Development of an autonomous solvent extraction system to isolate astatine-211 from dissolved cyclotron bombarded bismuth targets

Cyclotron-produced astatine-211 (211At) shows tremendous promise in targeted alpha therapy (TAT) applications due to its attractive half-life and its 100% α-emission from nearly simultaneous branched alpha decay. Astatine-211 is produced by alpha beam bombardment of naturally monoisotopic bismuth metal (209Bi) via the (α, 2n) reaction. In order to isolate the small mass of 211At (specific activity = 76 GBq·µg−1) from several grams of acid-dissolved Bi metal, a manual milliliter-scale solvent extraction process using diisopropyl ether (DIPE) is routinely performed at the University of Washington. As this process is complex and time consuming, we have developed a fluidic workstation that can perform the method autonomously. The workstation employs two pumps to concurrently deliver the aqueous and organic phases to a mixing tee and in-line phase mixer. The mixed phases are routed to a phase settling reservoir, where they gravity settle. Finally, each respective phase is withdrawn into its respective pump. However, development of a phase boundary sensor, placed in tandem with the phase settling reservoir, was necessary to communicate to the system when withdrawal of the denser aqueous phase was complete (i.e., the intersection of the two phases was located). The development and optimization of the autonomous solvent extraction system is described, and the 211At yields from several ~1.1 GBq-level 211At processing runs are reported.

Next, in-line mixing systems employing column and serpentine mixers were assembled and tested with a fluidic system to determine their effectiveness in Bi 3+ ion removal from the organic phase. Results are provided in Fig. 1. It was observed that both in-line mixing methods resulted in Bi 3+ contamination levels in the post-load organic phase that were considerably lower compared to the manual method (60 ± 2 µg and 125 ± 5 µg for the column and serpentine mixers, respectively). By the end of the triple-wash cycle, contamination levels had dropped to 8.1 ± 0.6 µg and 12.8 ± 0.5 µg, respectively. The three methods had approximately the same effectiveness at Bi 3+ ion removal across the load/wash sequence (each ending with <13 µg Bi); the manual solvent extraction method resulted in the highest Bi DF (~4.9 × 10 5 ), followed by the column and serpentine mixers (~3.5 × 10 5 and ~2.2 × 10 5 , respectively). 211 At solvent extraction during load/wash steps. Each of the two in-line mixing methods were subsequently evaluated for their effectiveness at 211 At extraction into DIPE. A surrogate dissolved target solution was prepared by spiking freshly isolated 211 At (~1.9 MBq) into freshly-dissolved Bi metal pieces (using 10 M HNO 3 ). The solution was evaporated to dryness, and the salts were dissolved in 8 M HCl (vide supra). A pair of syringe pumps was used to deliver the 211 At-/Bi-bearing solution and DIPE concurrently through a mixing tee and in-line mixer. At the conclusion of the delivery, the phases were allowed to gravity separate, and then a small volume of the DIPE solution (10 µL) was sampled and analyzed for 211 At activity levels. Next, each phase-separated liquid was withdrawn into its respective syringe pump, and the phase mixing→phase settling→DIPE sampling process was repeated three more times. We observed that 211 At extraction yields were nearly quantitative after the first pass, as shown in Table 1; the column and serpentine mixers provided extraction yields that were not statistically distinguishable from each other.
After confirming that both of the in-line mixers could effectively transfer 211 At into the organic phase during a load sequence, the complete forward extraction load/wash process was evaluated for the manual method and the two in-line mixing assemblies. Surrogate dissolved targets were prepared with spikes of 211 At (~1.9 MBq) added to Bi metal (~3 g), then dissolved and prepared as described above. Following each DIPE solution load and sequential wash step, a small volume of the aqueous phase was sampled and analyzed for 211 At activity concentration. This activity was corrected for aqueous phase volume and compared to the known activity initially present in the prepared surrogate target solution. The performance of each method was evaluated in triplicate.
The results in Table 2 present the calculated 211 At activity fraction remaining in the aqueous phase following each load and wash step (see Experimental Section and the ESM for details on load and wash step volumes for the in-line and manual methods, respectively). The manual method provided somewhat variable 211 At extraction during load, with 10.5 ± 5.8% of 211 At remaining in the aqueous phase. The subsequent 8 M HCl washes of the  At-loaded DIPE resulted in minor and slightly increasing 211 At losses at each stage. Overall, the manual method resulted in an extraction of all but 15.0 ± 5.9% of the 211 At activity originally present.
The in-line mixing methods were evaluated next. Surrogate 211 At-bearing dissolved target solution was delivered concurrently with DIPE two times through the in-line mixers prior to sampling for 211 At activity determination. Passage of the aqueous and organic phases through the column and serpentine mixers during the dual load steps succeeded at extraction of all but 4.3 ± 0.5% and 5.8 ± 1.7% of the 211 At, respectively. As with the manual method, each successive organic phase wash with 8 M HCl resulted in a small, incrementally increasing activity loss into the aqueous phase. Cumulative 211 At losses for both in-line methods were below ~11%; they were within experimental uncertainty at ±2 s. However, given the column mixer's slightly better performance relative to the serpentine mixer, it was selected to be integrated into the engineered fluidic system.

Phase boundary sensor (PBS) characterization. The development and integration of an aqueous/
organic PBS into the fluidic system would make it possible for the apparatus to trigger an event when the aqueous/organic phase boundary is identified. In this manner, the unpredictable volumes of the aqueous and organic phases in the PSR could be accommodated. The system aspirates the (dense) aqueous phase into the aqueous solution handling pump until the phase boundary is sensed (i.e., all aqueous solution has been withdrawn from the PSR). At this point, the aqueous solution handling pump ceases aspirating, and the organic phase handling pump aspirates the remaining organic phase from the PSR.
The PBS was designed to trigger an event based on a change in the electrical resistivity measured when a highly conductive aqueous solution (acid) passed through the monitored flow channel, followed by a poorly conductive organic liquid (DIPE). The resistivity measured across two electrical leads spikes to a "high" state when the organic solution enters the zone between the two leads. Below, the behavior of the PBS in acid/organic solutions is reported.
Given its well-established electrical conductivity and chemical resilience to strong acids, Pt wire was chosen as an electrode material to be tested in the PBS for the acid/organic phases. Using a data-logging potentiostat set up in open circuit potential (OCP) mode, we evaluated the Pt wire sensor performance as a function of time while it was repeatedly exposed to strong HCl and acid-contacted DIPE. In OCP mode, the potentiostat applies no current or voltage; it simply monitors the voltage across a circuit (in our case, the Pt wires positioned within the PBS flow channel). Using the potentiostat in this manner, the integrated switching logic of the digital input signal on our solvent extraction platform was monitored (i.e., input voltage level).
A testing program was set up wherein a syringe pump was programmed to aspirate liquid from a phase-separated reservoir of 8 M HCl and DIPE while logging the voltage levels across the Pt leads via the potentiostat. Each trial consisted of three withdrawals of HCl/DIPE phases from the PSR; data traces across three trials were recorded. The potentiostat traces are shown in Fig. 2.
When the Pt coil leads were exposed to acid, a low-resistivity condition was identified; the OCP recorded a low potential across the input logic circuit. When the input signal was below ~2.3 V potential (dashed line), the input circuit remained in the "low" state.
When the sensor signal rose above the ~2.3 V threshold potential, it switched to the "high" state. The high voltage is associated with a high resistivity between the Pt coil leads. Since DIPE is a vastly poorer electrical conductor than strong acid (vide supra), the resistivity across the sensor would be expected to increase significantly once the organic solution passed between the Pt coil electrodes within the monitored flow channel.
The data traces in Fig. 2 show that there is a brief period of sensor instability that occurs within a few seconds after 8 M HCl is introduced to the sensor (rapid fluctuation of signal above and below the sensor switching voltage). In order to assure that a premature switching event would not occur during aspiration of acid through the PBS, the software program was modified so that the PBS would exclude input data during this brief period of instability immediately following acid addition (3 s). Conversely, the voltage shift observed at the acid/DIPE interface did not exhibit the same instability; once the DIPE was in contact with the sensor, the voltage immediately went "high", assuring that the switching mechanism would be promptly activated.
Fluidic system performance testing on cyclotron bombarded Bi targets. After evaluations of in-line solvent mixing and phase boundary sensing were completed, a fully autonomous solvent extraction system capable of emulating the manual wet chemical 211 At isolation process was designed, constructed, and www.nature.com/scientificreports www.nature.com/scientificreports/ programmed. The programming of the system was initially refined while running non-radiological solutions, and then further refined on 211 At-spiked solutions in the UW glovebox. Ultimately, a series of nine performance tests were made on freshly-bombarded Bi cyclotron targets that contained 1.07 ± 0.02 GBq of 211 At (at end-of-bombardment (EOB)). The targets used in this series contained 4.8 ± 0.5 g of Bi metal.
For each GBq-level run, the Bi targets were dissolved using the automated target dissolution station that was previously described 38 . The dissolved Bi effluents were routed from the dissolution block to a heated distillation chamber that brought the solutions to dry Bi oxynitrate salts. After the salts were cooled, the fluidic system's pump 1 injected 8 M HCl onto the salt residue; a combination of magnetic mixing and aspiration/dispensation of the HCl solution (pump 1) resulted in complete dissolution of the salts after several cycles had been performed. The prepared target solutions were then ready for in-line solvent extraction.
The loss of 211 At to the aqueous phase was tracked at each stage of the processing runs (load and three successive washes). Each load and wash fraction was collected separately and analyzed after completion of the runs. The data presented in Fig. 3 shows the incremental 211 At loss at each stage of the forward extraction method for each target.
The data shows that the majority of the 211 At loss occurred during the prepared target/DIPE mixing ("Load") step. The average percentage of 211 At remaining in the aqueous phase following the Load step was 4.7 ± 0.9%. These results compare favorably to the manual processing runs, wherein the 211 At found in the post-contacted load solution was 10.5 ± 5.8% (Table 2).
After 211 At loading, some of the DIPE-extracted 211 At was incrementally lost to the three successive DIPE washes with 8 M HCl. The degree of 211 At loss increased slightly with each wash, resulting in 1.6 ± 0.6%, Figure 2. Voltage potentials measured across the Pt coil electrode terminals connected to the input logic board during alternating deliveries of 8 M HCl and DIPE solutions. Horizontal dashed line indicates approximate potential above which the sensor input logic is switched from "low" to "high" state. Arrows indicate a region of brief sensor instability upon exposure to acid. Figure 3. Observed 211 At losses in the aqueous phase following the prepared target/DIPE load step (4.7 ± 0.9% loss) and three successive washes with 8 M HCl (1.6 ± 0.6%, 2.4 ± 0.6%, and 2.7 ± 0.5% loss, respectively). (2019) 9:20318 | https://doi.org/10.1038/s41598-019-56272-7 www.nature.com/scientificreports www.nature.com/scientificreports/ 2.4 ± 0.6%, and 2.7 ± 0.5% lost in washes 1 through 3, respectively. These incremental 211 At losses were likewise observed in our earlier trials when comparing the manual and in-line mixing methods (Table 2).
Overall, the total 211 At loss to the aqueous phase was 11.4 ± 1.3% across the study. This was the fraction of 211 At activity that was not retained in the organic phase (routed to waste) during the pump 1/2 forward extraction operations that involved the packed column-based mixer/settler system. The cumulative 211 At recovery (obtained after target dissolution → target solution evaporation → recovered activity from the dissolved Bi salts → the above-described solvent extraction load/wash steps) was calculated to be 79.4 ± 4.5%.

Discussion
Necessity of the PBS. Development of a procedure that allows "automatic" solvent extraction in the isolation of 211 At is difficult, if not impossible, given the unpredictable nature of the aqueous/organic phase volume changes encountered during the target preparation and solvent extraction processes. Although the DIPE and 8 M HCl reagents used in the method were pre-contacted with each other prior to use (to minimize volume change during execution of the method), the precise volume of dissolved target/HCl/DIPE in each phase was not known. In the manual method, these volume inconsistencies are inconsequential since phase separation at each step is accomplished by pro-active (visual) means.
Due to tolerances in production of the Al backing for the cyclotron targets, and machining the Bi metal melted onto the Al backing, the Bi metal targets used for 211 At production had varying mass. In our initial studies, the Bi metal in the target assemblies ranged between 3.5 and 6.5 g. Later changes in machining tolerances, and adapting a weight-based removal of excess Bi from targets, reduced the Bi mass uncertainty substantially to 4.8 ± 0.5 g for targets employed in the GBq-level study. Because Bi metal mass in the target assemblies was determined by weight difference pre-and post-dissolution, the quantity of Bi metal could not be known in advance. Thus, dissolution of the Bi metal in nitric acid and subsequent heating/distillation of the acid resulted in unknown quantities of Bi-bearing salts. Further, differences in salt quantities affected the level of dryness obtained for a given thermal treatment interval, which in turn affected the chemical composition (e.g., bismuth:nitrate ratio and level of hydration) of the Bi oxynitrate salt [39][40][41][42] .
These unpredictable salt quantities and compositions resulted in unpredictable solution volumes when the salts were dissolved with 8 M HCl in preparation for 211 At solvent extraction. The HCl-dissolved target solution volumes varied as a function of starting Bi metal mass and the HCl volume added (see data presented in the ESM).
Additionally, during the initial contact of the dissolved target and DIPE solutions, some volume change between the phases was observed, the degree of which was generally driven by the ionic strength of the dissolved target solution. Phase volume changes during the three DIPE solution wash steps were nominal, since the 8 M HCl solution used in the washes was equilibrated with DIPE prior to introducing the reagent into the fluidic system.
The general issues with "automatic" solvent extraction from unknown input volumes are illustrated in Fig. 4. The top row shows three hypothetical conditions encountered upon delivery of three dissolved Bi targets and a fixed organic solution volume to a PSR. "Heavy" (A), "normal" (B), and "light" (C) Bi targets result in decreasing aqueous phase volumes, respectively.
The bottom row of Fig. 4 illustrates the effect of a fixed aqueous phase aspiration based on a pre-programmed "normal" target mass. In condition (B), the aspiration volume is sufficient to remove all of the dissolved target solution, thus enabling effective phase separation prior to initiation of the organic phase wash cycles. In condition (A), the same aspiration volume is insufficient to remove all of the dissolved target solution. This results in excess acid/Bi salt residing in the reservoir at the conclusion of the volume withdrawal. As a consequence, the subsequent wash steps with clean 8 M HCl are ineffective at removing Bi from the organic phase, since the remaining Bi-bearing target solution will simply be diluted at each wash step. In condition (C), the aspiration volume is excessive; all of the aqueous phase is withdrawn, along with a portion of the organic phase. Under this condition, some of the 211 At-bearing organic phase is sent to waste along with the post-contacted dissolved target solution. The 211 At yields will consequently suffer.
Given the unpredictable aqueous solution volumes going into the front end of the solvent extraction system, the process could not be achieved by performing a fixed sequence of pre-programmed ("automatic") commands. Our response to this issue was to develop a sensor that could identify the phase boundary between the two immiscible liquids. With such a sensor, a fully "autonomous", multi-step solvent extraction process became possible.
It is worth noting that the presented phase boundary sensing approach can conceivably result in the separation of arbitrary amounts of different aqueous/organic liquid phases from one another, so long as the liquid interface is well defined and the electrical conductivity of each liquid is sufficiently different. 211 At yields obtained from the autonomous fluidic system. The solvent extraction steps encompassing the loading of 211 At into the organic phase from the prepared target solution and the triple wash steps to remove Bi 3+ ions from the organic phase resulted in 88.6 ± 1.3% 211 At recovery in the DIPE. This was based on careful accounting of the 211 At present in the load and triple wash solutions (11.4 ± 1.3% 211 At loss in aqueous phase, Fig. 3).
When considering incremental 211 At losses in the steps leading up to the conclusion of the solvent extraction process, the cumulative 211 At yield was 79.4 ± 4.5%. This additional ~9.2% loss was attributed to several possible sources: (1) the inability to recover all 211 At from the aluminum target assembly that backs the Bi metal layer; (2)  www.nature.com/scientificreports www.nature.com/scientificreports/ only reported mean yields in the final 211 At product fraction (which included back-extraction of 211 At into 4 M NaOH). However, our yields at the conclusion of the acid → DIPE cycles (79.4 ± 4.5%, n = 9) compare favorably with those reported by Balkin et al. for the entire 211 At isolation process (78 ± 11%, n = 55). Further, the uncertainties associated with the autonomous vs. manual methods indicate that the presented method may be capable of providing more reproducible 211 At yields (although the number of trials was fewer in the current evaluation).
Fluidic system expansion. The autonomous acid/DIPE solvent extraction system described herein was further expanded to include the capability to perform an autonomous DIPE/4 M NaOH back-extraction step, during which the isolated 211 At in DIPE is transferred back to an aqueous phase. This back-extraction module allowed for execution of the complete, end-to-end solvent extraction process described by Balkin et al. 21 . The expanded system was comprised of three syringe pumps (pump 3 was for handling the NaOH-based back extractant) and a separate in-line mixer, PSR, and PBS. The PBS for DIPE/base phase identification employed the same electrical conductivity monitoring approach, although Pt electrodes were not practical and a new electrode material needed to be identified. Ultimately, the system dispensed the isolated 211 At product in a small volume of 4 M NaOH. A description of the back-extraction module development and its performance in the end-to-end process with clinical levels of 211 At will be described in a future article.
Bi pellets of 99.999% purity, used in the manufacture of Bi target assemblies 11 , were acquired from Alfa Aesar (Ward Hill, MA). Simulated dissolved Bi metal targets employed Bi metal pieces of the same purity (Sigma-Aldrich, St. Louis, MO). Platinum wire was 0.25 mm dia. and had a metal purity of 99.9% (Alfa Aesar). Coiled Pt electrodes were prepared by wrapping the wire around an 18 gauge hypodermic needle. The electrodes were secured and sealed into the PBS housing with ferruled 1/4-28 fittings. Manual method. The routine manual method for 211 At isolation from solution-prepared cyclotron bombarded Bi metal targets by solvent extraction has been described by Balkin et al. 21 . A brief summary of the manual method (including target preparation and solvent extraction steps) is presented in the ESM. In-line mixer/settler systems. The two liquid phases are delivered simultaneously from their respective syringe pump: aqueous solution from pump 1 and organic liquid from pump 2. The liquids intersect at a mixing tee and then pass into a phase mixer -a tortuous path through which the fluids are intimately co-mingled. Beyond the phase mixer is a PSR -a container used to collect the mixed phases and provide a static condition in which the phases can gravity separate (Fig. 5).
The first phase mixer evaluated was a 1 cm 3 SPE column (Sigma-Aldrich) packed with 212 µm dia. silanized glass beads (Sigma-Aldrich) (Fig. 5A). It was fitted with a custom-machined cap at the inlet end that enabled fluids to be delivered to the column via with a male luer adapter. Additionally, we evaluated a Super Serpentine Reactor ™ (GlobalFIA, Fox Island, WA), which was a 1.2 m length of 0.75 mm ID/1/16″ OD Teflon FEP tubing (0.53 mL internal volume) braided tightly through a perforated plate (Fig. 5B).
The flow rates of each pump were programmed so that the total volume of each respective phase was delivered towards the mixing tee over an equal time period. Hence, the syringe volume:dispensation flow rate ratio was the same between aqueous and organic phase syringes: typically 1.25 min per full syringe stroke (20 mL•min −1 for a www.nature.com/scientificreports www.nature.com/scientificreports/ 25 mL syringe; 8 mL•min −1 for a 10 mL syringe). The two solutions were merged at the mixing tee, and were then passed through the in-line mixer. A quick dispensation of air (at same flow rates as above) assured that the fluids were "chased" through the apparatus at the conclusion of the syringe stroke.
The tortuous path of the serpentine mixer causes in-line mixing to occur [43][44][45] . For the column mixer, the two liquids were delivered to the column of glass beads, thus creating intensively mixed phases as they were driven through the bed of small spheres. Upon exiting either the serpentine or column mixer, the biphasic mixture was delivered to a PSR (centrifuge tube or syringe barrel), where the two phases quickly separated. The phase settling interval was 30 s, which was ample time for the organic/aqueous phases to separate and for most of the fine solution-entrained bubbles from the air push to rise to the surface of the DIPE.
Next, tubing that connected each syringe pump's distribution valve to the bottom of the reservoir was used to withdraw first the (dense) aqueous phase, and then the organic phase, back into each respective pump (Fig. 5C). In this manner, the processing cycle was set to be repeated. Alternatively, the aqueous phase could be dispensed to waste, the syringe pump rinsed with clean 8 M HCl, and re-loaded with 8 M HCl rinse solution prior to the next phase mixing interval.

Phase boundary sensor (PBS).
The digital syringe pumps employed in the described fluidic processes are equipped with an external input signal processor board that allows voltage (0-5 V) to be monitored in real time; we took advantage of this feature to implement a PBS. The PBS body, which is machined out of a Teflon cylinder, is mounted to the base of a PSR (20 mL syringe barrel). The outlet of the PSR is connected to the inlet of the PBS with a luer/¼-28 coupler. Near the bottom of the PSR is a fluid channel in a "tee" configuration, which allows fluids to be withdrawn from the reservoir by either pump 1 or pump 2. Two electrodes project into the fluid channel, each held in place by ferruled ¼-28 fittings; they are positioned 2 cm apart. The electrodes are connected to the +5 V and ground terminals of the pump's input signal processor. Aqueous and organic liquids are simultaneously passed through a mixing tee and phase mixer from pump 1 and pump 2. Upon exiting the phase mixer, they are collected in a PSR perched atop the PBS (Fig. 6A).  Table 3.  Table 3. Identification of labels in Fig. 6.

Label Description
The fluid handling software was programmed to read the signals from a simple logic gate binary system offered by the pump's signal input board. After withdrawing 25 µL of aqueous solution through the PBS (at 40 mL•min −1 flow rate), the input board's signal is momentarily read. If the resistivity of the solution is small, then perform task 1; if the resistivity is large, then perform task 2. Task 1 instructs pump 1 to continue withdrawing solution through the sensor (at 25 µL increments), since acid is present between the two electrodes within the PBS's flow channel (sensor reads "low", Fig. 6B). Task 2 instructs the pump to cease withdrawing solution through the sensor once the organic phase has entered the sensor (e.g., the phase boundary has been detected, sensor reads "high" (Fig. 6C)). Once the phase boundary is sensed, pump 1 aspirates a small volume of the aqueous solution to clear the dead volume between the Pt electrodes and the tee at the base of the PBS (Fig. 6D). Finally, pump 2 withdraws the isolated organic phase from the PSR (Fig. 6E). Engineered solvent extraction system and sample processing. The fully engineered autonomous solvent extraction system is comprised of two digital syringe pumps, a forward extraction mixer/settler system (1 cm 3 column mixer), and a PBS (Fig. 7). The first pump handles the prepared target solution and acidic wash solutions; the second pump handles the DIPE solution. The syringe pumps were inverted, with the distribution valves below the syringe (note that Fig. 7 is not illustrated in this configuration). In this manner, liquid phases could be efficiently "chased" with air after each liquid delivery stroke.
The system was designed to have the syringe pumps and all peripheral components of the mixer/settler arrangement organized in a small space immediately in front of the pumps. In this compact configuration, the tubing connections are kept as short as possible, and they are less likely to be damaged by the cumbersome glovebox gloves that are necessary to be used when 211 At processing is underway. An image of the fluidic system is presented in the ESM. The sequence of high-level steps performed by the fluidic system is described in Table 4, and the delivered reagents and volumes at each step in the process is presented in Table 5.
Step 1 is performed in a fixed time interval, while the other steps have varying time duration.
Step 2 is performed until the salts are visually observed to be dry. This is primarily determined by observing the elapsed time between condensate droplets within a jacketed condenser positioned between the distillation and distillate chambers. Depending on the mass of Bi in the target assembly, the elapsed time of the combined target dissolution and nitric acid distillation was ~75-90 min (distillation performed at 180-190 °C). This automated acid distillation process takes longer than the manual distillation method (which requires ~30-45 min), since a temperature of 300 °C is used there 21 . The automated process cannot be performed at this high temperature, since severe Bi salt spatter begins to occur above ~200 °C. Salt spatter from acid boiling/bumping places much of the 211 At-bearing Bi oxynitrate salt out of reach of the 8 M HCl used to subsequently dissolve the saltcake (per step 3), and therefore needs to be avoided.
Upon completion of the distillation step (per a prompt of the software by the operator), the Bi salt conversion (step 3) is initiated. First, the heating block is rapidly cooled (by remote gating of chiller fluid through the block) to 75 °C. During the block cooling interval, the program pauses until a 75 °C heating block temperature is reached. Next, the still-warm Bi oxynitrate saltcake is dissolved by cycled dispensation/aspiration of 8 M HCl from pump 1 to/from the distillation chamber. A number of these cycles is required ensure the saltcake is fully www.nature.com/scientificreports www.nature.com/scientificreports/ dissolved (and the number of cycles performed is calibrated to successfully dissolve a saltcake resulting from the heaviest possible Bi target). Prior to initiation of the first solvent extraction step (step 4), the program ensures that the heating block temperature has been reduced to at least 35 °C via a second block cooling interval.
At the initiation of step 4, pump 1 aspirates the HCl-dissolved 211 At/Bi solution out of the distillation chamber and proceeds with the DIPE load step. During the solvent extraction process, the time to perform steps 4-5 varies slightly (14-17 min), depending on the volume of the dissolved Bi-bearing solution; a heavy Bi target solution, due to its larger volume, will require more time to pass through the PBS during the phase boundary determination cycles vs. a lighter target solution. Once the 211 At-depleted Bi solution is sent to waste and the wash cycles begin (steps 6-8), the elapsed times are very similar, since the aqueous and organic volume changes at this point are minimal. The autonomous solvent extraction process (steps 4-9) ranged between 29 and 32 min. Overall, a complete processing run required ~2.1 to ~2.4 elapsed hours between initiation of the Bi target dissolution and dispensation of the isolated 211 At product in DIPE. The autonomous solvent extraction operations represent only ~22% of the total 211 At processing time.

211
At activity quantification. All 211 At activity levels were decay corrected to EOB. Direct measurements of 211 At activity levels were obtained using a CRC-15R dose calibrator (Capintec, Inc., Ramsey, NJ). The instrument was calibrated for 211 At by UW; a setting of 040 was employed for 211 At-bearing solutions in polyethene vials. The setting was based on a cross-calibration to a high purity Ge (HPGe) detector (Ametek, Oak Ridge, TN) that had been calibrated against NIST-traceable gamma standards. It has been demonstrated that high Bi concentrations attenuate the weak 211 At and progeny X-ray and gamma emissions, thus resulting in negatively biased activity measurements 21 . Therefore, UW-determined correction factors were employed for samples containing well-known Bi concentrations. Otherwise, 211 At samples high in Bi were not reported by dose calibrator. Rather, liquid scintillation analysis (LSA) was employed.
LSA was performed on direct sample aliquots or serial dilutions of 211 At-bearing samples, depending on 211 At activity levels present in the sample. Typically, 20 µL aliquots of each fraction (direct or serial dilution) were Step # Step ID Description Step elapsed time, min (approx.)   Table 5. Reagent injections during the sequence of steps described in Table 4. a HCl-dissolved Bi-bearing salts were observed to increase in volume to between ~19 and ~22 mL. b DIPE was initially equilibrated with 8 M HCl. www.nature.com/scientificreports www.nature.com/scientificreports/ withdrawn and added to 4 mL scintillation cocktail that was pre-dispensed into a 2-dram polyethylene scintillation vial. The prepared samples were typically allowed to decay through one or more half-lives prior to alpha decay measurement by LSA (TriCarb 1900CA, PerkinElmer). Only LS measurements that exhibited ≤3500 cps were used, in order to assure linear LS analyzer response. If the sample measurement exceeded this count rate, it was allowed additional time to decay and then recounted. The counting region between 40 and 2000 channels, with tSIE quench correction activated, assured integration of the 211 At and 211 Po alpha emissions (5.87 and 7.45 MeV, respectively). Since 211 At branch decays to an alpha emission either via 211 At directly, or through 211 Po 6 , the sum of the two alpha peaks, adjusted to a 95% detection efficiency, yielded the activity of 211 At in the sample. The LS detection efficiency was determined as the ratio of the count rate of the LS alpha counting region and the 211 At disintegration rate reported by the cross-calibrated dose calibrator. All system components and effluents that came in contact with 211 At were sampled for 211 At activity during or at the completion of each run. Low Bi-bearing samples were analyzed by dose calibrator and LSA. Values and uncertainties shown on individual run results are the mean ± 1s obtained between the two analytical instruments. When the average result over multiple 211 At processing runs are reported, the uncertainty is the sample standard deviation (±1s) across that set of runs.
Bismuth analysis. The total mass of Bi in the isolated DIPE phase was determined following analysis of each sample by ICP-OES after undergoing the sample preparation method shown in Table 6.
Resulting solutions were analyzed using an iCap 6500 Duo (Thermo Scientific, Waltham, MA) ICP-OES with analysis performed in axial mode. An eight-point calibration curve was prepared for the Bi analysis by gravimetric dilutions of a 1000 µg/mL certified Bi standard purchased from High Purity Standards (Charleston, SC). Calibration curves used for the Bi standard concentrations had regression coefficients ≥0.9997; wavelengths of 190.2341, 222.8203, and 223.0602 nm were used in the analysis 46 . Analytical results were typically reported as being within ±5% at 2σ.
Step Treatment Notes