INTRODUCTION
The use of radiochemicals enriched with radiocarbon can contaminate work areas used for natural abundance measurements. The concentration of naturally occurring 14C is 10–12 and blanks measured by accelerator mass spectrometry (AMS) are <10–15. Therefore, commercially available radiochemicals containing 100% atom 14C are >15 orders of magnitude above blank levels and pose a catastrophic danger to AMS laboratories. Several laboratories have reported experiences of contamination and recovery from a single “hot” sample and we are aware of a number of other unreported examples (Jull et al. Reference Jull, Donahue and Toolin1990; Vogel et al. Reference Vogel, Southon and Nelson1990; Zhou et al. Reference Zhou, Wu, Lange, Lu, Cheng, Xiong, Cruz, Liu, Fu and Zhao2012). Practices for preventing contamination, evaluating, and monitoring potential workspaces and cleaning contaminated workspaces and laboratory equipment have been described for both natural abundance and bio-AMS preparation laboratories (Buchholz et al. Reference Buchholz, Freeman, Haack and Vogel2000; Zermeño et al. Reference Zermeño, Kurdyla, Buchholz, Heller, Kashgarian and Frantz2004).
In the case of a graphitization of a hot sample, it was found that the majority of the contamination occurred in the graphitization system where cross-talk between samples occurs due to extended manipulation of the gases during sample preparation. It was possible to recover from a contamination event from a single hot sample in the 10–6 to 10–9 range by extensive cleaning of the apparatus and replacement of parts that have been in direct contact with the hot sample. The ion source of the AMS system recovered quickly where it had been in operation with a hot sample for seconds but required extended cleaning when that time was longer. A general conclusion drawn from in all studies was that while an AMS laboratory could accommodate a wide range of 14C concentrations up to 10–11 and that recovery from a single severe contamination event was possible, it was not suitable to undertake natural abundance and enriched studies in the same area. It was also noted that refurbishment of contaminated laboratory space in one case had been unsuccessful and was abandoned.
It is known by those skilled in the art, that effective communication and a rigorous monitoring program is invaluable to ensure the isotopic fidelity of measurements. It is often difficult to know whether equipment, samples, or facilities have been affected by isotopically enriched materials; therefore, it is critical to have the capability to identify legacy contamination, screen for potentially hot samples, and monitor the status quo. Major AMS laboratories providing routine analysis ask clients if they are aware of the use of enriched 14C the vicinity of their work and can refer them to further testing services if necessary.
Testing for contamination can be done by liquid scintillation counting (LSC) or AMS and at this time the SWAB program at the University of Miami can check for gross contamination using LSC (see online Supplementary Material). An area is washed down with soapy water and an aliquot of the residue is mixed with scintillator and analyzed for 14C. AMS laboratories typically use sealed tube combustion and graphitization of samples taken by swabbing an area with a combusted quartz filter moistened with solvent. Dilution with 14C-free CO2 can be used for small or hot samples before graphitization. Additionally, solid graphite powder can be used to detect airborne contamination by sorption (Zermeño et al. Reference Zermeño, Kurdyla, Buchholz, Heller, Kashgarian and Frantz2004). These procedures are risky in themselves as they can contaminate equipment used for natural abundance measurements, so dedicated equipment is often used. In the case of AMS, this can be relatively costly as a separate vacuum line and graphitization system may be required.
In this study, we detail a new method for contamination detection using wet chemical oxidation preparation and AMS for analysis. The procedure is convenient, cost- and time-effective, and minimizes cross-contamination of equipment. We report on four contamination situations based on real events and the results of more than 800 analyses.
EXPERIMENTAL
A printable preparation protocol is given in the online Supplementary Material.
Sampling Apparatus
A sampling kit was prepared and kept on standby for use at any time by anyone. A carry basket contained freshly combusted Exetainer vials (12 mL, Labco, PN-9RK8W), metal tweezers, and 25-mm quartz fiber filters. Combustion was performed at 450°C for 2–3 hr. Also included were fresh Isopropanol (IPA) in a 100-mL Schott bottle, Kimtech Science Purple nitrile gloves (PN-90627), a roll of aluminium foil, metal vial rack, pen, notebook, and a copy of the protocol.
Swabbing Procedure
The work area was covered with aluminum foil and labeled vials were placed in the rack. A combusted quartz fiber filter was moistened by dipping it in the IPA with tweezers. Then, an area of 5–20 cm2 was wiped by hand with fresh gloves. Three blanks were prepared by placing a moistened filter into a vial without wiping anything. Sample details were noted using fresh gloves and when finished, the rack of vials was covered loosely with a single piece of foil.
Processing of the Samples
A dedicated area with dedicated equipment was used for drying of the samples and processing prior to AMS analysis to avoid possible contamination of the natural abundance laboratories. All preparatory work was conducted in a ventilated basement room in a separate building with restricted access. Care was taken to maintain the order of the vials and caps for clear sample tracking and to prevent cross-contamination. Fresh nitrile gloves were used for each sample to prevent cross-contamination.
The wet chemical oxidation method used for sample preparation was adapted from a method developed for analyzing dissolved organic carbon (Lang et al. Reference Lang, McIntyre, Bernasconi, Früh-Green, Voss, Eglinton and Wacker2016). The rack of vials and quartz filters were first dried in a dedicated oven overnight at 60°C to remove the IPA. The oxidant was prepared fresh by dissolving 1.5 g sodium persulfate (AR grade) in 50 mL of Milli-Q water and adding five drops of 85% H3PO4. The solution was stored in a combusted amber glass container and 1 mL of solution was added to each sample vial. The vials were capped and a fresh disposable 50-mm, 23-gauge syringe tip was put in each vial to allow gas to escape. The vials were purged one at a time for 1 min with 100 mL/min of high-purity helium. The helium supply was a cylinder and regulator fitted with a metering valve, flexible tubing, and a disposable syringe tip. The helium supply needle was inserted minimally (5–10 mm) through the septa and both needles withdrawn when purging was complete. The samples were immediately heated on a heater block at 100°C for 1 hr to complete the oxidation of organic carbon to CO2 and then cooled to room temperature. At this stage, the samples could be stored for months and shipped to an AMS facility for analysis.
AMS Analysis
The gas samples were analyzed for 14C using an AMS system fitted with a gas ion source, a gas interface system (GIS), and a carbonate handling system (MICADAS-GIS-CHS, Ionplus) (Wacker et al. Reference Wacker, Fahrni, Hajdas, Molnar, Synal, Szidat and Zhang2013). The autosampler can accommodate 42 Exetainer vials for the automated analysis of headspace CO2. The GIS was operated with parameters to maximize the throughput of samples. Samples were flushed onto the zeolite trap with helium for 30 s and the CO2 trapped at 100°C and desorbed at 400°C. Trap cleaning between samples was 30 s, and overall this allowed 12 samples/hr to be analyzed. The AMS was calibrated against 14C-free CO2 and CO2 from combusted standard oxalic acid (NIST SRM 4990C). Vials showing no contamination were acid-washed and reused, while contaminated vials were discarded.
Dilution of Samples
When necessary or if suspect, samples were diluted with 14C-free CO2 prior to analysis. Additional empty Exetainer vials were flushed with helium for 30 s at 100 mL/min using the CHS autosampler of the AMS system. Then, 0.1 mL of 14C-free CO2 was injected using a 1-mL insulin syringe and needle (Terumo U100), which was equivalent to 50 μg C. Samples could be diluted 1:12 or 1:120 v/v by injecting aliquots of 1 or 0.1 mL, respectively, into a vial containing blank CO2. Additional vials of blank CO2 were prepared to flush the system after hot samples if required.
RESULTS AND DISCUSSION
Samples and Blanks
The method described here was used to give a semi-quantitative “hot” or not result for screening for and monitoring of 14C contamination. Between January 2014 and May 2015, 27 batches of samples from eight institutions were analyzed comprising 750 samples and 80 blanks. This encompassed samples for screening of unknown areas, monitoring of work areas, and retesting of cleaned areas. Signs of use of enriched 14C were found at 7 of the 8 institutions checked, while the 8th institution was using enriched 13C. Each location was either being used for, or had plans to be used for, natural abundance 14C measurements.
The results showed that 14% of the samples (106) had an F14C>2 and 29% (216) had an F14C>1. The number of positive results is artificially high as the tests were largely targeting contaminated areas or were retesting cleaned areas. The highest value recorded was an F14C>2000 and the median value was F14C=0.9. The mean mass of carbon measured was 100±70 μg and the highest mass was 500 μg C. Based on a mean value of 100 μg C, the 1:12 and 1:120 v/v sample dilutions had a dilution of 1:6 and 1:60 w/w carbon, respectively. Typically, 20 s of data acquisition was enough to determine if a sample was hot or not. For samples that contained more than 100 μg C, the GIS would automatically reduce the size of the sample.
The 80 processing blanks had a measured mass of 13±10 μg with F14C=0.55±0.21 excluding four outliers with high mass and one with high F14C (due to exchange of a hot cap). Based on an upper limit at the 3σ error level, this is equivalent to a detection limit in the order of femtocuries (fCi), which is highly sensitive.
Performing a mass balance using the mean blank and sample data, any sample with an F14C>1 was considered to be suspicious. The variation of the blank appeared to be related to the type of glove being used to handle the moistened swab, with latex gloves giving higher values than other types of gloves. Different gloves were tested and it was found that the purple nitrile gloves (Kimtech PN-90627) gave the lowest mass and F14C values for the blanks.
A clear advantage of the method was that it could be comfortably used for small samples down to 5 μg C without having to add additional carrier carbon. The use of Exetainer vials and simple equipment for the preparation kept the costs and possibility of cross-contamination to a minimum. Based on the AMS throughput of 12 samples per hour, it was possible to analyze more than 100 samples in 3 days including collection (day 1), preparation (day 2), and analysis (day 3).
At the time of this study, the carryover of the system was estimated to be approximately 3% due to the shortened analysis method and a blockage in the GIS transfer line. Carryover predominantly occurred in the autosampler and GIS. Consistent with the findings of previous studies, the ion source could be brought back to normal within half an hour by running several fresh cathodes with blank gas. However, after the hottest sample with an F14C>2000, it took several days to bring the CHS and GIS system back to normal blanks levels, requiring repeated of cycles of heating, flushing with blank gas, and evacuation to remove excess 14C. After this event, risky samples were diluted before analysis.
Swabbing Strategies
Over the course of our swabbing program, we were able to refine our strategy for taking swabs. Additional advice and strategies can be found in the references and supplementary information. One key initiative that was implemented was a “swab before you start” program. Here, before a student or colleague commenced a new project, they were asked to swab test their equipment and facilities. This served several purposes including familiarization with the issues of contamination and ensuring that their equipment and facilities were clean. It was also possible to have them include additional samples from existing and new locations to extend our database of results. A quarantine area was set aside so that items and samples could be tested before being transferred into a 14C work area. Through this program, five new locations with evidence of previous tracer work were uncovered.
Consistent with previous studies, contamination could be found in commonly used areas and on commonly used equipment. Initially, a broad screening run of door handles, fume hoods, freeze dryers, and balance areas were targeted. Door handles were particularly useful as it was not necessary to enter a workspace to tell if something hot was inside. Multiple door handles could be swabbed with a single filter paper allowing whole buildings, floors, or corridors to be checked. Any suspect areas could be investigated further and 20–50 specific swabs of furniture, instrumentation, and apparatus could be taken. We would focus on switches, fume hoods, computer keyboards, bench tops, drawer handles, ovens, balances, fridges, and freezers. Using this procedure, we located point sources at most contaminated locations.
SCENARIOS
Here, four situations encountered are described without providing specific names or locations. These were the four most significant cases out of 10 that were studied.
Hot Gas Chromatograph in a Stable Isotope Laboratory
During the course of a compound-specific radiocarbon analysis (CSRA) project, a number of hot samples with F14C<10 were measured on the AMS, resulting in the loss of weeks of compound preparation work and unpublishable data. The contamination appeared to occur randomly and the laboratories used for sample preparation were refurbished just 5 yr previously, complete with new furniture and instrumentation. Broad screening of the laboratories located a point source at a gas chromatograph (GC) in a stable isotope laboratory used for CO2 quantification. Further swab tests narrowed this down to a set a drawers and a box of syringes that showed levels of F14C>100. One syringe had been previously used in an overseas laboratory that had conducted CSRA with labeled compounds more than 20 yr ago. This syringe had cross-contaminated the GC, samples, and the surrounding furniture. Cleanup was attempted but only reduced the contamination to F14C<10, which was still too high for the very low levels considered necessary for CSRA analysis. The area was abandoned for 14C work and access restricted by signage.
Tracer Experiment near a Sample Preparation Laboratory
During routine 14C sample preparation in a dedicated natural abundance laboratory, graphite blanks measured on the AMS increased overnight to F14C>0.2. It was found that a biology group in the same building had undertaken a tracer experiment the previous day using Na14CN. The Na14CN was prepared in a secure basement isotope laboratory before samples were transported to scintillation counters in laboratories above. Although the tracer experiment was performed carefully in a fume hood vented directly to the outside, it was speculated that dissolution of the Na14CN in aqueous solvent produced gaseous H14CN, which dispersed throughout the building, contaminating the natural abundance samples and blanks.
Operations in the AMS sample preparation laboratory were stopped and about a month’s worth of prepared graphite samples were contaminated and lost. The AMS preparation laboratory was moved to another building while cleaning and testing was undertaken. 14C levels in the building quickly decreased over several days after the event and after 1 month blank levels had mostly returned to workable levels. After 3 months, the preparation apparatus could be returned to its original building. In this case, it was fortunate that the contaminant was gaseous and was cleared by ventilation. Nonetheless, consequences of the event were a significant loss of commercial and research samples and time. In terms of revenue and labor, the monetary loss was estimated at more than $100K (USD). Prevention of future contamination relied on an agreement to cease further tracer experiments in the building and instigation of a 14C monitoring program.
Hot Gas Chromatograph in a Compound-Specific 14C Laboratory
Two students about to commence 14C projects were asked to swab the laboratories and equipment they planned to use. Initial results showed that several places such as the fume cupboards had low-level 14C contamination with F14C<20. It was also discovered that 13C tracer work was ongoing in the laboratories, which could potentially interfere with 14C analysis. The laboratories were wiped down and rearranged to isolate 14C operations into a dedicated section. A final swab of the laboratory found a previously untested preparative GC in the laboratory that had an F14C>2000. Although it was stated that the GC had never been used for tracer work, it had come from another institution that had used enriched 14C materials.
The preparative GC was removed and the laboratory was emptied and totally refurbished. The fume hood was professionally cleaned and the furniture was replaced and the laboratory was re-swabbed. The results showed that the area was more contaminated than before and it was revealed that the furniture was second-hand from the central repository. Despite extensive cleaning before putting it in the refurbished laboratory, it had not been tested first and had clearly been used near enriched materials. The furniture was removed and the laboratory professional cleaned a second time. Brand-new furniture was installed in the laboratory and a final batch of swabs found only two samples with F14C of 1–2. The whole process of setting up this lab took more than 1 yr, causing a significant delay to students and cost to the department. Local tracer work in the building was ongoing; however, an awareness lecture took place and restricted access and signage were implemented.
Laboratory Relocation to a Contaminated Area
Two students were about to start projects at a new institution. Upon discussion with the institution, it was revealed that a number of tracer experiments had been undertaken over the past 20–30 yr at that location and that enriched samples had been processed in several laboratories. Renovations were about to be undertaken during which time the laboratories would to be temporarily interchanged within a building. Screening of several laboratories showed areas of contamination with F14C>100 including a total organic carbon (TOC) analyzer, peristaltic pump, and freeze-dryer. High F14C levels were also found in fume hoods and balance areas. Discovery of these areas was fortunate, as it had been planned to relocate a clean preparation laboratory into one of these areas. As a result of these findings, it was recommended to abandon the contaminated areas permanently for 14C work. A newer and recently refurbished building was tested and a clean dedicated laboratory was reserved for natural abundance 14C work. One fortunate consequence of these tests was that samples destined for the calibration curve were saved from certain contamination.
Cleaning Strategies
The main question that was always asked was, “How do we clean up the contamination?” How clean is clean enough strongly depends on the size and age of the samples to be prepared. Larger modern samples are less sensitive to contamination than smaller older samples; therefore, in some situations a higher level of residual contamination could be considered tolerable. Laboratories for natural abundance CSRA require the cleanest conditions, i.e. ultra-trace levels. Other studies have reported cleaning strategies that have been successful ((Jull et al. Reference Jull, Donahue and Toolin1990; Vogel et al. Reference Vogel, Southon and Nelson1990; Zermeño et al. Reference Zermeño, Kurdyla, Buchholz, Heller, Kashgarian and Frantz2004; Zhou et al. Reference Zhou, Wu, Lange, Lu, Cheng, Xiong, Cruz, Liu, Fu and Zhao2012).
In general, it was found that for items with F14C<10, cleaning might be possible to acceptable levels of F14C<2. Above an F14C of 10, we had mixed success and for an F14C>100, we generally recommended abandoning the item for natural abundance 14C work. Typically, the first step of cleaning was washing with detergent and solvent such as IPA ,and the second step was a baking the items in a muffle furnace if possible. It was more difficult to clean complex apparatus such as a freeze-drier. This occurred where the freeze-drier was initially tested with F14C>300 and after very extensive washing, it was only reduced to F14C>100. In general, it can be recommended that anything disposable should be discarded and items to be kept should be cleaned and retested. When setting up a lab, we recommended that all areas and items should be new or proven clean. As discussed, a single hot item could contaminate an entire laboratory.
CONCLUSIONS
We have developed a convenient and cost-effective method that proved satisfactory for testing laboratories and equipment for enriched levels of 14C. We found that ongoing use of and residues from 14C tracer work were a problem and periodically impacted routine natural abundance operations. Transfer of tracer compounds was primarily by contact and could be mostly contained within a single laboratory. It was possible to clean contaminated areas; however, this depended the extent and levels of 14C contamination and on the complexity of the item to be cleaned. Consistent with previous studies, the mixing of tracer and natural abundance work was found to be not advisable. Good communication about prior use, thorough testing, and a monitoring program were essential to ensuring the isotopic fidelity of 14C measurements by AMS. Proven clean areas with restricted access and cessation of nearby tracer work were necessary for 14C AMS laboratories.
ACKNOWLEDGMENTS
Thanks to our colleagues at the various institutions who allowed us to perform testing and listened to our recommendations when given.
SUPPLEMENTARY INFORMATION
Attached is copy of the preparation protocol with illustrative photos. Currently, additional protocols and advice are available from the NOSAMS AMS facility at WHOI, the W.M. Keck Carbon Cycle AMS facility at UCI and the SWAB program at the University of Miami (http://www.whoi.edu/nosams/Submitting_Guidelines), (http://www.ess.uci.edu/researchgrp/ams/protocols) (http://www.rsmas.miami.edu/groups/tritium/swab/monitoring-of-shipboard-contamination/).
