL aliquots of 0.1 M HNO3 solutions) were analyzed using the SI-RSC separation format: (1) reagent (32) Technetium-99 in Water; Analytical Procedure TCW 01; EIChrom Industries, Inc., Darien, IL, 1995. (33) Technetium-99 in Soil; Analytical Procedure TCSO1; EIChrom Industries, Inc., Darien, IL, 1995. (34) Nevissi, A. E.; Silverston, M.; R. S., S.; Kaye, J. H. J. Radioanal. Nucl. Chem. Art. 1994, 177, 91-99. (35) Banavali, A. D.; Raimondi, J. M.; Moreno, E. M.; McCurdy, D. E. Radioact. Radiochem. 1995, 6, 26-35. (36) Hollenbach, M.; Grohs, J.; Mamich, S.; Kroft, M.; Denoyer, E. R. J. Anal. At. Spectrom. 1994, 9, 927-933.
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blank, (2) tank waste sample, (3) tank waste sample spiked with 2300 dpm/mL 99Tc(VII), and (4) tank waste sample spiked with 4350 dpm/mL of 99Tc(VII). Blank-subtracted detector count rate (cpm) plotted for the sample and two spiked samples against the added standard activity (dpm) gave a straight line (cpm ) 0.937 dpm +358, R ) 1.000). The sample 99Tc activity was determined from the x-axis intercept of the standard addition plot. The detection efficiency for the heterogeneous TEVA-resin slurry/ scintillation cocktail samples was estimated to be 95 ( 4%. The separation recovery was quantitative (99 ( 5%). The analysis result ((2σ, corrected for dilutions) for a tank waste sample obtained using the SI-RSC technique was 1240 ( 163 dpm/mL. This is in satisfactory agreement with the value of 1121 ( 146 dpm/mL determined by standard analysis procedures (see Experimental Section). These results demonstrate a unique capability of the SIRSC technique to perform automated separations that require recovery of spent sorbent for subsequent analytical steps. Discussion. In current practice, chromatographic and solidphase extraction separations are carried out with fixed beds of solid material. The columns or disks are either reused or manually disconnected and disposed of. With the development of flow injection approaches for manipulating and capturing suspensions of beads, it is now possible to generate renewable columns for automated separation procedures. We have developed a new technique for renewable column packing and disposal and illustrated its use in sorbent extraction separations of radionuclides. The method of bead capture and disposal shown in Figures 1 and 2 uses commercially available, chemically inert components and is very reliable. Column packing and sorbent disposal steps are accomplished fluidically using a frit restriction, rather than using a movable mechanical barrier with leaky tolerances. One of the potential concerns when using frit material is clogging. Under our experimental conditions, however, useful frit life was excellent. (If necessary, our instrument design provides for easy replacement of the frit material.) The only moving parts in our system are the commercial valves. There is no possibility of leakage of radioactive solutions past a seal associated with a moveable mechanical barrier used to trap beads. Used sorbent particles in our system design are discharged via a flow path that is separate from the path for eluted species. The particles do not pass through the flow path to the detector, and thus the SI-RSC method is compatible with a variety of detector instrumentation. Alternatively, eluted species or even the sorbent bed can be collected for analysis off-line. An important feature of our SI-RSC instrument is the flexibility in adjusting the column size to the particular application. The separation column can be scaled up from tens to several hundred microliters by changing only the dimensions of the column body tubing. This does not change either the pre- or postcolumn dead volume. Dead volume above the column bed is undesirable since (37) Lutz, E. S. M.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1997, 69, 4878-4884.
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mixing in this region can dilute the sample and create gradients between subsequent eluting solutions, which could broaden elution peaks. Similarly, minimizing dead volumes after the column is desirable to minimize peak dispersion after elution. Minimizing dead volumes is an essential feature for efficient column separations. In addition, the separation column bed has a fixed reproducible size for quantitative studies. The SI-RSC technique is also significant in that it represents an open architecture instrument design.5 Because separation materials, reagents, and samples can be loaded under computer control, a single instrument can be used for multiple analytical procedures which can be selected remotely via computer software. This feature adds a new degree of flexibility to SI separation methods. In general, the SI-RSC technique is highly suitable for the automation of separations that are subject to (1) analyte carryover on the sorbent; (2) limited useful column life, column material degradation, or eluent reagents that destroy the column material; (3) sample components that are difficult or impossible to elute; (4) recovery of the sorbent for subsequent analysis steps; or (5) assays where more than one solid-phase material is to be used for separation or evaluated. On this basis, we believe that the SI-RSC approach may find diverse applications in automation of various chromatographic, sorbent extraction, affinity capture, and solid-phase extraction methods. For example, the use of a flow injection system to screen bead-based combinatorial libraries prepared by solid-phase synthesis was recently proposed. 37 In proof of principle experiments, a slurry of the beads with immobilized analyte was mixed with a fluorescently labeled affinity protein and processed through a hollow fiber module to separate the beads from the supernatant. A fraction of the supernatant containing free label was analyzed with a fluorescence detector to evaluate the affinity characteristics of the bead-bound analyte. Our SI-RSC method has all the required characteristics to automate screening of spatially separated libraries of bead bound candidates: the beads do not go through the detector, all the supernatant does go through the detector (improving sensitivity as well as quantification), bed size is fixed and reproducible, and it is possible to load a different bead slurry into the system for each assay. ACKNOWLEDGMENT This work has been supported with funding from the Office of Biological and Environmental Research of the U.S. Department of Energy. The Pacific Northwest National Laboratory is a multiprogram national laboratory operated for the U.S. Department of Energy by Battelle Memorial Institute. Sandra Fadeff and David Blanchard are gratefully acknowledged for providing nuclear waste samples and helpful discussions. Received for review June 8, 1998. Accepted October 30, 1998. AC980623J
