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Detecting gravesoil with headspace analysis with adsorption on short porous layer open tubular (PLOT) columns
Forensic Science International 204 (2011) 156–161
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Detecting gravesoil with headspace analysis with adsorption on short porous layer open tubular (PLOT) columns§ Tara M. Lovestead, Thomas J. Bruno * Thermophysical Properties Division, National Institute of Standards and Technology, 325 Broadway, Boulder, CO 80305, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 December 2009 Received in revised form 12 May 2010 Accepted 25 May 2010 Available online 23 June 2010
Victims of crimes are often buried in clandestine graves. There are several techniques for finding buried bodies or the scattered remains of a victim; however, none of these methods are very reliable or work in all scenarios. One way to detect gravesoil is to detect the biochemical changes of the surrounding soil due to cadaver decomposition, for example, the release of nitrogenous compounds. A simple and low-cost way to detect these compounds is based on the reaction of alpha amino groups with ninhydrin to form Ruhemann’s purple. This test for ninhydrin-reactive nitrogen (NRN) has, to date, only been performed by direct solvent extraction of soil samples. Here, we present a method that detects trace quantities of NRN in the headspace air above gravesoil. Our method is based on an improved purge and trap method developed in our lab for sampling low volatility compounds, as well as volatile compounds at trace quantities, by applying low temperature collection on short alumina-coated porous layer open tubular (PLOT) columns. We modified this method to sample the headspace air above gravesoil with a motorized pipetter and a PLOT column at ambient temperatures. We generated gravesoil using rat cadavers and local soil. Trace quantities of NRN were successfully detected in the headspace air above gravesoil. We report the quantities of NRN recovered for buried rats, rats laid on top of soil, and blank graves (no rats) as a function of time (weeks to months). This work is the first (and thus far, only) example of a method for detecting NRN in the vapor phase, providing another tool for forensic investigators to aid in locating elusive clandestine graves. Published by Elsevier Ireland Ltd.
Keywords: Cadaver Clandestine graves Cryoadsorption Gravesoil Headspace Ninhydrin
1. Introduction A conviction for murder is much more easily obtained when the victim and the crime scene are located. Locating a clandestine grave or the exposed remains of a victim is sometimes a major obstacle for law enforcement personnel. Techniques for finding buried bodies include probing the soil and collecting soil samples, scanning with ground penetrating radar or geophysical electrical resistivity surveys, evaluating the landscape, and detecting the odor from cadaver decomposition with victim recovery (VR) canines [1–7]. Currently, none of these procedures for locating clandestine gravesites is very reliable and/or are useful for all scenarios. Thus, there is much interest in developing improved techniques and methods, and understanding better the science behind currently used methods for finding clandestine graves. The various stages of cadaver (including pig, rat, and human) decomposition have been studied in great detail by examining
§ Contribution of the United States Government; not subject to copyright in the United States. * Corresponding author. Tel.: +1 303 497 5158; fax: +1 303 497 5927. E-mail address: [email protected] (T.J. Bruno).
0379-0738/$ – see front matter . Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2010.05.024
insect colonization, microbial degradation of tissues, adipocere formation, and/or changes in the biochemistry of soil beneath a cadaver [8,9]. Additionally, the impacts of temperature, season, depth, and soil type on the aforementioned biochemical changes of the soil and the rate of decomposition have been examined [9–22]. Decomposition begins internally as the digestive enzymes and bacteria that were present in the body (i.e., intestines) prior to death feed off the internal organs and tissues [8–10]. The second stage of decomposition is putrefaction, or the bloating of the cadaver due to internal bacterial growth and activity [8,9]. Additionally, local bacteria, insects, flies, and maggots contribute to these initial stages of cadaver decomposition [8,9]. The later stages of cadaver decomposition include collapse of the bloated cadaver, drainage of the bodily fluids, consumption of the flesh by insects, cadaver fermentation and eventual drying out [8,9]. Changes in the biochemistry of soil surrounding a cadaver during decomposition can be detected by forensic scientists as a means to identify gravesoil to aid in locating clandestine graves. During the initial stages of decomposition, nitrogenous compounds are released into the surrounding area/soil [10,12,23–27]. Detection of an elevation in the concentration of nitrogenous compounds is one way to identify gravesoil. A simple and low-cost way to detect these compounds is based on the reaction of a-
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amino groups with ninhydrin to form Ruhemann’s purple [28]. Ninhydrin-reactive nitrogen (NRN) has to date been collected only by direct solvent extraction from soil samples. The ninhydrin reagent itself is commonly used by law enforcement personnel to detect latent fingerprints, and thus, crime scene technicians are already comfortable with its use. Extending the application to gravesoil detection has recently been under investigation [23]. Ninhydrin is a relatively safe chemical to work with and poses a danger only when ingested, inhaled, or splashed on the eyes or skin. These hazards are nearly ubiquitous with any non-food-grade chemical or reagent, and proper use of personal protective techniques (gloves, ventilation, and eye protection) reduces these hazards. Additional techniques for detecting clandestine graves are based on the release of volatile organic compounds (VOCs) during cadaver decomposition, i.e., detecting the odor signature of cadaver decomposition [1,4,7,29,30]. Victim recovery canines are able to locate clandestine graves by use of odor detection. Several VOCs have been identified below, above, and at the soil surface of shallow burial sites [7,30]; however, little work has been done to characterize compounds that have relatively low volatility or are present in only trace quantities. In this paper, we present a method that detects trace amounts of NRN in the headspace (HS) air above gravesoil. NRN has been found in mammalian and human gravesoil in trace quantities (ppm, mass/mass) [10,14,17,23,25]. Since trace quantities of NRN are typically recovered from gravesoil and most of the NRN compounds (amino acids and peptides; with the exception of ammonia) have extremely low vapor pressure, a method for detecting trace quantities of (typically) low vapor pressure compounds is required. Vapor detection methods are attractive forensic techniques because they are non-invasive (stand-off detection is possible) and can be very sensitive to the compound of interest. Detection and analysis of compounds with high vapor pressures (VOCs) is a relatively facile and routine process [31–35]. It is much more difficult for devices to detect analytes that have low volatility and/ or [(Fig._1)TD$IG] are at trace quantity. Headspace analysis techniques are often
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used to examine a gas that has been previously in contact with a condensed solid or liquid phase for the presence of VOCs to determine the safety and quality of drinking water, soils, foods and air [31,36–38]. Headspace sampling methods (often also called purge and trap methods) can be either static or dynamic. Static methods typically involve pressurizing a vial containing the sample with a gas and then sampling the gas with either a gas-tight syringe, a multiport sampling valve, or with solid phase microextraction (SPME) [39,40]. Dynamic methods typically employ a flow of carrier (or sweep) gas to the vial containing the sample. The sweep gas (and any solutes in the headspace) are passed out of the sample matrix and vial and through either a cryostat, adsorbent, or solvent to collect (or trap) the solutes. After the solutes are collected (trapped) they are concentrated, separated, and analyzed, often with gas chromatography (GC). As mentioned, headspace analysis is more difficult to apply to compounds that have low volatility, and especially those at trace quantity. Such applications of HS analysis typically require long collection periods to obtain sufficient sample mass for analysis and identification. Most commercially available purge and trap equipment were developed for measuring VOCs in aqueous samples; thus, these instruments typically have limited sweep periods for the purge cycle, and a limited temperature range for the sample holder (usually below 100 8C). These commercial instruments are nearly impossible to use for the analysis of low volatility compounds and/or compounds at trace quantity, to obtain precise quantitative measurements, and/or for analytical studies performed as a function of collection temperatures (i.e., studies aimed at obtaining data with predictive capability). To overcome these limitations of the commercially available HS analysis equipment, we have developed a high sensitivity, dynamic HS analysis technique that makes use of cryoadsorption on short alumina-coated PLOT columns (see Figs. 1 and 2) [41]. A detailed explanation of the experimental set-up has been described earlier [41]. We have used alumina as the adsorbent in most of our work with this technique, because it is robust and can be used with alkaline solvents if desired; however, when the measurement
Fig. 1. A schematic of the experimental apparatus used for quantitative and trace headspace analysis with an adaptation of cryoadsorption.
[(Fig._2)TD$IG]
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T.M. Lovestead, T.J. Bruno / Forensic Science International 204 (2011) 156–161 2. Experimental
Fig. 2. A schematic of the porous layer open tubular (PLOT) column used as a cryoadsorber trap. The PLOT column is coated with alumina, is approximately 3 m long, and is coiled at a 8 cm diameter to allow the column to be housed in the cryostat chamber (see Fig. 1).
warrants, one can easily use silica, porous polymers, clays, organoclays, and sol–gel phases as well [42,43]. Also, several different PLOT columns can be inserted into the sample vial’s septum at the same time to collect samples on multiple phases in parallel. It is important to mention that SPME, one of the most modern HS sampling techniques, is not always a viable method for readily obtaining quantitative data. Moreover, one must often salt-out the solutes of interest for SPME [44]. After HS collection, the cryoadsorber (PLOT) column is removed and the constituents are collected by desorption (usually solvent or thermal). Components of the HS are separated, identified and quantitated as a function of temperature, typically with GC coupled with mass spectrometry (GC–MS) [45,46]. We have applied this method to the temperature-dependent quantitative analysis of trace compounds and low volatility compounds in the headspace air above explosive/energetic materials (C-4, Semtex-H, Semtex-1A, detonating cord and sheet explosive) and spoiled chicken [47]. We modified this method for the ambient temperature collection of ninhydrin-reactive nitrogenous compounds from the headspace above decaying rats using alumina-coated PLOT columns and a portable motorized pipetter. This technique is the first (and thus far, only) technique for vapor detection of NRN, a technique that could aid forensic investigators in unusual or difficult clandestine grave searches. For example, if a body were suspected to lie underneath a concrete slab, a small hole could be drilled into the slab and the headspace air above the underlying soil and rip-rap in the settlement zone sampled (T. Clemmon, personal communication, National Institute of Standards and Technology, 2009). Thus, criminalists would determine whether demolition is necessary, and also more precisely locate the area to jackhammer and excavate. [(Fig._3)TD$IG]
Frozen dead feeder rats (Norwegian) with an average weight of 80 g and an average body length of 12.7 cm were obtained from a commercial supplier and were used intact, i.e., we did not cut them open. The rats were reported to be asphyxiated with CO2 and then flash frozen. The water used as a solvent in this work was HPLC (high performance liquid chromatography) grade. Potassium chloride (KCl, >99% purity) was obtained to make up a 2 M solution that was used to enhance desorption of the nitrogenous compounds from the alumina-coated PLOT columns [48]. The dl-leucine (CAS no. 61-90-5) used for standardization and the ninhydrin reagent solution (2%, v/v ninhydrin, CAS no. 485-47-2, with hydrindantin, CAS no. 5103-42-4, in DMSO and a lithium acetate buffer, pH 5.2) were purchased from a commercial supplier and used without further purification. The soil used in this study was obtained on-site at the NIST campus in Boulder, Colorado in May of 2009. According to the United States Department of Agriculture, the soil surrounding the NIST campus is classified as very cobbly sandy loam, which is approximately 58% sand, 32% silt, and 10% clay [49]. The soil pH was approximately 6.0. Pressed wood boxes with four separate compartments per box (for a total of 12 compartments) were used as burial sites (see Fig. 3). We placed approximately 8 cm of soil in each compartment (2 kg of soil). At least one frozen, dead feeder rat was placed in eight of the compartments. The rats in compartments 1–4 were placed on the surface of the soil and the rats in compartments 5–8 were buried in an additional 8 cm of soil. The remaining four compartments were blank graves (no rats, compartments 9–12). Two rats were placed in both gravesites 4 and 6 to investigate the impact of cadaver mass on the amount of NRN collected in the headspace above the gravesoil. The soil was used as is, and while it may have contained insects, neither insects nor water were added to the gravesites to minimize variables in this very controlled, initial study. We sealed the compartments (gravesites) with interference-fit lids to obtain nearly airtight seals (see Fig. 3b). The gravesite boxes were maintained outside, in two separate fume cabinets (one for compartments with gravesoil, 1–8, and the other for compartments with blank graves, 9–12), in a covered chemical storage facility. Each compartment lid had a septum port for sampling and an eye bolt for removing the lids after the study. At the time of sample collection, the septum was pierced with an activated alumina-coated PLOT column (see Fig. 2). The exposed end of the PLOT column was attached to a motorized pipetter, and the headspace air above the gravesoil was sampled at ambient temperature. The daily high and low temperatures for Boulder, CO, for the duration of this study (May 28–October, 2009) were obtained from the National Climate Data Center [50]. These data are displayed in Fig. 4. The average temperatures during this study were 17.2 8C for June, 21.1 8C for July, 20.9 8C for August, 17.3 8C for September, and 7.7 8C for October. A different PLOT column was used to sample the HS above each gravesite (compartment). After HS collection, the PLOT column was removed from the septum port and the adsorbed compounds were eluted by piercing one end through the crimp-cap septum of a vial with 0.5 mL of the 2 M KCl solution and the other end through the crimp-cap septum of a pre-weighed vial with 0.5 mL of 2% ninhydrin reagent. A blow gun pressurized with compressed air was used to force the KCl solution through the PLOT column and into the vial with the ninhydrin reagent. This latter vial was subsequently recapped and placed on a vortex stirring plate for 5 s and then incubated at 100 8C for 25 min. The sample vial was placed on the lab bench for 15 min to bring it to room temperature (approximately 23 8C) and then diluted with 10 mL 50% ethanol solution. The absorbance of this final solution was measured at 570 nm by use of a diode array UV–vis spectrophotometer. Twentyfive different alumina-coated PLOT columns were used to sample the gravesoil headspace throughout the study, and selected at random. Blanks were run periodically to ensure against carryover. The HS above the gravesoil in each compartments 1–12 was sampled at 1, 2, 3, 4, 5, 6, 10, and 20 weeks after the compartments were sealed. Vertical bars placed on the daily temperature graph in Fig. 4 indicate the headspace collection days. Calibration was done by use of dl-leucine standards of known concentrations in a 2 M KCl solution. Stock solutions (0 and 10 mg nitrogen per mL of the KCl solution)
Fig. 3. Eight of the twelve gravesites are shown. (a) At least one frozen, dead feeder rat was placed in each of the gravesites upon 8 cm of soil. An additional 8 cm of soil was placed on half of these gravesites. (b) The compartments (gravesites) after sealing with interference-fit lids are shown. Each compartment had a septum port for headspace collection and an eye bolt to aid in removal of the lids at the end of the study.
[(Fig._4)TD$IG]
[(Fig._5)TD$IG]
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Fig. 4. The daily high and low temperatures for the rat decomposition study are presented. Vertical bars are placed on the graph to indicate the headspace collection days. were prepared and used to make four concentrations of nitrogen in solution. The standards were run along with each sample set to calibrate the diode array spectrophotometer response. Values were reported as the total mass of NRN collected, corrected for the volume of 2 M KCl used to elute the PLOT column, and the volumes of both the ninhydrin reagent solution and the 50% ethanol solution added to each sample. The sources of uncertainty in evaluating the recovered mass of trace components from the HS are 2-fold; there is uncertainty in the spectrophotometer response, and in the calibration. The overall uncertainty (with a coverage factor k = 2), associated with the recovered mass for trace compounds, was determined to be 3% at the low concentrations encountered in this work.
3. Results NRN was not detected in the headspace above the gravesoil for the first month of sampling (weeks 1–4). This result was surprising, because other researchers have observed increases in NRN in the soil within the first month, although the decomposition rate is highly dependent on the size and the location of the cadaver, the season and the temperature [10,12,14,23]. Most likely the cool May/June weather in Colorado (see Fig. 4) delayed decomposition and the subsequent release and accumulation of NRN into the surrounding soil. Additionally, the rats were frozen when placed in the compartments, and the compartments in this study were sealed off from the environment (insects, moisture, and sunlight). In the previous studies that found indications of cadaver decomposition within the first month, the cadavers were buried or laid upon the ground outside, and thus, exposed to the elements, insects and scavengers [10]. Also, since most NRN compounds have very low vapor pressure, the concentration of NRN compounds in the headspace air above the soil is much lower than that in the soil, and thus, a larger period is required to establish a concentration of NRN in the HS air above gravesoil that can be detected with this method. It would be advantageous to establish the limit of detection of the headspace collection method used here and to determine the equilibrium constant for NRN in the soil and the vapor phase in future studies. Fig. 5 shows the recovered masses of ninhydrin-reactive nitrogen that were detected in the compartments/gravesites at weeks 5, 6, and 10. NRN was collected in trace quantities (total micrograms of nitrogen) from the HS above gravesoil in each compartment with decomposing rats (gravesites 1–8) at these time points. Fig. 5 also shows that at 5 and 6 weeks collection time (Fig. 5a and b, respectively), the lowest recovered masses of NRN were collected from the HS above the blank graves (plots 9–12). This result is also shown in Table 1, which presents the average recovered NRN masses for each plot grouping (rats laid on the soil, buried rats, and blank graves). Table 1 clearly shows that the
Fig. 5. The recovered masses of NRN collected from the headspace above the gravesoil in compartments 1–12 after 5 (a), 6 (b), and 10 (c) weeks of rat decomposition. Compartments 1–4 contained exposed rats, 5–8 contained buried rats, and 9–12 were blank graves. Gravesites 4 and 6 initially contained two frozen, dead feeder rats.
lowest average recovered masses for NRN were collected from the blank graves at all time points (5, 6, and 10 weeks) and that the difference in the values obtained from gravesites versus blank graves decreases throughout this study. This result is also Table 1 The average recovered masses for each group of rats (exposed, buried) and the blank graves at 5, 6, 10, and 20 weeks headspace collection. Compartments 1–4 are the exposed rats, compartments 5–8 are the buried rats, and compartments 9–12 are the blank gravesites. The values for 20 weeks are in italics because they were obtained using a significantly shorter headspace sampling period. The uncertainty is discussed in the text. Average recovered masses of NRN, mg
Exposed Buried Blank
5 weeks
6 weeks
10 weeks
20 weeks
1.4 1.8 0.1
1.0 1.0 0.2
0.4 0.6 0.2
0.5 0.4 0.1
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Fig. 6. The recovered masses of NRN collected from the headspace above the gravesoil in compartments 1–12 after 20 weeks of rat decomposition to illustrate the yes/no response of this method with only 5 min headspace sampling period. Compartments 1–4 contained exposed rats, 5–8 contained buried rats, and 9–12 were blank graves. Gravesites 4 and 6 initially contained two frozen, dead feeder rats.
illustrated in Fig. 5. Thus, a clear baseline value for ambient NRN in the HS above gravesoil is possible at 5 and 6 weeks of rat decomposition, while, at longer times, more uncertainty in the baseline NRN measurement is observed because the later stages of rat decomposition are occurring and NRN release into the soil has decreased significantly. Our observations are consistent with observations reported previously by Van Belle et al. [10] on the changes in the soil NRN concentrations during swine decomposition. These researchers observed a rapid decrease in the soil concentrations of NRN after 2 months decomposition time and the gravesoil NRN levels were equivalent to that of the blank graves by 5 months decomposition time. We also observed that at 5 and 10 weeks the average recovered masses of NRN is greater for the buried rats than for the rats placed on the surface of the soil. At 6 weeks HS collection times, the average recovered masses of NRN were equivalent for these two rat groups. In a previous study by Van Belle et al. [10], cadavers laid on the soil surface decomposed and released NRN into the surrounding soil more rapidly than buried cadavers. This study was conducted outside, and thus the accelerated decomposition rate of the cadavers placed on the soil surface could be due to environmental elements including, wind, rain, insects, scavengers, etc. In the current study, we sealed the gravesites off from the environment; thus, external cadaver decomposition was aided only by the microbes and insects initially present in the soil used. We hypothesize that more NRN was collected above the gravesoil of the buried rats because these cadavers were in direct contact with the soil on all surfaces, and thus, there was more contact with bacteria present in the soil. Different HS sampling periods were also investigated. As mentioned previously, the concentration of NRN in gravesoil is typically in the ppm (mass/mass) range. These compounds, with the exception of ammonia, have extremely low vapor pressures, and thus would be present in the HS above the gravesoil also in trace amounts, especially at ambient temperatures. HS collection periods of 2 h were chosen for this study based on our experience collecting trace compounds of low volatility at ambient temperature in previous work [47]. Fig. 5 shows that this headspace sampling period is adequate for quantitative detection of trace amounts of NRN in the HS above gravesoil. However, in practice, one is typically concerned only with determining whether or not the level of NRN exceeds the baseline level (that is, a go/no-go result), not necessarily the maximum amount of NRN present. Fig. 6 shows the amounts of NRN collected at week 20 from each
gravesites 1–12 with only a 5 min HS sampling period. It is interesting to note that some of the masses recovered at 20 weeks are comparable or higher than the masses collected at 10 weeks. This result shows that beyond 5 min headspace collection period, there is no advantage to increasing the collection period. In fact, it may be a disadvantage to use longer collection periods if NRN is potentially displaced from the PLOT column. Additionally, it is possible that the lower ambient temperature at week 20 improved the collection efficiency of the PLOT column. In the laboratory (where we have control over the headspace collection temperature) we typically use adsorber temperatures of approximately 0 8C. What is important to point out here is that after only 5 min, an NRN level above the baseline level (average amount detected in the blank graves, 0.06 mg of nitrogen) was collected in 6 of 8 of the gravesites. Thus, the headspace sampling period is not an important variable beyond 5 min; this is a promising result for the anticipated development of a field-ready, trace-vapor NRN detection method to aid investigators locate clandestine graves. Work on this aspect is ongoing. Interestingly, a significant difference in the amount of NRN collected and cadaver mass when comparing the gravesites with one versus two rats was not observed. We have already shown that increasing the headspace sampling period does not necessarily increase the amount of NRN collected from the headspace by use of this methodology. Thus, it is possible that beyond a minimum amount of cadaver decomposition, additional cadaver mass may not change the amount of NRN collected from the headspace above the gravesoil. Once again, for our purpose, it is more important to detect NRN levels above baseline values than to detect a specific amount. The influence of the environment and the burial conditions on the NRN released into the gravesoil has been shown in numerous papers. The impact of these conditions on the quantity and release of NRN into the headspace above gravesoil is an important aspect of this work to investigate further to help establish a fundamental understanding of this promising detection method. 4. Conclusions A method for the ambient temperature headspace collection of NRN above gravesoil, using a motorized pipetter and an activated alumina-coated PLOT column has been developed. The method is based on an improved purge and trap method for sampling compounds of low volatility, as well as volatile compounds at trace quantities, by applying low temperature collection on short alumina-coated PLOT columns. Gravesoil was generated with frozen, dead feeder rats and local soil. Half of the rats were placed on the surface of the soil, and half of the rats were buried. NRN was detected with only 5 min headspace collection period. The lowest average recovered masses for NRN were collected from the blank graves. Additionally, we observed a decrease in the average amount of NRN collected in each group (exposed, buried, and blank graves) throughout the study. We observed a slightly elevated recovered mass of NRN for buried rats compared with that recovered from rats laid upon the soil. No correlation of initial cadaver mass on NRN collected in the headspace above the gravesoil was observed. We have demonstrated that it is possible to detect trace quantities of NRN in the headspace above gravesoil, adding a unique method to aid law enforcement personnel and criminal investigators detect clandestine graves. Acknowledgments TML acknowledges a Professional Research Experiences Program and a National Academy of Sciences/National Research Council postdoctoral fellowship, both at NIST.
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[24] E. Zagal, C. Munoz, M. Quiroz, C. Cordova, Sensitivity of early indicators for evaluating quality changes in soil organic matter, Geoderma 151 (2009) 191–198. [25] D.O. Carter, D. Yellowlees, M. Tibbett, Using ninhydrin to detect gravesoil, in: 59th Annual Meeting of the American-Academy-of-Forensic-Science, San Antonio, TX, Feb, Blackwell Publishing, 2007, pp. 397–400. [26] M.R. Carter, Ninhydrin-reactive-N released by the fumigation-extraction method as a measure of microbial biomass under field conditions, Soil Biol. Biochem. 23 (1991) 139–143. [27] P.M. Mele, M.R. Carter, Estimation of microbial biomass by ninhydrin-reactive N using liquid chloroform, Can. J. Soil Sci. 76 (1996) 37–40. [28] H. Meyer, Ninhydrin reaction and its analytical applications, Biochem. J. 67 (1957) 333–340. [29] M.S. Macias, R.J. Harper, K.G. Furton, A comparison of real versus simulated contraband VOCs for reliable detector dog training utilizing SPME–GC–MS, Am. Lab. 40 (2008) 16. [30] A.A. Vass, R.R. Smith, C.V. Thompson, M.N. Burnett, D.A. Wolf, J.A. Synstelien, et al., Decompositional odor analysis database, J. Forensic Sci. 49 (2004) 760– 769. [31] C.E. Boswell, Fast and efficient volatiles analysis by purge and trap GC/MS, in: WTQA’99-15th Annual Waste Testing and Quality Assurance Symposium, Proceedings, 1999, pp. 190–194. [32] B.V. Ioffe, A.G. Vittenburg, I.A. Manatov, Head-Space Analysis and Related Methods in Gas Chromatography, Wiley-Interscience, 1984. [33] M.J. Preito, D. Berenguer, D. Marhuenda, A. Cardone, Purge and trap gas chromatographic determination of styrene in urine and blood: application to exposed workers, J. Chromatogr. B 741 (2000) 301–306. [34] A. Wasik, W. Janicki, W. Wardencki, J. Namiesnik, Device for isolation and preconcentration of volatile organic species by means of purging and cryotrapping analytes, Analusis 25 (1997) 59–64. [35] Z.C. Wu, Y.S. Fung, Isolation and determination of volatile organic compounds from water by dynamic purge and trap technique coupled with capillary gas chromatography, Int. J. Environ. Anal. Chem. 82 (2002) 431–442. [36] D. Helmig, Air analysis by gas chromatography, J. Chromatogr. A 843 (1999) 129– 146. [37] J. Namiesnik, B. Zygmut, Selected concentration techniques for gas chromatographic analysis of environmental samples, Chromatogr. Suppl. 56 (2002) S9–S18. [38] E. Olsen, G. Vogt, D. Ekeberg, M. Sandbakk, J. Pettersen, A. Nilsson, Analysis of the early stages of lipid oxidation in freeze-stored pork back fat and mechanically recovered poultry meat, J. Agric. Food Chem. 53 (2005) 338–348. [39] J. Pawliszyn, Solid Phase Microextraction: Theory and Practice, Wiley-VCH, New York, 1997. [40] S. Wercinski, Solid Phase Microextraction: A Practical Guide, CRC Press, Boca Raton, FL, 1999. [41] T. Bruno, Simple quantitative headspace analysis by cryoadsorption on a short alumina PLOT column, J. Chromatogr. Sci. 47 (2009) 569–574. [42] T.J. Bruno, A. Lewandowska, F. Tsvetkov, K.E. Miller, H.J.M. Hanley, Wall-coated open-tubular column chromatography on an organo-clay stationary phase, J. Chromatogr. A 973 (2002) 143–149. [43] K.E. Miller, T.J. Bruno, Thermally-treated clay as a stationary phase in liquid chromatography, J. Chromatogr. A 1042 (2004) 49–54. [44] T.J. Bruno, P.D.N. Svoronos, CRC Handbook of Basic Tables for Chemical Analysis, 2nd ed., CRC Press, Boca Raton, 2003. [45] T.J. Bruno, P.D.N. Svoronos, CRC Handbook of Fundamental Spectroscopic Correlation Charts, Taylor and Francis, CRC Press, Boca Raton, FL, 2005. [46] S.R.D., NIST/EPA/NIH Mass Spectral Database, SRD Program, National Institute of Standards and Technology, Gaithersburg, MD, 2005. [47] T.M. Lovestead, T.J. Bruno, Detection of poultry spoilage markers from headspace analysis with cryoadsorption on short alumina PLOT columns, Food Chem. 121 (2010) 1274–1282. [48] J.N. Ladd, M. Amato, Relationships between biomass 14Cc and soluble organic 14C of a range of fumigated soils, Soil Biol. Biochem. 20 (1988) 115–116. [49] N.R.C.S. United States Department of Agriculture, Web Soil Survey, http:// websoilsurvey.nrcs.usda.gov/app/WebSoilSurvey.aspx. [50] http://www.ncdc.noaa.gov/oa/ncdc.html.
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Detecting gravesoil with headspace analysis with adsorption on short porous layer open tubular (PLOT) columns§ Tara M. Lovestead, Thomas J. Bruno * Thermophysical Properties Division, National Institute of Standards and Technology, 325 Broadway, Boulder, CO 80305, United States
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 December 2009 Received in revised form 12 May 2010 Accepted 25 May 2010 Available online 23 June 2010
Victims of crimes are often buried in clandestine graves. There are several techniques for finding buried bodies or the scattered remains of a victim; however, none of these methods are very reliable or work in all scenarios. One way to detect gravesoil is to detect the biochemical changes of the surrounding soil due to cadaver decomposition, for example, the release of nitrogenous compounds. A simple and low-cost way to detect these compounds is based on the reaction of alpha amino groups with ninhydrin to form Ruhemann’s purple. This test for ninhydrin-reactive nitrogen (NRN) has, to date, only been performed by direct solvent extraction of soil samples. Here, we present a method that detects trace quantities of NRN in the headspace air above gravesoil. Our method is based on an improved purge and trap method developed in our lab for sampling low volatility compounds, as well as volatile compounds at trace quantities, by applying low temperature collection on short alumina-coated porous layer open tubular (PLOT) columns. We modified this method to sample the headspace air above gravesoil with a motorized pipetter and a PLOT column at ambient temperatures. We generated gravesoil using rat cadavers and local soil. Trace quantities of NRN were successfully detected in the headspace air above gravesoil. We report the quantities of NRN recovered for buried rats, rats laid on top of soil, and blank graves (no rats) as a function of time (weeks to months). This work is the first (and thus far, only) example of a method for detecting NRN in the vapor phase, providing another tool for forensic investigators to aid in locating elusive clandestine graves. Published by Elsevier Ireland Ltd.
Keywords: Cadaver Clandestine graves Cryoadsorption Gravesoil Headspace Ninhydrin
1. Introduction A conviction for murder is much more easily obtained when the victim and the crime scene are located. Locating a clandestine grave or the exposed remains of a victim is sometimes a major obstacle for law enforcement personnel. Techniques for finding buried bodies include probing the soil and collecting soil samples, scanning with ground penetrating radar or geophysical electrical resistivity surveys, evaluating the landscape, and detecting the odor from cadaver decomposition with victim recovery (VR) canines [1–7]. Currently, none of these procedures for locating clandestine gravesites is very reliable and/or are useful for all scenarios. Thus, there is much interest in developing improved techniques and methods, and understanding better the science behind currently used methods for finding clandestine graves. The various stages of cadaver (including pig, rat, and human) decomposition have been studied in great detail by examining
§ Contribution of the United States Government; not subject to copyright in the United States. * Corresponding author. Tel.: +1 303 497 5158; fax: +1 303 497 5927. E-mail address: [email protected] (T.J. Bruno).
0379-0738/$ – see front matter . Published by Elsevier Ireland Ltd. doi:10.1016/j.forsciint.2010.05.024
insect colonization, microbial degradation of tissues, adipocere formation, and/or changes in the biochemistry of soil beneath a cadaver [8,9]. Additionally, the impacts of temperature, season, depth, and soil type on the aforementioned biochemical changes of the soil and the rate of decomposition have been examined [9–22]. Decomposition begins internally as the digestive enzymes and bacteria that were present in the body (i.e., intestines) prior to death feed off the internal organs and tissues [8–10]. The second stage of decomposition is putrefaction, or the bloating of the cadaver due to internal bacterial growth and activity [8,9]. Additionally, local bacteria, insects, flies, and maggots contribute to these initial stages of cadaver decomposition [8,9]. The later stages of cadaver decomposition include collapse of the bloated cadaver, drainage of the bodily fluids, consumption of the flesh by insects, cadaver fermentation and eventual drying out [8,9]. Changes in the biochemistry of soil surrounding a cadaver during decomposition can be detected by forensic scientists as a means to identify gravesoil to aid in locating clandestine graves. During the initial stages of decomposition, nitrogenous compounds are released into the surrounding area/soil [10,12,23–27]. Detection of an elevation in the concentration of nitrogenous compounds is one way to identify gravesoil. A simple and low-cost way to detect these compounds is based on the reaction of a-
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amino groups with ninhydrin to form Ruhemann’s purple [28]. Ninhydrin-reactive nitrogen (NRN) has to date been collected only by direct solvent extraction from soil samples. The ninhydrin reagent itself is commonly used by law enforcement personnel to detect latent fingerprints, and thus, crime scene technicians are already comfortable with its use. Extending the application to gravesoil detection has recently been under investigation [23]. Ninhydrin is a relatively safe chemical to work with and poses a danger only when ingested, inhaled, or splashed on the eyes or skin. These hazards are nearly ubiquitous with any non-food-grade chemical or reagent, and proper use of personal protective techniques (gloves, ventilation, and eye protection) reduces these hazards. Additional techniques for detecting clandestine graves are based on the release of volatile organic compounds (VOCs) during cadaver decomposition, i.e., detecting the odor signature of cadaver decomposition [1,4,7,29,30]. Victim recovery canines are able to locate clandestine graves by use of odor detection. Several VOCs have been identified below, above, and at the soil surface of shallow burial sites [7,30]; however, little work has been done to characterize compounds that have relatively low volatility or are present in only trace quantities. In this paper, we present a method that detects trace amounts of NRN in the headspace (HS) air above gravesoil. NRN has been found in mammalian and human gravesoil in trace quantities (ppm, mass/mass) [10,14,17,23,25]. Since trace quantities of NRN are typically recovered from gravesoil and most of the NRN compounds (amino acids and peptides; with the exception of ammonia) have extremely low vapor pressure, a method for detecting trace quantities of (typically) low vapor pressure compounds is required. Vapor detection methods are attractive forensic techniques because they are non-invasive (stand-off detection is possible) and can be very sensitive to the compound of interest. Detection and analysis of compounds with high vapor pressures (VOCs) is a relatively facile and routine process [31–35]. It is much more difficult for devices to detect analytes that have low volatility and/ or [(Fig._1)TD$IG] are at trace quantity. Headspace analysis techniques are often
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used to examine a gas that has been previously in contact with a condensed solid or liquid phase for the presence of VOCs to determine the safety and quality of drinking water, soils, foods and air [31,36–38]. Headspace sampling methods (often also called purge and trap methods) can be either static or dynamic. Static methods typically involve pressurizing a vial containing the sample with a gas and then sampling the gas with either a gas-tight syringe, a multiport sampling valve, or with solid phase microextraction (SPME) [39,40]. Dynamic methods typically employ a flow of carrier (or sweep) gas to the vial containing the sample. The sweep gas (and any solutes in the headspace) are passed out of the sample matrix and vial and through either a cryostat, adsorbent, or solvent to collect (or trap) the solutes. After the solutes are collected (trapped) they are concentrated, separated, and analyzed, often with gas chromatography (GC). As mentioned, headspace analysis is more difficult to apply to compounds that have low volatility, and especially those at trace quantity. Such applications of HS analysis typically require long collection periods to obtain sufficient sample mass for analysis and identification. Most commercially available purge and trap equipment were developed for measuring VOCs in aqueous samples; thus, these instruments typically have limited sweep periods for the purge cycle, and a limited temperature range for the sample holder (usually below 100 8C). These commercial instruments are nearly impossible to use for the analysis of low volatility compounds and/or compounds at trace quantity, to obtain precise quantitative measurements, and/or for analytical studies performed as a function of collection temperatures (i.e., studies aimed at obtaining data with predictive capability). To overcome these limitations of the commercially available HS analysis equipment, we have developed a high sensitivity, dynamic HS analysis technique that makes use of cryoadsorption on short alumina-coated PLOT columns (see Figs. 1 and 2) [41]. A detailed explanation of the experimental set-up has been described earlier [41]. We have used alumina as the adsorbent in most of our work with this technique, because it is robust and can be used with alkaline solvents if desired; however, when the measurement
Fig. 1. A schematic of the experimental apparatus used for quantitative and trace headspace analysis with an adaptation of cryoadsorption.
[(Fig._2)TD$IG]
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T.M. Lovestead, T.J. Bruno / Forensic Science International 204 (2011) 156–161 2. Experimental
Fig. 2. A schematic of the porous layer open tubular (PLOT) column used as a cryoadsorber trap. The PLOT column is coated with alumina, is approximately 3 m long, and is coiled at a 8 cm diameter to allow the column to be housed in the cryostat chamber (see Fig. 1).
warrants, one can easily use silica, porous polymers, clays, organoclays, and sol–gel phases as well [42,43]. Also, several different PLOT columns can be inserted into the sample vial’s septum at the same time to collect samples on multiple phases in parallel. It is important to mention that SPME, one of the most modern HS sampling techniques, is not always a viable method for readily obtaining quantitative data. Moreover, one must often salt-out the solutes of interest for SPME [44]. After HS collection, the cryoadsorber (PLOT) column is removed and the constituents are collected by desorption (usually solvent or thermal). Components of the HS are separated, identified and quantitated as a function of temperature, typically with GC coupled with mass spectrometry (GC–MS) [45,46]. We have applied this method to the temperature-dependent quantitative analysis of trace compounds and low volatility compounds in the headspace air above explosive/energetic materials (C-4, Semtex-H, Semtex-1A, detonating cord and sheet explosive) and spoiled chicken [47]. We modified this method for the ambient temperature collection of ninhydrin-reactive nitrogenous compounds from the headspace above decaying rats using alumina-coated PLOT columns and a portable motorized pipetter. This technique is the first (and thus far, only) technique for vapor detection of NRN, a technique that could aid forensic investigators in unusual or difficult clandestine grave searches. For example, if a body were suspected to lie underneath a concrete slab, a small hole could be drilled into the slab and the headspace air above the underlying soil and rip-rap in the settlement zone sampled (T. Clemmon, personal communication, National Institute of Standards and Technology, 2009). Thus, criminalists would determine whether demolition is necessary, and also more precisely locate the area to jackhammer and excavate. [(Fig._3)TD$IG]
Frozen dead feeder rats (Norwegian) with an average weight of 80 g and an average body length of 12.7 cm were obtained from a commercial supplier and were used intact, i.e., we did not cut them open. The rats were reported to be asphyxiated with CO2 and then flash frozen. The water used as a solvent in this work was HPLC (high performance liquid chromatography) grade. Potassium chloride (KCl, >99% purity) was obtained to make up a 2 M solution that was used to enhance desorption of the nitrogenous compounds from the alumina-coated PLOT columns [48]. The dl-leucine (CAS no. 61-90-5) used for standardization and the ninhydrin reagent solution (2%, v/v ninhydrin, CAS no. 485-47-2, with hydrindantin, CAS no. 5103-42-4, in DMSO and a lithium acetate buffer, pH 5.2) were purchased from a commercial supplier and used without further purification. The soil used in this study was obtained on-site at the NIST campus in Boulder, Colorado in May of 2009. According to the United States Department of Agriculture, the soil surrounding the NIST campus is classified as very cobbly sandy loam, which is approximately 58% sand, 32% silt, and 10% clay [49]. The soil pH was approximately 6.0. Pressed wood boxes with four separate compartments per box (for a total of 12 compartments) were used as burial sites (see Fig. 3). We placed approximately 8 cm of soil in each compartment (2 kg of soil). At least one frozen, dead feeder rat was placed in eight of the compartments. The rats in compartments 1–4 were placed on the surface of the soil and the rats in compartments 5–8 were buried in an additional 8 cm of soil. The remaining four compartments were blank graves (no rats, compartments 9–12). Two rats were placed in both gravesites 4 and 6 to investigate the impact of cadaver mass on the amount of NRN collected in the headspace above the gravesoil. The soil was used as is, and while it may have contained insects, neither insects nor water were added to the gravesites to minimize variables in this very controlled, initial study. We sealed the compartments (gravesites) with interference-fit lids to obtain nearly airtight seals (see Fig. 3b). The gravesite boxes were maintained outside, in two separate fume cabinets (one for compartments with gravesoil, 1–8, and the other for compartments with blank graves, 9–12), in a covered chemical storage facility. Each compartment lid had a septum port for sampling and an eye bolt for removing the lids after the study. At the time of sample collection, the septum was pierced with an activated alumina-coated PLOT column (see Fig. 2). The exposed end of the PLOT column was attached to a motorized pipetter, and the headspace air above the gravesoil was sampled at ambient temperature. The daily high and low temperatures for Boulder, CO, for the duration of this study (May 28–October, 2009) were obtained from the National Climate Data Center [50]. These data are displayed in Fig. 4. The average temperatures during this study were 17.2 8C for June, 21.1 8C for July, 20.9 8C for August, 17.3 8C for September, and 7.7 8C for October. A different PLOT column was used to sample the HS above each gravesite (compartment). After HS collection, the PLOT column was removed from the septum port and the adsorbed compounds were eluted by piercing one end through the crimp-cap septum of a vial with 0.5 mL of the 2 M KCl solution and the other end through the crimp-cap septum of a pre-weighed vial with 0.5 mL of 2% ninhydrin reagent. A blow gun pressurized with compressed air was used to force the KCl solution through the PLOT column and into the vial with the ninhydrin reagent. This latter vial was subsequently recapped and placed on a vortex stirring plate for 5 s and then incubated at 100 8C for 25 min. The sample vial was placed on the lab bench for 15 min to bring it to room temperature (approximately 23 8C) and then diluted with 10 mL 50% ethanol solution. The absorbance of this final solution was measured at 570 nm by use of a diode array UV–vis spectrophotometer. Twentyfive different alumina-coated PLOT columns were used to sample the gravesoil headspace throughout the study, and selected at random. Blanks were run periodically to ensure against carryover. The HS above the gravesoil in each compartments 1–12 was sampled at 1, 2, 3, 4, 5, 6, 10, and 20 weeks after the compartments were sealed. Vertical bars placed on the daily temperature graph in Fig. 4 indicate the headspace collection days. Calibration was done by use of dl-leucine standards of known concentrations in a 2 M KCl solution. Stock solutions (0 and 10 mg nitrogen per mL of the KCl solution)
Fig. 3. Eight of the twelve gravesites are shown. (a) At least one frozen, dead feeder rat was placed in each of the gravesites upon 8 cm of soil. An additional 8 cm of soil was placed on half of these gravesites. (b) The compartments (gravesites) after sealing with interference-fit lids are shown. Each compartment had a septum port for headspace collection and an eye bolt to aid in removal of the lids at the end of the study.
[(Fig._4)TD$IG]
[(Fig._5)TD$IG]
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Fig. 4. The daily high and low temperatures for the rat decomposition study are presented. Vertical bars are placed on the graph to indicate the headspace collection days. were prepared and used to make four concentrations of nitrogen in solution. The standards were run along with each sample set to calibrate the diode array spectrophotometer response. Values were reported as the total mass of NRN collected, corrected for the volume of 2 M KCl used to elute the PLOT column, and the volumes of both the ninhydrin reagent solution and the 50% ethanol solution added to each sample. The sources of uncertainty in evaluating the recovered mass of trace components from the HS are 2-fold; there is uncertainty in the spectrophotometer response, and in the calibration. The overall uncertainty (with a coverage factor k = 2), associated with the recovered mass for trace compounds, was determined to be 3% at the low concentrations encountered in this work.
3. Results NRN was not detected in the headspace above the gravesoil for the first month of sampling (weeks 1–4). This result was surprising, because other researchers have observed increases in NRN in the soil within the first month, although the decomposition rate is highly dependent on the size and the location of the cadaver, the season and the temperature [10,12,14,23]. Most likely the cool May/June weather in Colorado (see Fig. 4) delayed decomposition and the subsequent release and accumulation of NRN into the surrounding soil. Additionally, the rats were frozen when placed in the compartments, and the compartments in this study were sealed off from the environment (insects, moisture, and sunlight). In the previous studies that found indications of cadaver decomposition within the first month, the cadavers were buried or laid upon the ground outside, and thus, exposed to the elements, insects and scavengers [10]. Also, since most NRN compounds have very low vapor pressure, the concentration of NRN compounds in the headspace air above the soil is much lower than that in the soil, and thus, a larger period is required to establish a concentration of NRN in the HS air above gravesoil that can be detected with this method. It would be advantageous to establish the limit of detection of the headspace collection method used here and to determine the equilibrium constant for NRN in the soil and the vapor phase in future studies. Fig. 5 shows the recovered masses of ninhydrin-reactive nitrogen that were detected in the compartments/gravesites at weeks 5, 6, and 10. NRN was collected in trace quantities (total micrograms of nitrogen) from the HS above gravesoil in each compartment with decomposing rats (gravesites 1–8) at these time points. Fig. 5 also shows that at 5 and 6 weeks collection time (Fig. 5a and b, respectively), the lowest recovered masses of NRN were collected from the HS above the blank graves (plots 9–12). This result is also shown in Table 1, which presents the average recovered NRN masses for each plot grouping (rats laid on the soil, buried rats, and blank graves). Table 1 clearly shows that the
Fig. 5. The recovered masses of NRN collected from the headspace above the gravesoil in compartments 1–12 after 5 (a), 6 (b), and 10 (c) weeks of rat decomposition. Compartments 1–4 contained exposed rats, 5–8 contained buried rats, and 9–12 were blank graves. Gravesites 4 and 6 initially contained two frozen, dead feeder rats.
lowest average recovered masses for NRN were collected from the blank graves at all time points (5, 6, and 10 weeks) and that the difference in the values obtained from gravesites versus blank graves decreases throughout this study. This result is also Table 1 The average recovered masses for each group of rats (exposed, buried) and the blank graves at 5, 6, 10, and 20 weeks headspace collection. Compartments 1–4 are the exposed rats, compartments 5–8 are the buried rats, and compartments 9–12 are the blank gravesites. The values for 20 weeks are in italics because they were obtained using a significantly shorter headspace sampling period. The uncertainty is discussed in the text. Average recovered masses of NRN, mg
Exposed Buried Blank
5 weeks
6 weeks
10 weeks
20 weeks
1.4 1.8 0.1
1.0 1.0 0.2
0.4 0.6 0.2
0.5 0.4 0.1
[(Fig._6)TD$IG]
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Fig. 6. The recovered masses of NRN collected from the headspace above the gravesoil in compartments 1–12 after 20 weeks of rat decomposition to illustrate the yes/no response of this method with only 5 min headspace sampling period. Compartments 1–4 contained exposed rats, 5–8 contained buried rats, and 9–12 were blank graves. Gravesites 4 and 6 initially contained two frozen, dead feeder rats.
illustrated in Fig. 5. Thus, a clear baseline value for ambient NRN in the HS above gravesoil is possible at 5 and 6 weeks of rat decomposition, while, at longer times, more uncertainty in the baseline NRN measurement is observed because the later stages of rat decomposition are occurring and NRN release into the soil has decreased significantly. Our observations are consistent with observations reported previously by Van Belle et al. [10] on the changes in the soil NRN concentrations during swine decomposition. These researchers observed a rapid decrease in the soil concentrations of NRN after 2 months decomposition time and the gravesoil NRN levels were equivalent to that of the blank graves by 5 months decomposition time. We also observed that at 5 and 10 weeks the average recovered masses of NRN is greater for the buried rats than for the rats placed on the surface of the soil. At 6 weeks HS collection times, the average recovered masses of NRN were equivalent for these two rat groups. In a previous study by Van Belle et al. [10], cadavers laid on the soil surface decomposed and released NRN into the surrounding soil more rapidly than buried cadavers. This study was conducted outside, and thus the accelerated decomposition rate of the cadavers placed on the soil surface could be due to environmental elements including, wind, rain, insects, scavengers, etc. In the current study, we sealed the gravesites off from the environment; thus, external cadaver decomposition was aided only by the microbes and insects initially present in the soil used. We hypothesize that more NRN was collected above the gravesoil of the buried rats because these cadavers were in direct contact with the soil on all surfaces, and thus, there was more contact with bacteria present in the soil. Different HS sampling periods were also investigated. As mentioned previously, the concentration of NRN in gravesoil is typically in the ppm (mass/mass) range. These compounds, with the exception of ammonia, have extremely low vapor pressures, and thus would be present in the HS above the gravesoil also in trace amounts, especially at ambient temperatures. HS collection periods of 2 h were chosen for this study based on our experience collecting trace compounds of low volatility at ambient temperature in previous work [47]. Fig. 5 shows that this headspace sampling period is adequate for quantitative detection of trace amounts of NRN in the HS above gravesoil. However, in practice, one is typically concerned only with determining whether or not the level of NRN exceeds the baseline level (that is, a go/no-go result), not necessarily the maximum amount of NRN present. Fig. 6 shows the amounts of NRN collected at week 20 from each
gravesites 1–12 with only a 5 min HS sampling period. It is interesting to note that some of the masses recovered at 20 weeks are comparable or higher than the masses collected at 10 weeks. This result shows that beyond 5 min headspace collection period, there is no advantage to increasing the collection period. In fact, it may be a disadvantage to use longer collection periods if NRN is potentially displaced from the PLOT column. Additionally, it is possible that the lower ambient temperature at week 20 improved the collection efficiency of the PLOT column. In the laboratory (where we have control over the headspace collection temperature) we typically use adsorber temperatures of approximately 0 8C. What is important to point out here is that after only 5 min, an NRN level above the baseline level (average amount detected in the blank graves, 0.06 mg of nitrogen) was collected in 6 of 8 of the gravesites. Thus, the headspace sampling period is not an important variable beyond 5 min; this is a promising result for the anticipated development of a field-ready, trace-vapor NRN detection method to aid investigators locate clandestine graves. Work on this aspect is ongoing. Interestingly, a significant difference in the amount of NRN collected and cadaver mass when comparing the gravesites with one versus two rats was not observed. We have already shown that increasing the headspace sampling period does not necessarily increase the amount of NRN collected from the headspace by use of this methodology. Thus, it is possible that beyond a minimum amount of cadaver decomposition, additional cadaver mass may not change the amount of NRN collected from the headspace above the gravesoil. Once again, for our purpose, it is more important to detect NRN levels above baseline values than to detect a specific amount. The influence of the environment and the burial conditions on the NRN released into the gravesoil has been shown in numerous papers. The impact of these conditions on the quantity and release of NRN into the headspace above gravesoil is an important aspect of this work to investigate further to help establish a fundamental understanding of this promising detection method. 4. Conclusions A method for the ambient temperature headspace collection of NRN above gravesoil, using a motorized pipetter and an activated alumina-coated PLOT column has been developed. The method is based on an improved purge and trap method for sampling compounds of low volatility, as well as volatile compounds at trace quantities, by applying low temperature collection on short alumina-coated PLOT columns. Gravesoil was generated with frozen, dead feeder rats and local soil. Half of the rats were placed on the surface of the soil, and half of the rats were buried. NRN was detected with only 5 min headspace collection period. The lowest average recovered masses for NRN were collected from the blank graves. Additionally, we observed a decrease in the average amount of NRN collected in each group (exposed, buried, and blank graves) throughout the study. We observed a slightly elevated recovered mass of NRN for buried rats compared with that recovered from rats laid upon the soil. No correlation of initial cadaver mass on NRN collected in the headspace above the gravesoil was observed. We have demonstrated that it is possible to detect trace quantities of NRN in the headspace above gravesoil, adding a unique method to aid law enforcement personnel and criminal investigators detect clandestine graves. Acknowledgments TML acknowledges a Professional Research Experiences Program and a National Academy of Sciences/National Research Council postdoctoral fellowship, both at NIST.
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