Analysis of Anions by Capillary Electrophoresis and Ion Chromatography for Forensic Applications, by M. Miller et al. (Forensic Science Communications, April 2001)
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April 2001 - Volume 3 - Number 2 |
Research and Technology
Analysis of Anions by Capillary Electrophoresis and
Ion Chromatography for Forensic Applications
Mark L. Miller
Research Chemist
Janet M. Doyle
Chemist
Federal Bureau of Investigation
Forensic Science Research Unit
Quantico, Virginia
Rip A. Lee
Professor
John Jay College of Criminal Justice
New York, New York
Robert Gillette
Visiting Scientist
George Mason University
Fairfax, Virginia
Abstract | Introduction | Explosives | Adulteration of Foodstuffs | Conclusion | References
Anion analyses can provide important leads in the forensic investigation of bombings and poisonings. The determination of these negative ions by ion chromatography or capillary electrophoresis provides information on the chemical species of the anions. In addition, these techniques provide a means for the separation of complex mixtures. Separating mixtures and providing speciation are two major advantages of ion chromatography and capillary electrophoresis over elemental analysis. The forensic application of analytical methods for anions found in explosives and poisons is discussed, and relevant examples are presented in this article.
Introduction
Inorganic salts and acids may be components or residue of explosives and poisons. The identification and characterization of the anions from compounds are of major significance for forensic applications. The advancement of chromatographic techniques such as ion chromatography and capillary electrophoresis has led to the ability to quickly profile and quantitate anionic species. This information can be used in forensic science to determine what substances may have been used to commit crimes and to associate evidence from criminal acts to suspects. Ion analysis has become prominent in forensic casework involving explosives residue (Doyle and McCord 1998; Kishi et al. 1998; Ruetter et al. 1983; Smith et al. 1999), illicit drug samples (Krawczeniuk and Bravenec 1998; Walker et al. 1996), tobacco products (Lu and Ralapati 1998), and biological specimens (Hortin et al. 1999; Wildman et al. 1991). Analysis techniques for anions of forensic interest are discussed in this article, and relevant examples are provided. This article presents past and current work on anion analysis at the Forensic Science Research Unit of the FBI Laboratory in Quantico, Virginia, and is meant to demonstrate the potential of ion determinations to practicing forensic scientists. The current research methods portion describes work in progress and is not to be viewed as finalized or validated operating procedures.
Explosives
The analysis of anions found in explosives and explosives residue has been of particular interest with the increase in terrorist bombings in recent years. The bombings of the World Trade Center in New York City, New York, and the Federal Building in Oklahoma City, Oklahoma, deserve mention because ion analysis played a significant role in both investigations. The proliferation of information in the public domain and its destructive use are of major concern to law enforcement. The recipes for explosive materials are readily obtained from sites on the Internet or from the literature. Obtaining the materials necessary to make improvised explosives devices is also easy. As an example, fertilizers and fuel oils used to formulate an ammonium nitrate/fuel oil explosive are commonplace and may be purchased without causing any suspicion to the sellers.
The detection of explosives residue after a bombing may be possible because not all of the explosive material is consumed during an explosion. Additionally, characteristic by-products may be formed by the chemical reactions that occur during an explosion. Inorganic explosives, mixtures of strong oxidizers and fuels, are used in the majority of improvised explosive devices in the United States (FBI Bomb Data Center 1997). Inorganic salts of chlorate, nitrate, and perchlorate are frequently used as oxidizers in low explosives. Common fuels include sources of carbon (e.g., charcoal, sugar, hydrocarbons), aluminum, and sulfur. Examples of anions of interest in pre- and post-blast materials include azide, chlorate, chloride, nitrate, nitrite, perchlorate, sulfate, and thiocyanate. However, samples containing low concentrations of these ions may be difficult to isolate and interpret in a massive pile of rubble after a bombing. The presence of high levels of the relevant anions, either alone or in combination, may be of significance when detected in post-blast residue. Bombs composed of an ammonium nitrate/fuel oil mixture or potassium nitrate/sulfur/sugar will both leave residue of nitrate, but the latter may also contain sulfate and thiocyanate as post-blast reaction products. Either chlorate or perchlorate is the major component in flash powders, which may be combined with sulfur and aluminum. Post-blast residue of flash powders may consist of chlorate or perchlorate ions in addition to chloride and sulfate.
The use of capillary electrophoresis has become more widespread in forensic science because of its micro-sampling capabilities, simple detection methods, and ease of sample preparation. The advantages of capillary electrophoresis in the analysis of anions in explosives residue include the following:
- Micro-sampling: Sample requirements are low so multiple analyses can be made with 50 nL injections from as little as 25 µL. This is very conducive to forensic casework because the best evidence in post-blast residue often may be found on the smallest samples.
- Ease of extraction: The extraction medium is water, which makes the extraction process for most anion analyses a “dilute and shoot” operation.
- Automation: As many as 20 to 60 samples may be run in sequence depending on the type and manufacturer of the capillary electrophoresis instrument used. This allows for greater throughput and flexibility because samples may be loaded into the instrument before an analyst leaves for the night. The results, which are waiting for interpretation, are available the following day.
- Elimination of gradients: The separation of solutes undergoing capillary electrophoresis is based on differences in the charge-to-size ratio (of the solvated ions) under the influence of an applied electric field. This mode of separation eliminates the need for gradients because it relies on migration, not elution (liquid chromatography).
- Fast: Most separations are performed at 15 to 30 kV. This allows for very fast separations. The anion separation described later in this article is performed in less than 16 minutes.
One procedure currently used in the FBI Laboratory for the detection of anions in post-blast residue is capillary electrophoresis analysis as described by McCord et al. (1994). The method employs a 70-cm x 75-µm I.D. fused silica capillary and a mobile phase consisting of 1.8 mM dichromate, 40 mM boric acid, and 2 mM borate. Adjustment of the pH to 7.65 is accomplished using diethylenetriamine. Aqueous extract samples are introduced into the capillary by hydrodynamic injection for 2 seconds. Electrophoretic separation of the anions takes place at –20 kV for approximately 16 minutes. Dual-wavelength ultraviolet detection is performed at 205 nm and 280 nm. Although all of the anions of interest are detected at the 280-nm wavelength, the nitrogen-containing anions are readily identified at 205 nm. Details and applications of this method are given in McCord et al. (1994).
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A complementary method for the detection of anions in explosives residue employed in the FBI Laboratory uses ion chromatography as described by Doyle et al. (2000). This method involves the separation of anions through a Waters® Anion HR™ (Milford, Massachusetts) polymethacrylate resin column containing a quaternary ammonium functionality. The mobile phase is composed of 2.75 mM boric acid, 0.37 mM D-gluconic acid, 1.05 mM lithium hydroxide, 1.25 mM glycerol, 5.5 mM octanesulfonic acid, 5 percent acetonitrile, and 0.6 mM tetrapropylammonium hydroxide, at pH 8.5. Sample load volume is 20 µL. Isocratic separations are performed at a flow rate of 1 mL/min. The chromatographic signal is collected using nonsuppressed conductivity detection. An example of the separation of anions in the extract from a post-blast test is illustrated in conjunction with an anion standard chromatogram in Figure 1. The observed residue of chloride, chlorate, and sulfate is expected from exploded improvised explosive devices such as potassium chlorate/sulfur/sugar pipe bombs. Other anions, which may be present in potassium chlorate/sulfur/sugar post-blast residue, include bicarbonate and hydrogen sulfide. One of the best features of this ion chromatography method is the ease of interpretation of the results due to the stable baseline. |
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One of the current research topics in the FBI Laboratory centers on developing methods for the detection of poisonous anions in foodstuffs. Specific analytical methods exist for well-known poisons such as cyanide, and elemental techniques can be used for metallic anions such as arsenate (Moffat 1986; Tanaka et al. 1996). However, it is desirable to have a general screening method for anions to reduce the number of tests required for unknowns and to detect small ions that are difficult to distinguish in a complex matrix. Therefore, the advantages of capillary electrophoresis, previously mentioned in this article, are being investigated for poisonous anions. The capillary electrophoresis method used for the analysis of anions in explosives residue cannot be used to determine the presence of some important poisonous anions in food because three components used in the mobile phase of the explosives residue method—namely borate, chromate, and dichromate—are anions of interest in food poisoning.
The capillary electrophoresis method recently under examination for the separation of poisonous anions in food consists of a 1.6-mM triethanolamine buffer (pH 7.7) containing 0.75 mM hexamethonium hydroxide and 2.25 mM pyromellitic acid (Harrold et al. 1993). The hexamethonium hydroxide is an electroosmotic flow modifier used to reverse the direction of flow (Harrold et al. 1993). The pyromellitic acid (1,2,4,5-benzenetetracarboxylic acid) is used as a visualization agent and carrier ion. Initial analysis of a panel of anions was conducted using a 57-cm x 75-µm I.D. fused silica capillary. Hydrodynamic injections of the ion standard and samples were performed for 1 second, and separation was accomplished at –30 kV for 12 minutes. The anions were observed using indirect ultraviolet detection at 250 nm. Wavelengths of 200 and 300 nm were also monitored.
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As seen in Figure 2, this method works well for the separation of the following anions: thiosulfate (S2O3–2), bromide (Br–), chromate (CrO4–2), iodide (I–), chloride (Cl–), sulfite (SO3–2), nitrite (NO2–), nitrate (NO3–), oxalate (C2O4–2), azide (N3–), thiocyanate (SCN–), chlorate (ClO3–), fluoride (F–), and formate (HCO2–). The chromate, iodide, and chloride peaks were found to coelute. However, the use of other wavelengths can spectrally resolve these three ions. The bromide, iodide, and nitrate peaks give negative deflections (positive absorbance) at 200 nm, which makes them easy to distinguish (Figure 2). Likewise, the chromate ion appears as a negative peak at 300 nm, but iodide and chloride do not. Thus, the elution order can be established as chromate, iodide, and chloride. Some difficulties in the determination of anions were noted during trials of the pyromellitic buffer system. For example, the analysis of mixtures of sulfite and sulfate revealed the coelution of these ions. Interference was observed when fluoride and bromate were at comparable concentrations (10 ppm). The ion migration time of bromate (BrO3–) is 0.2 minutes behind fluoride, but because of the tailing peak shape of fluoride, bromate is not resolved from it. |
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Chemical properties of some anions also present challenges to their analysis. For example, reactive ions such as hypochlorite and dichromate cannot be readily detected because their conversion to other species in solution is pH dependent. Additionally, the pKa of HOCl is 7.7; therefore, it is only half ionized in the pyromellitic buffer. Analysis of hypochlorite (ClO–) from a bottle of bleach (pH 12.7) exhibited peaks for chloride and chlorate. In alkaline solutions, a disproportionation reaction occurs to hypochlorite resulting in the formation of chloride and chlorate (Mahan 1969).
3 ClO– ® ClO3– + 2 Cl– (Equation 1)
A standard of dichromate (Cr2O7–2) indicated predominantly chromate in the electropherogram, but a small peak at 6.5 minutes is suspected to be dichromate. It is converted to chromate in a reaction where the equilibrium is increasingly shifted to products, as the pH is raised (Fritz and Schenk 1969).
Cr2O7–2 + H2O ® 2 H+ + 2 CrO4–2 (Equation 2)
Thiosulfate was observed in fresh solutions by capillary electrophoresis but not in older aqueous standards (2 months). Attempts to analyze cyanide (CN–) and arsenate (AsO4–3) were unsuccessful. The pKa of hydrogen cyanide is 9.21, and therefore, it is un-ionized in the pH 7.7 buffer. An injection of arsenate did not detect any peaks within a 25-minute run time. Arsenate is only partially ionized at pH 7.7 because the pKa’s of its acid form are 2.22, 6.98, and 11.53.
The use of a strongly alkaline system would be beneficial to the analysis of cyanide, arsenate, and arsenite (AsO3–3, pKa’s = 9.23, 12.13, and 13.4). A higher pH (12.1) capillary electrophoresis buffer system for these ions was effectively demonstrated by Soga et al. (2000) in a study on adulterated foods and beverages. A trial of the pH 12.1 Soga capillary electrophoresis buffer system resulted in short-lived capillary columns. Experiments were conducted with the pyromellitic system to determine if it could be used at a higher pH. Unstable baselines were observed during attempts to use the pyromellitic system adjusted to a pH of 10. However, the pyromellitic pH 7.7 buffer system was found to be stable and reproducible. Additionally, all of the samples in Table 1 were analyzed on a single capillary column.
Table 1. Beverages/Food Spiked at Detectable
Concentrations (Analysis Levels) of Adulteration
| Sample | Spike |
|---|---|
| Tap water | HNO3 & ClO3– 10 ppm |
| Sparkling wine | SCN– 5 ppm |
| Apple juice | F– 20 ppm |
| Cherry drink | HCl (drop pH 0.5) & 10 ppm S2O3–2 |
| Carbonated cola | HNO3 (drop pH 0.5) |
| Black tea | Br– 10 ppm |
| Black tea with sugar | N3– < 10 ppm & oxalate 10 ppm |
| Orange juice | ClO3– 10 ppm |
| Concorde grape juice | I– 10 ppm |
| Milk 1% fat | I– 10 ppm & CrO4–2 50 ppm |
| Table sugar 5000 ppm | SO3–2 10 ppm |
| Red grape extract | NO2– 10 ppm |
| Tropical punch drink | CrO4–2 10 ppm & formate 10 ppm |
A trial of the pyromellitic system was conducted to determine issues associated with the capillary electrophoresis analysis of anions in adulterated samples. The possibility of interference and interactions with samples was explored by spiking samples with low levels of toxicologically significant ions. The spiked levels were kept low as a measure of the performance of the method rather than as a representation of expected levels in forensic samples. Lethal levels would be much higher than the spiking experiments, but high concentrations of anions in toxic samples can be diluted serially with water until analytically useful data can be obtained. Capillary electrophoresis can be a quantitative technique, but response and linearity experiments for the pyromellitic system have not been conducted because it is intended for use as a screening tool.
Preliminary work with foodstuffs and beverages reveals that the pyromellitic pH 7.7 method can be adopted for routine screening of these anions in the part-per-million range (Table 1). However, trace analysis in the low parts-per-million range is not generally required for the detection of adulterated food and beverages as was found in actual cases in Japan (Soga et al. 2000). All of the liquids were prepared by diluting the sample twenty-fold with water. The milk sample was subjected to a 3,000 molecular weight cutoff centrifugal filtration (Corning Costar®, Spin-X® UF, Acton, Massachusetts) and then diluted one hundred-fold. The sugar sample was made at a concentration of 5,000 ppm. The stated levels of spiked substances are for the prepared analytical samples, not the prediluted liquids.
A pH comparison of a questioned beverage with an authentic item can reveal the type of adulteration and provides useful information in conjunction with capillary electrophoresis analysis. Although high concentrations of contaminates would be expected in adulteration cases, the tampering of a beverage with even small amounts of a corrosive or caustic substance will have a measurable effect on the pH and be detectable by capillary electrophoresis. Experiments were performed using small samples of acid to demonstrate the potential of this approach. For example, the cherry drink and carbonated cola (Table 1) had initial pH’s of less than three before they were spiked with 10 µL of concentrated hydrochloric and nitric acid. The pH of these samples dropped by 0.5 unit after the acid addition, which shows the pH of acidic beverages is still sensitive to small amounts of acid. Capillary electrophoresis analysis of the beverages showed elevated levels of chloride or nitrate. These combined results would indicate an unknown had been adulterated with hydrochloric or nitric acid. If an adulteration was a mischievous prank and salt like sodium chloride was used, then high levels of chloride would also be detectable but the pH would not change significantly.
| Azide adulteration is a challenge to detect because azide is short-lived and decomposes to nitrogen gas. An additional consideration is that the acid form of azide is volatile. It is isoelectronic with cyanide and disrupts oxidative metabolism in a similar manner. The pyromellitic capillary electrophoresis method is able to determine azide in a beverage. Figure 3 is one example of an electropherogram from a sample of black tea with sugar that was adulterated with azide (< 10 ppm). The experiment with azide spiked in tea was performed two times on separate days with the same positive results. The reactivity and volatility of azide make it difficult to quantitate, so the intensity may not be representative of the amount added. Although quantitative aspects have not been explored at this time, it was observed that older standards gave lower responses for azide than fresh solutions. The other peaks observed in the tea correspond to the migration times for chloride, sulfate, and oxalate. |
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The analytical characterization of anions in forensic science is important for the determination of explosives and poisons because these substances are often water-soluble inorganic chemicals. Simple aqueous extractions of complex matrices associated with forensic evidence such as bomb residue or food and beverages can be separated by ion chromatography or capillary electrophoresis. Water can be employed to wash or extract materials containing inorganic salts and small organic acids. Many inorganic bomb residues are distinguishable by the combination of ions present in their anion profile from ion chromatography or capillary electrophoresis. The determination of adulteration of foodstuffs is facilitated by comparison of suspect materials with reference substances. Although these separation methods cannot identify an anion individually, supportive information can be gathered by sample spiking experiments using anion standards. Subsequently, separate ion analysis systems or appropriate instrumental techniques such as X-ray crystallography or elemental analysis can be used. Ion chromatography and capillary electrophoresis for the analytical separation of anions in evidence are valuable complementary aids to other forensic methods for establishing investigative leads.
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