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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
Abstract
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).
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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Figure 1. Ion chromatography of an extract from a KClO3/S/sugar
pipe bomb fragment and an anion standard mixture for comparison.
Plates: (A; top) Standard:
(1) Cl, (2) NO2,
(3) ClO3, (4) NO3,
(5) SO42, (6) SCN,
and (7) ClO4;
(B; bottom) KClO3/S/sugar extract: (1) Cl,
(2) ClO3, (3) SO42
and (4) Unknown. Click for enlarged images: A and B.
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Adulteration
of Foodstuffs
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 methodnamely borate, chromate,
and dichromateare 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.
As seen in Figure 2, this
method works well for the separation of the following anions:
thiosulfate (S2O32), bromide
(Br), chromate (CrO42),
iodide (I), chloride (Cl),
sulfite (SO32), nitrite (NO2),
nitrate (NO3), oxalate (C2O42),
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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Figure 2. Capillary electrophoresis analysis of a 10
ppm standard mixture containing 14 anions (upper trace at 250
nm; lower trace at 200 nm; y-scale in mV). Peak assignments:
(1) S2O32, (2) Br,
(3) CrO42, (4) I,
(5) Cl, (6) SO32,
(7) NO2, (8) NO3,
(9) C2O42, (10) N3,
(11) SCN, (12) ClO3,
(13) F, and (14) HCO2.
Click for an enlarged
image.
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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
(Cr2O72) 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).
Cr2O72
+ H2O ® 2 H+ + 2 CrO42 (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 (AsO43) 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 (AsO33, 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
S2O32 |
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 & CrO42 50 ppm |
Table sugar 5000 ppm |
SO32 10
ppm |
Red grape extract |
NO2 10
ppm |
Tropical punch drink |
CrO42 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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Figure 3. Capillary electrophoresis analysis of black
tea with sugar (diluted 20×) and spiked with less than
10 ppm of sodium azide (trace at 250 nm; y-scale in mV).
Peak assignments: (1) Cl, (2) SO42,
(3) C2O42, (4) N3,
and (U) Unknown. Click
for an enlarged image.
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Conclusion
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.
References
Doyle, J. M. and McCord,
B. R. Novel electrolyte for the analysis of cations in low explosive
residue by capillary electrophoresis, Journal of Chromatography
B (1998) 714:105111.
Doyle, J. M., Miller, M.
L., McCord, B. R., McCollam, D. A., and Mushrush, G. W. A multicomponent
mobile phase for ion chromatography applied to the separation
of anions from the residue of low explosives, Analytical Chemistry
(2000) 72:23022307.
FBI Bomb Data Center. FBI
Bomb Data Center Report: 1997 Bombing Incidents [Online].
(September 2000). Available: http://www.fbi.gov/programs/lab/org/1997bombrep.pdf
Fritz, J. S. and Schenk,
G. H. Quantitative Analytical Chemistry. 2nd ed., Allyn
and Bacon, Boston, 1969, p. 82.
Harrold, M. P., Wojtusik,
M. J., Riviello, J., and Henson, P. Parameters influencing the
separation and detection of anions by capillary electrophoresis,
Journal of Chromatography (1993) 640:463471.
Hortin, G. L., Dey, S. K.,
Hall, M. B., and Robinson, C. A. Detection of azide in forensic
samples by capillary electrophoresis, Journal of Forensic
Sciences (1999) 44:13101313.
Kishi, T., Nakamura, J.,
and Arai, H. Application of capillary electrophoresis for the
determination of inorganic ions in trace explosives and explosive
residues, Electrophoresis (1998) 19:35.
Krawczeniuk, A. S. and Bravenec,
V. A. Quantitative determination of cocaine in illicit powders
by free zone capillary electrophoresis, Journal of Forensic
Sciences (1998) 43:738743.
Lu, G. H. and Ralapati, S.
Application of high-performance capillary electrophoresis to
the quantitative analysis of nicotine and profiling of other
alkaloids in ATF-regulated tobacco products, Electrophoresis
(1998) 19:1926.
Mahan, B. H. University
Chemistry. 2nd ed., Addison-Wesley, Reading, Massachusetts,
1969, p. 650.
McCord, B. R., Hargadon,
K. A., Hall, K. E., and Burmeister, S. G. Forensic analysis of
explosives using ion chromatographic methods, Analytica Chimica
Acta (1994) 288:4356.
Moffat, A. C., ed. Clarke's
Isolation and Identification of Drugs. 2nd ed., Pharmaceutical
Press, London, 1986, pp. 57, 65.
Ruetter, D. J., Buechele,
R. C., and Rudolph, T. L. Ion chromatography in bombing investigations,
Analytical Chemistry (1983) 55:1468A1472A.
Smith, K. D., McCord, B.
R., MacCrehan, W. A., Mount, K., and Rowe, W. F. Detection of
smokeless powder residue on pipe bombs by micellar electrokinetic
capillary electrophoresis, Journal of Forensic Sciences
(1999) 44:789794.
Soga, T., Tajima, I., and
Heiger, D. N. Capillary electrophoresis for the determination
of forensic anions in adulterated foods and beverages, American
Laboratory (2000) 32(3):124128.
Tanaka, T., Hara, K., Tanimoto,
A., Kasai, K., Kita, T., Tanaka, N., and Takayasu, T. Determination
of arsenic in blood and stomach contents by inductively coupled
plasma/mass spectrometry (ICP/MS), Forensic Science International
(1996) 81:4350.
Walker, J. A., Marche, H.
L., Newby, N., and Bechtold, E. J. A free zone capillary electrophoresis
method for the quantitation of common illicit drug samples, Journal
of Forensic Sciences (1996) 41:824829.
Wildman, B. J., Jackson,
P. E., Jones, W. R., and Alden, P. G. Analysis of anion constituents
of urine by inorganic capillary electrophoresis, Journal of
Chromatography (1991) 546:459466.
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