Final Report Daad13 02 C 0015 Part5 App L P

CRC Book Chapter 5 Draft
Appendix L

Surface-enhanced Raman detection of chemical agents in water
Steven Christesen, Kevin Spencer, Stuart Farquharson,
Frank Inscore, Kristina Gonser, and Jason Guicheteau

I. INTRODUCTION
In 1994 and 1995, sarin gas was released in the Japanese cities of Matsumoto and Tokyo,1 respectively by
members of the AUM Shinrikyo religious cult. Later in 1995, the same group attempted to poison commuters in
Tokyo with cyanide.1,2 Fortunately, the cyanide producing devices were discovered before they could be used.
Although unsophisticated by military standards, the two sarin attacks resulted in 19 deaths and almost 6000 people
injured. More than 30 deliberate chemical releases were reported in Japan in 1998 alone causing more deaths and
injuries.
The agent used in the Tokyo and Matsumoto attacks (sarin or GB) is one of a class of toxic organophosphorus
nerve agents that include soman (GD), tabun (GA), cyclo-sarin GF and VX (no common name). These nerve agents
are particularly toxic and have LD50’s ranging from 24 milligrams per kilogram of body weight for sarin to 0.07
mg/kg for VX, where the LD50 is defined as the lethal dose of a liquid agent causing death in 50% of a given
population via percutaneous liquid exposure on bare skin.3 Organophosphorus nerve agents are toxic because they
bind to acetylcholinesterase enzymes (AChE) thereby inactivating them and allowing the neurotransmitter
acetylcholine to accumulate at synapses. Symptoms of nerve agent poisoning include twitching, pinpointed pupils,
convulsions, coma, and eventually death if the level of exposure is high enough.
The threat of terrorism within the United States became a reality in 2001 with the attacks on the Pentagon
building and the World Trade Center towers. The mailing of anthrax, shortly thereafter, and the salmonella
poisonings of salad bars in Oregon by the followers of Bhagwan Shree Rajneesh are just two examples of biological
terrorism in the US. The variety of terrorist attacks and the widespread use of GA, GB, and GF in the Iraq/Iran war
suggest that chemical warfare agents must be considered as a potential weapon against the US. Although the
majority of these incidents have involved vapor or aerosol dispersal of the agent, the threat to water supplies is
obvious and techniques for quickly and accurately identifying the threat are needed.
The Army’s water quality standards for allowable chemical agent contamination levels in drinking water are
published in the Department of the Army’s Technical Bulletin TB MED 577 Sanitary Control and Surveillance of
Field Water Supplies.4 This document is in the process of being updated to include new standards for chemical agent
contamination. The new standards are expected to conform generally to the recommendations of the National
Academy of Sciences (NAS) Subcommittee on Guidelines for Military Field Drinking-Water Quality,5 which are
shown in Table 1. In the case of nerve agents, these limits are based on a modeled 25% inhibition of AChE, and
represent a “no observed adverse effect level” (NOAEL).
Another class of chemical agents is the vesicants that include sulfur mustard (HD), three variations of nitrogen
mustard (HN-1, HN-2, and HN-3), and lewisite (L). The ingestion of low concentrations of HD in water is expected
to result in gastrointestinal irritation. Based on toxicity studies on rats, the military guideline limits were set at 47
µg/L and 140 µg/L for water consumption rates of 15 and 5 liters/day, respectively. Although used in World War I,
and by the Aum Shinrikyo terroists, hydrogen cyanide is not considered a militarily significant agent due to its high
volatility and rapid detoxification by humans. Environmental cyanide contamination of water, however, can occur
as the result of industrial processes such as electroplating and metal polishing. Cyanide is not nearly as toxic as the
nerve agents or mustard, but its ready availability lends itself to possible use by terrorists to poison water supplies.
The military’s M272 water testing kit, first fielded in 1984, is currently used to detect and identify chemical
agents in treated and source water. Agents are detected via color changing reactions with sensitivities of 0.02 mg/L
for nerve agents, 2 mg/L for mustard and lewisite, and 20 mg/L for cyanide.6 In general, the M272 is very sensitive
and meets the current requirements listed in TB MED 577, but it is not sufficiently sensitive to ensure that water
meets the recommended water quality standards shown in Table 1. In addition, the vials and chemicals used in the
kit are not easily manipulated when wearing a protective suit, mask, and gloves.
In an effort to meet military and national security needs for detecting chemical agents in water,
sophisticated laboratory methods have been investigated with reasonable success. More than a decade ago, Black et
al. demonstrated the ability of combining gas chromatography with mass spectrometry detection (GC/MS) to
measure sarin and mustard.7 Sega at al. used GC with a phosphorous-selective flame ionization detector to analyze
nerve agent hydrolysis products in groundwater,8 while several researchers used capillary electrophoresis (CE) to
measure chemical warfare agents and their hydrolysis products.9, 10, 11 The sensitivity of these techniques has
improved by two orders of magnitude from 1 mg/L to 0.01 mg/L in 10 years. A comprehensive development of

1


these techniques was undertaken by Creasy et al. in analyzing chemical weapon decontamination waste from the
Johnston Atoll.12,13 These researchers used GC/MS for nerve agents, GC coupled atomic emission detection for
arsenic compounds, LC/MS for mustard compounds, and CE with ultraviolet absorption detection for alkyl
phosphonic acids. Detection limits of 0.02 and 0.140 mg/L were reported for nerve agents and mustard,
respectively. Detection of the alkyl phosphonic acids have proven more difficult, and Liu, Hu and Xie recently used
GC/MS to detect mg/L concentrations of these degradation products.14 However, they concede that all of these
separation methods require extraction, derivatization, and repeated column calibration, making them labor intensive,
time consuming (typically 30 to 60 minutes), and less than desirable for field use. Another variant of these
separation/mass detection technologies is ion mobility spectrometry (IMS).15 This technology has been successfully
developed to measure explosives in air samples, and commercial products can be found at most airports.16 Eiceman
et al. have investigated the ability of IMS to measure organophosphorous compounds in air,17 while Steiner et al.
have investigated IMS to measure chemical agent simulants in water.18 In the latter case, electrospray ionization
was coupled to the sample entry point of an IMS, and a time-of-flight MS was added as an orthogonal detector.
Water samples spiked with 10 mg/L diisopropylmethylphosponate and thiodiglycol could be measured in 1-min,
once sample pretreatment was accomplished. It is worth noting that with proper care these MS-based technologies
are likely to detect chemical agents with virtually no false-positives, but detection limits are still insufficient by 1 to
2 orders of magnitude in the case of nerve agents and their hydrolysis products.
Raman spectroscopy, using both NIR19 and UV20 laser excitation, has found military application for the
detection and identification of chemical agents. The highly selective nature of the Raman spectrum makes this
technology ideal for non-intrusive and/or short range remote detection of highly concentrated samples. But the trace
level detection sensitivity required for chemical agents in water is beyond the capabilities of normal Raman
spectroscopy. Improved sensitivity, however, can be achieved with surface-enhanced Raman spectroscopy (SERS).
Work by Vo-Dinh on the SERS detection of organophosphorus insecticides21 and organophosphonate vapors22 using
silver coated spheres and oxidized silver foils, respectively, demonstrated the potential efficacy of SERS for nerve
agent detection. More recently, two different SERS substrates, one produced by electrochemical roughening of
silver or gold foils23 and one produced by gold- or silver-doped sol-gels24,25 were used to measure the primary
hydrolysis of nerve agents, methyl phosphonic acid, with detection limits of 50 to 100 µg/L, respectively. In order
to develop a SERS sensor for detecting chemical agents in water, however, sensitivity and reproducibility need to be
demonstrated for actual chemical agents in all classes. To this end, results on the detection of VX, HD, CN, and
their hydrolysis products using SERS these substrates are presented.

II. EXPERIMENTAL

A. SERS Substrates
1. Electrochemically Roughened Silver Foils
The electrochemically roughened silver substrate foils (EIC Laboratories, Inc., Norwood, MA) were prepared as
described in Reference 26 from silver foil coupons (Surepure) cut to a size of ¼” x ½” on a dedicated jeweler’s saw.
The cut edges and faces were smoothed to mirror flatness using 0.3 µm Al2O3 polish then washed and stored in 0.1
M KOH solution prior to electrochemical roughening in 0.1 M KCl using a platinum gauze counter electrode.
Roughening was accomplished by cycling 20 times at a sweep rate of 10 mV/s with upper and lower limits of 0.25
and -0.6V versus silver/silver chloride, respectively. The silver substrate foils were electrochemically cleaned at
cathodic potential to remove chemical impurities and then soaked overnight in water to remove excess chloride ion.
These foils were then electrochemically cycled in sodium hydroxide to create a thick hydroxide layer, which
stabilizes the silver chemistry and reduces its propensity for oxidation. The gold and silver foils were shipped to US
Army Chemical Biological Center (ECBC) for agent testing and generally used within a week of manufacture. All
measurements were made by either dipping the foil directly into the solution or by spotting the foil with less than 10
µL of the analyte in water. In both cases, the sample was allowed to dry on the substrate foil before recording the
SER spectrum.

2. Silver- or Gold-Doped Sol-Gels
The sol-gel coated vials were prepared by Real-Time Analyzers, Inc. (RTA, Middletown, CT) using procedures
previously published by Farquharson et al.27 Silver-doped sol-gels were formed by adding ammonium hydroxide to
a solution of silver nitrate, tetramethyl orthosilicate (TMOS), and methanol. Gold-doped sol-gels were coated by

2


adding nitric acid to a solution of gold tetrachloride, TMOS and methanol. The two precursor solutions were
prepared, mixed, and transferred to 2-ml glass vials, dried and heated. After sol-gel formation, the incorporated
metal ions were reduced with dilute sodium borohydride (1 mg/ml), followed by a water wash to remove residual
reducing agent. The sol-gel vials were produced at RTA and shipped to ECBC for testing. In all cases, the vials
were filled with the desired agent in solution, capped, and then the SER spectrum was recorded.

B. Sample Preparation
Stock solutions of HD, VX, and EA2192 were prepared at the ECBC by using Chemical Agent Analytical
Reference Material (CASARM)-Grade neat agent (95+ % purity). Cyanide solutions were made by dissolving KCN
(Aldrich) in DI water. For the measurements using the roughened metal foils, the samples were prepared by
dissolving the neat agent in distilled, deionized (DI) water at approximately 1 mg/ml and making serial,
volumetrically dilutions to achieve the lower concentrations. For the measurements using silver- and gold-doped
sol-gel coated vials the neat agents were dissolved in 2-propanol prior to the addition of DI water. In this case,
samples of several concentrations were prepared just prior to the start of each test. The percentage of 2-propanol
was kept constant throughout the volumetric dilution series. Typically, the HD and VX samples contained 1.1 %
and 2.0 % 2-propanol, respectively.

C. Instrumentation
Tests were preformed using two different Raman instruments. For the measurements with roughened silver
foils, a dispersive Raman spectrometer, comprised of a 785 nm diode laser (300 mW), an echelle spectrograph, and
a TE cooled CCD camera operating at approximately 45°C below ambient (model RS2000, InPhotonics, Norwood,
MA), was used to acquire 1 cm-1 resolution spectra.19 The laser was fiber optically coupled to a sample chamber
box using a RamanProbeTM (InPhotonics). The probe both conveyed the laser light to the sample and served as an
optical filtering device to remove background signals arising in the fiber optic cable.
For measurements using sol-gel coated vials, a Fourier transform Raman spectrometer (model IRA-785, Real-
Time Analyzers) equipped with a silicon photo-avalanche detector (Perkin Elmer model C30902S, Stamford, CT)
was used to acquire spectra at a resolution of 8 cm-1. A 785 nm diode laser (model 785-600, Process Instruments,
Salt Lake City, UT) delivered approximately100 mW of power to the sample through a 30 foot fiber optic cable,
such that the instrument could be located outside the lab for added safety. The sample system consisted of an XY
positioning stage (Conix Research, Springfield, OR) on which the vials were mounted horizontally just inside the
focal point of an f/0.7 aspheric lens. This lens and the other optics within the fiber optic probe have been previously
described.27

III. RESULTS AND DISCUSSION

A. HD and TDG
Sulfur mustard (bis(2-chloroethyl) sulfide) hydrolyzes to form mustard chlorohydrin (2-chloroethyl 2-hydroxyethyl
sulfide), which then hydrolyses to thiodiglycol (TDG). These reactions progress through cyclic sulfonium ion
intermediates as shown in Figure 1. Although HD rapidly hydrolyzes to TDG (Table 2), the rate is limited by its
low solubility and solvation rate.28 HD hydrolysis approaches an SN1 mechanism via the pathway shown in Figure 1
only when predissolved in organic solvent and at low concentrations (less than 0.001 M or 160 mg/L).29 At the
interface of the HD droplet with water, the sulfonium ions can also interact with TDG to form stable sulfonium salts,
such as HD-TDG, CH-TDG, and H-2TDG (Figure 2). The presence of multiple stable HD conformers and the
formation of the sulfonium salts complicate the analysis of the SER spectra of sulfur mustard in treated water.
The SER spectra of HD in water are dominated by a broad peak with a maximum intensity at 620 cm-1, for both
electrochemically roughened silver foils and silver-doped sol-gel coated vials, and at 610 cm-1 for gold-doped sol-
gel vials. This broad peak aligns with the series of peaks observed between 600 and 800 cm-1 in the normal Raman
spectrum of HD and can be ascribed to the C-S and C-Cl stretching modes (Figure 3).30,31,32 At least two additional
peaks appear in the spectra at 1003 and 1292 cm-1 for the silver foils, and at 1007 and 1290 cm-1 for the gold-doped
sol-gels. Based on the Raman peaks at 1038 and 1292 cm-1, these peaks are assigned to a C-C stretching mode and a
CH2 bend.33
The shift in the C-C stretch is also observed in the C-S or C-Cl stretch. These shifts are consistent with those
reported in the literature by Joo et al.34 for diethyl sulfide (DES) on silver. They report an approximate 20 cm-1 shift
of the C-S stretching modes of the different conformers, and suggest that this is due to DES binding to the surface

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via the sulfur atom. The general shift here suggests that it is likely that HD also binds via the sulfur atom. The
silver foil spectra also contain a peak at 722 cm-1, which is absent or at least weak in both sol-gel spectra. There are
at least four possible explanations as to the source of this peak. 1) In the case of the silver foils, the samples were
measured dried, while the sol-gels were measured in solution, and the 722 cm-1 may represent a different conformer
on the surface. 2) The HD was dissolved in water for the foils, and in a combination water and organic solvent for
the sol-gel vials. The latter co-solvent should dramatically increase solvation, expedite hydrolysis and the removal
of the terminal chlorines. If so, then the 722 cm-1 peak may be assigned to a C-Cl stretching mode. This is
supported by the fact that thiodiglycol does not have this peak, at least under some measurement conditions. 3) The
722 cm-1 peak could be due to the formation of one of the sulfonium salts (Figure 2), but these salts are more likely
to contribute new peaks to the sol-gel spectra, not the foil spectra. 4) It was also noted that this peak was more
intense than the 620 cm-1 peak in some foil measurements, and it may alternatively be assigned to a photo-
degradation product. This is supported by the generation of a peak at the same frequency in photo-degraded TDG.35
Furthermore, at high laser powers and long exposures (several minutes) additional peaks (e.g. 668 cm-1) also
appeared in HD SER spectra using the gold-doped vials, supporting the assignment of the 722 cm-1 peak to photo-
degradation.
HD was consistently measured at approximately 50 mg/L using the silver foils (e.g. Figure 3C), and
occasionally at 100 mg/L for the silver-doped sol-gels. Both substrates provided repeatable measurements of 1 g/L
for HD (e.g. Figure 3B). However, the reproducibility of the SER signal intensity for HD was highly variable at 777
mg/L using the silver foils, but considerably better at 1000 mg/L using the silver-doped sol-gels.36 It is not clear,
however, how much of the variability in the former is the result of non-reproducibility in the substrate or the drying
process, or how much might be explained by the complex and changing mixture of mustard hydrolysis products.
The SER spectrum of thiodiglycol was only measured using silver-doped sol-gels. Initial measurements at 1000
mg/mL yielded a high quality spectrum that was consistent with SERS of HD and the Raman spectrum of TDG
(Figure 4). The SER spectrum is dominated by three peaks at 627, 715, and 1008 cm-1, while the Raman spectrum is
dominated by peaks at 652 and 1005 cm-1, which can be assigned to S-C and C-C stretching modes, respectively.
Although the 715 cm-1 peak could correspond to either of two weak Raman peaks at 680 and 728 cm-1, it was absent
in recent SER measurements of TDG in a flowing stream.35 Here, measurements at low laser powers (15 mW,
Figure 4B) confirm that this peak, as well as the 1008 cm-1 peak, are likely due to photo-degradation. This
conclusion also supports the assignment of the 722 cm-1 peak in the HD SER spectra to a TDG photo-degradation
product.

B. VX, EA2192, and EMPA
VX (ethyl S-2-diisopropylamino ethyl methylphosphonothioate) hydrolyzes in distilled water via the pathways
shown in Figure 537,38,39 with a half-life of greater than 3 days.40 The hydrolysis product EA2192 is itself highly
toxic and much more stable in water, although it will eventually hydrolyze to 2-(diisopropylamino) ethanethiol
(DIASH). The ethyl methylphosphonic acid (EMPA) product of Reaction Pathway 1 can also undergo further
hydrolysis to methylphosphonic acid (MPA). Both the silver foils and sol-gels produced SER spectra that had many
spectra features in common with each other and the normal Raman spectrum (Figure 6). In particular, at least three
overlapping peaks are seen in all three spectra between 425 and 575 cm-1. Farquharson et al. have assigned the
peaks at 460, 485, and ~530 cm-1 to a POn bending mode, an NC3 stretching mode, and a POnS bending mode,
respectively (Table 3).41 Neither substrate provided good sensitivity for VX. The sol-gels provided good
reproducibility at 1000 mg/L (Figure 6B), but only occasionally was a spectrum observed at 100 mg/L, whereas the
foils produced spectra at 100 mg/L on most attempts (Figure 6A). In the case of the latter, a large background
contribution above 800 cm-1, was removed by baseline correction.
EA2192 (ethyl S-2-diisopropylamino methylphosphonothioate) the primary hydrolysis product of VX following
Reaction Pathway 2 (Figure 5) produced very similar SER spectra using silver foils or silver-doped sol-gels (Figure
7). In fact, a high quality spectrum is obtained using the foils at 113 mg/L with peaks at 481, 584, 622, 700, 743,
780, 810, 830, 942, 974, 1040, 1120, 1366, 1442, and 1461 cm-1. Many of these peaks have been assigned
previously (Table 3).41 The SER spectra are however, considerably different than the Raman spectrum of EA2192
(Figure 7C). Most notably, the 1055 and 1185 cm-1 peaks, assigned to a PO2S stretch and NC stretch, in the Raman
spectrum are absent in the SER spectra. It is surprising that the PO2S stretch is not SER-active, and its assignment
may be in doubt. It is also worth noting that the most intense peak in the SER spectra at 942 cm-1, assigned to an
NC3 stretch, also dominated the SER spectrum of DIASH.41

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The silver foils provided reasonable sensitivity for EA2192 and spectra were obtained at a number of
concentrations to as low as 33 mg/L. The silver-doped sol-gels successfully measured 1000 mg/L, but no lower
concentrations were tried.
The SER spectra for EMPA are more interesting (Figure 8), in that both the sol-gel and foil spectra appear to
contain photo-degradation products. In the case of silver-doped sol-gels, a spectrum can be obtained that has many
of the same features as found in the normal Raman spectrum, as long as low laser powers are used (30 mW, Figure
8B). Peaks occur at 505, 730, 792, 893, 1047, 1098, 1293, 1420, and 1454 cm-1 in both spectra, which all have been
assigned.41 The primary difference between the two spectra is that a second intense peak occurs at 745 cm-1, albeit
the normal Raman spectrum contains a shoulder at close to this frequency. As soon as the laser power is increased
to 100 mW, this peak increases in intensity substantially, indicating photo-degradation (Figure 8A). This effect is
even more dramatic for the silver foils, where the this peak completely replaces the 730 cm-1 peak, especially at low
concentrations (100 µg/L, Figure 8D). Additional peaks also grow in at 610, 915, and 955 cm-1. It is reasonable to
assign the 745 cm-1 peak to the formation of methyl phosphonic acid, since it has a dominant peak at 755 cm-1, but
MPA does not contain the latter three peaks, and it is unclear as to composition of the degradation product. It is
clear from the data that EMPA degrades rapidly in the presence of silver and laser irradiation.

C. Cyanide
Hydrogen cyanide (AC) is a highly volatile liquid (boiling point of 25.7 °C) belonging to a class of chemical
agents known as blood agents. It has commercial uses in the extraction of gold from ore and in the manufacture of
other chemicals, such as acrylonitrile and methyl methacrylate. AC is highly soluble in water and hydrolyzes slowly
to ammonia and formic acid. Hydrogen cyanide and its potassium and sodium salts release toxic free cyanide (CN)
when dissolved in weak acids such as water. Cyanide’s toxicity results from its attack on the enzyme cytochrome
oxidase thereby preventing cell respiration and the normal transfer of oxygen from the blood to body tissues.
Research in the past focused on the gold mining and textile industries’ concerns over cyanide leakage into the
groundwater and the EPA’s limit of 1 part per million or less of CN in detoxified industrial waste. The majority of
these SERS techniques not only yield detection limits below the EPA mandate but well into the low part per billion
range. Detection of cyanide has also become somewhat of a standard assessment of a SERS substrate’s capability
and sensitivity. SERS applications utilizing gold nanostructures,42,43,44 silver electrodes,45,46,47,48,49 sol-gels,25,50 and
others51,52 have reported great success at detecting CN in a variety of forms. The success can be attributed to the
high binding efficiency of –(C≡N) to the metal surface resulting in a distinct band between 2120-2150 whose
position is dependent on pH and concentration.25,42
Both silver- and gold-doped sol-gel vials have been used to measure cyanide in water to 1 mg/L and below
(Figure 9A). In fact very reproducible measurements have been made at 10 mg/L. Both electrochemically
roughened silver and gold foils have successfully been used to measure low concentrations of cyanide, and in the
case of the latter, consistent measurements down to 20 µg/L. The SER signal strength is highly variable, however,
as seen for cyanide in water at 200 µg/L (Figure 9B). Data collected on the same day (but different gold foils) were
more consistent than measurements on different days, indicating that the substrates may be changing in storage or
shipment. This is illustrated by a fit of the data from the two days by a Langmuir adsorption isotherm (Figure 10).
This isotherm can be used to describe both physical and chemical adsorption:53

Kc
θ= (1)
Kc + 1

The cyanide concentration is given by c, the fractional surface coverage by θ , and the adsorption equilibrium
constant by K. The fractional coverage is calculated as the ratio of the SERS peak intensity divided by the intensity
at full surface coverage (I/Imax). In this analysis, both K and Imax were fit using a nonlinear regression technique
(DataFit, Oakdale Engineering, Oakdale, PA). The calculated adsorption equilibrium constants for the two days are
a factor of 10 different (0.4 L/mg vs. 0.04 L/mg).

IV. CONCLUSIONS

The potential sensitivity and selectivity of SERS coupled with the lack of strong water interference make it an
attractive technique for chemical detection in aqueous solution. Two very different SERS-active substrates,

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electrochemically roughened gold and silver foils, and gold- and silver-doped sol-gels both proved capable of
measuring sulfur mustard, VX, cyanide and several hydrolysis products of these chemical agents. However, only in
the case of cyanide was sensitivity sufficient for a SERS-based chemical agent water monitor. Substantial
improvements in sensitivity are required for the other agents. Furthermore, low laser powers are required to
minimize photo-degradation. Finally, the ability to manufacture substrates that yield reproducible results remains
elusive. Nevertheless, the detection limits for some of the phosphonic acid nerve agent hydrolysis products and
cyanide show promise. Efforts to improve sensitivity and reproducibility will continue to be pursued.

Table 1. Recommended Field Drinking Water Guidelines
CONSUMPTION RATE

Chemical Agent 5 L/day 15 L/day
Cyanide (µg/L) 6000 2000
Sulfur mustard (µg/L) 140.0 47.0
Nerve agents (µg/L)
GA 70 22.5
GB 13.8 4.6
GD 6.0 2.0
VX 7.5 2.5

Table 2. Hydrolysis half-lives and solubilities of chemical agents and their primary hydrolysis products.
Chemical Water Solubility (at
Hydrolysis ½ life
Agent 25°C)
Sarin (GB) 39 hr (pH 7) completely miscible
IMPA stable but can hydrolyze to MPA 4.8 g/L
MPA very stable >1000 g/L
VX >3 days (pH 7) 150 g/L
EA2192 > 10 x VX ∞ sol.
DIASH stable ca. 1000 g/L
EMPA >8 days 180 g/L
MPA very stable >1000 g/L
HD 5 min 0.648 g/L*
Mustard 3 min**
Chlorohydrin
TDG stable 6900 g/L
*Seidell A. 1941. Solubilities of organic compounds. A compilation of quantitative solubility data from the
periodical literature. Vol. 11, 3rd Edition. New York: D. Van Nostrand Company, Inc. 241-242.
**Ogston, A. G.; Holiday, E. R.; Philpot, J. St. L.; Stocken, L. A., Trans. Faraday Soc., 1948, 44, 45-52.

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Table 3. Tentative vibrational mode assignments for EA2192 and VX:41 Normal Raman, Sol-gel SER, and
Roughened Ag SER.
EA2192 VX
Sol-Gel Roughened Sol-Gel Roughened
NR NR Tentative Assignments
SER* Ag SER SER* Ag SER
386 387 372 376 SPO bend
418 413 CC or CN bend
453 456 461 458 441 POn bend
484 481 481 484 484 487 NC3 breathing
499 POn bend
513 526 523 528 539 532 POn(S) bend
587 584 586 NCn bend
645 623 667 622 PSC bend
CSH bend
693 700 696 682 CS stretch
732 735 743 744 731 735 PC stretch + backbone (CPOCC)
769 769 771 PC stretch and/or backbone
814 811 811 805 SC stretch + NC3 breathing
831 830 830 836 820
863 856 CH3 bend
905 891 883 891 885 889 OPC stretch / CCN stretch
925
947 939 943 931 939 940 NC3 stretch
966 971 975 965 POn stretch
1010 1006 1015 1006 1009 POn or CH3 bend
1043 1040 1029 1031 SCCN bend
1054 PO2(S) stretch
1100 1100 1101 1096 1098 OC or CC stretch
1132 1125 1128 1121 1125 NC stretch
1183 1183 1170 NC stretch
1219 1214 1220 NC stretch
1229 1228 1237 CH2 bend
1306 1300 1300 1301 CH3 bend
1329 1327 1329
1343 CN bend + CC bend
1366 1365 1369 1366
1399 1399 1394 1400 CH3 bend / NC3 stretch
1418 CH3 bend
1427 1423 1443 1439 1439 CH2 bend
1451 1446 CHn bend
1460 1464 1463 1462 1462 1461 CHn bend
1493

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+
S H2O S H2O S
Cl Cl Cl + Cl- Cl OH + HCl
I Mustard
HD Chlorohydrin
CH
+
S H2O S -
H2O S
Cl OH HO + Cl HO OH + HCl
II TDG
Figure 1. HD hydrolysis pathway.

S S
Cl S+ HO S+
HO OH HO OH

H-TDG CH-TDG
OH
S
S + S+
HO HO OH

H-2TDG
Figure 2. Sulfonium salts produced in reaction of I and II (Figure 1) with TDG.

8


Figure 3. SERS of HD using A) gold-doped sol-gel, B) silver-doped sol-gel, and C) electrochemically roughened
silver foil. D) Raman spectrum of HD. Conditions: A) and B) 1 g/L in isopropanol/water, 100 mW of 785 nm, 1-
min, C) 0.777 g/L in distilled water, 100 mW of 785 nm, 0.5-min, D) neat HD, 300 mW of 785 nm, 1-min.

Figure 4. SERS of TDG using silver-doped sol-gels using A) 100 and B) 15 mW of 785 nm excitation, and C)
Raman of neat TDG. Conditions: A) and B) 1 g/L, and C) 300 mW.

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OH
1 H N + O P O
S
34%-37%
DIASH EMPA
O
S O
P N H2O 2 S
P N
O + EtOH
42%-50% O
VX H
EA2192

3 S-
N
~10%
O P O + HO

O-ethyl 2-(diisopropylamino)
methylphosphonothioate ethanol
Figure 5. VX hydrolysis pathways.

Figure 6. SERS of VX using A) roughened silver foil and B) silver-doped sol-gel, and C) Raman spectrum of neat
VX. Conditions: A) 1 g/mL B) 129 mg/L.

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Figure 7. SERS of EA2192 using A) silver-doped sol-gel vial and B) roughened silver foil, and C) Raman spectrum
of solid EA2192. Conditions: A) 1 g/L, B) 113 mg/L. The substrate was dipped into the solution for 2 minutes prior
to collecting a 0.5 min spectrum.

Figure 8. SERS of EMPA in silver-doped-sol-gel vials using A) 100 and B) 30 mW of 785 nm excitiation. SERS of
EMPA on electrochemically roughened silver foils for D) 0.1 and E) 1 mg/L samples. Raman spectra of neat EMPA
in C) and E) for comparison. Conditions A) and B) 1 g/L, ...

11


Figure 9. SER spectra of sodium cyanide A) in three different silver-doped sol-gel coated vials at 1, 10, and 100
mg/L and B) on four different roughened gold foils all at 0.2 mg/L. Conditions: A) 100 mw 785 nm, 1-min, 8, B)
300 mw 785 nm, 0.5-min, 1. The top two traces were measured on the same day, as were the bottom two traces.

1.0

0.8

0.6

0.4

0.2

0.0

-0.2
1 10 100 1000 10000 100000 1000000
Concentration (µg/L)

Figure 10. Plot of surface coverage (θ) vs. concentration for CN on electrochemically roughened gold. The
diamonds and open circles are results from two different days, and each point represents data from a different
substrate. The solid line and dashed line are fits of the data to the Langmuir adsorption isotherm and have R2 values
of 0.971 and 0.944, respectively.

12


References

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Government Affairs Permanent Subcommittee on Investigations, October 31, 1995 Staff Statement,
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bin/getarticle.pl5?nn20000718a1.htm.
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Agents, Nat. Acad. Press (Washington, D.C.) 1997.
4 http://chppm-www.apgea.army.mil/documents/TBMEDS/TBMED577.pdf
5 Committee on Toxicology. Guidelines for Chemical Warfare Agents in Military Field Drinking Water, Nat.
Acad. Press (Washington, D.C.) 1995
6 McKone, T.E., Huey, B.M., Downing, E., and Duffy, L.M., Strategies to Protect the Health of Deployed U.S.
Forces: Detecting, Characterizing, and Documenting Exposures, Division of Military Science and Technology
and Board on Environmental Studies and Toxicology, National Research Council, NATIONAL ACADEMY
PRESS, Washington, D.C.,207, 2000. http://www.nap.edu/books/0309068754/html.
7
. R.M. Black, R.J. Clarke, R.W. Read, M.T. Reid: J. Chromat. 662, 301 (1994)
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. S.A. Oehrle, P.C. Bossle: J. Chromat. A 692, 247 (1995)
10
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12
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13
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18
. W.E. Steiner, B.H. Clowers, L.M. Matz,W.F. Siems, H.H. Hill Jr.: Anal. Chem. 74, 4343 (2002)
19 Christesen, S., MacIver, B., Procell, L., Sorrick, D., Carrabba, M., and Bello, J., Non-intrusive analysis of
chemical agent identification sets (CAIS) using a portable fiber-optic Raman spectrometer, Appl. Spectr., 53,
850, 1999.
20 Sedlacek III, A.J., Christesen, S.D., Chyba, T., and Ponsardin, P., Application of UV-Raman spectroscopy to
the detection of chemical and biological threats, in Proc. SPIE Vol. 5269, Sedlacek III, A.J, Christesen, S.D.,
Colton, R. and Vo-Dinh, T., Eds., SPIE, Bellingham, Washington, 2004, 23.
21 Alak, A.M. and Vo-Dinh, T., Surface-enhanced Raman spectrometry of organophosphorus chemical agents,
Anal. Chem., 59, 2149, 1987.
22 Taranenko, N., Alarie, J-P., Stokes, D.L., VoDinh, T., Surface-enhanced Raman detection of nerve agent
simulant (DMMP and DIMP) vapor on electrochemically prepared silver oxide substrates, J. Raman Spectrosc.,
27, 379, 1996.
23 Spencer, K., Sylvia, J., Clauson, S. Janni, J., Surface-enhanced Raman as a water monitor for warfare agents, in
Proc. SPIE Vol. 4577, Christesen, S.D. and Sedlacek III, A.J, Eds., SPIE, Bellingham, Washington, 2002, 158
24 Farquharson, S., P. Maksymiuk, K. Ong, and S. Christesen, Chemical agent identification by surface-enhanced
Raman spectroscopy, in Proc. SPIE Vol. 4577, Christesen, S.D. and Sedlacek III, A.J, Eds., SPIE, Bellingham,
Washington, 2002, 166.
25 Farquharson, S., Gift, A., Maksymiuk, P., Inscore, F. E., and Smith, W. W., pH dependence of methyl
phosphonic acid, dipicolinic acid, and cyanide by surface-enhanced Raman spectroscopy, in Proc. SPIE Vol.
5269, Sedlacek III, A.J, Christesen, S.D., Colton, R. and Vo-Dinh, T., Eds., SPIE, Bellingham, Washington,
2004, 117.
26 Spencer, K.M., Sylvia, J.M., Marren, P.J., Bertone, J.F., and Christesen, S.D., Surface-enhanced Raman
spectroscopy for homeland defense, in Proc. SPIE Vol. 5269, Sedlacek III, A.J, Christesen, S.D., Colton, R. and
Vo-Dinh, T., Eds., SPIE, Bellingham, Washington, 2004, 1.
27 Farquharson, S, Gift, A., Maksymiuk, P. and Inscore, F., Appl. Spectrosc. 58, 351 (2004).
28 Ogsten, A.G.; Holiday, E.R.; Philpot, J. St. L.; Stocken, L. A., The replacement reactions of b,b'-dichlorodiethyl
sulphide and of some analogues in aqueous solution: the isolation of b-chloro-b'-hydroxydiethyl disulphide,
Trans. Faraday Soc., 44, 45, 1948.

13


29 Yang, Y-C., Szafraniec, L.L, Beaudry, W.T., and Ward, J.R., Kinetics and mechanism of the hydrolysis of 2-
chloroethyl sulfides, J. Org. Chem., 53, 3293, 1988.
30 Sosa, C., Bartlett, R.J., KuBulat, K. and Person, W.B., A theoretical study of the harmonic vibrational
frequencies and infrared intensities of XCH2CH2SCH2CH2X and XCH2CH2SH (X=H, Cl), J. Phys. Chem.,
93, 577, 1989.
31 Donovan, W.H. and Famini, G.R., Conformational analysis of sulfur mustard from molecular mechanics,
semiempirical, and ab initio methods, J. Phys. Chem., 98, 3669, 1994.
32 Christesen, S.D., Vibrational spectra and assignments of diethyl sulfide, 2-chlorodiethyl sulfide and 2,2’-
dichlorodiethyl sulfide, J. Raman Spectrosc., 22, 459, 1991.
33 Donovan, W.H. and Famini, G.R., Jensen, J.O., and Hameka, H.F., Phosphorus, Sulfur, and Silicon, 80, 47,
1993.
34 Joo, T.H., Kim, K. and Kim, M.S., Surface-enhanced Raman study of organic sulfides adsorbed on silver, J.
Mol. Struct., 162, 191, 1987.
35 Inscore, F., and Farquharson, S., Detecting hydrolysis products of blister agents in water by surface-enhanced
Raman spectroscopy, in Proc. SPIE Vol. 5993, Vo-Dinh, T., Lieberman, R.A., and Gauglitz, G., Eds., SPIE,
Bellingham, Washington, 2005, 19.
36 Farquharson, S., A. Gift, P. Maksymiuk, F. Inscore, W. Smith, K. Morrisey, and S.D. Christesen, Chemical
agent detection by surface-enhanced Raman spectroscopy, in Proc. SPIE Vol. 5269, Sedlacek III, A.J,
Christesen, S.D., Colton, R. and Vo-Dinh, T., Eds., SPIE, Bellingham, Washington, 2004, 16.
37 www.mitretek.org/home.nsf/homelandsecurity/VX
38 Szafraniec, L. J.; Szafraniec, L. L.; Beaudry, W. T.; Ward, J. R., On the Stoichiometry of Phosphonothiolate
Ester Hydrolysis, CRDEC-TR-212, July 1990, AD-A250773
39 Epstein, J.; Callahan, J. J.; Bauer, V. E., The kinetics and mechanisms of hydrolysis of phophonothiolates in
dilute aqueous solution, Phosphorus, 1974, 4, 157-163.
40 Yang, Y-C., Chemical detoxification of nerve agent VX, Acc. Chem. Res., 1999, 32, 109-115
41 Farquharson, S, Gift, A., Maksymiuk, P. and Inscore, F., Surface-Enhanced Raman Spectra of VX and its
Hydrolysis Products, Appl. Spectrosc., 59, 654, 2005.
42 Tessier, P. M., Christesen, S. D., Ong, K. K., Clemente, E. M., Lenhoff, A. M., Kaler, E. W., and Velev, O. D.,
On-line spectroscopic characterization of sodium cyanide with nanostructured gold surface-enhanced Raman
spectroscopy substrates, Applied Spectroscopy, 56, 1524, 2002.
43 Kuncicky, D. M., Christesen, S. D., and Velev, O. D., Role of the micro- and nanostructure in the performance
of SERS substrates assembled from gold nanoparticles, Appl. Spectrosc., 59, 401, 2005.
44 Tessier, P. M., Christesen, S. D., Ong, K. K., Clemente, E. M., Lenhoff, A. M., Kaler, E. W., and Velev, O. D.,
Assembly of gold nanostructured films templated b colloidal crystals and used in surface-enhanced Raman
spectroscopy, in Proc. SPIE Vol. 4577, Christesen, S.D. and Sedlacek III, A.J, Eds., SPIE, Bellingham,
Washington, 2002, 53.
45 Shelton, R. D., Hass, J. W., and Wachter, E. A., Surface-enhanced Raman detection of aqueous cyanide, Appl.
Spectrosc., 48, 1007, 1994.
46 Mahoney, M. R., and Cooney, R. P., The evidence for stoichiometric silver oxide-cyanide phase in cyanide in
cyanide SERS from silver electrodes, Chem. Phys. Lett., 117, 71, 1985.
47 Benner, R. E., Dornhaus, R., Chang, R. K., and Laube, B. L., Correlations in the Raman spectra of cyanide
complexes adsorbed on silver electrodes with voltammograms, Surf. Sci., 101, 341, 1980.
48 Kellogg, D. S. and Pemberton, J. E., Effects of solution conditions on the surface-enhanced Raman scattering of
cyanide species at Ag electrodes, J. Phys. Chem., 91, 1120, 1987.
49 Billmann, J., Kovacs, G., and Otto, A., Enhanced Raman effect from cyanide adsorbed on a silver electrode,
Surf. Sci., 92, 153, 1980.
50 Premasiri, W. R., Clarke, R. H., Londhe, S., and Womble, M. E., Determination of cyanide in waste water by
low-resolution surface enhanced Raman spectroscopy on sol-gel substrates, J. Raman Spetrosc. 30, 827, 1999.
51 Vo-Dinh, T., Surface-enhanced Raman spectroscopy using metallic nanostructures, Trends Anal. Chem., 17,
557, 1998.
52 Wachter, E. A., Storey, J. M. E., Sharp, S. L., Carron, K. T., and Jiang, Y., Hybrid substrates for real-time
SERS-based chemical sensors, Appl. Spectrosc., 48, 193, 1995.
53 Adamson, A.W. and Gast, A.P., Physical Chemistry of Surfaces (Wiley Interscience, New York, 1997), 6th ed.,
p. 599.

14

Springer Book Kneipp Editor Appendix M Draft 1

25. Detecting chemical agents and their hydrolysis products in water
Stuart Farquharson, Frank E. Inscore and Steve Christesen
Real-Time Analyzers, Middletown, CT, 06457

25.1 INTRODUCTION

The use of chemicals as weapons was introduced during World War I. It is estimated that chlorine, phosgene and
sulfur-mustard (HD) resulted in an estimated death of 100,000 soldiers and 1 million injuries [1]. Over the next 20
years, chemicals designed specifically for warfare were developed; this included the substantially more toxic nerve
agents, tabun, sarin, and soman (GA, GB, and GD, respectively). Fortunately, these abhorrent chemicals were not
used in WWII, as world leaders feared reprisal attacks on their cities. During the Iran-Iraq war in the 1980s, the
Iraqis used HD, GA, GB, and GF (cyclo-sarin), and in 1988, Saddam Hussein used mustard and possibly nerve
agents in killing several thousand Kurds [1].

In more recent years, chemical agents have been used by terrorists. In Japan, the Aum Shinrikyo religious cult
released GB within the Tokyo subway system in 1995 [2]. The release of GB in this confined space had devastating
effects resulting in 12 fatalities and hospitalization of thousands. This event and the mailing of anthrax causing
spores through the US Postal System in 2001 demonstrated that deployment of chemical and biological agents do
not require sophisticated delivery systems, and a wide range of attack scenarios must be considered. Among these
scenarios is the deliberate poisoning of drinking water. This includes water supplies used in military operations and
water delivered to major cities from reservoirs and through distribution systems. Countering such an attack requires
detecting poisons in water rapidly, and at very low concentrations.

The required detection sensitivity for each agent depends on several factors, such as toxicity and hydrolysis (Table
25.1). In the case of cyanide (AC) it known that 4 milligrams per liter of water produces detectable changes in
human blood chemistry and 8 mg L-1 causes severe, but reversible symptoms [3]. The military has used this and
other toxilogical data to set a field drinking water standard (FDWS) for cyanide at 2 mg L-1 [4]. The FDWS
represents the maximum allowable concentration that is assumed safe when 15 L of water per day is consumed over
5 days (expected soldier intake in arid climates). Human toxicity data for the other chemical warfare agents in water
have, in general, not been determined. The normal route of exposure for chemical warfare agents is inhalation, and
most of the toxicity data is given as the LCt50s [1], the concentration that is lethal to 50% of an exposed population
as a function of exposure time. In the case of mustard, animal studies along with the inhalation LCt50, the oral lethal
dosage of 0.7 mg per kg of body mass (LD50), and modeling studies [5], have been used to set the FDWS at 0.047
mg L-1. Similar analyses of LCt50s and LD50s for GB and VX have been used to set their FDWS at 0.0046 and
0.0025 mg L-1, respectively. The FDWS concentrations have also been used by the military to set the minimum
detection requirement for poisons in water.

Table 25.1. Military field drinking water standard [4], lethal exposures and dosages [1,3,5], and water properties for
selected chemical warfare agents.
Chemical FDWS LCt50 LD50 Water Hydrolysis Hydrolysis
5-day/15L inhalation oral Solubility Half-Life* Product
(mg L-1) (mg-min m-3) (mg kg-1) at 25°C
HCN (AC) 2 2000 - - CN
NaCN 480 g L-1
Mustard (HD) 0.047 900 0.7 0.92 g L-1 2-30 hours TDG
Sarin (GB) 0.0046 70 2 completely 20-40 hours IMPA, MPA
miscible
VX 0.0025 35 0.07 150 g L-1 82 hours DIASH, EMPA,
EA2192, MPA

In order to detect these poisons in water, their properties in water must also be considered, i.e. the solubility, rate of
hydrolysis, and hydrolysis products formed. In the case of cyanide, as HCN, KCN, or NaCN, all of these chemicals
are extremely soluble in water (completely miscible, 716, and 480 g L-1, respectively) [6]. In solution the cyanide

Springer Book Kneipp Editor Draft 2

ion is formed in equilibrium with the conjugate acid, HCN (Figure 25.1A), according to the Ka of 6.15x10-10 [7 ]. In
the case of cyanide then it is important to know the pH, if one form of the chemical is to be detected versus the
other. For example, if 2 mg L-1 of NaCN is added to water (the FDWS), then 1.25 mg L-1 of CN- and 0.75 mg L-1 of
HCN will be present.

A

B

C

D

Figure 25.1. Hydrolysis reaction pathways for A) CN, B) HD, C) GB, and D) VX.

In the case of sulfur-mustard, the situation is somewhat more complex. It is marginally soluble in water tending to
form droplets, and hydrolysis occurs at the droplet surface. This property has made measuring the hydrolysis rate
constant difficult, and half-lives anywhere from 2 to 30 hours are reported [8]. Chemically, the hydrolysis of HD
involves the sequential replacement of the chlorine atoms by hydroxyl groups through cyclic sulfonium ion
intermediates to form thiodiglycol (TDG, Figure 25.1B) [9]. If a median hydrolysis rate is assumed, then early
detection of poisoned water will require measuring HD, while post-attack or downstream monitoring will require
measuring TDG. For sarin, the analysis is more straightforward, since it dissolves readily into water and it is stable
for a day or more. In this case, detecting poisoned water will largely require measuring sarin, while monitoring the
attack will require detecting its sequential hydrolysis products, isopropyl methylphosphonic acid (IMPA) and methyl
phosphonic acid (IMPA, MPA, respectively, Figure 25.1C) [8,10,11]. The other hydrolysis products, hydrofluoric
acid and 2-propanol, are too common to provide definitive evidence of water poisoning and their measurement
would be of limited value. VX is reasonably soluble, and like sarin, is fairly persistent with a hydrolysis half-life
greater than 3 days [12]. Unfortunately, one of its hydrolysis products, known as EA2192, is considered just as
toxic as VX, more soluble and more persistent in water [13]. Consequently, detecting the early stages of poisoning
water should focus on measuring VX, while longer term monitoring should focus on EA2192.

The earliest technologies developed for CWA detection were based on electrochemical, ionization, or colorimetric
analysis. Examples of the latter include phosgene, M8 and M9 tape, which change color when in contact with a
sample like pH paper. Although these tapes are easy to use, they are not generally agent specific and suffer from a
high percentage of false-positives [14]. For example, M8 changes color when in contact with common solvents such
as acetonitrile, ethanol, methanol, or common petroleum products such as brake fluid, lighter fluid, or WD-40 [15].
More rigorous laboratory methods have been successfully developed to detect chemical agents with minimum false-
positive responses. More than a decade ago, Black et al. demonstrated the ability of combining gas chromatography


with mass spectrometry detection (GC/MS) to measure sarin and mustard [16]. Sega at al. used GC with a
phosphorous-selective flame ionization detector to analyze nerve agent hydrolysis products in groundwater [17],
while several researchers used capillary electrophoresis (CE) to measure chemical warfare agents and their
hydrolysis products [18,19,20]. The sensitivity of these techniques has improved by two orders of magnitude from 1
mg L-1 to 0.01 mg L-1 in 10 years. A comprehensive development of these techniques was undertaken by Creasy et
al. in analyzing chemical weapon decontamination waste from the Johnston Atoll [11,21]. These researchers used
GC/MS for nerve agents, GC coupled atomic emission detection for arsenic compounds, LC/MS for mustard
compounds, and CE with ultraviolet absorption detection for alkyl phosphonic acids. Detection limits of 0.02 and
0.140 mg L-1 were reported for nerve agents and mustard, respectively. Detection of the alkyl phosphonic acids
have proven more difficult, and Liu, Hu and Xie recently used GC/MS to detect mg L-1 concentrations of these
degradation products [22]. However, they concede that all of these separation methods require extraction,
derivatization, and repeated column calibration, making them labor intensive, time consuming (typically 30 to 60
minutes), and less than desirable for field use. Another variant of these separation/mass detection technologies is ion
mobility spectrometry (IMS) [23]. This technology has been successfully developed to measure explosives in air
samples, and commercial products can be found at most airports [24]. Eiceman et al. have investigated the ability of
IMS to measure organophosphorous compounds in air [25], while Steiner et al. have investigated IMS to measure
chemical agent simulants in water [26]. In the latter case, electrospray ionization was coupled to the sample entry
point of an IMS, and a time-of-flight MS was added as an orthogonal detector. Water samples spiked with 10 mg
L-1 diisopropylmethylphosponate and thiodiglycol could be measured in 1-min, once sample pretreatment was
accomplished. It is worth noting that with proper care these MS-based technologies are likely to detect chemical
agents with virtually no false-positives, but detection limits are still insufficient by 1 to 2 orders of magnitude in the
case of nerve agents and their hydrolysis products.

More rapid analysis of agents in the solid, liquid and gas phase has been demonstrated by vibrational spectroscopy
[27-31]. Hoffland et al. reported infrared absorbance spectra and absolute Raman cross sections for several
chemical agents [27], while Christesen measured Raman cross sections for sarin, tabun, mustard gas, and VX [28].
Again, however these technologies also have limitations. Raman spectroscopy is simply not a very sensitive
technique, and detection limits are typically 0.1% (1000 ppm). And infrared spectroscopy would have limited value
in analyzing poisoned water, since the very strong infrared absorption of water would obscure most other chemicals
present. Nevertheless, efforts to overcome these limitations have been demonstrated. Braue and Pannella quantified
the G-series nerve agents (tabun, sarin, and soman) in terms of infrared attenuated total reflectance using a circle-
cell [29].

Enormous improvements in sensitivity for Raman spectroscopy can be achieved through surface-enhancement [32].
The interaction of surface plasmon modes of metal particles with target analytes can increase scattering efficiency
by as much 14-orders of magnitude, although 6-orders of magnitude are more common. The details of surface-
enhanced Raman spectroscopy (SERS) can be found in the beginning of this book. The utility of SERS to measure
chemical agents was first demonstrated by Alak and Vo-Dinh by measuring several organophosphonates as
simulants of nerve agents on a silver-coated microsphere substrate [33]. Spencer, et al. used SERS to measure
cyanide, MPA, HD and EA2192 on electrochemically roughed gold or silver foils [34,35,36]. However, in all of
these measurements, the sample needed to be dried on the substrates to obtain the best sensitivity (e.g. 0.05 mg L-1
for MPA). More recently, Tessier et al. obtained SERS of 0.04 mg L-1 cyanide in a stream flowing over a substrate
formed by a templated self-assembly of gold nanoparticles [37]. However, optimum sensitivity required
introduction of an acid wash and the measurements were irreversible.

In the past few years, we have also been investigating the ability of SERS to measure chemical agents at 0.001 mg
L-1 in water and with sufficient spectral uniqueness to distinguish the agent and its hydrolysis products [38-43]. In
our work, we have developed silver-doped sol-gels as the SERS-active medium. These sol-gels can be coated on the
inside walls of glass vials, such that water samples can be added to perform point-analysis, or they can be
incorporated into glass capillaries, such that flowing measurements can be performed [44]. Here, both sampling
devices were used to measure and compare SER spectra of AC, HD, VX and several of their hydrolysis products,
TDG, EA2192, EMPA, and MPA. In addition, a field-usable Raman analyzer was used to measure 0.01 mg L-1
cyanide flowing in water with a detection time of less than 1-min.


25.2 EXPERIMENTAL
Sodium cyanide, 2-hydroxyethylethyl sulfide (HEES), 2-chloroethylethyl sulfide (CEES) and methylphosphonic
acid (MPA) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Ethyl methylphosphonic
acid (EMPA), isopropyl methylphosphonic acid (IMPA), 2-(diisopropylamino) ethanethiol (DIASH), and
thiodiglycol (TDG, bis(2-hydroxyethyl)sulfide) were purchased from Cerilliant (Round Rock, TX). Highly distilled
sulfur mustard (HD, bis(2-chloroethyl)sulfide), isopropyl methylphosphonofluoridate (GB), ethyl S-2-
diisopropylamino ethyl methylphosphonothioate (VX), and ethyl S-2-diisopropylamino methylphosphonothioate
(EA2192) were obtained at the U.S. Army’s Edgewood Chemical Biological Center (Aberdeen, MD) and measured
on-site. All samples were initially prepared in a chemical hood as 1000 parts-per-million (1 g L-1 or 0.1% by
volume, Environmental Protection Agency definition) in HPLC grade water (Fischer Scientific, Fair Lawn, NJ) or in
some cases methanol or ethanol (Sigma-Aldrich) to minimize hydrolysis.

Once prepared, the samples were transferred into 2-ml glass vials internally coated with a silver-doped sol-gel
(Simple SERS Sample Vials, Real-Time Analyzers, Middletown, CT) or drawn by syringe or pump into 1-mm
diameter glass capillaries filled with the same SERS-active material [45,46,47]. In the case of flow measurements, a
peristaltic pump (variable flow mini-pump, Control Co., Friendswood, TX) was used to flow the various cyanide
solutions through a SERS-active capillary at 1 mL min-1. The vials or capillaries were placed on aluminum plates
machined to hold the vials or capillaries on a standard XY positioning stage (Conix Research, Springfield, OR),
such that the focal point of an f/0.7 aspheric lens was positioned just inside the glass wall. The probe optics and
fiber optic interface have been described previously [40]. SER spectra were collected using a Fourier transform
Raman spectrometer equipped with a 785 nm diode laser and a silicon photo-avalanche detector (IRA-785, Real-
Time Analyzers). All spectra were nominally collected using 100 mW, 8 cm-1 resolution, and 1-min acquisition
time, unless otherwise noted. Complete experimental details can be found in Reference 48. For added safety, all
samples were measured in a chemical hood. In the case of actual agents measured at Edgewood, the FT-Raman
instrument was placed outside the laboratory and 30 foot fiber optic and electrical cables were used to allow remote
SERS measurements and plate manipulation.

25.3 RESULTS AND DISCUSSION

25.3.1 Cyanide. Sodium cyanide completely dissolves in water forming the ions in equilibrium with the conjugate
acid, HCN as described above. Concentrations of 1.0, 0.1, and 0.01 mg L-1 result in CN- concentrations of 0.52,
0.016, and 0.00021 mg L-1 as the corresponding pH decreases from just above the pKa of 9.21 at 9.24 to 8.48 and
7.54. This is significant in that only CN- appears to interact sufficiently with silver to produce a SER spectrum, and
no spectral signal is observed below pH 7 (except on electrodes at specific potential conditions [49]). The SER
spectra of cyanide are dominated by an intense, broad peak at 2100 cm-1 attributed to the C≡N stretch (Figure 25.2).
This mode occurs at 2080 cm-1 in Raman spectra of solutions, and the frequency shift in SER spectra is attributed to
a strong surface interaction, which is supported by the appearance of a low frequency peak at 135 cm-1 due to a Ag-
CN stretch (not shown). It is also observed that as the concentration decreases, the CN peak shifts to 2140 cm-1.
This shift has been attribute to the formation of a tetrahedral Ag(CN)32- surface structure [50], as well as to CN
adsorbed to two different surface sites [51]. Alternatively, it has also been suggested that at concentrations near and
above monolayer coverage, the CN- species is forced to adsorb end-on due to crowding, and at lower concentrations
the molecule can reorient to lie flat. This suggests that the 2100 and 2140 cm-1 peaks correspond to the end-on and
flat orientations, respectively. However, a previous concentration study of cyanide on a silver electrode observed
the reverse trend, i.e. greater intensity was observed for the 2100 cm-1 peak at low concentration [49].

Repeated measurements of cyanide in the SERS-active vials consistently allowed measuring 1 mg L-1 (1 ppm), but
rarely below this concentration (Figure 25.2A). Nevertheless, this sensitivity is in general sufficient for point
sampling of water supplies. In the case of continuous monitoring of water, the capillaries are a more appropriate
sampling format, and they also allowed routine measurements at 0.01 mg L-1 and repeatable measurements at 0.001
mg L-1 (1 ppb, Figure 25.2B). Employing this format, a 50 mL volume of 0.01 mg L-1 cyanide solution was flowed
at 2.5 mL min-1 through a SERS-active capillary, and spectra were recorded every 20 seconds. As Figure 25.3
shows, the cyanide peak was easily discerned as soon as the solution entered the capillary and remained relatively
stable over the course of the experiment. It is worth noting, as indicated above, that the SERS peak in Figure 25.3 is
in fact due to 210 ng L-1!


A B

Figure 25.2. Surface enhanced Raman spectra of CN in water in silver-doped sol-gel A) coated glass vials and B)
filled glass capillaries. All spectra were recorded using 100 mW of 785 nm in 1-min and at a resolution of 8 cm-1.

Figure 25.3. 2100 cm-1 peak height measured during continuous flow of a 0.01 mg L-1 (10 ppb) cyanide in water.
Surface-enhanced Raman spectra are shown for 1 and 6 min after sample introduction. A 2.5 mL min-1 flow rate
was used and spectra were recorded every 20 sec using 100 mW of 785 nm.


25.3.2 HD and CEES. The surface-enhanced Raman spectrum of HD is dominated by a peak at 630 cm-1 with an
extended high frequency shoulder composed of at least two peaks evident at 695 and 830 cm-1, as well as a
moderately intense peak at 1045 cm-1 (Figure 25.4A). The latter peak is assigned to a CC stretching mode, based on
the assignment for a peak at 1040 cm-1 in the Raman spectrum of HD [52]. The assignment of the 630 cm-1 peak is
less straightforward, since the Raman spectrum of HD contains five peaks in this region at 640, 655, 700, 740, and
760 cm-1 [40,52]. Theoretical calculations for the Raman spectrum of HD indicate that the first three peaks are due
to CCl stretching modes, and the latter two peaks to CS stretching modes [53]. Based on these calculations, and the
expected interaction between the chlorine atoms and the silver surface, it is reasonable to assign the 630 cm-1 SERS
peak to a CCl mode [40]. However, recent SERS measurements of diethyl sulfide produced a very simple spectrum
with an intense peak at 630 cm-1 [54,55], strongly suggesting CS or CSC stretching modes as the appropriate
assignment for this peak [56]. The authors of the theoretical treatment concede that the CCl and CS assignments
could be reversed [53]. The CS assignment also indicates that HD interacts with the silver surface through the sulfur
electron lone pairs. But, interaction between chlorine and silver is still possible and may be responsible for the 695
cm-1 peak. The 830 cm-1 peak is left unassigned.

A

B

Figure 25.4. Surface-enhanced Raman spectra of A) HD in methanol and B) TDG in water. Spectral conditions as
in Fig. 25.2, samples were 1 g L-1.

The surface-enhanced Raman spectrum of TDG is also dominated by a peak at 630 cm-1 with minor peaks at 820,
930, 1210, and 1275 cm-1 (Figure 25.4B). Again, the 630 cm-1 peak is preferably assigned to a CSC stretching mode
versus a CCl mode, especially since the chlorines have been replaced by hydroxyl groups. Furthermore, the lack of
a 695 cm-1 peak in the TDG spectrum supports the assignment of this peak in the HD spectrum to a CCl mode. The
930, 1210 and 1275 cm-1 SERS peaks are assigned to a CC stretch with CO contribution, and two CH2 deformation
modes (twist, scissors, or wag) based on the assignments for the corresponding peaks at 940, 1230 and 1290 cm-1 in
the Raman spectrum of TDG [52,54 ]. It is worth noting that irradiation at high laser powers or for extended periods
produces peaks at 715 and 1010 cm-1, which are attributed to a degradation product, such as 2-hydroxy ethanethiol
[54].

The SERS of CEES is very similar to HD, dominated by a peak at 630 cm-1 that is accordingly assigned to a CS or
CSC stretching mode (Figure 25.5A). This peak also has a high frequency shoulder centered at 690 cm-1, and a third
peak appears at 720 cm-1 in this region. Again, these can be assigned to CCl or CS modes. The quality of this
spectrum also reveals weak peaks at 1035, 1285, 1410, and 1445 cm-1. Peaks at 1035, 1285, 1425, and 1440 cm-1


appear in the Raman spectrum of CEES, and the previous peak assignments are used here [52], i.e. the first peak is
assigned to a CC stretch, while the remaining peaks are assigned to various CH2 deformation modes.

A

B

Figure 25.5. Surface-enhanced Raman spectra of A) CEES and B) HEES. Spectral conditions as in Fig. 25.2,
samples were 1 g L-1 in methanol.

Replacing the chlorine atom of CEES by a hydroxyl group in forming HEES produces SER spectral changes
analogous to those cited above for HD and TDG. Again, the SER spectrum is dominated by an intense peak at 630
cm-1 attributed to a CS or CSC stretching mode, and the other CEES peaks in this region, specifically the 720 cm-1
peak, disappear (Figure 25.5B). Peaks with modest intensity at 1050 and 1145 cm-1 are assigned to a CC stretching
mode and CH2 deformation, respectively. A new peak at 550 cm-1 is likely due to a skeletal bending mode, such as
CSC, SCC, or CCO. Finally, it is worth stating that HD, TDG, CEES, and HEES all produce moderately intense
peaks at 2865 and 2925 cm-1 (not shown), that can be assigned to symmetric and asymmetric CH2 stretching modes.

Only a limited number of measurements of HD were performed to evaluate sensitivity, due to the safety
requirements. HD was repeatedly observed at 1 g L-1 and usually observed at 0.1 g L-1 (100 ppm) in the SERS-
active vials [40] But even at the latter concentration, substantial improvements in sensitivity are required to
approach the required 0.05 mg L-1 (50 ppb) sensitivity. More extensive experiments were performed on HD’s
hydrolysis product, TDG since this chemical is safely handled in a regular chemical lab. Flowing TDG through
SERS-active capillaries allowed repeatable measurements at 10 mg L-1, and routine measurements at 1 mg L-1 (1
ppm) [55]. These SERS measurements of TDG suggest that the required HD sensitivity may be achievable using
this technique. Similar flowing measurements in capillaries for HD, CEES, and HEES have not been performed.

25.3.3 Sarin. SERS measurements of GB have not been made, but its primary hydrolysis products, IMPA and
MPA, have been measured using the SERS-active vials. The SERS of IMPA is very similar to its Raman spectrum
[42], which in turn is very similar to the Raman spectrum of sarin [28]. The SER spectrum is dominated by a peak
at 715 cm-1 (Figure 25.6A), which is assigned to a PC or PO plus skeletal stretching mode, as is a weak peak at 770
cm-1. These assignments are also consistent with a theoretical treatment of the Raman spectrum for sarin [57].
Similarly, a modest peak at 510 cm-1 can be assigned to a PC or PO plus skeletal bending mode. Other SERS peaks
of modest intensity occur at 875, 1055, 1415, and 1450 cm-1, and based on the spectral analysis of sarin and the
Raman spectrum of IMPA with peaks at 880, 1420, and 1455 cm-1, are assigned to a CCC bend, a PO3 stretch, a CH3
bend, and a CH2 rock, respectively.


A

B

C

Figure 25.6. Surface-enhanced Raman spectra of A) IMPA, B) MPA, and C) EMPA. Spectral conditions as in Fig.
25.2, samples were 1 g L-1 in water.

MPA has been well characterized by infrared and Raman spectroscopy [58,59], as well as normal coordinate
analysis [60], and the literature assignments are used here for the SERS of MPA. The SER spectrum is dominated
by a peak at 755 cm-1, which is assigned to the PC symmetric stretch (Figure 25.6B). In comparison to IMPA, it is
clear that removing the isopropyl group shifts this frequency substantially (40 cm-1), as the mode becomes a purer
PC stretch. Additional peaks with comparatively little intensity occur at 470, 520, 960, 1040, 1300, and 1420 cm-1,
and are assigned to a PO3 bending mode, a C-PO3 bending mode, a PO3 stretching mode, another PO3 bending
mode, and two CH3 deformation modes (twisting and rocking).

SERS-active vials allowed repeatable measurements of MPA at 10 mg L-1 and routine measurements at 1 mg L-1,
and repeatable measurements of IMPA at 100 mg L-1 and routine measurements at 10 mg L-1. Again, however,
substantial improvements in sensitivity are required to achieve the minimum requirement of 0.004 mg L-1.

25.3.4 VX. The hydrolysis of VX can occur along two pathways (Figure 25.1D) [11,22], either being converted to
DIASH and EMPA or EA2192 and ethanol with the former pathway favored four to one. These products also
hydrolyze, and EMPA forms MPA and ethanol, while EA2192 forms DIASH and MPA. Here the SER spectra of
VX, EA2192 and DIASH are compared, while EMPA is compared to IMPA and MPA.

The SER spectrum of VX is similar to its Raman spectrum with corresponding peaks at 375, 460, 540, 730, 1095,
1300, 1440, and 1460 cm-1 (Figure 25.7A). Since a computer predicted Raman spectrum contains most of the
measured Raman spectral peaks [43,61], it is used to assign the above SERS peaks respectively to an SPO bend, a
CH3-P=O bend, a PO2CS wag, an OPC stretch, a CC stretch, and three CHn bends. As previously described for
CEES and HD, the 730 cm-1 peak could alternatively be assigned to a CS stretch, but the SER spectra of these
chemicals suggest otherwise.

The SER spectrum of EA2192 is somewhat different than VX with the PO modes having limited intensity and the
NC3 modes having significant intensity (Figure 25.7B). Specifically, the EA2192 spectrum has moderately intense
peaks at 480, 585, 940, and 1125 cm-1 that can be assigned to an NC3 breathing mode, an NCC bending mode,
another NC3 stretching mode, and a NCC stretching mode. Two additional peaks with significant intensity at 695


and 735 cm-1 are assigned to a CS stretching mode and an OPC stretching mode, respectively. Two peaks of modest
intensity at 525 and 970 cm-1 are attributed to a PO2S bending mode and a PO2 stretching mode.

A

B

C

Figure 25.7. Surface-enhanced Raman spectra of A) VX, B) EA2192, and C) DIASH. Spectral conditions as in Fig.
25.2, samples were 1 g L-1 in water.

The SER spectrum of DIASH contains most of the NC3 modes cited previously for EA2192 (Figure 25.7C),
specifically peaks appear at 480, 585, 940, and 1120 cm-1, and can be assigned as above. Additional peaks at 740,
810, and 1030 cm-1, are assigned to CH bending, a combination of SC stretching and NC3 bending, and SCCN
bending modes, based on the Raman spectrum of DIASH [43]. A broad peak centered at 695 cm-1 also occurs that
has previously been assigned to an SC stretch, but the frequency and intensity of this mode in the HD and CEES
spectra above, makes this assignment less certain.

It is worth noting the similarity between the EA2192 and DIASH SER spectra, the principle difference being the
addition of the SCCN bending mode at 1030 cm-1 for the latter. This may simply be due to the fact that both
molecules interact through the sulfur with the metal surface to similar extents resulting in similar spectra. However,
it is also possible that the EA2192 spectrum is of DIASH formed either by hydrolysis or photo-degradation. Since
the sample was measured within one hour of preparation, and the hydrolysis half-life is on the order of weeks [12],
the former explanation seems unlikely. Since the peak intensities did not change during these measurements, photo-
degradation catalyzed by silver also seems unlikely. Further experiments are required to clarify this point.

The SER spectrum of the other hydrolysis product formed from VX, EMPA, is shown in Figure 25.6. It is included
with MPA and IMPA, the hydrolysis products of GB, for convenient spectral comparison of these structurally
similar chemicals. The spectrum is dominated by a peak at 745 cm-1 with a substantial low frequency shoulder at
725 cm-1. Both are assigned, similarly to IMPA, to PC or PO plus skeletal stretching modes. In fact, virtually all of
the peaks in the SER spectrum correspond to peaks of similar frequency in the SER spectrum of IMPA, and are
assigned as follows: the peaks at 480 and 500 cm-1 to PC or PO plus skeletal bends; 890, 1415, and 1440 cm-1 to
CHn deformations; 945 and 1060 cm-1 to POn stretches; and 1095 to a CO or CC stretch. A peak at 1285 cm-1 is
assigned to a CHn deformation based on the MPA spectral assignment for a peak at 1300 cm-1.

In this series of chemicals VX and EA2192 were routinely measured at 100 mg L-1, and on occasion at 10 mg L-1
using the SERS-active vials. Again, however, only a limited number of measurements were attempted. More


extensive measurements of EMPA using the SERS-active capillaries allowed repeatable measurements of 10 mg L-1
and routine measurements of 1 mg L-1. No concentration studies of DIASH were undertaken.

25.4 Conclusions

The ability to obtain surface-enhanced Raman spectra of several chemical agents and their hydrolysis products has
been demonstrated using silver-doped sol-gels. Two sampling devices, SERS-active vials and capillaries, provided
a simple means to measure water samples containing chemical agents. No sample pretreatment was required and all
spectra were obtained in 1 minute. It was found that the SER spectra can be used to identify chemical agents by
class. Specifically, cyanide contains a unique peak at 2100 cm-1, HD and CEES both have a unique peak at 630
cm-1, while VX has a unique peak at 540 cm-1. In the case of HD and CEES, their hydrolysis products produce very
similar spectra, and it may be difficult to determine relative concentrations in an aqueous solution. In the case of the
VX hydrolysis products, EA2192 and DIASH were spectrally similar, as was IMPA and MPA. However, there
appears to be sufficient differences when comparing entire spectra, such that chemometric approaches might allow
successful compositional analysis of aqueous solutions.

The SERS-active vials and capillaries provided sufficient sensitivity to measure cyanide below the required 2 mg L-1
sensitivity either as a point measurement or as a continuous flowing stream measurement. Measurements of TDG
suggest that the sensitivity requirements for it and HD may be attainable with modest improvements. In contrast,
the vials and capillaries did not provide sensitivity sufficient to meet the requirements of VX. In this case
substantial improvements in sensitivity are required and are being pursued.

25.5 Acknowledgements

The authors are grateful for the support of the U.S. Army (DAAD13-02-C-0015, Joint Service Agent Water Monitor
program) and the Environmental Protection Agency (EP-D-05-034). The authors would also like to thank Mr.
Chetan Shende for sol-gel chemistry development.

25.6 References

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3 Committee on Toxicology: Review of Acute Human-Toxicity Estimates for Selected Chemical-Warfare Agents.
(Nat Acad Press, 1997)
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5 T.C. Marrs, R.L. Maynard, F.R. Sidell: Chemical Warfare Agents: Toxicology and Treatment. (John Wiley and
Sons, 1996)
6 Material Safety Data Sheets, available at www.msds.com
7 D.R. Lide, Ed: Handbook of Chemistry and Physics: (CRC Press, 1997) p. 8-43
8 N.B. Munro, S.S. Talmage, G.D.Griffin, L.C. Waters, A.P. Watson, J.F. King, V. Hauschild: Environ. Health
Perspect. 107, 933 (1999)
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10 G. Wagner, Y. Yang: Ind. Eng. Chem. Res. 41, 1925 (2002)
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12 Y. Yang: Acc. Chem. Res. 32, 109 (1999)
13 Y. Yang, J. Baker, J. Ward: Chem. Rev. 92, 1729 (1992)
14 B. Erickson: Anal. Chem. News & Features, 397A (1998)
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21 W.R. Creasy: Am. Soc. Mass. Spectrom. 10, 440 (1999)
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24 See products from Smiths Detection, Bruker Daltronics, etc.
25 N. Krylova, E. Krylov, G.A. Eiceman: J. Phys. Chem. 107, 3648 (2003)
26 W.E. Steiner, B.H. Clowers, L.M. Matz,W.F. Siems, H.H. Hill Jr.: Anal. Chem. 74, 4343 (2002)
27 L.D. Hoffland, R.J. Piffath, J.B. Bouck: Opt. Eng. 24, 982 (1985)
28 S.D. Christesen: Appl. Spectrosc. 42, 318 (1988)
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30 C-H. Tseng, C.K. Mann, T.J. Vickers: Appl. Spectrosc. 47, 1767 (1993)
31 S. Kanan, C. Tripp: Langmuir 17: 2213 (2001)
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33 A.M. Alak, T. Vo-Dinh: Anal. Chem. 59, 2149 (1987)
34 K.M. Spencer, J. Sylvia, S. Clauson, J. Janni: Proc. SPIE 4577,158 (2001)
35 S.D. Christesen, M.J. Lochner, M. Ellzy, K.M. Spencer, J. Sylvia, S. Clauson: 23rd Army Sci. Conf. (2002)
36 S.D. Christesen, K.M. Spencer, S. Farquharson, F.E. Inscore, K. Gosner, J. Guicheteau: In: S. Farquharson, Ed.
Applications of Surface-Enhanced Raman Spectroscopy. (CRC Press, in preparation)
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(2002)
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40 S. Farquharson, A. Gift, P. Maksymiuk, F. Inscore, W. Smith, K. Morrisey, S. Christesen: Proc. SPIE 5269, 16
(2004)
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Appendix N

Detecting hydrolysis products of blister agents in water
by surface-enhanced Raman spectroscopy
Frank Inscore and Stuart Farquharson
Real-Time Analyzers, Middletown, CT, 06457

ABSTRACT

Protecting the nation’s drinking water from terrorism, requires microg/L detection of chemical agents and their
hydrolysis products in less than 10 minutes. In an effort to aid military personnel and the public at large, we have been
investigating the ability of surface-enhanced Raman spectroscopy (SERS) to detect microgram per liter (part-per-billion)
concentrations of chemical agents in water. It is equally important to detect and distinguish the hydrolysis products of
these agents to eliminate false-positive responses and evaluate the extent of an attack. Previously, we reported the SER
spectra of GA, GB, VX and most of their hydrolysis products. Here we extend these studies to include the chemical
agent sulfur-mustard, also known as HD, and its principle hydrolysis product thiodiglycol. We also report initial
continuous measurements of thiodiglycol flowing through a SERS-active capillary.

Keywords: chemical warfare agent detection, CWA, hydrolysis, SERS, Raman spectroscopy

1. INTRODUCTION

The July 2005 terrorist bombings of the London transit system are a stark reminder that such attacks on the United
Kingdom and the United States will continue. Countering such attacks requires recognizing likely deployment scenarios
and having the required technology to rapidly detect the deployment event. In addition to the expected use of chemical
agents released into the air, terrorists may also poison water supplies with chemical warfare agents (CWAs). The
National Strategy for Homeland Security designates the Environmental Protection Agency with the task of securing the
nations drinking water.1 Presently, the EPA employs several field test kits to monitor drinking water supplies, and gas
chromatography coupled with mass spectrometry in supporting laboratories to confirm positive responses.2
Unfortunately, these test kits are prone to false-positive responses, and follow-up analysis typically takes a day. This is
entirely inadequate for the prevention of widespread illness and potential fatalities.

In the past several years we have been investigating the use of surface-enhanced Raman spectroscopy (SERS) to be used
as a field-usable analyzer that can detect chemical agents in water at the required microg/L sensitivity and 10 minute
timeframe.3,4,5,6,7 The expected success of SERS is based on the million-fold or more Raman signal increase obtained
when a molecule interacts with surface plasmon modes of metal nanoparticles.8 In the case of cyanide, an industrial-
based CWA and methyl phosphonic acid, the final hydrolysis product for the nerve agents, we have measured at or
below 10 microg/L in one minute.9 The expected success of SERS is also based on the unique set of Raman spectral
peaks associated with the molecular vibrational modes of each molecule. The unique SER spectra should not only
reduce false-positive responses, but also allow discriminating hydrolysis products of CWAs. This is important, since
CWAs can hydrolyze rapidly in the presence of water,10 and detection of the hydrolysis products could allow
determining 1) the state of an attack (ratio of CWA to hydrolysis product(s)), 2) the point of attack initiation, and 3) the
continued extent and severity of the CWA attack throughout a water distribution system.

Previously, we used SERS to measure sarin, tabun, VX, and EA2192, and their respective hydrolysis products.3,4,6,7 Here
we extend these studies to include the chemical warfare agent sulfur-mustard, designated HD, and its primary hydrolysis
product thiodiglycol (TDG, Figure 1). The physical and chemical properties of this blister agent are well known. It’s
solubility in water is 0.92 g/L with a hydrolysis half-life of 8.5 min (both at 25 C).10 HD has an oral LD50 of 0.7 mg/kg
in humans,11 and the military drinking water guideline places the 5-day 5L limit at 100 microg/L.12 TDG is relatively
non-toxic, very water soluble at 690 g/L, and stable in water with a hydrolysis half-life of approximately 6 days.
Accordingly, a reasonable sensitivity goal to ensure safe water is placed at 10 microg/L for HD and an equivalent goal to
map HD usage is placed at 10 microg/L for TDG.13

SPIE-2005-5993 19

H2O
+ 2HCl

Figure 1. Hydrolysis of bis(2-chloroethyl)sulfide (HD) to bis(2-hydroxyethyl)sulfide (TDG).

2. EXPERIMENTAL

Highly distilled sulfur mustard, designated HD (bis(2-chloroethyl)sulfide), was measured at the U.S. Army’s Edgewood
Chemical Biological Center (Aberdeen, MD). Thiodiglycol, designated TDG here (bis(2-hydroxyethyl)sulfide), was
purchased as an analytical reference material from Cerilliant (Round Rock, TX). TDG was measured at Real-Time
Analyzers, Inc. (RTA, Middletown, CT). All solvents, including methanol, ethanol, and HPLC water, as well as all sol-
gel precursor chemicals including AgNO3, tetramethyl orthosilicate, methyltrimethoxysilane, HNO3 and NaBH4, were
purchased from Sigma-Aldrich (St. Louis, MO). HD samples prepared for SERS analysis consisted of 0.1% v/v HD in
methanol. The methanol was used to minimize hydrolysis. The final concentration is 1000 parts-per-million (ppm, EPA
definition). TDG samples were prepared for SERS analysis using methanol for static measurements and HPLC grade
water for flow measurements. All HD measurements were performed in SERS-active vials (Simple SERS Sample Vials,
RTA),14 while all TDG measurements were performed in SERS-active capillaries (1-mm diameter glass capillaries filled
with silver-doped sol-gels).15,16 In the case of flow measurements, a peristaltic pump (variable flow mini-pump, Control
Co., Friendswood, TX) was used to flow the 1 and 10 ppm TDG samples through a SERS-active capillary at 1 mL per
min.

The vials or capillaries were mounted horizontally on an XY positioning stage (Conix Research, Springfield, OR), such
that the focal point of an f/0.7 aspheric lens was positioned just inside the glass wall. The probe optics and fiber optic
interface have been described previously.16 In all cases a 785 nm diode laser was used to deliver ~100 mW of power to
the SERS samples and 300 mW to the Raman samples. A Fourier transform Raman spectrometer equipped with a
silicon photo-avalanche detector (RTA, model IRA-785), was used to collect both the RS and SERS at 8 cm-1 resolution.

3. RESULTS AND DISCUSSION

The surface-enhanced and normal Raman spectra of HD have been measured and are shown in Figure 2. The SER
spectrum is dominated by a peak at 630 cm-1 with an extended high frequency shoulder composed of two or more peaks
(695, 830 cm-1), as well as a moderately intense peak at 1045 cm-1. It is possible to assign these peaks based on the
normal Raman spectrum of HD, and previous assignments.17 Theoretical studies assigned the 640, 655, 700 cm-1 peaks
to C-Cl stretching modes and the 740, and 760 cm-1 peaks to C-S stretching modes. Additional peaks are observed at
1040, 1190, 1270, 1295, 1410, 1425, and 1440 cm-1. The first peak is assigned to a C-C stretch, while the remaining
peaks are all CH2 deformation modes (scissors, twists, and wags). Based on these assignments, then only the C-Cl peak
maintains significant intensity in the SER spectrum occurring at 630 cm-1. If the C-Cl assignments are correct, then the
SER spectra suggest that the molecule to metal interaction is strongest through the chlorine end groups. Alternatively,
the electron lone pairs of the tetrahedrally coordinated sulfur of HD could interact with the silver surface. Consequently,
the 630 cm-1 SERS peak could be assigned to CS or CSC stretching modes (see below).18

The surface-enhanced and normal Raman spectra of TDG have been measured and are shown in Figure 3. The SER
spectrum is dominated by three peaks at 630, 715, and 1010 cm-1 with minor peaks at 400, 820, 930, 1210, 1275, 1410,
and 1460 cm-1. Similarly, the Raman spectrum contains two intense peaks at 660 and 1010 cm-1, while moderately
intense peaks occur at 400, 680 (shoulder), 735, 770, 830, 950, 1040, 1230, 1290, 1420, and 1465 cm-1. In both spectra,
the assignment of the peaks near 1000 cm-1 can be confidently assigned to C-C stretching modes, while the peaks from
1200 to 1465 cm-1 can be confidently assigned to various CH2 deformation modes. Here, however, it is difficult to
assign the 630 cm-1 SERS peak to a C-Cl mode, since the chlorines have been replaced by hydroxyl groups.

SPIE-2005-5993 20

Consequently, in the case of HD and TDG, assigning the 630 cm-1 peak to a CS or a CSC stretch, is favored. Although
the 620-680 cm-1 peaks are normally assigned to C-Cl modes, and the 700-750 cm-1 peaks to CS modes, most authors
concede that the reverse assignments are possible.

A A

B B

Figure 2. A) SERS and B) RS of HD. A) 0.1% v/v (1000 Figure 3. A) SERS and B) RS of TDG. A) 0.1% v/v in
ppm) in MeOH in a SERS-active vial, 100 mW of 785 nm, MeOH in SERS-active capillary, 100 mW of 785 nm, 1-
1-min, B) neat sol. in glass container, 300 mW of 785 nm, min, B) neat sol. in glass capillary, 300 mW of 785 nm,
5-min. 5-min.

Of possibly greater importance, is that the TDG SER spectrum is of high quality, with three distinct peaks. With the
goal of detecting this hydrolysis product of HD in water, a number of samples of decreasing concentration were prepared
and measured. As Figure 4 shows, these peaks are evident even at 10 ppm (0.001% v/v in methanol). However,
repeated measurements of 1 ppm did not yield any discernable peaks (lowest trace in Figure 4). Notwithstanding,
measurements were also performed in a flowing stream. Initial measurements of a 10 ppm sample yielded quality
spectra and prompted measurements of a 1 ppm sample. As Figure 5 shows, reasonable spectra are obtained, even at 1
minute resolution. It is worth stating that the 630 cm-1 peak was evident in all spectra collected over a 12 minute period.
There is an important difference between the TDG spectra recorded for static and flowing samples, namely that the 715
cm-1 peak is noticeably more intense in the static sample. This suggests that it may represent a photo-degradation
product. Further studies are required to clarify this point.

Figure 4. SERS of 1000, 100, 10 and 1 ppm TDG in Figure 5. SERS of 1 ppm TDG in water flowing
water (top to bottom). All in SERS-active capillaries, through a SERS-active capillary at 1, 2, 3, 4, and 5 min.
100 mW of 785 nm, 1-min. (top to bottom), 100 mW of 785 nm, 1-min each.

SPIE-2005-5993 21

Final Report Daad13 02 C 0015 Part5 App L P

Final Report Daad13 02 C 0015 Part5 App L P

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